kai yang - university of toronto

THE DIFFERENTIAL REGULATION OF SUBTYPES OF N-METHYL-D-ASPARTATE RECEPTOR IN CA1 HIPPOCAMPAL NEURONS BY G

PROTEIN COUPLED RECEPTORS

by

Kai Yang

A thesis submitted in conformity with the requirements

of Doctor of Philosophy

Graduate Department of Physiology

University of Toronto

Copyright by Kai Yang (2011)

ii



By

Kai Yang

Doctor of Philosophy

Department of Physiology University of Toronto

2011

The role of NMDAR subtypes in synaptic plasticity is very controversial partially

caused by the lack of specific GluN2A containing NMDA receptor (GluN2AR)

antagonists Here we took a novel approach to selectively modulate NMDAR subtype

activity and investigated its role in the induction of plasticity Whole cell recording in

both acutely isolated CA1 cells and hippocampal slices demonstrated that pituitary

adenylate cyclase activating peptide 1 receptors (PAC1 receptors) which are Gαq

coupled receptors selectively recruited Src kinase and enhanced currents mediated by

GluN2ARs In addition biochemical experiments showed that the activation of PAC1

receptors phosphorylated GluN2ARs specifically In contrast vasoactive intestinal

peptide receptors (VPAC receptors) which are Gαs coupled receptors selectively

stimulated Fyn kinase potentiated currents mediated by GluN2B containing NMDARs

(GluN2BRs) Furthermore dopamine D1 receptor activation (another Gαs coupled

receptor) specifically phosphorylated GluN2BRs Interestingly field recording

experiments showed that PAC1 receptor activation lowered the threshold for LTP whilst

iii

LTD was enhanced by dopamine D1 receptor activation In conclusion the activity of

GPCRs can signal through different pathways to selectively modulate absolute

contribution of GluN2ARs versus GluN2BRs in CA1 neurons via Src family kinases

Furthurmore Epac activated by some Gαs coupled receptors also modulated NMDAR

currents via a PKCSrc dependent pathway but whether it selectively modulates

NMDAR subtypes and has capacity to change the induction of plasticity requires further

study

By this means we can investigate the role of NMDAR subtypes in the direction

of synaptic plasticity by selectively modulating the activity of GluN2ARs or GluN2BRs

In addition based on my work some interfering peptides and drugs can be designed and

used to selectively inhibit the activity of GluN2BRs and GluN2ARs by interrupting Fyn-

and Src - mediated signaling cascade respectively It will provide new candidate drugs for

the treatment of some neurological diseases such as Alzheimer disease (AD) and

schizophrenia

iv

ACKNOWLEDGEMENTS

First I would like to express my deepest gratitude to my supervisor Dr

JFMacdonald for providing me the opportunity to pursue PhD degree in his lab I have

learned many valuable skills and techniques during my time in the lab This experience

will offer me new exciting prospects for my future Without his support encouragement

and patience I donrsquot think I could have gotten PhD degree I also acknowledge my

supervisory committee members Dr Michael Salter Lu-Yang Wang and John Roder for

their assistance and suggestion during my graduate study

I thank all the past and present members in the Macdonaldrsquos lab Especially I

would like to acknowledge Dr Michael Jackson for his technical assistance and advices

I am also very thankful to Lidia Brandes Natalie Lavine Catherine Trepanier Dr

Hongbin Li Gang Lei Oies Hussein Jillian Roberts and Cristi Orth for their help in the

lab

Finally from the bottom of my heart I appreciate the incredible support from my

parents Without their help I would not get through all the difficulties I met

v

TABLE OF CONTENTS

A Abstract ii B Acknowledgements iv C Table of Contents v D List of Figures viii E Abbreviations xi VI Section 1 ndash Introduction

11 Excitatory Synaptic Transmissin in the hippocampus 111 AMPAR 2 112 LTP and LTD 4 113 Physiological functions of LTP and LTD 7

12 NMDARs 9 13 NMDAR subunits

131 GluN1 subunits 10 132 GluN2 subunits 11 133 GluN3 subunits 18 134 Triheteromeric GluN1GluN2AGluN2B receptors 19

14 The modulation of NMDARs by SerineTheronine kinases and phosphatases 141 The modulation of NMDARs by serinetheronine kinases 21 142 The modulation of NMDARs by serinetheronine phosphatases 26

15 The modulation of NMDARs by Src family kinases and tyrosine phosphatases 151 The structure of Src family kinases 27 152 The modulation of NMDARs by Src family kinases 31 153 The modulation of NMDARs by tyrosine phosphatases 35 154 The regulation of LTP by SFKs 36

16 The regulation of NMDARs by GPCRs 37 17 Distinct functional roles of GluN2 subunits in synaptic plasticity 40 18 Metaplasticity 41 19 PACAPVIP system

191 PACAP and VIP 43 192 PACAPVIP receptors 45 193 Signaling pathway initiated by the activation of PACAPVIP 47 receptors 1104 The mechanism of NMDARs modulation by PACAP 48

110 The hippocampus 49 111 The pharmacology of GluN2 subunits of NMDARs 50 112 GluN2 subunit knockout mice 52 113 Overall hypothesis 55

VII Section 2 ndash Methods and Materials

vi

21 Cell isolation and whole cell recording 59 22 Hippocampal slice preparation and recording 61 23 Immunoprecipation and western blotting 63 24 Animals 64 25 Drugs and Peptides 64 26 Statistics 65 VIII Section 3 ndash Results

Project 1 The activation of PAC1 receptors (Gαq coupled receptors) selectively targets GluN2ARs and favours LTP induction

311 Hypothesis 67

312 Results 67 Project 2 The activation of VPAC receptors (Gαs coupled receptors) selectively targets GluN2BRs 321 Hypothesis 91 322 Results 91

X Section 4 - Discussion

41 The differential regulation of NMDAR subtypes by GPCRs 105 42 GPCR activation induces metaplasticity 107 43 The involvement of SFKs in the synaptic transmission

431 The differential regulation of NMDAR subtypes by SFKs 113 442 The trafficking of NMDARs induced by SFKs 114 443 The role of the scaffolding proteins on the potentiation of 116 NMDARs by SFKs 444 The involvement of SFKs in the synaptic plasticity in the 117 Hippocampus 445 The specificity of Fyn inhibitory peptide (Fyn (39-57)) 119

44 The functions of PACAPVIP in the CNS 441 The mechanism of NMDAR modulation by VIP 120

442 The regulation of synaptic transmission by PACAPVIP 123 System 443 The involvement of PACAPVIP system in learning and 126 Memory

444 The other functions of PACAPVIP system in the CNS 127 45 Significance 129

46 Future experiments 130 XI Section 5 ndash Appendix

Project 3 Epac activated by Gαs coupled receptors also modulates

vii

NMDARs

1 Introduction

51 cAMP effector Epac 136 52 Epac and Gαs coupled receptors 139 53 Epac mediated signaling pathways 139 54 Compartmentalization of Epac signaling 141 55 Epac selective agonist 8-pCPT-2prime-O-Me-cAMP 142 56 Epac mediates the cAMP dependent regulation of ion channel 144 Function 57 Hypothesis 145

2 Results 147

3 Discussion

58 The regulation of NMDARs by Epac 160 59 A role for Epac in the regulation of intracellular Ca2+ signaling 162 510 Epac reduces the presynaptic release 163 511 Epac and learing and memory 165

XII Section 6 ndash References 61 References 169

viii

LIST OF FIGURES Fig 11 The unique domains between Src kinase and Fyn kinase are not conserved 30

Fig 12 The structure of Src family kinases 32

Fig 13 PACAP selectively enhanced peak of NMDAR current 57

Fig 21 Representation of rapid perfusion system in relation to patched pyramidal 60

CA1 neurons

Fig 311 The activation of PAC1 receptors selectively modulated GluN2ARs 78

over GluN2BRs in acutely isolated CA1 cells

Fig 312 The activation of PAC1 receptors selectively targeted GluN2ARs 79

Fig 313 Src targeting GluN2ARs increased NMDAR currents in acutely isolated 80

CA1 cells

Fig 314 Quantification of NMDAR currents showed that Src selectively 81

modulates GluN2ARs over GluN2BRs

Fig 315 Fyn (39-57) blocked the enhancement of NMDAR currents by Fyn kinase 82

specifically

Fig 316 GluN2BRs were involved in the increase of NMDAR currents by Fyn 83

Fig 317 the activation of PAC1 receptors selectively phosphorylated the tyrosine 84

residues of GluN2A

Fig 318 The application of PACAP increased Src activity 85

Fig 319 The activation of PAC1 receptor by PACAP (1 nM) enhanced 86

NMDAREPSCs via SrcGluN2AR pathway

Fig 3110 PACAP (1 nM) could not reduce the threshold of LTP induced 87

by high frenquency protocol or theta burst stimulation

ix

Fig 3111 The application of PACAP (1 nM) converted LTD to LTP induced by 88

10 Hz protocol (600 pulses)

Fig 3112 The application of PACAP shifted BCM curve to the left and reduced 89

the threshold for LTP inducition

Fig 321 Low concentration of VIP (1nM) enhanced NMDAR currents via VPAC 97

receptors in isolated CA1 cells

Fig 322 The activation of both VPAC1 receptor and VPAC2 receptor enhanced 98

NMDAR currents

Fig 323 PKA was involved in the potentiation of NMDARs by the activation of 99

VPAC receptors

Fig 324 PKC was not required for the VIP (1 nM) effect while the increase of 100

intracellular Ca2+ was necessary

Fig 325 VIP (1 nM) effect could be accentuated by PDE4 inhibitor and required 101

AKAP scaffolding protein

Fig 326 Src was not required for VIP (1 nM) effect on NMDAR currents 102

Fig 327 The activation of VPAC receptors enhanced NMDAR currents via Fyn 103

and GluN2BRs

Fig 41 The activation of PAC1 receptor selectively modulated GluN2ARs 111

over GluN2BRs by signaling through PKCCAKβSrc pathway

Fig 42 The activation of Gαs coupled receptors such as dopamine D1 receptor 112

and VPAC receptor increased NMDAR currents through PKAFyn signaling

pathway In addition they all selectively modulated GluN2BRs over GluN2ARs

Fig 51 Application of an Epac selective agonist (10 microM) 8-pCPT-2prime-O-Me-cAMP 152

x

to acutely isolated CA1 pyramidal neurons increased NMDAR currents

Fig 52 PKA was not involved in the potentiation of NMDARs by Epac 153

Fig 53 PLC was involved in the potentiation of NMDARs by Epac 154

Fig 54 PKCSrc dependent signaling pathway mediated the potentiation of 155

NMDARs by Epac

Fig 55 The elevated Ca2+ concentration in the cytosol was required for the 156

potentiation of NMDAR currents by Epac

Fig 56 In the presence of Epac agonist 8-pCPT-2prime-O-Me-cAMP pair-pulse 157

facilitation was increased

Fig 57 Bath application of Epac agonist 8-pCPT-2prime-O-Me-cAMP reduced 158

NMDAREPSCs

Fig 58 In the presence of this membrane impermeable Epac agonist 159

8-OH-2prime-O-Me-cAMP NMDAREPSCs was significantly increased

xi

ABBREVIATIONS AND ACRONYMS

α7AChR - α7-nicotinic acetylcholine receptor

ABD ndash agonist binding domain

AC ndash adenylyl cyclase or adenylate cyclase

aCSF ndash artificial cerebrospinal fluid

AD ndash Alzheimerrsquos disease

ADNF ndash activityndashdependent neurotrophic factor

A2AR - adenosine A2A receptors

AHP ndash afterhyperpolarization

AKAP ndash Andashkinase anchor proteins

AMPA ndash α-amino-3-hydroxy-5-methyl-4-isoxazdepropionic acid

APP ndash amyloid precursor protein

ARAP3 ndash Arf and Rho GAP adapter protein

ARF ndash ADPndashribosylation factor

BBM ndash brush border membrane

BDNF ndash brain derived neruotrophic factor

BFA ndash brefeldin-A

CAKβPyk2 ndash cell adhesion kinase βproline rich tyrosine kinase 2

CaM ndash calciumcalmodulin

CaMKII ndash α-calcium-calmodulin-dependent protein kinase II

cADPR - cADP-ribose

cAMP ndash cyclic adenosine monophosphate

CBD ndash cAMP binding domain

CBP ndash CREB binding protein

CD35 ndash the complement receptor 1

CDC25HD ndash CDC25 homology domain

CDK5 - cyclin-dependent kinase 5

Chk - Csk homology kinase

CKII - caesin kinase II

CNS ndash central nervous system

CNTF ndash ciliary neurotrophic factor

xii

CRE ndash cAMP response element

CREB ndash cyclic AMP response element binding protein

Csk ndash C-terminal Src kinase

DAG ndash diacylglycerol

DEP ndash Dishevelled Egl-10 and Pleckstrin domain

DH ndash dorsal hippocampus

DNA-PK ndash DNA dependent protein kinase

DARPP-32 - dopamine- and cAMP-regulated neuronal phosphoprotein

EPAC ndash exchange protein activated cAMP

ECF ndash extracellular fluid

ENaC - amiloride-sensitive Na+ channels

EPSC ndash excitatory postsynaptic current

EPSP ndash excitatory postsynaptic potential

ER ndash endoplasmic reticulum

ERK ndash extracellular singalndashregulated kinase

FMRP - fragile X mental retardation protein

FPRL1 ndash formyl peptide receptorndashlike 1

GABA ndash gamma ndash aminobutyric acid

GAP ndash GTPase-activating peptide

GEF ndash guanine nucleotide exchange factor

GFAP - glial fibrilary acidic protein

GLAST ndash glutamate ndashaspartate transport

GluA ndash AMPAR subunit

GluN ndash NMDAR subunit

GPCR ndash G-protein coupled receptor

GRF ndash Guanine nucleotide releasing factor

GRIP12 ndash glutamate receptor interacting protein frac12

HCN - hyperpolarization-activated cyclic nucleotide gated channels

HFS ndash high frequence stimulation

I-1 ndash Inhibitor 1

IP3 ndash inositol trisphosphate

xiii

JNKSAPK ndash Jun N-terminal kinasestress activated protein kinase

KATP channels - ATP-sensitive K+ channels

LVs ndash large dense core vesicles

LC1 ndash light chain 1

LFS ndash low frequency stimulation

LIF ndash long term facilitation

LIVBP ndash Leucine isoleucine valine binding protein

LPA ndash lysophosphatidic acid

LTDLTP ndash long term depressionlong term potentiation

MAGUK ndash membrane associated guanylate kinase

mAKAP ndash muscle specific AKAP

MAP1 ndash microtubule associated protein

MAP1B - microtube-associated protein 1B

MAPK ndash mitogen activated protein kinase

MDM ndash monocyte ndash derived macrophage

mEPSC ndash miniature EPSC

mGluR ndash metabatropic glutamate receptor

MMP-9 ndash Matrix metalloproteinase ndash 9

NAc - Nucleus accumbens

NADDP - Nicotinic acid adenine dinucleotide phosphate

ND2 - NADH dehydrogenase subunit 2

NHE3 - Na+ndashH+ exchanger 3

NMDA ndash N-methyl-D-aspartate

NO - nitric oxide

NR1 ndash NMDA receptor subunit 1



NRC ndash NMDA receptor complex

NRG1 ndash neuregulin 1

NTD ndash Nndashterminal domain

OA ndash Okadaic acid

xiv

Po - channel open probability

PA ndash phosphatidic acid

PACAP ndash pituitary adenylate cyclase activating peptide

PAC1 receptor ndash PACAP receptor

PC - Prohormone convertases

PDBu ndash phorbol ester

PDE4 ndash phosphodiesterase 4

PDGF - platelet-derived growth factor

P38 MAPK ndash p38 mitogenndashactivated protein kinase

PHI - Peptide histidine isoleucine

PKA ndash cAMP dependent protein kinaseprotein kinase A

PKB ndash protein kinase B

PKC ndash protein kinase C

PKM - Protein kinase Mζ

PICK1 ndash protein interacting with C kinase ndash1

PIP2 - phosphatidylinositol 45-bisphosphate

PI3K ndash Phosphatidylinositol 3-kinases

PLC ndash phospholipase C

PLD ndash phospholipase D

PP1 ndash serinethreonine protein phosphatase 1

PP2A ndash protein phosphatase 2A

PP2B ndash protein phosphatase 2B

PPF ndash paired pulse facilitation

PPI ndash prepulse inhibition

PPR ndash paired pulse ratio

PRP - PACAP related peptide

PSD93 ndash postsynaptic density 93


PTP ndash protein tyrosine phosphatase

PTPα ndash protein tyrosine phosphatase α

RA ndash Ras associating domain

xv

RACK1 ndash receptor for activated C kinase 1

RapGAP ndash Rap GTPase activating protein

RasGRF1 - Ras protein-specific guanine nucleotide-releasing factor 1

REM ndash Ras exchange motif

RGS ndash regulator of G-protein signaling

RyRs - ryanodine receptors

SAP102 - synapse-associated protein 102

SAP97 ndash synapse-associated protein 97

SD ndash sleep deprivation

SFK ndash Src family kinase

SH1 - Src homology 1

SH2 ndash Src homology 2



SHP12 - Src homology-2-domain-containing phosphatases 12

SNARE - Synaptosome-associated-protein receptor

SNAP25 - Synaptosomal-associated protein 25

STDP ndash spike timing dependent plasticity

STEP61 ndash Striatal-enriched protein tyrosine phosphatase 61

SVs ndash small vesicels

SynGAP - Synaptic Ras GTPase activating protein

TARP ndash transmembrane AMPAR regulatory protein

Tiam1 ndash T-cell lymphoma invasion and metastasis

TrkA ndashtyrosine kinase receptor A

VIP ndash Vasoactive intestinal peptide

VGCCs - Voltage-gated Ca2+ channels

VPAC ndash VIPPACAP receptor

VTA ndash Ventral tegmental area

7TM ndash seven transmembrane

1

Section 1

Introduction

2

In the central nervous system (CNS) glutamate is the major excitatory

neurotransmitter (Kennedy 2000) In response to the presynaptic release of glutamate

glutamate receptors at postsynaptic sites generate excitatory postsynaptic potentials

(EPSPs) (Dingledine et al 1999 Traynelis et al 2010) Glutamate receptors consist of

two classes ionotropic and metabotropic glutamate receptors Metabotropic glutamate

receptors (mGluRs) are G-protein coupled receptors (GPCRs) and consist of eight

subtypes Ionotropic glutamate receptors are ligand gated ion channels and include three

subtypes α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR)

N-methyl-D-aspartate receptors (NMDAR) and kainate receptors (Dingledine et al 1999

Traynelis et al 2010)

11 Excitatory Synaptic Transmission in the hippocampus

When glutamate binds to its receptors these receptors are activated and generate

EPSPs The EPSPs often consist of both NMDAR and AMPAR-mediated components

However the basal EPSP and its underlying excitatory postsynaptic current (EPSC) are

largely mediated by AMPARs since NMDARs are blocked by extracellular Mg2+ at

resting conditions (Mayer et al 1984) When glutamate is released AMPARs are

activated although K+ efflux through AMPARs more Na+ influx It generates inward

currents and results in membrane depolarization which is sufficient to relieve the

inhibition of NMDARs by Mg2+ The activated NMDARs are permeable to Ca2+

resulting in the elevation of [Ca2+]i which mediates most of the physiological effects of

NMDAR activity ((Perkel et al 1993)

111 AMPAR

3

AMPARs are the major glutamate receptors which mediate fast excitatory

neurotransmission in the hippocampus They have four subunits (GluA1-GluA4) which

are transcribed from four different genes Each AMPAR subunit can be alternatively

spliced into flip and flop (Derkach et al 2007 Kessels and Malinow 2009) Most

AMPARs are tetramers their subunit composition varies in different brain regions for

instance at mature hippocampal excitatory synapses most AMPARs are GluA1GluA2

and GluA2GluA3 receptors (Derkach et al 2007 Kessels and Malinow 2009)

The subunit compositions of AMPARs determine the functional properties of

receptors After the GluA2 subunit is transcribed the arginine (R) codon replaces the

glutamine (Q) codon at residue 607 by RNA editing this modification suppresses the

Ca2+ permeability of GluA2 subunit (Derkach et al 2007 Kessels and Malinow 2009)

In the adult hippocampus most of AMPARs are impermeable to Ca2+ only AMPARs

without GluA2 subunits are Ca2+ permeable (Derkach et al 2007 Kessels and Malinow

2009) In addition the subunit compositions of AMPARs determine receptor trafficking

In the absence of synaptic activity GluA2GluA3 receptors continuously move in and out

of the membrane whereas the trafficking of GluA1GluA2 and GluA4GluA2 receptors

is regulated by synaptic activity (Hayashi et al 2000 Zhu et al 2000)

Additionally the functions of AMPARs can be regulated by the phosphorylation

of receptor subunits (Derkach et al 2007 Kessels and Malinow 2009) For example

calciumcalmodulin (CaM) ndash dependent protein kinase II (CaMKII) phosphorylates Ser-

831 of GluA1 subunits this phosphorylation significantly increases both the activity and

surface expression of AMPARs (Derkach et al 1999 Lee et al 2000) In contrast

4

protein kinase C (PKC) phosphorylates Ser-880 of GluA2 subunits resulting in the

removal of GluA2 containing receptors from synapses (Boehm et al 2006)

AMPAR functions such as gating and trafficking are modulated by the recently

discovered protein stargazin which belongs to the transmembrane AMPAR regulatory

protein (TARP) family (Milstein and Nicoll 2008 Nicoll et al 2006 Sager et al 2009)

The interaction of stargazin and AMPARs in the endoplasmic reticulum (ER) enhances

the trafficking of AMPARs to the plasma membrane Then by lateral surface diffusion

these complexes move to synaptic sites by the interaction of stargazin and postsynaptic

density 95 (PSD95) (Milstein and Nicoll 2008 Nicoll et al 2006 Sager et al 2009) In

addition stargazin has the ability to modulate the electrophysiological properties of

AMPARs (Milstein and Nicoll 2008 Nicoll et al 2006 Sager et al 2009) Recently

two members of the Cornichon transmembrane protein family were found by proteomic

analysis to interact with AMPARs Similar to stargazin cornichons increased surface

expression of AMPARs and changed channel gating by slowing deactivation and

desensitization kinetics (Schwenk et al 2009 Shi et al 2010b)

112 LTP and LTD

In the early 1970s Bliss et al (Bliss and Lomo 1973) discovered that in the

hippocampus repetitive activation of excitatory synapses resulted in an enhancement of

synaptic strength This enhancement could last for hours or even days (Bliss and Lomo

1973) this phenomenon was named long term potentiation (LTP) Later long term

depression (LTD) was discovered by Mark Bear (Dudek and Bear 1992) LTD refers to

the persistent decrease of synaptic strength induced by low frequency stimulation Both

5

LTP and LTD are two forms of synaptic plasticity Synaptic plasticity also includes other

two forms homeostatic plasticity (Nelson and Turrigiano 2008 Turrigiano 2008) and

metaplasticity (Abraham 2008 Abraham and Bear 1996)

1121 LTP

To date two distinct types of LTP have been identified they are NMDAR-

dependent LTP and hippocampal mossy fiber LTP

NMDAR-dependent LTP can be induced by high frequency stimulation (HFS)

Robust excitation resulting for example from repetitive stimulation at high frequencies

(gt50 Hz) is required to strongly depolarize dendritic spines and relieve the voltage-

dependent block of NMDARs by Mg2+ The resulting large increase of [Ca2+]i evoked by

such stimulation activates CaMKII leading to phosphorylatation of AMPARs This

phosphorylation of AMPARs increases both channel conductance and surface expression

of AMPARs and induces LTP (Malenka and Bear 2004 Malinow and Malenka 2002)

Another mechanistically distinct form of LTP hippocampal mossy fiber LTP

which is NMDAR independent also exists in the hippocampus It occurs at mossy fiber

synapses between the axons of dentate gyrus granule cells and the dendrites of CA3

pyramidal cells (Nicoll and Malenka 1995) The expression of mossy fiber LTP is

presynaptic When HFS is applied presynaptic voltage dependent calcium channels open

resulting in an increase in [Ca2+]i The increase in presynaptic Ca2+ activates a CaM

dependent adenylyl cyclase (AC) and protein kinase A (PKA) The activation of PKA

phosphorylates several important presynaptic proteins and enhances the neurotransmitter

release (Nicoll and Schmitz 2005) Both Rab3A (a small GTPase) (Castillo et al 1997)

6

and Rim1α (an active zone protein) (Castillo et al 2002) are proposed PKA substrates

for the enhancement of neurotransmitter release

1122 LTD

So far at least two types of LTD have been discovered they are NMDAR-

dependent LTD and mGluR-dependent LTD

NMDAR-dependent LTD is often induced by low frequency stimulation (LFS)

Compared to LTP Ca2+ influx through NMDARs in the postsynaptic dendritic spine by

LFS is smaller A prolonged but modest Ca2+ influx activates phosphatases including

protein phosphatase 1 (PP1) and protein phophatase 2B (PP2B) (Collingridge et al 2010

Malenka and Bear 2004 Malinow and Malenka 2002) thereby dephosphorylating

AMPARs The dephosphorylation of AMPAR then results in LTD (Collingridge et al

2010 Malenka and Bear 2004 Malinow and Malenka 2002)

Under some experimental conditions LFS also induces mGluR-dependent LTD

which is mechanistically different from NMDAR-dependent LTD In the hippocampus

mGluR-dependent LTD is dependent on protein synthesis (Gladding et al 2009 Luscher

and Huber 2010) In mice without fragile X mental retardation protein (FMRP) mGluR-

dependent LTD is enhanced in both the hippocampus (Huber et al 2002) and the

cerebellum (Koekkoek et al 2005) suggesting that FMRP plays an important role in

regulating activity-dependent synaptic plasticity in the brain The detailed mechanism

underlying mGluR-dependent LTD expression is controversial Either a presynaptic

component or a postsynaptic component or both might contribute to the expression of this

kind of LTD (Gladding et al 2009 Luscher and Huber 2010)

7

113 Physiological functions of LTP and LTD

Since the discovery of LTP and LTD many studies have linked LTP and LTD to

many different types of experience-dependent plasticity Understanding the mechanism

of synaptic plasticity may provide us novel therapeutic approaches to treat a number of

neuropsychiatric disorders

1131 Hippocampus-dependent learning and memory

The role of LTP in hippocampus-dependent learning and memory has been well

demonstrated For example when NMDAR antagonist AP5 was infused into the

hippocampus both LTP and some types of spatial learing were impaired (Morris et al

1986) In addition after the infusion of a PKMζ inhibitor to the hippocampus the

maintence of LTP and long-lasting spatial memory were blocked (Pastalkova et al 2006)

The involvement of LTD in hippocampus-dependent learning and memory has

recently been demonstrated with the use of transgenic mice LTD induction was

facilitated when rats explored complex environment which contained novel objects

(Kemp and Manahan-Vaughan 2004) Additionally in transgenic mice in which protein

phosphatase 2A (PP2A) was inhibited in the forebrain not only NMDAR-LTD was

blocked but also Morris water maze and a delayed nonmatch to place T-maze task

showed deficits (Nicholls et al 2008) Furthermore in freely moving adult rats the

injection of LTD-blocking GluN2BR antagonist impaired spatial memory consolidation

indicating LTD in the hippocampal CA1 region was required for the consolidation of

spatial memory (Ge et al 2010)

8

1132 Fear conditioning in amygdale

Pavlovian fear conditioning relies on the amygdale for its induction and

maintenance (Sigurdsson et al 2007) In the lateral amygdale both NMDAR-dependent

LTP and LTD could be induced (McKernan and Shinnick-Gallagher 1997 Yu et al

2008) In addition fear conditioning also induced LTP (Rogan et al 1997) These studies

established a direct link between LTP and fear conditioning in amygdale

Furthermore the extinction of Pavlovian fear memory required NMDAR-

dependent LTD and the endocytosis of AMPARs (Dalton et al 2008) When LTD

induction in the amygdale was blocked by a peptide which blocked AMPAR endocytosis

the extinction of Pavlovian fear memory was disrupted (Dalton et al 2008) Additionally

the application of a PKMζ inhibitor inhibited the amygdale LTP maintenance and erased

fear memory in rats (Migues et al 2010)

1133 Drug addiction

So far many forms of LTP and LTD induction have been demonstrated at

excitatory synapses in the ventral tegmental area (VTA) and nucleus accumbens (NAc) of

mesolimbic dopamine system (Kauer and Malenka 2007 Kelley 2004) Synaptic

plasticity occurring in the VTA and NAc is proposed to induce or mediate some drug-

induced behavioral adaptions For example when the GluA1 subunit of AMPARs was

overexpressed by viral mediated infection in the NAc the extinction of cocaine-seeking

responses was facilitated (Sutton et al 2003) In addition after repeated injections of

amphetamine animals often showed some behavioral sensitization but the injection of

9

the peptide which blocked the endocytosis of AMPARs and LTD induction also blocked

this effect (Brebner et al 2005)

The work in this thesis focuses on NMDARs so the information about NMDARs

is described in detail NMDARs are tetramers composed of two GluN1 (formerly NR1)

subunits and two GluN2 (formerly NR2) subunits or in some cases an GluN2 and an

GluN3 subunit (Cull-Candy and Leszkiewicz 2004) Structurally NMDAR subunits are

composed of two domains in the extracellular region including N-terminal domain (NTD)

and agonist-binding domain (ABD) the membrane region consisting of three

transmembrane segments and a re-entrant loop the C-terminal tail which interacts with

various intracellular proteins (McBain and Mayer 1994)The NTD of NMDAR subunits

plays an important role in subunit assembly (Herin and Aizenman 2004) In GluN2A and

GluN2B subunits it also binds to allosteric inhibitors such as Zn2+ and Ro25-25-6981

(Mony et al 2009 Paoletti and Neyton 2007) The ABD is an agonist binding domain

When the agonists bind they stabilize a closed conformation of the two lobes and open

the receptor In contrast competitive antagonists bind the same cleft but impede cleft

closure and prevent channel activation (Furukawa et al 2005 Kussius et al 2009)

12 NMDARs

Not only has the involvement of NMDARs in learning and memory been well

demonstrated the dysfunction of NMDAR is also found in many neurological disorders

such as stroke schizophrenia and Alzheimers disease (AD) In stroke and AD patients

the activity of NMDAR maybe abnormally high (Lipton 2006 Plosker and Lyseng-

10

Williamson 2005) while NMDAR activity is low in schizophrenia patients (Kristiansen

et al 2007)

131 GluN1 subunits

13 NMDAR subunits

GluN1 is expressed ubiquitously in the brain its gene (Grin1) consists of 22

exons Alternative splicing of three exons (exons 5 21 and 22) generates eight different

isoforms (Zukin and Bennett 1995) Exon 5 encodes a splice cassette at N terminus of

extracellular domain of GluN1 subunit (termed N1) whereas exons 21 and 22 encode

two splice cassettes at C terminus of intracellular domain of GluN1 subunit (termed C1

and C2 respectively) (Zukin and Bennett 1995) The splicing of the C2 cassette removes

the first stop codon and encodes a different cassette (termed C2rsquo) (Zukin and Bennett

1995) GluN1 subunits did not form functional receptors alone but their cell surface

expression relied on the splice variant (Wenthold et al 2003) Trafficking of the GluN1

subunit from the ER to the plasma membrane was regulated by alternative splicing

because the C1 cassette contained a ER retention motif (Wenthold et al 2003) When the

GluN1 isoform which contains N1 C1 and C2 was expressed in heterologous cells it

was retained in the ER (Standley et al 2000) In contrast other variants had the ability to

traffick to the cell surface (Standley et al 2000) since the C2rsquo cassette could mask the

ER retention motif in the C1 cassette (Wenthold et al 2003) In addition when the

GluN1 subunit bound to GluN2 subunit this ER retention motif was also masked then

GluN1GluN2 receptor was released from ER and moved to the surface (Wenthold et al

2003) Furthermore alternative splicing of GluN1 subunit contributes to the modulation

11

of NMDARs by PKA and PKC the serine residues of the C1 cassette of GluN1 subunit

can be phosphorylated by both PKA and PKC (Tingley et al 1997) PKC

phosphorylation relieved ER retention caused by the C1 cassette and enhanced the

surface expression of the GluN1 subunit (Scott et al 2001) This action required the

coordination from PKA phosphorylation of an adjacent serine (Tingley et al 1997)

GluN1 splicing isoforms also confer different kinetic properties to NMDARs

(Rumbaugh et al 2000) Furthermore GluN1 isoforms without the exon 5 derived

domain were inhibited by protons and Zn2+ and potentiated by polyamines whereas those

containing this region in GluN1 isoforms lacked these properties (Traynelis et al 1995

Traynelis et al 1998) The exon5 derived domain might form a surface loop to screen the

proton sensor and Zn2+ binding site

132 GluN2 subunits

In contrast to GluN1 isoforms four GluN2 subunits (GluN2A-D) are transcribed

from seperate genes Although the family of GluN2 subunits consists of GluN2A

GluN2B GluN2C and GluN2D GluN2C subunits are often expressed in the cerebellum

while the expression of GluN2D subunits is mainly restricted to brainstem (Kohr 2006)

Most adult CA1 pyramidal neurons express GluN2A and GluN2B subunits (Cull-Candy

and Leszkiewicz 2004) During the development the expression of GluN2B and

GluN2D subunits is abundant early and decreases during maturation whereas the

expression of GluN2A and GluN2C subunits increases (Cull-Candy and Leszkiewicz

2004) At mature synapses in the hippocampus GluN2A subnits occupy synapses

12

whereas GluN2B subunits predominate at extrasynaptic sites (Cull-Candy and

Leszkiewicz 2004)

1321 Electrophysiological characterization of GluN2 subunits

The composition of GluN2 subunits determines many biophysical properties of

NMDARs (Cull-Candy and Leszkiewicz 2004) GluN1GluN2A receptors have the

shortest deactivation time constant while GluN1GluN2B and GluN1GluN2C receptors

exhibit intermediate deactivation time and GluN1GluN2D receptors display the slowest

deactivation kinetics (Cull-Candy and Leszkiewicz 2004) In addition other important

properties of NMDARs also depend on GluN2 subunits Although all of the GluN2

subunits are highly permeable to Ca2+ only GluN1GluN2A and GluN1GluN2B

receptors show a relatively high single channel conductance and Mg2+ sensitivity

whereas both GluN1GluN2C and GluN1GluN2D receptors have relatively low single

channel conductance and the sensitivity of Mg2+ inhibition is also low (Cull-Candy and

Leszkiewicz 2004)

1322 Synaptic and extrasynaptic NMDARs

Whether or not the subunit compositions of NMDARs are different between

synaptic and extrasynaptic sites is controversial Using the glutamate-uncaging technique

both synaptic and extrasynaptic sites demonstrated the same sensitivity to GluN2BR

antagonists (Harris and Pettit 2007) But studies examining extrasynaptic NMDAR

subunit compositions using NMDA bath applications have drawn inconsistent

conclusions Some studies suggested that GluN2B subunits were mostly expressed

13

extrasynaptically (Stocca and Vicini 1998 Tovar and Westbrook 1999) while other

studies suggested that both GluN2A and GluN2B subunits exist at extrasynaptic sites

(Mohrmann et al 2000)

Nevertheless NMDARs were found both at synaptic and extrasynaptic locations

and coupled to distinct intracellular signaling pathways in the hippocampus (Hardingham

et al 2002 Hardingham and Bading 2002 Hardingham and Bading 2010 Ivanov et al

2006) While the activation of synaptic NMDAR strongly induced cyclic AMP response

element binding protein (CREB)-dependent gene expression extrasynaptic NMDAR

stimulation reduced the CREB-dependent gene expression (Hardingham et al 2002) In

addition synaptic NMDARs activated the extracellular signal-regulated kinase (ERK)

pathway whereas extrasynaptic NMDARs inactivated ERK (Ivanov et al 2006)

Furthermore synaptic NMDARs activated a variety of pro-survival genes such as Btg2

and Bcl6 (Zhang et al 2007) Btg2 was a gene which suppresses apoptosis (El-Ghissassi

et al 2002) while Bcl6 was a transcriptional repressor that inhibited the expression of

p53 (Pasqualucci et al 2003) In contrast extrasynaptic NMDARs induced the

expression of Clca1 (Zhang et al 2007) a presumed Ca2+-activated Cl- channel that

induced the proapoptotic pathways (Elble and Pauli 2001) In neurons relatively low

concentrations of NMDA activated synaptic NMDAR signaling and increased action-

potential firing In contrast relatively high concentrations of NMDA strongly suppressed

firing rates and did not favour synaptic NMDAR activation (Soriano et al 2006) In

addition the NMDAR-mediated component of synaptic activity enhanced the antioxidant

defences of neurons by a triggering a series of appropriate transcriptional events In

14

contrast extrasynaptic NMDAR failed to enhance antioxidant defenses (Papadia et al

2008)

Recently it was proposed that GluN2B containing NMDARs (GluN2BRs)

promoted neuronal death irrespective of location while GluN2A containing NMDARs

(GluN2ARs) promoted survival (Liu et al 2007) In addition GluN2ARs and GluN2BRs

played differential roles in ischemic neuronal death and ischemic tolerance (Chen et al

2008) The specific GluN2AR antagonist NVP-AAM077 enhanced neuronal death after

transient global ischemia and abolished the induction of ischemic tolerance (Chen et al

2008) In contrast the specific GluN2BR antagonist ifenprodil attenuated ischemic cell

death and enhanced preconditioning-induced neuroprotection (Chen et al 2008)

Additionally NMDA-mediated toxicity in young rats was caused by activation of

GluN2BRs but not GluN2ARs (Zhou and Baudry 2006) In contrast another study (von

et al 2007) suggested that GluN2BRs were capable of promoting both survival and

death signaling Moreover in more mature neurons (DIV21) GluN2ARs were recently

shown to be capable of mediating excitotoxicity as well as protective signaling (von et al

2007) Additionally both GluN2ARs and GluN2BRs were found to be involved in the

induced hippocampal neuronal death by HIV-1-infected human monocyte-derived

macrophages (HIVMDM) (ODonnell et al 2006) Taken together these studies indicate

that GluN2BRs and GluN2ARs may both be capable of mediating survival and death

signaling

1323 The distinct functional roles of GluN2 subunits

15

Functionally the composition of the GluN2 subunits within NMDARs imparts

distinct properties to the receptor For example GluN1GluN2B (2 GluN1 and 2 GluN2B)

receptors have a higher affinity for glutamate and glycine than GluN1GluN2A receptors

(2 GluN1 and 2 GluN2A) GluN1GluN2A receptor mediated currents exhibit faster rise

and decay kinetics than those by generated GluN1GluN2B receptors (Lau and Zukin

2007) The longer time constant of decay for currents generated by GluN1GluN2B

receptors allows a greater relative contribution of Ca2+ influx compared to that by

GluN1GluN2A receptors This suggests the potential of distinct Ca2+ signaling via the

two subtypes of NMDARs (Lau et al 2009) So at the low frequencies typically used to

induce LTD GluN1GluN2B receptors make a larger contribution to total charge transfer

than do GluN1GluN2A receptors However with high-frequency tetanic stimulation

which is often used to induce LTP the charge transfer mediated by GluN1GluN2A

receptors exceeds that of GluN1GluN2B receptors (Berberich et al 2007) This

highlights the potential for distinct Ca2+ signaling via the these two subtypes of

NMDARs (Erreger et al 2005)

1324 Ca2+ permeability of GluN2 subunits

NMDARs are non-selective cation channels which are permeable to Na+ K+ and

Ca2+ The current carried by Ca2+ only consists of 10 total NMDAR current

(Schneggenburger et al 1993) But the volume of the spine head is very small so the

activation of NMDARs will likely induce a large rise of Ca2+ inside the spine

When individual spines were stimulated using the glutamate uncaging technique

the contribution of GluN2ARs and GluN2BRs to NMDAR currents and Ca2+ transients

16

inside the spine varied depending on individual spine examined (Sobczyk et al 2005)

Furthermore when GluN2BRs were repetitively activated the influx of Ca2+ stimulated a

serinethreonine phosphatase resulting in the reduction of Ca2+ permeability of these

channels (Sobczyk and Svoboda 2007) In addition dopamine D2 receptor activation

selectively inhibited Ca2+ influx into the dendritic spines of mouse striatopallidal neurons

through NMDARs and voltage-gated Ca2+ channels (VGCCs) The regulation of Ca2+

influx through NMDARs depended on PKA and adenosine A2A receptors (A2AR) In

contrast Ca2+ entry through VGCCs was not modulated by PKA or A2ARs (Higley and

Sabatini 2010)

These results were consistent with a previous report that the Ca2+ permeability of

NMDARs was regulated by a PKA-dependent phosphorylation of the receptors For

example one study implied that PKA activation increased the Ca2+ permeability of

GluN2ARs (Skeberdis et al 2006) since PKA inhibitor reduced Ca2+ permeability

mediated by these receptors

1325 Interaction with downstreram signaling pathways

Furthermore GluN2ARs and GluN2BRs couple to different signaling pathways

upon activation The GluN2B subunit has many unique binding protens For example

GluN2B subunit indirectly interacts with synaptic Ras GTPase activating protein

(SynGAP) through synapse-associated protein 102 (SAP102) SynGAP is a novel Ras-

GTPase activation protein which selectively inhibits ERK signaling (Kim et al 2005)

But another study demonstrated that GluN2B subunit specifically bound to Ras protein-

specific guanine nucleotide-releasing factor 1 (RasGRF1) a CaM dependent Ras guanine

17

nucleotide releasing factor this action might also regulate ERK activation (Krapivinsky

et al 2003)

GluN2A and GluN2B subunits also bound to active CaMKII with different

affinities (Strack and Colbran 1998) CaMKII bound to GluN2B subunits with high

affinity but the interaction between CaMKII and GluN2A was weak (Strack and Colbran

1998) When CaMKII was activated by CaM it moved to the synapses and bound to

GluN2B strongly (Strack and Colbran 1998) Even if Ca2+CaM was dissociated from

CaMKII later CaMKII remained active (Bayer et al 2001) In addition both CaMKII

activation and its association with GluN2B were required for LTP induction (Barria and

Malinow 2005)

Recently one study demonstrated that GluN2A subunit co-immunoprecipitates

with neuronal nitric oxide (NO) synthase (Al-Hallaq et al 2007) but this interaction is

possibly indirect In addition whether this interaction is involved in some GluN2A-

mediated signaling pathways requires further study

Furthermore the C-terminus of both GluN2A and GluN2B subunits has PDZ-

binding motifs so they have ability to interact with membranendashassociated guanylate

kinase (MAGUK) family of synaptic scaffolding proteins such as PSD95 postsynaptic

density 93 (PSD93) synapse-associated protein 97 (SAP97) and SAP102 (Kim and

Sheng 2004) It was proposed that GluN2A subunits selectively bound to PSD95 while

GluN2B subunits preferentially interacted with SAP102 (Townsend et al 2003) but

recent study demonstrated that diheteromeric GluN1GluN2A receptors and

GluN1GluN2B receptors interacted with both PSD95 and SAP102 at comparable levels

(Al-Hallaq et al 2007)

18

133 GluN3 subunits

The newest member of NMDAR family the GluN3 subunit includes two

subtypes GluN3A and GluN3B subunits they are encoded by two different genes

Although attention has focused on the role of GluN2 subunits in neural functions

recently the physiological roles of GluN3 subunits have began to be elucidated

(Nakanishi et al 2009) Both GluN3A and GluN3B subunits were widely expressed in

the CNS (Cavara and Hollmann 2008 Henson et al 2010 Low and Wee 2010) The

expression of GluN3A subunits occurred early after birth and during development

GluN3B subunit expression increased into adulthood (Cavara and Hollmann 2008

Henson et al 2010 Low and Wee 2010) GluN3 subunits could be assembled into two

functional receptor combinations the triheteromeric GluN3 containing NMDARs and the

diheteromeric GluN3 containing receptors (Henson et al 2010 Low and Wee 2010)

GluN3 containing NMDA receptors have unique properties that differ from the

conventional GluN1GluN2 receptors Surprisingly the presence of GluN3 subunit in

NMDARs (GluN1GluN2GluN3) decreased Mg2+ sensitivity and Ca2+ permeability of

receptors and reduces agonist-induced currents (Cavara and Hollmann 2008 Das et al

1998 Perez-Otano et al 2001) When coassembling with GluN1 subunits alone GluN3

formed a glycine receptor (GluN1GluN3) and it was insensitive to by glutamate and

NMDA (Chatterton et al 2002)

Recently several studies demonstrated that the GluN3A subunit influenced

dendritic spine density (Roberts et al 2009) synapse maturation (Roberts et al 2009)

memory consolidation (Roberts et al 2009) and cell survival (Nakanishi et al 2009)

The neuroprotective role for GluN3A has been studied using GluN3A knockout and

19

transgenic overexpression mice the loss of GluN3A exacerbated the ischemic-induced

neuronal damage while the overexpression of GluN3A reduced cell loss (Nakanishi et al

2009) The dominant negative effect of GluN3A on current and Ca2+ influx through

NMDARs has also been shown to affect synaptic plasticity (Roberts et al 2009) The

extension of expression of GluN3A using reversible transgenic mice that prolonged

GluN3A expression in the forebrain inhibited glutamatergic synapse maturation and

decreased spine density Furthermore inhibition of endogenous GluN3A using siRNA

accelerated synaptic maturation (Roberts et al 2009) In addition learning and memory

were also impaired when the expression of GluN3A was prolonged (Roberts et al 2009)

134 Triheteromeric GluN1GluN2AGluN2B receptors

Several studies suggested that in addition to diheteromeric NMDARs (GluN1

GluN1 GluN2x GluN2x) triheteromeric NMDARs (GluN1 GluN1 GluN2x GluNy (or

GluN3x)) may exist in some brain areas One study demonstrated the existence of

triheteromeric GluN1GluN2BGluN2D receptors in the cerebellar golgi cells By

measuring the kinetics of single channel current in isolated extrasynaptic patches

triheteromeric GluN1GluN2BGluN2D was proposed to be located at extrasynaptic sites

of cerebellar golgi cells (Brickley et al 2003) Furthermore a new paper proposed that

triheteromeric GluN1GluN2CGluN3A receptors also were located in oligodendrocytes

Firstly coimmunoprecipitation demonstrated the interaction between GluN1 GluN2C

and GluN3A subunits Secondly the inhibition of NMDAR currents by Mg2+ in

oligodendrocytes was similar to that mediated by GluN1GluN2CGluN3A receptors and

significantly different from that mediated by GluN1GluN2C receptors (Burzomato et al

20

2010) But whether or not these triheteromeric NMDARs represented surface expressed

and or functional synaptic receptors remains unknown

So far no study showed that functional triheteromeric receptors existed in CA1

synapse although they have been implicated in developing neurons in culture (Tovar and

Westbrook 1999) CA1 pyramidal neurons predominantly expressed dimeric

GluN1GluN2A and GluN1GluN2B receptors (Al-Hallaq et al 2007) one study

demonstrated that triheteromeric GluN1GluN2AGluN2B receptors were much less that

of dimeric GluN1GluN2A and GluN1GluN2B receptors (Al-Hallaq et al 2007) In

addition triheteromeric NMDARs had different pharmacological properties compared to

diheteromeric NMDARs For example triheteromeric GluN1GluN2AGluN2B receptors

demonstrated an ldquointermediaterdquo sensitivity to both GluN2AR and GluN2BR antagonists

(Hatton and Paoletti 2005 Neyton and Paoletti 2006 Paoletti and Neyton 2007)

All NMDAR subunits have a large intracellular C-terminal tail This domain

contains many serine and threonine residues that are potential sites of phosphorylation by

PKA PKC cyclin-dependent kinase 5 (CDK5) casein kinase II (CKII) and CaMKII

Although it was not known how phosphorylation of NMDAR modulates channel

properties it was proposed that NMDAR phosphorylation levels were correlated with

receptor activity (Taniguchi et al 2009) Various kinases phosphorylated NMDAR

subunits and regulate its activity trafficking and stability at synapses (Chen and Roche

2007 Lee 2006 Salter and Kalia 2004)

14 The modulation of NMDAR by serinethreonine kinases and phosphatases

21

141 The modulation of NMDAR by serinethreonine kinases

1411 PKA regulation of NMDARs

Both PKA and PKC are well studied in the regulation of NMDARs PKA is one

of the downstream effectors of cyclic AMP (cAMP) PKA consists of two catalytic

subunits and two regulatory subunits When cAMP binds to the regulatory subunits PKA

activity is increased

Multiple PKA phosphorylation sites have been identified on GluN2A GluN2B

and GluN1 subunits of NMDARs (Leonard and Hell 1997) PKA activated by cAMP

analogs or by the catalytic subunit of PKA have been shown to increase NMDAR

currents in spinal dorsal horn neurons (Cerne et al 1993) In addition the activation of

PKA through β-adrenergic receptor agonists increased the amplitude of synaptic

NMDAR mediated EPSCs currents (NMDAREPSCs) (Raman et al 1996)

The regulation of NMDARs by PKA in neurons was also highly controlled by

serinethreonine phosphatases such as PP1 and by the A kinase anchoring proteins

(AKAPs) For example Yotiao a scaffolding protein belonging to AKAP family

targeted PKA to NMDARs and the disruption of this interaction reduced NMDAR

currents expressed in HEK293 cells (Westphal et al 1999) In addition the inhibitory

molecule Inhibitor 1 (I-1) which targeted the PP1 was also a key substrate of PKA By

this means PKA activation led to inhibition of PP1 and decreased dephosphorylation

(enhanced phosphorylation) of NMDARs (Svenningsson et al 2004)

Recent studies suggested that in addition to regulate the gating of NMDARs PKA

phosphorylation also modulated the Ca2+ permeability of GluN2ARs (Skeberdis et al

2006)

22

In some conditions PKA may decrease NMDAR currents In inside-out patches

from cultured hippocampal neurons catalytic PKA failed to increase NMDAR currents

instead it inhibited Src potentiation of NMDARs (Lei et al 1999) This inhibition might

be mediated by c-terminal Src kinase (Csk) as this kinase was regulated by PKA and it

reduced Src kinase activity (Yaqub et al 2003) But whether the direct phosphorylation

of NMDARs by PKA modulates NMDA channel function requires further study Some

studies have shown that PKA signals indirectly via stimulation of Fyn kinase to regulate

NMDARs (Dunah et al 2004 Hu et al 2010)

PKA activation also regulates the trafficking of NMDARs For example

activation of PKA induced synaptic targeting of NMDARs (Crump et al 2001) In

addition together with PKC PKA phosphorylation of ER retention motif of GluN1

subunit enhanced the release of GluN1 from ER and increased the surface expression of

GluN1 (Scott et al 2003) Recently several studies demonstrated that the activation of

PKA by dopamine D1 receptor agonists also induced trafficking of GluN2B subunit to

the membrane surface (Dunah et al 2004 Hu et al 2010)

1412 PKC regulation of NMDARs

There is conceived evidence demonstrating that PKC has ability to regulate

NMDARs Recent studies showed that two different PKC isoforms phosphorylated

GluN1 subunit in cerebellar granule cells (Sanchez-Perez and Felipo 2005) PKCλ

preferentially phosphorylated Ser-890 while PKCα specifically phosphorylated Ser-896

(Sanchez-Perez and Felipo 2005) Protein C kinases can be divided into three groups

The conventional PKCs are activated by Ca2+ and diacylglycerol (DAG) while the novel

23

PKCs which lack a Ca2+ binding domain are only stimulated by DAG In contrast the

atypical PKCs are only sensitive to phospholipids both Ca2+ and DAG fail to activate

them When PKC is activated it will translocate to the membrane from the cytosol

(Steinberg 2008)

PKC activation increased NMDAR currents in isolated and cultured hippocampal

neurons (Lu et al 1999a) in isolated trigeminal neurons PKC potentiated NMDAR

mediated currents through the reduction of voltage-dependent Mg2+ block of channels

(Chen and Huang 1992) In addition the constitutively active protein kinase C (PKM)

potentiated NMDAR currents in cultured hippocampal neurons (Xiong et al 1998) In

cerebellar granule cells the phosphorylation of GluN2C subunit modulated the

biophysical properties of NMDARs when Ser-1244 of GluN2C was mutated to Alanine

(Ala) it accelerated the kinetics of NMDARs currents (Chen et al 2006) But the

phosphorylation of this site did not regulate the surface expression of GluN2C (Chen et

al 2006)

Biochemical studies have shown that GluN1 GluN2A GluN2B and GluN2C

subunits can be phosphorylated by PKC in vivo and in vitro (Chen et al 2006 Jones and

Leonard 2005 Liao et al 2001 Tingley et al 1997) In addition in Xenopus oocytes

transfected with GluN1 and GluN2B subunits if Ser-1302 or Ser-1323 of GluN2B were

mutated to Ala the potentiation of NMDAR currents by PKC was significantly reduced

(Liao et al 2001) Insulin also failed to potentiate GluN1GluN2B receptors when these

sites of GluN2B subunit were mutated to Ala (Jones and Leonard 2005) Furthermore

when Ser-1291 and Ser-1312 of GluN2A subunit were mutated to Ala insulin lost its

ability to potentiate GluN1GluN2A receptors (Jones and Leonard 2005) However

24

other studies (Zheng et al 1999) demonstrated that when PKC phosphorylation sites of

NMDAR were mutated to Ala PKC still potentiated NMDAR currents indicating that

PKC acted through another signaling molecule to regulate NMDAR currents (Zheng et

al 1999) Later our laboratory demonstrated that this signaling molecule was Src When

Src inhibitory peptide (Src (40-58)) was applied in the patch pipette PKC failed to

increase NMDAR currents in acutely isolated cells (Lu et al 1999a)

Surprisingly in acutely isolated hippocampal CA1 cells PKC activation enhanced

peak NMDAR currents while steady-state NMDAR currents were depressed indicating

that PKC also enhanced the desensitization of NMDARs (Lu et al 1999a Lu et al

2000) This PKC induced desensitization of NMDARs was unrelated to the PKCSrc

signaling pathway instead it depended on the concentration of extracellular Ca2+ (Lu et

al 2000) It was proposed that the C0 region of the GluN1 subunit competitively

interacted with actin-associated protein α-actinin2 and CaM (Ehlers et al 1996

Wyszynski et al 1997) When Ca2+ influxed through NMDAR it activated CaM and

displaced the binding of α-actinin2 from GluN1 subunit resulting in the desensitization

of NMDARs (Wyszynski et al 1997) PKC activation also enhanced the glycine-

insensitive desensitization of GluN1GluN2A receptors in HEK293 cells but when all the

previously identified PKC phosphorylation sites in GluN1 and GluN2A subunits were

mutated to Ala this kind of desensitization was still induced by PKC (Jackson et al

2006) In addition the phosphorylation of Ser-890 of GluN1 subunit disrupted the

clustering of this subunit resulting in the desensitization of NMDARs (Tingley et al

1997)

25

PKC modulates channel activity not only by changing physical properties of

receptors but also by the regulation of receptor trafficking PKC induced the increase of

surface expression of NMDARs via SNARE (synaptosome-associated-protein receptor)

dependent exocytosis in Xenopus oocytes (Carroll and Zukin 2002 Lan et al 2001 Lau

and Zukin 2007) Furthermore interaction of NMDARs with PSD95 and SAP102

enhanced the surface expression of NMDARs and occludes PKC potentiation of channel

activity (Carroll and Zukin 2002 Lin et al 2006)

1413 The regulation of NMDARs by other serinethreonine kinases

In addition to PKC and PKA another serinetheroine kinase Cdk5 modulated

NMDAR as well Cdk5 kinase is highly expressed in the CNS unlike other cyclin-

dependent kinases CdK5 kinase is not activated by cyclins instead it has its own

activating cofacotrs p35 or p39 It phosphorylated NR2A at Ser-1232 and increased

NMDA-evoked currents in hippocampal neuron (Li et al 2001) Inhibition of this

phosphorylation protected CA1 pyramidal cells from ischemic insults (Wang et al 2003)

Additionally Cdk5 kinase facilitated the degradation of GluN2B by directly interacting

with calpain (Hawasli et al 2007)

Similar to PKA CKII kinase consists of α αrsquo or β subunits the α and αrsquo subunits

are catalytically active whereas the β subnit is inactive In addition CKII kinase can not

be directly activated by Ca2+ CKII kinase also directly phosphorylated GluN2B subunit

at Ser-1480 this phophorylation disrupted its interaction with PSD95 and resulted in the

internalization of NMDARs (Chung et al 2004)

26

The modulation of NMDAR by CaMKII has also been investigated The CaMKII

kinase includes an N-terminal catalytic domain a regulatory domain and an association

domain In the absence of CaM the catalytic domain interacts with the regulatory domain

and CaMKII activity is inhibited Upon activation by CaM the regulatory domain is

released from the catalytic domain and CaMKII kinase is activated When CaMKII

bound to GluN2B CaMKII remained active even after the dissociation of CaM (Bayer et

al 2001) By this way CaMKIIα enhanced the desensitization of GluN2BRs (Sessoms-

Sikes et al 2005) providing a novel mechanism to negatively regulate GluN2BRs by the

influx of Ca2+

Recently GluN2C was found to be phosphorylated by protein kinase B (PKB) at

Ser-1096 (Chen and Roche 2009) The phosphorylation of this site regulated the binding

of GluN2C to 14-3-3ε In addition the treatment of growth factor increased the

phosphorylation of GluN2C at Ser-1096 and surface expression of NMDARs (Chen and

Roche 2009) Furthermore in cerebellar neurons PKB activated by cAMP

phosphorylated Ser-897 of GluN1 subunits and activated NMDARs (Llansola et al

2004)

142 The modulation of NMDARs by serinetheronine phosphatases

In the brain the majority of serinethreonine phosphatases consist of PP1 PP2A

PP2B and protein phosphatases 2C (PP2C) (Cohen 1997) PP1 and PP2A are

constitutively active while PP2B known as calcineurin is activated by CaM but the

activity of PP2C is only dependent on Mg2+ (Colbran 2004)

27

In inside-out patches from hippocampal neurons the application of exogenous

PP1 or PP2A decreased the open probability of NMDAR single channels Consistently

phosphatase inhibitors enhanced NMDAR currents (Wang et al 1994) In addition PP1

also exerted its inhibition on NMDARs by interaction with yotiao (Westphal et al 1999)

Furthermore the regulation of NMDARs by PKA acted through PP1 as well PKA

activation inhibited the activity of dopamine- and cAMP-regulated neuronal

phosphoprotein (DARPP-32) (Svenningsson et al 2004) or I-1 (Shenolikar 1994)

resulting in the inhibition of PP1 activity and enhancement of NMDAR phosphorylation

Additionally using cell attached recordings in acutely dissociated dentate gyrus

granule cells the inhibition of endogenous PP2B by okadaic acid or FK506 prolonged the

duration of single NMDA channel openings and bursts This action depended on the

influx of Ca2+ via NMDARs (Lieberman and Mody 1994) PP2B was also demonstrated

to be involved in the desensitization of NMDAR induced by synaptic desensitization

(Tong et al 1995) In HEK 293 cells transfected with GluN1 and GluN2A subunits Ser-

900 and -929 of GluN2A were found to be required for the modulation of desensitization

of NMDAR by PP2B (Krupp et al 2002)

151 The structure and regulation of SFKs

15 The modulation of NMDAR by Src family kinases (SFKs) and protein tyrosine

phosphatises (PTPs)

Since SFKs have ability to regulate NMDAR currents their structure and

regulation are introduced

28

SFKs were first proposed as proto-oncogenes (Stehelin et al 1976) They could

regulate cell proliferation and differentiation in the developing CNS (Kuo et al 1997) in

the developed CNS SFKs played other functions such as the regulation of ion channels

(Moss et al 1995) Five members of the SFKs are highly expressed in mammalian CNS

including Src Fyn Yes Lck and Lyn (Kalia and Salter 2003) In my thesis I focus on

Src and Fyn These SFKs each possess a regulatory domain at the C terminus a catalytic

domain (SH1) domain a linker region a Src homology 2 (SH2) domain a Src homology

3 (SH3) domain a Src homology 4 (SH4) domain and a unique domain at the N terminal

(Ingley 2008 Roskoski Jr 2005 Salter and Kalia 2004 Thomas and Brugge 1997)

SFKs are conserved in most of domains except the unique domain at the N-

terminus Salter et al designed the peptide which mimicked the region of unique domain

of Src and found that it selectively blocked the potentiation of NMDARs by Src (Yu et al

1997) Using a similar approach we synthesized a peptide Fyn (39-57) which is

corresponding to a region of the unique domain of Fyn (Fig 11) The unique domain are

important for selective interactions with proteins that are specific for each family member

(Salter and Kalia 2004) acting as the structural basis for their different roles in many

cellular functions mediated by SFKs For example the unique domain of Src specifically

bound to NADH dehydrogenase subunit 2 (ND2) and loss of ND2 in neurons prevented

the enhancement of NMDAR activity by Src (Gingrich et al 2004)

The SH4 domain of SFKs is a very short motif containing the signals for lipid

modifications such as myrisylation and palmitylation (Resh 1993) The importance of

this domain was illustrated by observations that the specificity of Fyn in cell signaling

depended on its subcellular locations (Sicheri and Kuriyan 1997) The SFK SH3 domain

29

is a 60 amino acids sequence and it interacts with proline rich motifs of a number of

signaling molecules and mediates various protein-protein interactions (Ingley 2008

Roskoski Jr 2005 Salter and Kalia 2004) The SH2 domain has around 90 amino acids

and binds to phosphorylated tyrosine residues of interacting protein Between the SH2

domain and SH1 domain is the linker region which is involved in the regulation of SFKs

The SH1 domain is highly conserved among SFKs it includes an ATP binding

site which is required for the phosphoryation of SFK substrates SFKs inhibitor PP2 binds

to this site and inhibits the phosphorylation of SFK substrates (Osterhout et al 1999)(Fig

11) It also contains an important tyrosine residue (for example Y416 in Src) in the

activation loop the phosphoryation of this residue is necessary for the SFK activation

(Salter and Kalia 2004) Its importance was demonstrated by that striatal enriched

tyrosince phosphatase 61 (STEP61) dephosphorylated this residue and inhibited Fyn

activity (Braithwaite et al 2006 Nguyen et al 2002)

The C-terminal of SFK has a specific tyrosine residue (for example Y527 in Src)

when it is phosphorylated it interacts with SH2 domain and SFK activity is inhibited

Two kinases including Csk (Nada et al 1991) and Csk homology kinase (Chk)

phosphorylate SFK on this site (Chong et al 2004) This site can also be

dephosphorylated by some protein tyrosine phosphatases (PTPs) including protein

tyrosine phosphatase α (PTPα) and Src homology-2-domain-containing phosphatases 12

(SHP12)

30

Figure 11 The unique domains between Src kinase and Fyn kinase are not

conserved Based on the sequence of Src inhibitory peptide (Src (40-58)) after sequence

alignment we designed Fyn inhibitory peptide (Fyn (39-57)

31


The dephosphorylation of this residue will result in the disruption of the interaction

between SH2 and C terminus of SFKs and activate SFKs (Fig 12)

SFKs are kept low at basal condition by two intramolecular interactions Here I

use Src kinase as an example one interaction is between the SH3 domain and the linker

region The other is between the SH2 domain and the phosphorylated Y527 in the C-

terminal SFK activation requires the dephosphorylation of Y527 andor

autophosphorylation of Y416 Y416 phosphorylation is taken as representive of the degree

of SFK activation SFKs can be activated in several ways the first way is to inhibit Csk

activity or increase the activity of phosphatase such as PTPα so the phosphorylation of

Y527 is reduced thus disrupting the interaction between SH2 domain and C-terminus and

activates SFKs The second way is to interrupt the binding of SH2 domain to the C-

terminal using a SH2 domain binding protein and enhance SFK activity The third way is

to weaken SH3 domain interacting with the linker region of SFK resulting in the increase

of SFK acitivy (Fig 11)

152 The modulation of NMDARs by SFKs

NMDARs can be regulated not only by serinetheronine kinase but also by SFKs

(Src and Fyn) (Chen and Roche 2007 Salter and Kalia 2004)

The regulation of NMDARs by Src has been well studied (Salter and Kalia 2004

Yu et al 1997) When Src activating peptide was applied directly to inside-out patches

taken from cultured neurons the open probability of NMDAR channels was increased

This effect was blocked by Src inhibitory peptide (Src (40-58)) suggesting

32

Figure 12 The structure of Src family kinases

33

that Src has ability to change the gating of GluN2ARs (Yu et al 1997) In contrast

neither Src nor Fyn altered the gating of recombinant GluN2BRs in HEK293 cells (Kohr

and Seeburg 1996) indicating that Fyn may enhance GluN2BR trafficking without

changing gating

In addition both tyrosine kinases and phosphatases can modulate NMDAR

activity through SFKs For example endogenous SFK activity could also be regulated by

Csk a tyrosine kinase which phosphorylated Y527 and inhibited SFK activity (Xu et al

2008) A recent study demonstrated that the application of recombinant Csk depressesed

NMDARs in acutely isolated cells This inhibitory effect was dependent on SFK activity

since it was occluded by SFK inhibitor PP2 (Xu et al 2008)

The GluN2A subunit is phosphorylated on a number of tyrosine residues such

studies have identified Y1292 Y1325 and Y1387 in the GluN2A C-tail as potential sites for

Src-mediated phosphorylation Another study showed that in HEK293 cells point

mutation Y1267F or Y1105F or Y1387F of GluN2A abolished Src potentiation of

NMDAR currents Additionally Src also failed to change the Zn2+ sensitivity of receptors

with any one of these three tyrosine mutations (Zheng et al 1998) although Xiong et al

(1999) did not agree (Xiong et al 1999) In addition Y842 of GluN2A was also

phosphorylated and dephosphorylation of this residue may regulate the interaction of

NMDARs with the AP-2 adaptor (Vissel et al 2001) This downregulation of interaction

was prevented by the inclusion of Src kinase in the pipette or by application of tyrosine

phosphatase inhibitors indicating that it was dependent on tyrosine phosphorylation

(Vissel et al 2001) Tyrosine phosphorylation of GluN2A subunits might also prevent

the removal of GluN2A by protecting the subunits against degradation from calpain

34

(Rong et al 2001) Src-mediated tyrosine phosphorylation of residues 1278-1279 of

GluN2A C-terminus inhibited calpain-mediated truncation and provided for the

stabilization of the NMDARs in postsynaptic structures (Bi et al 2000) Y1325 of

GluN2A was highly phosphorylated not only in the cultured cells but also in the brain

The phosphorylation of Y1325 was found to be critically involved in the regulation of

NMDAR channel activity and in depression-related behavior (Taniguchi et al 2009)

Up to now a number of studies demonstrated that Y1252 Y1336 and Y1472 were

potential sites of GluN2B phosphorylation by Fyn but Y1472 was the major site for

phosphorylation (Nakazawa et al 2001) What might be the function of phosphorylation

of GluN2B by Fyn The first is the trafficking of GluN2BR Y1472 was within a tyrosine-

based internalization motif (YEKL) which bound directly to the AP-2 adaptor

Phosphorylation of GluN2B Y1472 disrupted its interaction with AP-2 thereby resulting in

inhibition of the endocytosis of GluN2BR (Lavezzari et al 2003 Roche et al 2001)

The second is ubiquitination of GluN2BR After tyrosine residue Y1472 was

phosphorylated by Fyn the interaction between E3 ubiquitin ligase Mind bomb-2 (Mib2)

with GluN2B subunit was enhanced This led to the down-regulation of NMDAR activity

(Jurd et al 2008) This negative regulation of NMDARs may be one of the protective

mechanisms which neurons use to countertbalance the overactivation of the NMDARs

After NMDARs were phosphorylated and activated by Fyn if the hyperactivity of

NMDARs lasted for a long time it was detrimental to the neurons

Fyn phosphorylation of GluN2B is also involved in physiological functions such

as learning and memory as well as pathological functions such as pain One study

demonstrated that the level of Y1472 phosphorylation of GluN2B was increased after

35

induction of LTP in the hippocampus In addition in Fyn -- mice the phosphorylation of

Y1472 of GluN2B was reduced (Nakazawa et al 2001) Another phosphorylation site

Y1336 of GluN2B was very important for controlling calpain-mediated GluN2B cleavage

In cultured neurons the phosphorylation of GluN2B by Fyn potentiated calpain mediated

GluN2B cleavage But when Y1336 was mutated to Phenylalanine (Phe) Fyn failed to

increase the cleavage of GluN2B by calpain (Wu et al 2007) For the maintenance of

neuropathic pain Fyn kinase-mediated phosphorylation of GluN2B subunit of NMDAR

at Y1472 was found to be required (Abe et al 2005) Additionally mice with a GluN2B

Tyr1472Phe knock-in mutation exhibited deficiency of fear learning and amygdaloid

synaptic plasticity NMDAR mediated CaMKII signaling was also impaired in these

mutant mice (Nakazawa et al 2006)

153 The modulation of NMDARs by PTPs

The activity of NMDARs is regulated by tyrosine phosphorylation and

dephosphorylation (Wang and Salter 1994) Several studies have demonstrated that some

PTPs such as STEP61 (Pelkey et al 2002) and PTPα can regulate NMDAR activity (Lei

et al 2002) All members of the PTP family have at least one highly conserved catalytic

domain (Fischer et al 1991) the cysteine (Cys) residue within this motif is required for

PTP catalytic activity and mutation of this residue completely abolishes the phosphatase

activity (Pannifer et al 1998)

PTPα has two phosphatase domains and a short highly glycosylated extracellular

domain with no adhesion motif (Kaplan et al 1990) Biochemical studies indicated that

PTPα interacted with NMDAR through PSD95 PTPα enhanced NMDAR activity by

36

regulating endogenous SFK activity in cultured neurons It dephosphorylated Y527 in the

regulatory domain of SFKs and increased SFK activity (Lei et al 2002) By contrast

inhibiting PTPα activity with a functional inhibitory antibody against PTPα reduced

NMDAR currents in neurons (Lei et al 2002)

STEP family members are produced by alternative splicing consisting of

cytosolic (STEP46) and membrane-associated (STEP61) isoforms (Braithwaite et al

2006) SFK activity was also modulated by STEP61 which dephosphorylated Y416 After

the dephosphorylation by STEP61 SFK activity was decreased (Pelkey et al 2002)

Indeed exogenous STEP61 depressed NMDAR currents whereas inhibiting endogenous

STEP61 enhanced these currents but all of these effects were prevented by the inhibition

of Src (Pelkey et al 2002) In addition the reduced NMDAR activity by STEP61 was

mediated at least in part by the internalization of NMDARs (Snyder et al 2005b)

STEP61 dephosphorylated Y1472 of GluN2B subunit resulting in the endocytosis of

NMDARs (Snyder et al 2005b) Amyloid β (Aβ) was proposed to increase the

endocytosis of NMDARs through this pathway (Snyder et al 2005b) Recently Aβ was

found to increase the expression of STEP61 by inhibiting its ubiquitination resulting in

increased internalization of GluN2B subunits which may contribute to the cognitive

deficits in AD (Kurup et al 2010)

154 The regulation of LTP by SFKs

Our lab has demonstrated that the activity of NMDARs can be amplified by Src

family kinases (Src and Fyn) to trigger LTP (Huang et al 2001 Lu et al 1998

Macdonald et al 2006) Src and Fyn kinases have both been involved in the induction of

37

LTP at CA3-CA1 synapses (Grant et al 1992 Lu et al 1998a) In hippocampal slices

Src activating peptide caused an NMDAR-dependent enhancement of basal EPSPs and

occluded the subsequent LTP induction In contrast Src inhibitory peptide (Src (40-58))

inhibited the induction of LTP Therefore Src can act as a ldquocorerdquo molecule for LTP

induction (Lu et al 1998b) Tyrosine phosphatases and kinase also serve as ldquocorerdquo

molecules for LTP induction by regulating Src activity For example Pyk2 induced both

NMDAR and Ca2+ dependent increase of basal EPSPs and this enhancement could be

blocked by Src (40-58) (Huang et al 2001) In addition the tyrosine phosphatase

STEP61 blocked the induction of LTP by inactivating Src (Pelkey et al 2002) In

contrast Inhibitors of endogenous PTPanother different phosphatase which stimulated

Src by dephosphorylating Y524 of Src blocked the induction of LTP (Lei et al 2002)

Recently our lab has shown that during basal stimulation Src was continuously inhibited

by Csk Relief of Src suppression by a functional inhibitory antibody against Csk was

sufficient to induce LTP which was Src and NMDAR dependent (Xu et al 2008)

16 The regulation of NMDARs by GPCRs

GPCRs are the largest family of receptors in the cell membrane and a target of

currently available therapeutics agents (Jacoby et al 2006) These receptors are

characterized by their 7TM configuration (Pierce et al 2002) as well as by their

activation via heterotrimeric G proteins When a GPCR is activated its conformation

changes and allows the receptor to interact with G proteins The exchange of GTP for

GDP dissociates Gα from Gβγ subunits subsequently resulting in the activation of

various intracellular effectors (Gether 2000) The activation of G protein can be

38

terminated by regulators of G protein signaling (RGS) proteins resulting in the cessation

of signaling pathways induced by GPCRs (Berman and Gilman 1998) In addition more

and more studies indicate that some GPCR induced signaling does not depend on G

proteins (Ferguson 2001)

GPCRs include three distinct families A B and C based on their different amino

acid sequences Family A is the largest one and is divided into three subgroups Group

1a contains GPCRs which bind small ligands including rhodopsin Group 1b is activated

by small peptides and group 1c contains the GPCRs which recognize glycoproteins

Family B has only 25 members including PACAP (pituitary adenylate cyclase activating

peptide) and VIP (Vasoactive intestinal peptide) Family C is also relatively small and

contains mGluR as well as some taste receptors All of them have a very large

extracellular domain which mediates ligand binding and activation (Pierce et al 2002)

The Gα subunit that couples with these receptors is also used to classify receptors

They can be divided into four families Gαs Gαio Gαq11 Gα1213 The Gαs pathway

usually stimulates AC activity whereas the Gαio family inhibits it The Gαq pathway

activates PLCβ to produce inositol trisphosphate (IP3) and DAG while G1213 stimulates

Rho (Neves et al 2002)

NMDAR activity at CA3-CA1 hippocampal synapses is regulated by cell

signaling activated by various GPCRs and non-receptor tyrosine kinases such as Pyk2

and Src (Lu et al 1999a Macdonald et al 2005) We have shown that a variety of Gαq

containing GPCRs including mGluR5 M1 and LPA receptors enhanced NMDAR-

39

mediated currents via a Ca2+-dependent and sequential enzyme signaling cascade that

consisted of PKC Pyk2 and Src (Kotecha et al 2003 Lu et al 1999a) Furthermore

PACAP acted via the PAC1 receptor to enhance NMDA-evoked currents in CA1

transduction cascade rather than by stimulating the typical Gs AC and PKA pathway

(Macdonald et al 2005) Mulle et al (2008) also demonstrated that at hippocampal

mossy fiber synapses postsynaptic adenosine A2A receptor (a Gαq coupled receptor)

activation possibly regulated NMDAEPSCs via G proteinSrc pathway and was involved in

the LTP of NMDAEPSCs induced by HFS (Rebola et al 2008) Recently acetylcholine

(ACh) was shown to induce a long-lasting synaptic enhancement of NMDAEPSCs at

Schaffer collateral synapses this action was mediated by M1 receptors and the activation

of these receptors stimulated the PKCSrc signaling pathway to increase NMDAEPSCs

(Fernandez de and Buno 2010) Furthermore the activation of Gαq containing GPCRs

such as mGluR1 receptors also increased the surface trafficking of NMDARs (Lan et al

2001)

In addition Gαs containing GPCRs signals through PKA to modulate NMDAR

function For example β-adrenergic receptor agonists increased the amplitude of

EPSCNMDAs (Raman et al 1996) This increase in NMDAR currents was caused by the

increased gating of NMDARs Recent studies have shown that the Ca2+ permeability of

NMDARs was under the control of the cAMP-PKA signaling cascade and PKA

inhibitors reduced the relative fraction of Ca2+ influx through NMDARs (Skeberdis et al

2006) Similar to Gαq containing receptors Gαs containing receptor activation also

enhance the trafficking of NMDARs to the membrane surface For example dopamine

D1 receptor activation increased surface expression of NMDARs in the striatum This

40

interaction required the Fyn kinase since in Fyn -- mice dopamine D1 receptor agonist

failed to do so (Dunah et al 2004 Hallett et al 2006) Consistently the activation of

dopamine D1 receptors increased the surface expression of GluN2B subunits in cultured

PFC neurons (Hu et al 2010)

GluN2 subunits couple to distinct intracellular signaling complexes and play

differing roles in synaptic plasticity as the C-terminal domain of the subunits interacts

with various cytosolic proteins

17 Distinct Functional Roles of GluN2 subunits in synaptic plasticity

It was proposed that GluN2ARs are required for the induction of LTP while

GluN2BRs are responsible for LTD induction (Liu et al 2004 Massey et al 2004) This

proposal soon raised a lot of criticisms three research groups demonstrated that blocking

GluN1GluN2B receptors did not prevent the induction of LTD (Morishita et al 2007)

Another study even suggested that GluN2BR antagonist ifenprodil enhanced the

induction of LTD in the CA1 region of the hippocampus (Hendricson et al 2002) These

studies demonstrated that the induction of LTD did not require activation of GluN2BRs

Other electrophysiological studies have shown indeed in several regions of the

brain GluN2BRs promoted the induction of LTP induced by a number of stimulation

protocols GluN2B mediated LTP by directly associating with CaMKII (Barria and

Malinow 2005) In addition studies in transgenic animals showed that LTP could still be

induced in GluN2A subunit knockout mice while mice with overexpression of GluN2B

subunit demonstrated enhanced LTP (Tang et al 1999 Weitlauf et al 2005)

Additionally a recent paper demonstrated that for LTP induction the physical presence of

41

GluN2B and its cytoplasmic tail were more important than the activation of GluN2BRs

indicating GluN2B might function as a mediator of protein interactions independent of its

channel activity (Foster et al 2010)

So far many studies indicated that both GluN2AR and GluN2BR contributed to

the induction of LTP and LTD It was not surprising that the role of these receptor

subtypes in synaptic plasticity was more complicated Instead the ratio of GluN2AR

GluN2BR was proposed to determine the LTPLTD threshold In the kitten cortex a

reduction in GluN2ARGluN2BR ratio by visual deprivation was associated with the

enhancement of LTP (Cho et al 2009 Philpot et al 2007) This change has been

attributed to the reduction of GluN2A surface expression (Chen and Bear 2007) In

addition in hippocampal slices electrophysiological manipulation can change the ratio of

GluN2ARGluN2BR by different protocols The reduction of GluN2ARGluN2BR ratio

was associated with LTP enhancement whilst increasing this ratio favors LTD (Xu et al

2009)

It is well known that the threshold for the induction of LTP and LTD can be

influenced by prior activity In 1992 Malenka et al discovered that high frequency

stimulation induced LTP (Huang et al 1992) but if a weak stimulation was applied first

the subsequent LTP induction was inhibited In addition if an NMDAR antagonist APV

was added during the prestimulation the inhibition of subsequent LTP induction was

relieved This study demonstrated that this kind of metaplasticity was mediated by

NMDARs (Huang et al 1992)

18 Metaplasticity

42

Bear proposed that the ratio of GluN2ARGluN2BR determined the direction of

synaptic plasticity and anything that altered this ratio would serve as a mechanism of

ldquometaplasticityrdquo which is referred to as ldquoplasticity of plasticityrdquo (Abraham 2008

Abraham and Bear 1996 Yashiro and Philpot 2008) Bienenstock Cooper and Munro

(BCM model) (Bienenstock et al 1982) developed a theoretical model of metaplasticity

based upon observations of experience-dependent plasticity in the kitten visual cortex

Shifts to the right or left of the BCM ldquocurvesrdquo indicate metaplastic changes in plasticity

(θM the inflection point when LTD becomes LTP) In visually deprived kittens the

curves are shifted to the right indicative of a reduced value for θM (elevated LTP

threshold) (Yashiro and Philpot 2008) Recently metaplasticity was also demonstrated

in the hippocampus although its mechanism still remained unknown (Xu et al 2009

Zhao et al 2008)

Although many experimental protocols have been developed to investigate the

mechanism of metaplasticity they all required a prior history of activation before the

subsequent induction of synaptic plasticity This prior history may be induced by

electrical pharmacological or behavioral stimuli and is often dependent upon activation

of NMDARs Our lab has demonstrated that a lot of GPCRs had ability to regulate

NMDAR activity It is not surprising that the activation of GPCRs may changes the

threshold of subsequent LTP induction or LTD induction thus resulting in metaplasticity

As I mentioned before basal synaptic transmission at the CA1 synapse is mainly

mediated AMPARs because of the voltage-dependent block of NMDARs by Mg2+ In

fact the relief of Mg2+ block by depolarization alone cannot induce enough Ca2+ influx

through NMDARs for the induction of LTP The activity of NMDARs must also be

43

amplified by SFKs Our lab has shown that the recruitment of NMDARs during basal

transmission was limited not only by Mg2+ but also by Csk (Xu et al 2008) Additionally

SFKs were also involved in the NMDAR-mediated LTD Src kinases inhibited LTD in

cerebellar neurons (Tsuruno et al 2008) although their role in LTD has not been

examined at CA1 synapses In conclusion SFKs may govern the induction of LTP and

LTD through their regulation of NMDARs

In this dissertation I chose two different types of GPCRs as examples to

investigate this possibility One was PACAP receptor (PAC1 receptor) which is Gαq

coupled receptor The other were VIP receptors (VPAC12 receptors) they were Gαs

coupled receptor These receptors were highly expressed in the hippocampus and their

deficit in transgenic mice showed memory impairment (Gozes et al 1993 Otto et al

2001 Sacchetti et al 2001) In addition the activation of these receptors signaled

through different pathways

191 PACAP and VIP

19 PACAPVIP system

Almost 40 years ago VIP was isolated from pig small intestine by Said and Mutt

when they tried to identify the vasoactive substance which reduces blood pressure (Said

and Mutt 1969) The VIP gene contains 7 introns and 6 exons five of which have coding

sequences It can be translated into a 170 amino acid precursor peptide preproVIP This

precursor includes VIP and peptide histidine isoleucine (PHI) PHI is structurally related

to VIP and shares many of its biological actions but it is less potent than VIP After

44

several cleavages by enzymes both PHI and VIP can be produced from preproVIP

(Fahrenkrug 2010)

Since its discovery many studies have investigated the distribution of VIP in the

body It is mainly found in both the brain and the periphery In the CNS VIP is widely

distributed throughout the brain with highly expression in the cerebral cortex

hippocampus amygdala suprachiasmatic nucleus (SCN) and hypothalamus (Dickson and

Finlayson 2009 Vaudry et al 2000 Vaudry et al 2009)

In 1989 PACAP38 was discovered in ovine hypothalamus by Arimura (Miyata et

al 1989) In the same year a second peptide PACAP27 was purified This peptide is a

C-terminally truncated form of PACAP38 Both PACAPs show 68 sequence homology

with VIP and they all belong to the VIPglucagonsecretin superfamily (Dickson and

Finlayson 2009 Harmar et al 1998) In addition PACAP38 has more than 1000-fold

higher ability to activate AC compare to VIP (Miyata et al 1990) Multiple factors are

known to stimulate PACAP38 gene expression including phorbol esters and cAMP

analogues (Suzuki et al 1994 Yamamoto et al 1998) The PACAP gene consists of

five exons and four introns Exon 5 encodes PACAP38 while exon 4 encodes PACAP

related peptide (PRP) Translation of the PACAP mRNA produces a 176 amino acid

peptide prepro PACAP After they are cleaved by prohormone convertases (PC) both

PACAP38 and PRP are yielded (Dickson and Finlayson 2009 Vaudry et al 2000

Vaudry et al 2009)

PACAP38 a dominant isoform of PACAPs in the brain is highly expressed in the

CNS Its expression is very high in the hypothalamus the amygdala the cerebral cortex

and hippocampus Although PACAP expression in neurons has been well demonstrated

45

it is also expressed in astrocytes (Dickson and Finlayson 2009 Vaudry et al 2000

Vaudry et al 2009)

Both PACAP and VIP can be co-released with classical transmitters by electrical

stimulation For example activation of the postganglionic parasympathetic nerves that

innervate blood vessels releases both VIP and ACh (Fahrenkrug and Hannibal 2004)

Furthermore in retinal ganglion cells that project to the SCN PACAP can be released

with glutamate together to adjust the circadian rhythm (Michel et al 2006) In addition

to acting as neurotransmitter both PACAP and VIP can regulate the release of some

neurotransmitters by acting as neuromodulators Recently one study demonstrates that

PACAP modulates acetylcholine release at neuronal nicotinic synapses (Pugh et al

2010)

192 PACAP VIP receptors

Three receptors for PACAP and VIP have been identified all of which belong to

family B of GPCRs PAC1 receptor exhibits a higher affinity for PACAP than VIP

whereas VPAC1 receptor and VPAC2 receptor have similar affinities for PACAP and

VIP (Harmar et al 1998) The difference between these receptors is illustrated by the

observation that secretin has a higher affinity for the VPAC1 receptor than for the

VPAC2 receptor

In 2001 Murthy and co-workers identified a new VIP receptor in guinea-pig

smooth muscle cells In contrast to VPAC receptors this receptor could only be activated

by VIP but not PACAP (Teng et al 2001) Several other groups confirmed the existence

of this selective VIP receptor Gressens and colleagues demonstrated that this selective

46

VIP receptor mediated the neuroprotective effects by VIP following brain lesions in

newborn mice (Gressens et al 1994 Rangon et al 2005) This action could only be

mimicked by VPAC2 receptor agonists and PHI whereas VPAC1 receptor agonists and

the PACAP peptides had no effect (Rangon et al 2005) In addition Ekblad and

colleagues showed that this specific VIP receptor was also only activated by VIP in the

mouse intestine (Ekblad et al 2000 Ekblad and Sundler 1997)

Although all of these receptors are highly expressed in the hippocampus PAC1

receptor is more abundant and widely distributed compared to VPAC1 receptor and

VPAC2 receptor (Dickson and Finlayson 2009 Vaudry et al 2000 Vaudry et al 2009)

To date 4 variants of VPAC receptors have been described although the PAC1

receptor has more than 7 splice variants (Dickson and Finlayson 2009) The first two

VPAC receptor variants were VPAC1R 5-TM and VPAC2R 5-TM They lack the third

IC loop the third EC loop and the TM domains 6-7 and have the poor ability to stimulate

the cAMP dependent pathway (Bokaei et al 2006) In addition two deletion variants of

the VPAC2 receptor have also been identified One was VPAC2de367-380 which deletes

14 amino acid from 367 to 380 at its C-terminal end (Grinninger et al 2004) so the

ability of this mutant to activate cAMP was weak The second VPAC2 receptor variant

(VPAC2de325-438(i325-334)) had a deletion in exon 11 which created a frame shift and

introduced a premature stop codon these changes impaired its ability to induce signaling

pathways (Miller et al 2006)

In the rat five splice variants of the PAC1 receptor were produced by alternative

splicing in the third intracellular loop region They were null hip hop1 hop2 and

hiphop1 (Spengler et al 1993) Their differences lay in the presence of two 28 amino

47

acid cassettes (hip and hop) in the third loop (Journot et al 1995) The presence of the

hip cassette impaired the ability of PAC1 receptor to stimulate AC and PLC activity

(Spengler et al 1993) In addition three other splice variants in the N-terminal

extracellular domain have been identified The full length PAC1 variant was called

PAC1normal (PAC1n) the second variant named PAC1short (PAC1s) (residues 89-109)

had 21 amino acid deletion and the third variant PAC1veryshort (PAC1vs) lacked 57

amino acids (residues 53-109) (Dautzenberg et al 1999) PAC1s showed the same

affinity for PACAP38 PACAP27 and VIP While PAC1vs bound PACAP38 and

PACAP27 with lower affinity compared to PAC1n (Dautzenberg et al 1999) Another

PAC1 splice variant (PAC1TM4) lacked transmembrane regions 2 and 4 Binding of

PACAP27 to PAC1TM4 opens L-type Ca2+ channels (Chatterjee et al 1996)

193 Signaling pathways initiated by the activation of PACAPVIP receptors

The activation of PAC1 receptors signals either through Gαq11 to PLC or to AC

pathway via Gαs (Dickson and Finlayson 2009 Harmar et al 1998 McCulloch et al

2002 Spengler et al 1993) So PACAP stimulates both PKA and PKC dependent

signaling pathways (Dickson and Finlayson 2009 Harmar et al 1998) In contrast the

VPAC receptor activation only couples to Gαs and thus only activates AC dependent

signaling pathways (Spengler et al 1993)

In addition to cAMP the activation of both PAC1 receptor and VPAC receptors

can stimulate the increase of intracellular Ca2+ ([Ca2+]i) (Dickson et al 2006 Dickson

and Finlayson 2009) Using a VPAC2 agonist R025-1553 it was demonstrated that

VPAC2 receptors were involved in increasing [Ca2+]i (Winzell and Ahren 2007)

48

Furthermore additional signaling pathways that are not G-protein-mediated may also

exist For example the activation of VPAC receptors also modulated the activity of

phospholipase D (PLD) (McCulloch et al 2000) which was dependent on the small G-

protein ARF (ADP-ribosylation factor) (McCulloch et al 2000)

194 The mechanism of NMDAR modulation by PACAP

Previous studies have shown that PACAP enhanced NMDAR activity in the

hippocampal CA1 regions (Liu and Madsen 1997 Michel et al 2006 Wu and Dun

1997 Yaka et al 2003) However Liu and Madsen (1997) proposed that this modulation

was independent of intracellular second messengers possibly acting through the glycine

binding site (Liu and Madsen 1997) In contrast the Ron group proposed PAC1 receptor

activation increased NMDAR-mediated currents through a PKAFynGluN2BR signaling

pathway (Yaka et al 2003) They showed that this enhancement was abolished in the

presence of the specific GluN2BR antagonist ifenprodil Furthermore in slices from Fyn

knockout mice (Fyn --) they reported that PACAP failed to potentiate NMDAR-

mediated field EPSPs (Yaka et al 2003) Critical to this interpretation was the use of

peptides designed to interfere with the binding of GluN2BR and Fyn to receptor of

activated protein kinase C1 (RACK1) Salter pointed out a flaw in that one of the

peptides targeted a region that was not unique to Fyn this peptide would modulate Src as

well as Fyns interactions with RACK1 (Salter and Kalia 2004)

The activation of PAC1 receptors can couple the Gαs pathway in addition to the

Gαq pathway our lab therefore re-examined pathways by which PAC1 receptors

regulated NMDARs Individual CA1 pyramidal neurons acutely isolated from brain

49

slices were recorded from using whole-cell voltage-clamp Using a rapid perfusion

system the exact drug concentration applied to the cell was precisely controlled In

addition the resolution of both peak and steady state of NMDAR currents could be easily

determined by this method (Macdonald et al 2005 Macdonald et al 2001) The

application of PACAP (1 nM) increased NMDA-evoked current in acutely isolated CA1

pyramidal neurons This potentiation induced by PACAP was blocked by a specific

PAC1 receptor antagonist PACAP (6-38) confirming that this enhancement was

mediated by the PAC1 receptor (Macdonald et al 2005) Additionally in contrast to

Liursquos finding (Liu and Madsen 1997) heterotrimeric G-proteins were found to be

involved since using GDP-β-S a competitive inhibitor for the GTP binding site

abolished this potentiation (Macdonald et al 2005) The G-protein subtype involved in

this signaling pathway was Gαq as the application of a specific RGS2 protein which

selectively prevented the binding of Gαq to GPCRs eliminated the PACAP induced

enhancement (Macdonald et al unpublished data) In mice lacking PLCβ the

enhancement of NMDARs was significantly attenuated A role for PKC signaling in this

pathway was implicated because bisindolymaleimide I an inhibitor of PKC blocked the

PACAP effect In addition applications of the functionally dominant-negative form of

recombinant CAKβ CAKβ 457A and the Src specific inhibitor Src (40-58) both blocked

the potentiation of NMDAR currents by PACAP These results confirmed that the PAC1

receptor activation could enhance NMDAR currents via a GαqPLCβ1PKCPyk2Src

signal cascade (Macdonald et al 2005)

110 The Hippocampus

50

The hippocampus is one of the most widely studied regions in the brain and is

very important for learning and memory the patient who has hippocampus impairment

demonstrated memory deficit (Milner 1972) Additionally the function of the

hippocampus is disrupted in many neurological diseases such as Alzheimerrsquos disease and

schizophrenia (Terry and Davies 1980) The hippocampal formation includes two

interlocking C-shaped regions the hippocampus and the dentate gyrus It forms three

important fiber pathways One is the perforant pathway which links the entorhinal cortex

to the hippocampus The second is the mossy fibre pathway which runs from the dentate

gyrus to the CA3 region The last is the schaffer collaterals which connects the CA3

region pyramidal neurons with those in the CA1 region

In this dissertation all the work has been done using rodent hippocampus There

are several reasons One is that it is easy to dissect the rodent hippocampus In addition

it has a highly structured and clearly laminar cellular organization so it it easy to identify

and isolate neurons from the hippocampus for acutely isolated cell recordings

Furthermore transverse slices from the hippocampus preserve normal neuronal circuitry

so field recording and whole cell recording in the slices can be done in vitro Overall the

relatively accessible nature of the hippocampus for in vivo studies and ease of slice

preparation and maintenance for in vitro studies make the hippocampus an attractive

model system

111 The Pharmacology of GluN2 subunits of NMDARs

In my thesis I used several different specific GluN2 containing NMDAR

antagonists to investigate if Src and Fyn selectively modulated GluN2AR and GluN2BR

51

respectively So the properties of these GluN2 containing NMDAR antagonists were

introduced here

There are several agents which selectively inhibit GluN2 containing NMDARs

Although selective GluN2BR antagonists such as ifenprodil and Ro25-6981 are available

a selective GluN2AR antagonist is still lacking Ifenprodil bound with GluN2BRs having

about 400 fold selectivity for GluN2BR over GluN2AR (Williams 1993) Another

GluN2BR antagonist Ro 25-6981 had about 5000-fold selectivity for GluN2BR over

GluN2AR (Fischer et al 1997) Although early reports claimed NVP-AAM077

displayed strong selectivity for GluN2ARs over GluN2BRs (Auberson et al 2002) later

it was demonstrated that it had only 9-fold selectivity for GluN2AR over GluN2BR in

Xenopus oocytes and HEK293 cells (Bartlett et al 2007 Berberich et al 2005 Neyton

and Paoletti 2006) In addition NVP-AAM077 could also block GluN2C- and GluN2D-

containing receptors (GluN2CR and GluN2DR respectively) (Feng et al 2004)

Although ifenprodil shows high selectivity for GluN2BR over GluN2AR there

are still several drawbacks to its use Firstly ifenprodil primarily inhibited NMDARs

when a high concentration of glutamate was present (it is a non-competitive antagonist)

In contrast with very low glutamate concentrations ifenprodil could actually potentiate

NMDAR currents (Kew et al 1996) Secondly ifenprodil could not totally block

GluN2BRs It only partially inhibited at most 80 of the current mediated by GluN2BRs

(Williams 1993) Thirdly ifenprodil also affected triheteromeric GluN12A2B receptors

(Neyton and Paoletti 2006) The most potent and selective inhibitor of GluN2ARs is

Zn2+ (Paoletti et al 1997 Paoletti et al 2000 Paoletti et al 2009 Rachline et al 2005)

But this GluN2AR antagonist also has some problems firstly it partially inhibited

52

GluN2AR mediated currents (Paoletti et al 2009) secondly Zn2+ also inhibited

triheteromeric GluN1GluN2AGluN2B receptors (Paoletti et al 2009) and thirdly it

had a lot of other targets besides NMDARs (Smart et al 2004) so it could not be used in

slices or in vivo (Neyton and Paoletti 2006)

In addition specific GluN2CRGluN2DR antagonists are also available PPDA

displayed some selectively for GluN2CRGluN2DR over GluN2ARGluN2BR although

this selectivity was weak (Feng et al 2004) Recently a new selective

GluN2CRGluN2DR antagonist quinazolin-4-one derivatives has been identified which

had 50-fold selectiviey over GluN2ARGluN2BR (Mosley et al 2010)

There are several uncompetitive NMDAR antagonists available as well

(Macdonald et al 1990 Macdonald et al 1991 Macdonald and Nowak 1990 McBain

and Mayer 1994 Traynelis et al 2010) These compounds included phencyclidine

(PCP) ketamine MK-801 and memantine they were open channel blockers Only when

NMDARs were open they blocked NMDAR channels (Macdonald et al 1990

Macdonald et al 1991 Macdonald and Nowak 1990 McBain and Mayer 1994

Traynelis et al 2010) All of these compounds had high affinity for NMDARs except

memantine they induced psychotomimetic-like effect in animals and were used to induce

schizophrenia symptoms in rodents (Neill et al 2010) In contrast memantine

demonstrated low affinity for NMDARs and had fast on-and-off kinetics (Chen and

Lipton 2006 Lipton 2006) Now memantine is used in clinical to treat memory deficit

in moderate to severe Alzheimerrsquos disease (Chen and Lipton 2006 Lipton 2006)

112 GluN2 subunit knockout mice

53

There has been great interest and controversy about the role of GluN2 subunits in

synaptic plasticity Much of the argument came from the selectivity of GluN2AR

antagonist Therefore genetically modified mice in which GluN2 subunit is selectively

maniputed provide an alternative way

So far global GluN2B (GluN2B --) and GluN1 knockout (GluN1 --) mice cannot

survive after birth (Forrest et al 1994 Kutsuwada et al 1996) but global GluN2A

(GluN2A --) GluN2C (GluN2C --) and GluN2D knockout (GluN2D --) mice are viable

(Ebralidze et al 1996 Miyamoto et al 2002 Sakimura et al 1995) only recently

conditional GluN2B -- mice are generated (Akashi et al 2009 von et al 2008)

Because GluN1 subunits were required for the formation of functional NMDARs

GluN1 -- mice died after birth (Forrest et al 1994) but GluN1 knockdown mice could

survive In these mutant mice the expression of GluN1 subunit was reduced so the

quantity of functional NMDARs produced was only 10-20 of normal levels The

residual NMDARs in GluN1 knockout mice might explain why they avoided the lethality

and survived (Ramsey et al 2008 Ramsey 2009)

In GluN2A -- mice both NMDAR current and hippocampal LTP were

significantly reduced at the CA1 synapses In addition learning and memory were

impaired in these mutants (Sakimura et al 1995) At the commissuralassociational CA3

synapse these knockout mice demonstrated reduced EPSCNMDAs and LTP (Ito et al 1997)

Recently when these knockout mice were exposed to a lot of behavior tests they

demonstrated normal spatial reference memory water maze acquisition but their spatial

working memory was impaired (Bannerman et al 2008)

54

Global GluN2B -- mice cannot survive to adult because GluN2B is very

important for the development In the hippocampus of these mutant mice synaptic

NMDA responses and LTD were also abolished (Kutsuwada et al 1996) Consistently

in GluN2B overexpression mice both hippocampal LTP and learning and memory were

enhanced (Tang et al 1999) Additionally at the fimbrialCA3 synapses both

EPSCNMDAs and LTP were diminished in these GluN2B -- mice (Ito et al 1997)

Recently several conditional GluN2B -- mice were generated (Akashi et al 2009 von

et al 2008) these transgenic mice demonstrated significant deficits in synaptic plasticity

and some behaviours

In addition GluN2C subunits were mostly expressed in the cerebellum in

GluN2C -- mice NMDAR currents at mossy fibergranule cell synapses were increased

but non-NMDA component of the synaptic currents was reduced (Ebralidze et al 1996)

Despite these changes the GluN2C -- mice showed no deficit in motor coordination tests

(Kadotani et al 1996) However when GluN2C -- and GluN2A -- were crossed to

produce doubled knockout mice (GluN2C -- GluN2A --) these mutants had no

NMDARs in the cerebellum and EPSCNMDAs also disappeared In addition motor

coordination of these mutants was also impaired (Kadotani et al 1996)

No abnormal phenotype was found in GluN2D -- mice but their monoaminergic

neuronal activities were upregulated Additionally the spontaneous locomotor activity of

these mutant mice was reduced In the elevated plus-maze light-dark box and forced

swimming tests these mice demonstrated less sensitivity to stress (Miyamoto et al

2002)

55

As I mentioned above the C-terminus of GluN2 subunits were very important

since they mediated interactions of the NMDARs with many signaling molecules In

order to investigate the role of C-terminus of GluN2 subunits in synaptic plasticity

transgenic mice which expressed NMDARs without the C-terminus of GluN2A or

GluN2B or GluN2C were generated (Sprengel et al 1998) Mice expressing truncated

GluN2B subunits died perinatally while mice with truncated GluN2A subunits were able

to survive but their synaptic plasticity and contextual memory were impaired (Sprengel

et al 1998) In addition all of these transgenic mice including mice containg truncated

GluN2C mice displayed deficits in motor coordination (Sprengel et al 1998)

Our lab has demonstrated that the activation of PAC1 receptors which are Gαq

coupled receptors increases NMDAR activity through a PKCCAKβSrc signaling

pathway During the analysis of our data we noticed that the activation of PAC1

receptors by low concentration of PACAP (1 nM) enhanced the peak of NMDA currents

to a greater extent than the steady-state of NMDA-evoked currents (Fig 13) Due to

kinetic differences between the activation rates of NMDARs composed of either

GluN2AR or GluN2BR NMDA peak currents are more likely to be contributed by

GluN2ARs while GluN2BRs contribute more strongly to the sustained or steady-state

component of the currents (Macdonald et al 2001) This led us to propose that Gαq

couple receptor such as PAC1 receptor activation may specifically targets GluN2AR via

GαqPKCSrc pathway

113 Overall hypothesis

56

In contrast Gαs coupled receptor may selectively modulate GluN2BR over

GluN2AR via GαsPKAFyn pathway Bear has proposed that the change of

GluN2ARGluN2BR ratio induced metaplasticity (Abraham 2008 Abraham and Bear

1996) So different GPCRs may have the ability to regulate the ratio of

GluN2ARGluN2BR and induce metaplasticity

57

10 min afterPACAP

Baseline

1s200pA

1a

A

091

1112131415161718

PACAPPeak

PACAPSS

Norm

alize

d Cu

rrent

Figure 13 PACAP selectively enhanced peak of NMDAR currents A Sample traces

from the same cell before baseline and after the application of PACAP (1 nM) B

PACAP selectively enhanced peak of NMDA current over its steady state

B

58

Section 2

Methods and Materials

59

Hippocampal CA1 neurons were isolated from postnatal rats (Wistar 14-22 days)

or postnatal mice (28-34 days) using previously described procedures (Wang and

Macdonald 1995) To control for variation in response recordings from control and

treated cells were made on the same day Following anesthetization and decapitation the

brain was transferred to ice cold extracellular fluid (ECF) The extracellular solution

consisted of (in mM) 140 NaCl 13 CaCl2 5 KCl 25 HEPES 33 glucose and 00005

tetrodotoxin (TTX) with pH 74 and osmolarity between 315 and 325 mOsm TTX was

added in order to block voltage-gated sodium channels and reduce neuronal excitability

The hippocampus was rapidly isolated and transverse slices were cut by hand Then

hippocampal slices were stored in oxygenated ECF at room temperature for 45 minutes

later papain was added to digest hippocampal slices for 30 minutes Slices were then

washed three times in fresh ECF and allowed to recover in oxygenated ECF at room

temperature (20-22ordmC) for two hours before use Before the recording hippocampal slices

were transferred to a cell culture dish and placed under a microscope Fine tip forceps

were used to isolated neurons by gently abrading the pyramidal CA1 area of the slices

This action caused dissociation of neurons from the specific area being triturated

21 Cell isolation and whole Cell Recordings

Cells were patch clamped using glass recording electrodes (resistances of 3-5

MΩ) these recording electrodes were constructed from borosilicate glass (15 microm

diameter WPI) using a two-stage puller (PP83 Narashige Tokyo Japan) and filled with

intracellular solution that contained (in mM) 140 CsF 11 EGTA 1 CaCl2 2 MgCl2 10

HEPES 2 tetraethylammonium (TEA) and 2 K2ATP pH 73 (osmolarity between 290

and 300 mOsm) Upon approaching the cell negative pressure (suction) was

60

Figure 21 Representation of rapid perfusion system in relation to patched

pyramidal CA1 neurons A Several acutely isolated CA1 hippocampal pyramidal

neurons under phase contrast microscopy B the representation of multi-barrel system

and typical NMDA evoked current All the barrels contain glycine and only one barrel

includes NMDA Shifting barrels to the NMDA-containing barrel by computer control

evokes NMDAR current

61

applied to the patch pipette to form a seal After the formation of a tight seal (gt1 GΩ)

negative pressure was then used to rupture the membrane and form whole cell

configuration When the whole-cell configuration is formed the neurons were voltage

clamped at -60 mV and lifted into a stream of solution supplied by a computer-controlled

multi-barreled perfusion system (Lu et al 1999a Wang and Macdonald 1995) To

monitor access resistance a voltage step of -10 mV was made before each application of

NMDA When series resistance varied more than 15 MΩ the cell was discarded Drugs

were included in the patch pipette or in the bath Recordings were conducted at room

temperature (20-22degC) Currents were recorded using MultiClamp 700B amplifiers

(Axon Instruments Union City CA) and data were filtered at 2 kHz and acquired using

Clampex (Axon Instruments) All population data are expressed as mean plusmn SE The

Students t-test was used to compare between groups and the ANOVA test was used to

analyze multiple groups

Transverse hippocampal slices were prepared from 4- to 6-week-old Wistar rats

using a vibratome (VT100E Leica) After dissecting hippocampal slices were placed in

a holding chamber for at least 1 hr before recording in oxygenated (95 O2 5 CO2)

artificial cerebrospinal fluid (ACSF) (in mM 124 NaCl 3 KCl 13 MgCl2-6H2O 26

CaCl2 125 NaH2PO4-H2O 26 NaHCO3 10 glucose osmolarity between 300-310

mOsm) A single slice was then transferred to the recording chamber continually

superfused with oxygenated ACSF at 28-30degC with a flow rate of 2 mLmin Synaptic

responses were evoked with a bipolar tungsten electrode located about 50 μm from the

22 Hippocampal Slice Preparation and Recording

62

cell body layer in CA1 Test stimuli were evoked at 005 Hz with the stimulus intensity

set to 50 of maximal synaptic response For voltage-clamp experiments the patch

pipette (4ndash6 MΩ) solution (in mM 1325 Cs-gluconate 175 CsCl 10 HEPES 02

EGTA 2 Mg-ATP 03 GTP and 5 QX 314 pH 725 290 mOsm) Patch recordings

were performed using the ldquoblindrdquo patch method 10uM bicuculline methiodide and 10uM

CNQX was added into ACSF to isolate NMDA receptor mediated EPSCs Cells were

held at -60 mV and series resistance was monitored throughout the recording period

Only recordings with stable holding current and series resistance maintained below 30

MΩ were considered for analysis Signals were amplified using a MultiClamp 700B

sampled at 5 KHz and analyzed with Clampfit 102 software (Axon Instruments Union

City CA)

Field excitatory postsynaptic potentials (fEPSPs) were evoked at a frequency of

005 Hz by electrical stimulation (100 μs duration) delivered to the Schaffer-collateral

pathway using a concentric bipolar stimulating electrode (25 μm exposed tip) and

recorded using glass microelectrodes (3-5 MΩ filled with ACSF) positioned in the

stratum radiatum layer of the CA1 subfield Electrode depth was varied until a maximal

response was elicited (approximately 175 microm from surface) The input-output

relationship was first determined in each slice by varying stimulus intensity (10-1000 microA)

and recording the corresponding fEPSP Using stimulus intensity that evoked 30-40 of

the maximal fEPSP paired-pulse responses were measured every 20 s by delivering two

stimuli in rapid succession with intervals (interstimulus interval ISI) varying from 10-

1000 ms Following this protocol fEPSPs were evoked and measured for twenty minutes

at 005 Hz using the same stimulus intensity to test for stability of the response At this

63

time plasticity was induced by 1 10 50 or 100 Hz stimulation with train pulse number

constant at 600 Any treatments were added to ACSF and applied to the slice for the ten

minutes immediately prior to the induction of plasticity

Hippocampal slices were prepared from Wistar rats (2 weeks to 3 weeks) and

incubated in ACSF saturated with 95 O2 and 5 CO2 for at least 1h at room

temperature This was followed by treatment with either PACAP (1 nM for 15 min) and

their vehicles for control After wash with cold PBS 3 times slices were homogenized in

ice-cold RIPA buffer (50 mM TrisndashHCl pH 74 150 mM NaCl 1 mM EDTA 01 SDS

05 Triton-X100 and 1 Sodium Deoxycholate) supplemented with 1 mM sodium

orthovanadate and 1 protease inhibitor cocktail 1 protein phosphatases inhibitor

cocktails and subsequently spun at 16000 rcf for 30 min at 4degC (Eppendorf Centrifuge

5415R) The supernatant was collected and kept at -70degC For immunoprecipitation the

sample containing 500 microg proteins was incubated with antibodies (see below) at 4degC and

gently shaken overnight Antibodies used for immunoprecipitation were anti-GluN2A

and GluN2B (3 microg rabbit IgG Enzo Life Sciences 5120 Butler Pike PA) anti-Src (1

500 mouse IgG Cell Signaling Technology (CST) 3 Trask Lane Danvers MA) The

immune complexes were collected with 20 microl of protein AGndashSepharose beads for 2 h at

4degC Immunoprecipitants were then washed 3 times with ice-cold PBS resuspended in 2

times Laemmli sample buffer and boiled for 5 min These samples were subjected to SDSndash

PAGE and transferred to a nitrocellulose membrane The blotting analysis was performed

by repeated stripping and successive probing with antibodies anti-pY(4G10) (12000

23 Immunoprecipitation and Western blotting

64

mouse IgG Millipore Corp 290 Concord Rd Billerica MA 01821) anti-GluN2A and

anti-GluN2B (11000 rabbit IgG CST 3 Trask Lane Danvers MA) pSrcY416 (11500

rabbit IgG CST 3 Trask Lane Danvers MA)

All animal experiments were conducted in accordance with the policies on the

Use of Animals at the University of Toronto GluN2A -- mice were provided by Ann-

Marie Craig (University of British Columbia Vancouver Canada) Both wild type and

GluN2A -- mice (5-6 weeks old) used in all experiments have a C57BL6 background

24 Animals

The drugs for this study are as follows NMDA glycine BAPTA Tricine ZnCl2

and R025-6981 from Sigma (St Louis MO USA) PACAP VIP Rp-cAMPS PKI14-22

U73122 U73343 bisindolylmaleimide I and phosphodiesterase 4 inhibitor (35-

Dimethyl-1-(3-nitrophenyl)-1H-pyrazole-4-carboxylic acid ethyl ester) from Calbiochem

(San Diego CA USA) Src (p60c-Src) and Fyn (active) (Upstate Biotechnology CA

USA) InCELLect AKAP St-Ht31 inhibitor peptide from Promega (Madison WI USA)

Bay55-9877 [Ala11 22 28]VIP [Ac-Tyr1 D-Phe2]GRF (1-29) and CNQX from Tocris

(Ellisville MI USA) 8-pCPT-2prime-O-Me-cAMP Sp-8-pCPT-2prime-O-Me-cAMPS and 8-OH-

2prime-O-Me-cAMP (Biolog life science institute Bremen Germany) Src (40-58) and

scrambled Src (40-58) were provided by Dr M W Salter (Hospital for Sick Children

Toronto Canada) Maxadilan and M65 were a gift from Dr Ethan A Lerner (Harvard

University Boston USA) NVP-AAM077 was provided by Dr YP Auberson (Novartis

25 Drugs and Peptides

65

Pharma AG Basel Switzerland) Peptides were synthesized by the Advanced Protein

Technology Centre (Toronto Ontario Canada) with the following sequences Fyn

inhibitory peptide (Fyn (39-57)) (YPSFGVTSIPNYNNFHAAG Fyn amino acids 39-57)

scrambled Fyn inhibitory peptide (Scrambled Fyn (39-57)) (PSAYGNPGSAYFNFT

-NVHI)

All population data are expressed as mean plusmn SE Studentrsquos t-test was used to

compare between two groups and the ANOVA test was used to analyze among multiple

groups

26 Statistics

66

Section 3 Results

Project 1 The activation of PAC1 receptors (Gαq coupled receptors) selectively modulates GluN2ARs and favours

LTP induction

67

Activation of PAC1 receptors by low concentration of PACAP (1 nM) enhanced

NMDAR currents via PKCCAKβSrc pathway rather than by PKA and Fyn (Macdonald

et al 2001) In preliminary and unpublished experiments it was shown that both Src and

low concentrations of PACAP (1 nM) preferentially enhanced the peak of NMDAR-

evoked currents in a small subset of recordings but only provided very rapid applications

of NMDA were achieved (Macdonald et al unpublished data) Also the effects of Src

were blocked by a relatively selective GluN2AR antagonist (Macdonald et al

unpublished) Given the more rapid kinetics of GluN2AR versus GluN2BR we

hypothesized that Src might also selectively target GluN2ARs and not GluN2BRs as

proposed by Ronrsquos group (Yaka et al 2003) Therefore we propose that PAC1 receptor

activation in CA1 pyramidal neurons of the hippocampus specifically targets GluN2ARs

over GluN2BRs to enhance the effects of the GluN2A over the GluN2B subtype of

NMDARs

311 Hypothesis

PACAP (1 nM) enhances NMDA evoked current via the PAC1 receptors

(Macdonald et al 2005) In order to examine if the effect of PAC1 receptor activation by

PACAP is mainly mediated by GluN2A NMDAR currents were evoked once every 60

seconds using a three second exposure to NMDA (50 microM) and glycine (05 μM) After 5

minutes of stable baseline recording I applied PACAP (1 nM) in the bath for 5 minutes

after which it was washed out The applications of PACAP produced a rapid and robust

increase in peak NMDA evoked currents In order to determine if PACAP (1 nM)

312 Results

68

selectively modulates GluN2AR over GluN2BR a series of experiments were performed

using GluN2R antagonists in all extracellular solutions If during the application of a

GluN2AR antagonist the PACAP modulation of NMDAR currents is inhibited we can

conclude that GluN2ARs are required for this modulation but if no block of the PACAP

effect is observed we can conclude that GluN2ARs are not required The same

conclusions can be reached for GluN2BRs using GluN2BR antagonists Ro 25-6981 is

the most potent and selective blocker of GluN2BRs having about a 5000-fold selectivity

for GluN2BR over GluN2AR (Fischer et al 1997) While GluN2AR selective antagonist

NVP-AAM077 displays considerably lower selectivity It has only about 9-fold

selectivity for GluN2AR over GluN2BR (Neyton and Paoletti 2006) Due to the fact that

at a concentration of 400 nM NVP-AAM077 almost entirely blocked NMDAR currents

in acutely isolated cells (Yang et al unpublished data) all the experiments were

performed with a lower concentration of NVP-AAM077 (50 nM) this concentration was

specifically recommended by George Kohr in his paper (Berberich et al 2005) When I

added GluN2AR antagonist NVP-AAM077 (50 nM) or GluN2BR antagonist Ro 25-6981

(100 nM) in the extracellular solutions tbe basal absolute NMDAR currents was

significantly reduced compared to the control solutions without these drugs (Yang et al

unpublished data) In order to keep the basal absolute NMDAR currents in the presence

of GluN2R antagonists the same as that in the control solution I applied NMDA (100

microM) and glycine (1 μM) to evoke NMDAR currents when I added these GluN2R

antagonists to the extracellular solutions (Yang et al unpublished data) The use of NVP-

AAM077 (50 nM) in all external solutions blocked the ability of PACAP to increase

normalized NMDAR peak currents In contrast the inclusion of Ro 25-6981 (100 nM) in

69

the bath had no effect on the ability of PACAP to increase normalized NMDAR mediated

peak currents (1 nM PACAP plus NVP-AAM077 24 plusmn 16 n=6 1 nM PACAP plus

284 plusmn 49 n=5 1 nM PACAP 385 plusmn 52 n=6) These results suggested that

GluN2BRs were not involved in the increase of NMDAR currents by PACAP (1 nM)

although NVP-AAM077 has ability to block GluN2ARs it also antagonizes GluN2CR

and GluN2DR (Fig 311)

Next in order to exclude the involvement of GluN2CR and GluN2DR in the

potentiation of NMDAR by PACAP (1 nM) a more specific GluN2AR antagonist Zn2+

was chosen to block GluN2ARs In the nanomolar range Zn2+ is highly potent at

inhibiting GluN2ARs displaying strong selectivity for GluN2ARs over all other

GluN1GluN2 receptors (gt100 fold) (Paoletti et al 1997) Zn2+ chelator tricine was used

to buffer Zn2+ and Zn2+ (300 nM) in the solution was applied to selectively antagonize

GluN2ARs as recommended by Paoletti (Paoletti et al 1997 Paoletti et al 2009

Paoletti and Neyton 2007) Tricine has many interesting properties firstly it has very

good solubility in aqueous solutions secondly it has an intermediate affinity for Zn2+

thirdly it does not bind Ca2+ and Mg2+ (Paoletti et al 2009) Thus tricine has the

features to act as a rapid Zn2+ specific chelator (Chu et al 2004 Traynelis et al 1998)

But we should keep in mind the following points Firstly at selective concentrations it

produces only partial inhibition secondly Zn2+ appears also to inhibit triheteromeric

NMDARs and thirdly besides NMDARs it also inhibits γ-aminobutyric acid receptor

subtype A (GABAA receptors) and other channels (Draguhn et al 1990) so it cannot be

used in the brain slices or in vivo (Paoletti et al 2009) In the presence of Zn2+ (300 nM)

70

the application of PACAP (1 nM) failed to increase normalized NMDAR peak currents

(23 + 35 n=6) (Fig 312)

Although Zn2+ can be used as a very specific antagonist for GluN2ARs in acutely

isolated cells it still has several limitations (Paoletti et al 2009) So we also studied if

PACAP lost its ability to potentiate NMDAR currents in mice with a genetic deletion of

GluN2A In GluN2A -- mice the expression level of GluN1 and GluN2B is normal

compare to that of wild type mice although GluN2A expression disappears (Philpot et al

2007) but whether PAC1 receptorsPKCSrc signaling pathway is changed in these

GluN2A -- mice remains unknown In wildtype mice the application of PACAP (1 nM)

in the patch pipette increased normalized NMDAR peak currents up to 428 + 6 (N=5)

but this potentiation induced by the application of PACAP (1 nM) was abolished in

GluN2A -- mice (-67 + 64 n=5) These results demonstrated that GluN2ARs were

the main targets for PACAP to increase NMDAR currents (Fig 312)

Our lab has demonstrated that the activation of PAC1 receptors by PACAP (1 nM)

enhances NMDAR currents via Src so next I investigated if Src modulates NMDAR

currents via GluN2ARs but not GluN2BRs In acutely isolated CA1 hippocampal

neurons recombinant Src kinase (30 Uml) was included in the patch pipette To

determine if Src selectively modulates GluN2ARs over GluN2BR GluN2 antagonists

were used The use of NVP-AAM077 (50 nM) in all external solutions completely

blocked the ability of Src to increase normalized NMDAR peak currents (Src plus NVP-

AAM077 -06 plusmn 29 compared to baseline n = 7) By comparison the presence of Ro

25-6981 (100 nM) in the external solution had no effect on the ability of Src to enhance

normalized NMDAR mediated peak currents (Src 511 plusmn 76 n = 8 Src plus Ro 25-

71

6981 715 plusmn 103 n = 6) These results demonstrated that Src modulation of

NMDARs was likely via GluN2ARs (Fig 313) In addition the presence of Zn2+ (300

nM) abolished the increase of normalized NMDAR peak current induced by Src (218 +

89 n = 5) Further evidence for a role of GluN2ARs came from an examination of

GluN2A -- mice In GluN2A -- mice the application of recombinant Src could not

potentiate normalized NMDA mediated peak current In contrast this potentiation of

NMDAR currents still could be seen after the treatment of Src in wildtype mice (GluN2A

WT 718 + 151 n=6 GluN2A KO 34 + 43 n = 6) (Fig 314)

Several studies have shown that some GPCRs such as dopamine D1 receptor

activation could singal through Fyn to increase the surface trafficking of GluN2BRs

(Dunah et al 2004 Hallett et al 2006 Hu et al 2010) whether Fyn selectively

modulates GluN2BRs over GluN2ARs was also investigated Given that there are no

specific Fyn inhibitors available we designed a specific Fyn inhibitory peptide (Fyn (39-

57)) based on the sequence of Src (40-58) Src (40-58) and Fyn (39-57) mimic the unique

domain of Src and Fyn respectively Src (40-58) was proposed to interfere with the

interaction between Src and ND2 and inhibit the ability of Src to regulate NMDAR

currents (Gingrich et al 2004) We proposed Fyn (39-57) had the same capacity to

modulate the regulation of NMDAR currents by Fyn Electrophysiologcal methods were

initially used to test the specificity of Fyn (39-57) There are no specific peptides or drugs

which can activate endogenous Fyn directly so recombinant Fyn (1 Uml) and Fyn (39-57)

(25 microgml) were mixed and added to the patch pipette In this condition normalized

NMDAR mediated peak currents only showed slight increase Compare to the control

group their differences were not significant (Fyn 587 plusmn 51 n = 4 Fyn plus Fyn (39-

72

57) 211 plusmn 104 n = 10 p lt 001 Fyn (39-57) -93 plusmn 85 n = 6) (Figure 315) In

contrast scrambled Fyn (39-57) (25 microgml) had no effect on the potentiation of NMDAR

peak currents induced by exogenous Fyn kinase (Fyn plus Fyn (39-57) 679 plusmn 123 n

= 7) (Figure 315) it implied that Fyn (39-57) could inhibit the potentiation of NMDAR

induced by exogenous Fyn in acutely isolated hippocampal CA1 cells Since Fyn (39-57)

could only be dissolved in DMSO we also investigated whether DMSO alone had effect

on NMDAR currents results showed that in the presence of DMSO alone normalized

NMDAR peak currents was not changed (DMSO -63 plusmn 42 n = 6) In addition the

application of Fyn (39-57) (25 microgml) alone also failed to change normalized NMDAR

peak currents (Figure 315) Furthermore Fyn (39-57) (25 microgml) and recombinant Src

kinase (30 Uml) were mixed and added to the patch pipette In the presence of Fyn (39-

57) the application of Src kinase still could increase normalized NMDAR peak currents

in acutely isolated CA1 cells (Src 422 plusmn 71 n = 5 Src plus Fyn (39-57) 373 plusmn

25 n = 4) (Figure 315) These results confirmed the specificity of Fyn (39-57) we

designed

In addition the specificity of Src (40-58) was also investigated recombinant Fyn

kinase (1 Uml) and Src (40-58) (25 microgml) were mixed and added to the patch pipette

the result showed that Src (40-58) could not prevent the increase of normalized NMDAR

peak currents induced by recombinant Fyn kinase in acutely isolated hippocampal CA1

cells (Fyn plus Src (40-58) 373 plusmn 25 n = 4) (Figure 315)

Next I studied if Fyn selectively modulated GluN2BR over GluN2AR Both

GluN2AR antagonist NVP-AAM077 and GluN2BR antagonist Ro 25-6981 were used

The application of recombinant Fyn kinase in the patch pipette induced an increase in

73

normalized NMDA evoked peak currents in acutely isolated CA1 hippocampal neurons

The presence of Ro 25-6981 completely blocked the increase of normalized NMDA

mediated peak currents induced by Fyn kinase but NVP-AAM077 application only

slightly reduced this increase (Fyn 697 plusmn 103 n = 6 Fyn plus NVP-AAM077 505 plusmn

53 n = 6 Fyn plus Ro 25-6981 0 plusmn 22 n = 6) (Fig 316) We also investigated if

recombinant Fyn kinase could also potentiate normalized NMDAR peak currents in the

presence of Zn2+ (300 nM) which preferentially blocked GluN2AR The presence of

Zn2+ in the external solution failed to block the increase of normalized NMDAR peak

currents induced by recombinant Fyn kinase (616 plusmn 98 n = 7) (Fig 316) In addition

in GluN2A -- mice the inclusion of recombinant Fyn kinase in the patch pipette could

still potentiate normalized NMDAR peak currents (Fyn WT 603 + 87 n = 4 Fyn KO

723 + 93 n = 5) These results provided solid evidences to demonstrate that Fyn

modulation of NMDAR was mainly mediated by GluN2BRs (Fig 316)

Many studies have demonstrated that the phosphorylation of the receptor is

correlated with changes in receptor function (Chen and Roche 2007 Taniguchi et al

2009) Therefore I performed biochemical experiments to determine if the activation of

PAC1 receptors by PACAP (1 nM) caused selective phosphorylation of GluN2A subunits

but not GluN2B subunits We monitored the phosphorylation of the total tyrosine

residues of GluN2A subunits and GluN2B subunits using antibody which can detect

phosphotyrosine (Druker et al 1989) After the hippocampus was isolated from rat brain

it was cut into several slices and treated with PACAP (1 nM) for 15 minutes The slices

were then homogenized and the samples were immunoprecipitated using anti-GluN2A

antibody or anti-GluN2B antibody Next the blots were probed using pan antibody which

74

can detect the phosphorylated tyrosine residues After the treatment of PACAP (1 nM)

the tyrosine phosphorylation of GluN2A subunits was significantly increased by 984 +

65 (N=4) whereas tyrosine phosphorylation of GluN2B subunits was unchanged (Fig

317) We also studied if PACAP (1 nM) activated Src activity in the hippocampal slices

There are two critical tyrosines residues in Src Y416 the phosphorylation of which

increases Src activity and Y527 the phosphorylationof which inhibits Src activity (Salter

and Kalia 2004) In our experiment we used the antibody which specifically recognizes

the phosphorylation of Y416 of Src as a tool to monitor the phosphorylation of this residue

Usually the phosphorylation of Y416 in Src can be used as a representive of Src activity

The application of PACAP (1 nM) for 15 minutes increased Y416 phosphorylation of Src

(546 + 54 N=4) (Fig 318) indicating that Src activity was increased after PACAP

application in the hippocampus This method was not perfect since the phosphorylation

of Y527 is also important for Src activity (Salter and Kalia 2004) in the future more

experiments will be done to confirm that this residue is not phosphorylated by PACAP

Collectively using acutely isolated CA1 cells in the hippocampus these results

demonstrated that the activation of PAC1 receptors induced a PKCCAKβSrc signaling

pathway to differentially regulate GluN2ARs NMDAR currents recorded in acutely

isolated CA1 cells are mixtures of both synaptic NMDAR currents and extrasynaptic

NMDAR currents In orde to study whether the activation of PAC1 receptors by PACAP

(1 nM) increased synaptic NMDAR mediated EPSCs currents (NMDAREPSCs) pyramidal

neurons were patch clamped in a whole cell configuration at a holding voltage of -60 mV

Schaffer Collateral fibers were stimulated every 30 s using constant current pulses (50-

100 micros) to evoke NMDAREPSCs A previous study in our lab showed that PACAP (1 nM)

75

increased the amplitude of NMDAREPSCs at CA1 synapses in the brain hippocampal

slices and this potentiation was abolished by Src (40-58) (Macdonald et al 2005) But in

the presence of Fyn inhibitory peptide (Fyn (39-57)) (25 microgml) bath application of

PACAP (1 nM) still increased NMDAREPSCs (PACAP plus Fyn (39-57) 159 plusmn 015 n =

5) suggesting that Src but not Fyn was required for the potentiation of NMDAREPSCs by

PACAP (1 nM) Furthermore to investigate if PACAP induced enhancement of

NMDAREPSCs was mediated by GluN2ARs I recorded in the continued presence of Ro

25-6981 in order to block GluN2BRs NMDAREPSCs were still augmented by PACAP (1

nM) (Fig 319)

Wang et al (Liu et al 2004) proposed that the direction of NMDAR dependent

synaptic plasticity was determined by NMDAR subtypes GluN2AR was required for

LTP induction while GluN2BR was necessary for LTD induction (Liu et al 2004) But

Bear et al (Philpot et al 2001 Philpot et al 2003 Philpot et al 2007) claimed that the

ratio of GluN2ARGluN2BR determined the direction of synaptic plasticity mediated by

NMDARs If the ratio of GluN2ARGluN2BR was high LTD was more easily induced

If the ratio was low LTP induction was favored (Philpot et al 2001 Philpot et al 2003

Philpot et al 2007) This hypothesis did not distinguish relative changes from absolute

changes in one or the other subtype of receptor The direction of plasticity change is

likely determined not only by the activation ratio of each subpopulation but also by the

absolute level of synaptic NMDAR activation achieved The activation of PAC1

receptors by PACAP preferentially augments the function of synaptic GluN2ARs but not

GluN2BRs by enhancing Src kinase activity I and Bikram Sidu (Masterrsquos graduate

student) therefore examined the consequences of enhancing GluN2ARs on synaptic

76

plasticity using field recording technique We stimulated the Schaffer collateral pathway

at 005 Hz with 01 ms pulses to evoke synaptic response in the hippocampal slices After

the maximal synaptic response was achieved by adjusting the position of the recording

electrode the baseline was chosed to yield a one-third maximal response by changing the

stimulation intensity In control slices baseline was monitored for a minimum of 20

minutes before the induction of synaptic plasticity In drug treated slice baseline

responses were monitored for 10 minutes before applying PACAP (1 nM) Drug

treatment was continued for 10 minutes before the induction of synaptic plasticity I did

several experiments to determine the effect of PACAP on the direction of synaptic

plasticity I found that baseline field EPSPs were unaffected by the application of PACAP

(Fig 3110) In addition the application of PACAP (1 nM) had no effect on the LTP

induction by both high frequency stimulation and theta burst stimulation (Fig 3110)

But when I stimulated hippocampal slices using an intermediate frenquency (10 Hz 600

pulses) the application of PACAP (1 nM) induced LTP although in the control slices

this protocol induced LTD (Fig 3111)

Then Bikram Sidhu examined whether PACAP (1 nM) had ability to change the

synaptic plasticity induced by a range of frequencies Hippocampal slices were stimulated

at frequencies of 1 10 20 50 and 100 Hz The number of stimulation pulses was kept

constant (600 pulses per stimulation freqency) After 20 min baseline recording standard

protocols were used to induce either LTP or LTD in hippocampal CA1 slices In

untreated slices HFS (100 Hz and 50 Hz) induced LTP whereas LFS (10 Hz and 1 Hz)

induced LTD the direction of plasticity changed from LTD to LTP at induction

frequencies greater than 20 Hz When PACAP was applied in the bath solution for 10

77

min before the stimulation the HFS protocol (100 Hz and 50 Hz) still induced LTP

similar to control (Fig 3112) but the application of PACAP induced LTP by

intermediate frenquecies of stimulation (10 Hz and 20 Hz) In the control slices this

protocol induced LTD (Fig 3111) In conclusion PACAP shifted the modification

threshold to the left thus reducing the threshold for LTP induction (Fig 3112)

78

Figure 311 The activation of PAC1 receptors selectively modulated GluN2ARs

over GluN2BRs in acutely isolated CA1 neurons The application of PACAP (1 nM)

increased NMDA evoked currents in acutely isolated CA1 hippocampal neurons (385 +

52 n = 6) In the presence of the GluN2AR antagonist NVP-AAM077 (50 nM)

PACAP failed to increase NMDAR currents (24 plusmn 16 n = 6) In contrast the

presence of Ro 25-6981 (100 nM) had no effect on the ability of PACAP to modulate

NMDAR mediated currents (284 plusmn 49 n = 5) Sample traces from the cells with

PACAP or PACAP plus Ro25-6981 or PACAP plus NVP-AAM077 were shown at the

beginning (t = 3min) and the end of the recording (t = 26min)

79

Figure 312 The activation of PAC1 receptors selectively targeted GluN2A

Quantification data demonstrated that in the presence of NVP-AAM077 or Zn2+ PACAP

had no ability to potentiate NMDAR currents Furthermore PACAP coul not increase

NMDAR currents in GluN2A KO mice In contrast the GluN2BR antagonists Ro25-

6981 and ifenprodil could not prevent the potentiation of NMDAR currents by PACAP

80

Figure 313 Src targeting GluN2ARs increased NMDAR currents in acutely isolated

CA1 cells Applications of Src in patch pipette produced an increase in NMDA evoked

currents (511 + 76 n = 8) The use of NVP-AAM077 (50 nM) completely blocked the

ability of Src to increase NMDAR currents (-06 + 29 n = 7) By comparison the

presence of Ro 25-6981 (500 nM) had no effect on the ability of Src to modulate

NMDAR mediated currents (715 + 103 n = 6) Sample traces from the cells with Src

or Src plus Ro25-6981 or Src plus NVP-AAM077 were shown at the beginning (t = 3min)

and the end of the recording (t = 26min)

81

Figure 314 Quantification of NMDAR currents showed that Src selectively

modulates GluN2ARs over GluN2BRs Nanomolar concentration of Zn2+ inhibited the

increase of NMDAR currents in acutely isolated CA1 cells In the presence of Zn2+ (300

nM) inclusion of Src in the patch pipette could not increase NMDAR currents (21 +

89 n=5) The potentiation induced by Src in the patch pipette was abolished in

GluN2A -- mice (-34 + 43 n = 6) In contrast GluN2BR antagonist Ro25-6981

blocked the Src modulation of NMDARs

82

Figure 315 Fyn (39-57) blocked the enhancement of NMDAR currents by Fyn

kinase specifically (A) Fyn (39-57) abolished the increase of NMDAR currents by Fyn

Sample traces from the neurons treated with Fyn or Fyn plus Fyn (39-57) were shown at

the beginning (t = 3min) and the end of the recording (t = 26 min) (B) Only Fyn (39-57)

blocked Fyn effect on NMDAR currents but scrambled Fyn (39-57) Src (40-58) and

scrambled Src (40-58) failed to do so In addition Fyn (39-57) could not inhibit effects of

Src on NMDAR currents

83

Figure 316 GluN2BRs were involved in the increase of NMDAR currents by Fyn

(A) Fyn also enhanced NMDAR currents in acutely hippocampal CA1 cells and this

potentiation was blocked by Ro 25-6981 Sample traces from the cells with Fyn or Fyn

plus Ro25-6981 or Fyn plus NVP-AAM077 were shown at the beginning (t = 3 min) and

the end of the recording (t = 26 min) (B) Quantification of NMDAR currents

demonstrated that only Ro25-6981 blocked the increase of NMDAR currents by Fyn but

NVP-AAM077 and Zn2+ failed In addition Fyn still potentiated NMDAR currents in

GluN2A KO mice

84

IP GluN2A

pTyr

GluN2A

Ctrl PACAP

Glu

N2A

pho

spho

ryla

tion

Ctrl PACAP

pTyr

GluN2B

IP GluN2B

A B

C D

Figure 317 The activation of PAC1 receptors selectively phosphorylated the

tyrosine residues of GluN2A A PACAP treatment increased the tyrosine

phosphorylation of GluN2A B the application of PACAP failed to enhance the tyrosine

phosphorylation of GluN2B Right (C and D) the relative density of pTyr for GluN2A

and GluN2B was quantified from immunoblots (n = 4) for each of the conditions shown

indicates p lt 001

85

pSrcY416

Src

Ctrl PACAP

Figure 318 The application of PACAP increased Src activity Antibody which

specifically recognizes the phosphorylation of Y416 of Src was used to monitor the

phosphorylation of this residue indicating Src activity The application of PACAP (1 nM)

increased Y416 phosphorylation of Src indicating that Src activity was increased after

PACAP application

86

Figure 319 The activation of PAC1 receptor by PACAP (1 nM) enhanced

NMDAREPSC via SrcGluN2A pathway PACAP (1 nM) increased NMDAREPSC in the

hippocampal slices and this increase of NMDAREPSCs by PACAP was unaffected by

Ro25-6981 or by Fyn (39-57)

87

-40 -20 0 20 40 6005

10

15

20

25

Control (N=6) 1nM PACAP38 (N=8)

Norm

alize

d fE

PSP

Slop

e

time (min)

-20 0 20 40 6005

10

15

20

25

Norm

alize

d fE

PSP

Slop

e

time (minutes)

Control (N=7) 1 nM PACAP38 (N=7)

Figure 3110 PACAP (1 nM) had no effect on LTP induction induced by high

frequency protocol or theta burst stimulation Both high frequency protocol and theta

burst protocol induced LTP in the control slices In the presence of PACAP (1 nM) LTP

induction was not changed

88

-40 -30 -20 -10 0 10 20 30 40 50 60 70

06

07

08

09

10

11

12

13 PACAP applicationNo

rmali

zed

fEPS

P Sl

ope

time (min)


Figure 3111 The application of PACAP (1 nM) converted LTD to LTP induced by

10 Hz protocol (600 pulses) In control slices this protocol induced LTD but in the

presence of PACAP (1nM) LTP was induced

89

06

08

10

12

14

16

Nor

mal

ized

Fiel

d Am

plitu

de

Stimulus Frequency (Hz)

1 10 20 50 100

Figure 3112 The application of PACAP (1 nM) shifted BCM curve to the left and

reduced the threshold for LTP induction The effect of PACAP (1 nM) on synaptic

plasticity was monitored by repetitive stimulation at varying frequencies For control and

PACAP treated slices post-induction fEPSPs from each treatment group were normalized

to baseline responses and plotted versus the stimulation frequency (1-100 Hz) used

during the induction of plasticity The application of PACAP shifted BCM curve to the

left and favoured LTP induction

90

Project 2 The activation of VPAC receptors (Gαs coupled receptors) selectively targets GluN2BRs

91

Using in situ hybridization autoradiography and immunohistochemistry VPAC1

receptors and VPAC2 receptors have been identified within the hippocampus (Joo et al

2004) These receptors are best known for their ability to stimulate Gαs AC cAMP

production and subsequently activate PKA (Harmar et al 1998) Cunha-Reis et al (2005)

reported that VPAC2 receptors enhanced transmission via the anticipated stimulation of

PKA but VPAC1 receptor did so as a consequence of PKC activation (Cunha-Reis et al

2005) In addition VIP plays very important roles in the CNS such as neuronal

development and neurotoxicity (Vaudry et al 2000 Vaudry et al 2009) We proposed

that the activation of VPAC receptors enhance NMDAR currents through

cAMPPKAFyn pathway In addition this modulation is largely mediated GluN2BR

321 Hypothesis

In order to examine the effects of VIP on NMDAR-mediated currents a

concentration of VIP (1 nM) was initially chosen to selectively activate VPAC receptors

and not PAC1 receptor This concentration was based on the EC50 of VIP for VPAC

receptors (Harmar et al 1998) Initially individual CA1 pyramidal cells were acutely

isolated from slices cut from rat hippocampus Using acutely isolated cells drugs were

directly and rapidly applied to individual cells using a computer driven perfusion system

Unlike the situation of CA1 neurons in situ the concentrations of applied agents are

tightly controlled NMDAR currents were evoked every 60 seconds using a three-second

exposure to NMDA (50 microM) and glycine (05 μM) After establishing a stable baseline

of peak NMDA-evoked current amplitude VIP was applied to isolated CA1 hippocampal

neurons continuously for five minutes Applications of VIP (1 nM) induced a substantial

322 Results

92

and long-lasting increase in normalized NMDA evoked peak currents that far outlasted

the application of VIP (Fig 321) This increase (39 plusmn 4 n = 6) reached a plateau

twenty five minutes after the commencement of the VIP application (20 minutes after

terminating its application) To exclude the involvement of receptors other than VPAC1

and VPAC2 receptors in this enhancement of NMDA-evoked currents [Ac-Tyr1 D-Phe2]

GRF (1-29) was co-applied with VIP in a separate series of recordings Co-applications

of [Ac-Tyr1 D-Phe2] GRF (1-29) (01 μM) a peptide that can selectively block VPAC12

receptors (Waelbroeck et al 1985) together with VIP (1 nM) prevented the increase in

NMDA-evoked currents induced by VIP (1 nM) (4 plusmn 2 n = 6) (Fig 41) In contrast

similar recordings done in the presence of M65 (01 μM) a specific PAC1-R antagonist

(Moro et al 1999) failed to alter the VIP (1nM)-induced enhancement of NMDA-

evoked currents (39 plusmn 7 n= 5) (Fig 321)

In order to confirm the involvement of both the VPAC1 receptor and VPAC2

receptor in the enhancement of NMDA-evoked currents the actions of both the VPAC1-

selective agonist [Ala112228]VIP (Nicole et al 2000) and the VPAC2-selective agonist

Bay55-9837 (Tsutsumi et al 2002) were examined Application of [Ala112228]VIP (10

nM) caused an increase in NMDA-evoked currents (27 plusmn 2 n = 6) and this effect was

eliminated in the presence of the VPAC12 receptor antagonist [Ac-Tyr1 D-Phe2] GRF

(1-29) (01 μM) (-7 plusmn 2 n = 5) (Fig 322) Similarly application of Bay55-9837 (1

nM) also resulted in a significant potentiation of NMDA-evoked currents of 44 plusmn 8 (n =

6) In turn this potentiation was blocked by co-application of Bay55-9837 (1 nM)

together with [Ac-Tyr1 D-Phe2] GRF (1-29) (01 μM) (4 plusmn 3 n = 5) (Fig 322)

93

We then investigated the role of the cAMPPKA pathway in the potentiation of

NMDA-evoked currents based on the observations that VPAC12 receptors most often

signal through Gαs to cAMPPKA (Harmar et al 1998) Rp-cAMPS binds to the

regulatory subunit of PKA and inhibits dissociation of the catalytic subunit from the

regulatory subunit Inclusion of this competitive cAMP inhibitor (500 μM) in the patch

pipette blocked the subsequent effect of VIP (4 plusmn 3 n = 6) but itself had no effect on

NMDA-evoked currents in isolated CA1 neurons (5 plusmn 2 n = 5) (Fig 323) Unlike

RpCAMPS PKI14-22 binds to catalytic subunit of PKA to inhibit its kinase activity

Application of this highly selective PKA inhibitory peptide PKI14-22 (03 μM) attenuated

the VIP-induced potentiation of NMDA-evoked currents (VIP + PKI14-22 1 plusmn 4 n = 6)

compared to VIP alone (40 plusmn 5 n = 6) In contrast PKI14-22 alone had no effect on

NMDA-evoked currents (1 plusmn 3 n = 5) (Fig 323)

Some VIP-mediated actions in the nervous system have also been associated with

an increase in PKC activity (Cunha-Reis et al 2005) Therefore I used the PKC inhibitor

bisindolylmaleimide I (bis-I) (500 nM) to test whether the VIP-induced potentiation of

NMDA-evoked currents in the CA1 area of the hippocampus was also PKC-dependent

Application of this inhibitor (500 nM) had no effect on the amplitudes of baseline

responses (8 plusmn 1 n = 5) and it also failed to alter the VIP-induced potentiation of

NMDA-evoked currents (50 plusmn 10 n = 6) (Fig 324) In addition one study showed

that Ca2+ transients in colonic muscle cells are enhanced by VIP acting via a cAMPPKA-

dependent enhancement of ryanodine receptors (Hagen et al 2006) In pancreatic acinar

cells VPAC-Rs also evoke a Ca2+ signal by a mechanism involving Gαs (Luo et al

1999) To test whether the modulation of NMDA-evoked currents by VIP required an

94

elevation of internal Ca2+ high concentrations of the fast Ca2+ chelator BAPTA (20 mM)

were included in the patch pipette BAPTA blocked the effect of VIP (1 nM) (5 plusmn 3 n

= 6) The application of BAPTA by itself caused no time-dependent change in

normalized peak NMDAR currents (1 plusmn 4 n = 7) (Fig 324) Recent studies have

demonstrated that the BAPTA actually bound to Zn2+ with a substantially higher affinity

than Ca2+ (Hyrc et al 2000) Further study using more specific Ca2+ chelater is required

cAMP specific phosphodiesterase 4 (PDE4) which catalyzes hydrolysis of

cAMP plays a critical role in the control of intracellular cAMP concentrations it is

highly expressed in the hippocampus (Tasken and Aandahl 2004) Pre-treatment with

PDE4-selective inhibitors blocks memory deficits induced by heterozygous deficiency of

CREB-binding protein (CBP) (Bourtchouladze et al 2003) and PDE4 is also involved in

the induction of LTP in the CA1 sub region of the hippocampus (Ahmed and Frey 2003)

To investigate if PDE4 is involved in the VIP (1 nM) effect on NMDA-evoked currents I

included an inhibitor of PDE4 termed ldquoPDE4 inhibitorrdquo (35-Dimethyl-1-(3-

nitrophenyl)-1H-pyrazole-4-carboxylic acid ethyl ester) in the patch pipette (100 nM)

This compound is a specific inhibitor of phosphodiesterases 4B and 4D (Card et al

2005) It accentuated the VIP-induced enhancement of NMDA-evoked currents (PDE4 +

1 nM VIP 58 plusmn 3 n = 6 1 nM VIP 32 plusmn 3 n = 6) In a separate set of recordings

PDE4 inhibitor (100 nM) on its own had no time-dependent effect on normalized peak

NMDAR currents (5 plusmn 2 n = 6) (Fig 325)

Targeting of PKA by the scaffolding protein AKAP is required for mediation of

the biological effects of cAMP (Tasken and Aandahl 2004) For example disruption of

the PKA-AKAP complex is associated with a reduction of AMPA receptor activity

95

(Snyder et al 2005a) In addition AKAPYotiao targets PKA to NMDARs and

interference with this interaction reduces NMDAR currents expressed in HEK293 cells

(Westphal et al 1999) To determine if AKAP was required for VIP (1 nM) modulation

of NMDA-evoked currents in hippocampal neurons I included the St-Ht31 inhibitor

peptide (10 μM) in the patch pipette This inhibitor mimics the amphipathic helix that

binds the extreme NH2 terminus of the regulatory subunit of PKA and thereby dislodges

PKA from AKAP and consequently from its substrates Because of this property it has

been extensively used to study the functional implications of AKAP in several systems

(Vijayaraghavan et al 1997) Inclusion of St-Ht31 inhibitor peptide (10 μM) blocked

the ability of the VIP to increase NMDA-evoked currents (12 plusmn 3 n = 6) This peptide

(10 μM) alone has no time-dependent effect on NMDA-evoked currents (6 plusmn 1 n = 6)

(Fig 325)

Our lab has shown that low concentrations of PACAP enhance NMDA-evoked

currents in CA1 hippocampal neurons via a PKCSrc signal transduction cascade

(Macdonald et al 2005) Therefore I also studied the involvement of Src in the VIP (1

nM)-mediated increase of NMDA-evoked currents Intracellular application of the Src

inhibitory peptide Src (40-58) did not block the effect of VIP (49 plusmn 7 n = 6) (Fig

326) By itself Src (40-58) had no time-dependent effect on the amplitude of NMDA-

evoked currents (data not shown) Instead many studies have demonstrated that PKA

could stimulate Fyn directly (Yeo et al 2010) or indirectly through STEP61 (Paul et al

2000) Next I investigated if Fyn was involved in the potentiation of NMDARs by the

activation of VPAC receptors I added Fyn (39-57) (25 microgml) in the patch pipette and

determined its effects on the response to VIP Under these conditions the application of

96

VIP (1 nM) failed to increase NMDA evoked current in acutely isolated cells (1 nM VIP

429 + 45 n = 5 1 nM VIP plus Fyn (39-57) 02 + 25 n = 6) This result indicated

that the activation of VPAC receptors signaled through Fyn to potentiate NMDARs

(Figure 327)

I have shown that Fyn activation selectively modulated GluN2BRs Next in order

to investigate if the enhancement of NMDARs by VIP (1 nM) was mediated by

GluN2BRs I applied the GluN2BR antagonist Ro25-6981 in the medium In the presence

of Ro25-6981 VIP (1 nM) fails to potentiate NMDARs (1 nM VIP 423 + 97 n = 5 1

nM VIP plus Ro25-6981 -02 + 48 n = 6) (Figure 327)

97

0 5 10 15 20 25 30 35

09

10

11

12

13

14

15

16

VIP application

1nM VIP VIP+M65 VIP+GRF

Norm

alized

Peak

Curre

nt

Time Course (min)

1nM VIP

2

1

200pA

1s

1nM VIP+GRF

2

1

200pA

1s

1nM VIP+M65

2

1

100pA

1s Figure 321 Low concentration of VIP enhanced NMDAR currents via VPAC

receptors in acutely isolated cells Application of VIP (1 nM) to acutely isolated CA1

pyramidal neurons increased NMDA-evoked peak currents (39 plusmn 4 n = 6) throughout

the recording period But in the presence of [Ac-Tyr1 D-Phe2] GRF (1-29) (01 μM) a

specific VPAC-R antagonist the VIP effect on NMDA-evoked peak currents was

inhibited (4 plusmn 2 n = 6) But the addition of M65 (01 μM) a specific PAC1-R

antagonist could not prevent the increase of NMDA-evoked currents (39 plusmn 7 n = 5) In

addition sample traces from the same cells with VIP or VIP + [Ac-Tyr1 D-Phe2] GRF

(1-29) or VIP + M65 in the bath solution were shown at baseline (t = 3 min) and after

drug application (t = 28 min)

98

0 5 10 15 20 25 30 3508

10

12

14

[Ala112228]VIP application

[Ala112228]VIP [Ala112228]VIP+GRF

Nor

mal

ized

Peak

Cur

rent

Time Course (min)

0 5 10 15 20 25 30 3508

10

12

14

16

Bay 55-9877 application

Control Bay 55-9877 Bay 55-9877+01uM GRF

Nor

mal

ized

Peak

Cur

rent

Time Course (min)

Figure 322 The activation of both VPAC1 receptor and VPAC2 receptor enhanced

NMDAR currents Addition of [Ala112228]VIP (10 nM) caused an enhancement in

NMDA-evoked currents (27 plusmn 2 n = 6 data obtained at 30 min of recording) but the

existence of [Ac-Tyr1 D-Phe2] GRF (1-29) (01 μM) blocked the potentiation of NMDA-

evoked currents (-7 plusmn 2 n = 5) by [Ala112228]VIP (10 nM) In addition application of

Bay55-9837 (1 nM) also increased NMDA evoked currents (44 plusmn 8 n = 6 data

obtained at 30 min of recording) but the coapplication of [Ac-Tyr1 D-Phe2] GRF (1-29)

(01 μM) with Bay55-9837 (1 nM) had no effect on NMDA-evoked currents (4 plusmn 3 n

= 5)

99

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

VIP application

1nM VIP VIP+Rp-cAMPs Rp-cAMPs

Norm

aliz

edPe

akC

urre

nt

Time Course (min)

0 5 10 15 20 25 30 35

09

10

11

12

13

14

15

16

VIP application

1nM VIP VIP+PKI PKI

Nor

mal

ized

Peak

Curre

nt

Time Course (min)

Figure 323 PKA was involved in the potentiation of NMDARs by the activation of

VPAC receptors Intracellular administration Rp-cAMPs (500 μM) blocked the effect of

VIP (4 plusmn 3 n = 6 data obtained at 30 min of recording) and is similar to Rp-cAMPs

alone (5 plusmn 2 n = 5 data obtained at 30 min of recording) Addition of PKI14-22 (03 μM)

in all extracellular solutions blocked the potentiation of NMDA-evoked currents induced

by VIP (1 nM) (PKI14-22 plus VIP 1 plusmn 4 n = 6 VIP alone 40 plusmn 5 n = 6 data

obtained at 30 min of recording)

100

0 5 10 15 20 25 30 35

08

10

12

14

16

18

VIP application

1nM VIP Bis VIP+Bis

Norm

alize

dPe

akCu

rrent

Time Course (min)

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

VIP application

1nM VIP BAPTA VIP+BAPTA

Norm

alize

dPe

akCu

rrent

Time Course (min)

Figure 324 PKC was not required for the VIP (1 nM) effect while the increase of

intracellular Ca2+ was necessary A Application of the 500 nM Bis (a specific PKC

inhibitor) in all extracellular solutions could not block the VIP-induced potentiation of

NMDAR currents (Bis plus VIP 50 plusmn 10 n = 6 Bis alone 8 plusmn 1 n = 5 data obtained

at 30 min of recording) B Intracellular application of 20 mM BAPTA blocked the effect

of VIP (1 nM) on the NMDA-evoked currents (BAPTA plus VIP 5 plusmn 3 n = 6 BAPTA

alone 1 plusmn 4 n = 7 data obtained at 30 min of recording)

101

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

16

17

VIP application

1nM VIP PDE4 inhibitor VIP+PDE4 inhibitor

Norm

aliz

edPe

akC

urre

nt

Time Course (min)

0 5 10 15 20 25 30 35

08

09

10

11

12

13

14

15

16

17

VIP application

1nM VIP Ht31 VIP+Ht31

Norm

aliz

edPe

akC

urre

nt

Time (minutes)

Figure 325 VIP (1 nM) effect could be accentuated by PDE4 inhibitor and

required AKAP scaffolding protein Inclusion of PDE4 (100 nM) inhibitor augmented

the VIP-induced increase of NMDA-evoked currents (PDE inhibitor plus VIP 58 plusmn 3

n = 6 VIP alone 32 plusmn 3 n = 6 PDE inhibitor alone 5 plusmn 2 n = 6 data obtained at 30

min of recording) In the presence of St-Ht31 inhibitor peptide (10 μM) VIP (1 nM)

could not induce an increase in NMDA peak currents (St-Ht31 inhibitor peptide plus VIP

12 plusmn 3 n = 6 St-Ht31 inhibitor peptide alone 6 plusmn 1 n = 6 data obtained at 30 min of

recording)

102

0 5 10 15 20 25 30 35

08

09

10

11

12

13

14

15

16

17

VIP application

1nM VIP VIP+Src (40-58)

Nor

mal

ized

Peak

Cur

rent

Time Course (min)

Figure 326 Src was not required for VIP (1 nM) effect on NMDA-evoked currents

Intracellular administration of the Src inhibitory peptide Src (40-58) could not inhibit 1

nM VIP effect (49 plusmn 7 n = 6 data obtained at 30 min of recording)

103

0 5 10 15 20 25 30 35

08

10

12

14

16

18VIP

2 sec

500 p

A15

0 pA

21

21

Ro25-6981 control

norm

alized

I NMDA

time (min)

+ Ro2

5-698

1

+ Scra

mbled Ipe

p

+ Fyn(

39-57

)

VIP

08

10

12

14

16

18

B

A

norm

alized

I NMDA

Figure 327 The activation of VPAC receptors enhanced NMDAR currents via Fyn

and GluN2B (A) VIP increased NMDAR currents in acutely hippocampal CA1 neurons

and Ro25-6981 blocked this potentiation Sample traces from the cells with VIP or VIP

plus Ro25-6981 were shown at the beginning (t = 3 min) and the end of the recording (t =

26 min) (B) Quantification data indicates that the potentiation of NMDAR currents by

VIP was inhibited by Fyn (39-57) and Ro25-6981 but not by scrambled Fyn (39-57)

104

Section 4

Discussion

105

Discussion

In my experiments three lines of evidence suggested that the activation of the

PAC1 receptors preferentially increased the activity of GluN2ARs Firstly NVP-

AAM077 blocked NMDAR potentiation induced by the PAC1 receptors but Ro25-6981

failed to do so Secondly Zn2+ a selective inhibitor of GluN2ARs at nanomolar

concentrations blocked the potentiation of NMDARs induced by the PAC1 receptors

Finally in the GluN2A -- mice the activation of the PAC1 receptors failed to increase

NMDAR currents

41 The differential regulation of NMDAR subtypes by GPCRs

My study suggested that triheteromeric NMDAR (GluN1GluN2AGluN2B) in

the hippocampal CA1 neurons played little or no role in the regulation of NMDARs by

SFKs Paoletti et al (Hatton and Paoletti 2005) demonstrated that triheteromeric

NMDAR were blocked by both GluN2AR and GluN2BR antagonists although the

efficacy of the inhibition was greatly reduced For example only about 14 to 38 of

triheteromeric receptors were inhibited by Zn2+ (300 nM) while in the presence of

ifenprodil (3 microM) triheteromeric NMDARs showed 20 inhibiton (Hatton and Paoletti

2005) In my experiments the potentiation of NMDARs by PAC1 receptor activation was

totally blocked by NVP-AAM077 and Zn2+ while Ro25-6981 had no effect on NMDAR

potentiation induced by the PAC1 receptors If trihetermeric NMDARs were involved in

the potentiation of NMDAR by the activation of the PAC1 receptors this potentiation

should have been inhibited by Ro25-6981 as well Consistent with this there is currently

no evidence for functional triheteromeric NMDARs at CA1 synapses Indeed in the CA1

region the content of triheteromeric NMDARs was much less than that of dimeric

106

GluN2ARs and GluN2BRs (Al-Hallaq et al 2007) and most GluN2A and GluN2B

subunits did not coimmunoprecipitate (Al-Hallaq et al 2007)

Previous studies showed that the activation of the PAC1 receptors was coupled to

Gαq proteins (Vaudry et al 2000 Vaudry et al 2009) and that they increased NMDAR

currents via the PKCCAKβSrc signaling pathway (Macdonald et al 2005) Other

GPCRs including muscarinic receptors LPA receptors and mGluR5 receptors which also

initiated signaling pathway via Gαq proteins likely enhanced NMDAR currents through

the same pathway (Kotecha et al 2003 Lu et al 1999a) In this study I further showed

that PAC1 receptor activation selectively potentiated GluN2ARs but it remains to be

shown whether or not other GPCRs coupled to Gαq proteins also selectively target

GluN2ARs

In addition although the activation of the PAC1 receptors stimulated Src activity

the application of PACAP (1 nM) did not induce any change on the basal synaptic

responses In contrast activation of endogenous Src by Src activating peptide increased

basal synaptic responses and induced LTP (Lu et al 1998) The activation of Src by the

PAC1 receptors during basal stimulation likely was suppressed by endogenous Csk (Xu

et al 2008) In contrast when Src activating peptide was applied it would have

interfered with the interaction between the SH2 domain and the phosphorylated Y527 in

the C-terminus of Src resulting in the persistent activation of Src So if endogenous Csk

phosphorylated Y527 the phosphorylated Y527 failed to interact with the SH2 domain

and Src was still active

My results also demonstrated that distinct from the PKCCAKβSrc cascade

induced by Gαq proteins the activation of Gαs coupled receptors such as VPAC

107

receptors enhanced NMDAR currents through a PKAFyn signaling pathway

Furthermore this potentiation of NMDAR currents was only mediated by GluN2BRs

One PhD student in our lab Catherine Trepanier has demonstrated that the activation of

dopamine D1 receptor another Gαs coupled receptor also signaled through

PKAFynGluN2BR to potentiate NMDARs

Based on these results we proposed that different signaling mechanisms may

regulate GluN2ARs versus GluN2BRs so GPCRs which coupled to different Gα

subtypes may regulate different subtypes of NMDARs Some other studies also indirectly

supported this hypothesis For example the application of orexin increased the surface

expression of GluN2ARs but not GluN2BRs in VTA which was dependent on OXR1

receptorsGαqPKC signaling pathway (Borgland et al 2006) Further another study

demonstrated that dopamine D5 receptor activation caused the recruitment of GluN2BRs

from cytosol to synaptic sites thereby leading to the potentiation of NMDAR currents

Dopamine D5 receptor activation was coupled to Gαs and cAMPPKA signaling pathway

(Schilstrom et al 2006) But these studies did not show if the differential regulation of

GluN2ARs and GluN2BRs by these GPCRs required SFK or not Additionally a recent

study demonstrated that dopamine D15 receptor enhanced LTP induction by PKA

activation and this enhancement was also mediated by SFK and GluN2BRs (Stramiello

and Wagner 2008)

A number of studies have demonstrated that NMDARs were required for the

induction of metaplasticity in the visual cortex (Philpot et al 2001 Philpot et al 2003

42 GPCR activation induces metaplasticity

108

Philpot et al 2007) Light deprivation decreased the ratio of GluN2ARGluN2BR and

induced a more slowly deactivating NMDAR current in neurons in layer 23 of visual

cortex In contrast exposure to visual stimulation increased the ratio and induced a more

rapid NMDAR current (Philpot et al 2001) These changes in the ratio of

GluN2ARGluN2BR were accompanied to changes in LTPLTD induction or

metaplasticity In addition in GluN2A -- mice metaplasticity in the visual cortex was

lost (Philpot et al 2007) Metaplasticity can also be modulated by mild sleep deprivation

Mild (4-6h) sleep deprivation (SD) selectively increased surface expression of GluN2AR

in adult mouse CA1 synapses favouring LTD induction But in the GluN2A -- mice this

metaplasticity was absent (Longordo et al 2009)

In addition to regulation by experience the ratio of GluN2ARGluN2BR is also

modulated by pre-stimulation A recent study demonstrated that the regulation of

GluN2ARGluN2BR ratio using GluN2AR or GluN2BR antagonist controled the

threshold for subsequent activity dependent synaptic modifications in the hippocampus

Additionally priming stimulations across a wide range of frequencies (1-100Hz) changed

the ratio of GluN2ARGluN2BR resulting in changes of the levels of LTPLTD

induction (Xu et al 2009) This study demonstrated that LTDLTP thresholds could be

regulated by factors which alter the ratio of GluN2ARGluN2BR If the ratio of

GluN2ARGluN2BR was elevated LTD induction was favoured While the ratio of

GluN2ARGluN2BR was low the threshold for LTP induction was reduced

Pre-stimulation may have the capacity to modulate not only the ratio of

GluN2ARGluN2BR but also the tyrosine phosphorylation of NMDARs through SFKs

Consequently even if prior activity does not itself cause substantial NMDAR activation

109

such activity could nevertheless cause the activation of several GPCRs which in turn

regulate NMDAR function and thus the ability to subsequently induce plasticity Indeed

our lab has demonstrated that the activation of several GPCRs can regulate the function

of NMDARs through SFKs (Kotecha et al 2003 Lu et al 1999a) thus having the

ability to subsequently induce metaplasticity

In my thesis I confirmed this possibility When I activated the PAC1 receptors

which are Gαq coupled receptors the BCM curve shifted to the left indicating that the

threshold for LTP induction was reduced In contrast when Gαs coupled dopamine D1

receptors were stimulated the BCM curve moved to the right and the threshold for LTD

induction was reduced (unpublished data) These results indicate that the enhancement of

GluN2ARs versus GluN2BRs by GPCRs at CA1 synapses differentially regulate the

direction of synaptic plasticity It is consistent with the hypothesis proposed by Yutian

Wang (Liu et al 2004) that GluN2AR is required for LTP induction while GluN2BR is

for LTD But my results showed that enhancing GluN2A favored LTP over LTD and

GluN2B favored LTD over LTP Our results do not exclude the possibility that both

subtypes of receptors contribute to both forms of synaptic plasticity

Our results are less consistent with Mark Bearrsquos ratio hypothesis He proposed

that when the ratio of Glun2ARGluN2BR was decreased LTP induction was favored

But if the ratio of GluN2ARGluN2BR was increased it would favor LTD induction In

my study when GluN2AR activity was selectively enhanced over GluN2BR (increased

Glun2ARGluN2BR) I observed a leftward shift in the BCM curve whereas Bearrsquos

hypothesis would have predicted a rightward shift There are several possibilities to

explain this difference Firstly Bearrsquos study only investigated the relative change of

110

GluN2AR and GluN2BR For example although the ratio of GluN2ARGluN2BR was

reduced after monocular deprivation at the beginning the expression of GluN2BR was

increased but later a reduction of GluN2AR expression was observed (Chen and Bear

2007) In contrast we selectively augmented the absolute activity of GluN2AR or

GluN2BR while presumably keeping the activity of the other subtype constant The

relative changes of GluN2AR and GluN2BR might result in different outcomes from

absolute changes in the activity of these subtypes Secondly we manipulated the ratio of

GluN2ARGluN2BR acutely by GPCR activation but they changed this ratio by using

chronic visual deprivation for several days Acute pharmacologically-induced changes of

GluN2ARGluN2BR might differ mechanistically from the chronical changes in the

visual cortex after monocular deprivation Thirdly we adjusted the ratio of

GluN2ARGluN2BR by the selective phosphorylation of subtypes while they changed it

by changing the relative surface expression of GluN2AR and GluN2BR After the

phosphorylation by the activation of GPCRs through SFKs the gating of GluN2AR and

GluN2BR might be changed (Kohr and Seeburg 1996) It might result in the change of

their contribution to LTPLTD induction In contrast monocular deprivation only

modulated the relative number of GluN2AR and GluN2BR at the synapses their gating

had no change

111

Figure 41 The activation of PAC1 receptor selectively modulated GluN2AR over

GluN2BR by signaling through PKCCAKβSrc pathway

112

Figure 42 The activation of Gαs coupled receptors such as dopamine D1 receptor and

VPAC receptor increased NMDAR currents through PKAFyn signaling pathway In

addition they all selectively modulated GluN2BR over GluN2AR

113

43 The involvement of SFKs in the synaptic transmission

431 The differential regulation of NMDAR subtypes by SFKs

My study suggested that Src preferentially upregulates the activity of GluN2ARs

Firstly NVP-AAM077 blocked NMDAR potentiation induced by Src Secondly Zn2+ a

selective GluN2AR antagonist at nanomolar concentrations blocked the Src mediated

potentiation of NMDARs Finally in the GluN2A -- mice the inclusion of Src in the

patch pipette failed to increase NMDAR currents The involvement of triheteromeric

NMDARs in the enhancement of NMDAR currents by Src was also unlikely since the

GluN2BR antagonist Ro25-6981 had no ability to block this potentiation induced by Src

In addition our data suggests that Fyn selectively regulates the activity of

GluN2BR NVP-AAM077 failed to inhibit the potentiation of NMDARs when I included

recombinant Fyn in the patch pipette In addition Zn2+ did not block the increase of

NMDAR currents induced by Fyn In the GluN2A -- mice the inclusion of Fyn in the

patch pipette still increased NMDAR currents Only in the presence of GluN2BR

antagonist Ro 25-6981 was the ability of Fyn to regulate NMDAR currents lost

Triheteromeric NMDARs were also not involved since in the presence of NVP-AAM077

and Zn2+ Fyn still increased NMDAR currents

A previous study demonstrated that when Src activating peptide was applied to

inside-out patches from culture neurons the open probability of NMDAR channels was

increased (Yu et al 1997) In addition this enhancement was mediated by Src since the

Src inhibitory peptide ((Src (40-58)) blocked this effect (Yu et al 1997) Furthermore

my study has demonstrated that Src selectively modulated GluN2ARs indicating that Src

might alter the gating of GluN2ARs Recently several papers suggested that PKC

114

increased the surface expression of NMDARs by directly phosphorylating synaptosomal-

associated protein 25 (SNAP25) in cultured hippocampal neurons (Lau et al 2010) This

increase of NMDAR surface expression occurred mostly at extrasynaptic regions (Suh et

al 2010) If Src is also involved in the enhancement of NMDAR trafficking requires

further study

Furthermore a previous study has shown that in HEK293 cells neither Src nor

Fyn changed the gating of GluN2BRs (Kohr and Seeburg 1996) Fyn may just increase

GluN2BR trafficking instead of altering gating Consistently after dopamine D1 receptor

was activated the surface expression of GluN2B was enhanced via Fyn (Hu et al 2010)

In addition the acute application of Aβ induced the endocytosis of GluN2B likely via

activation of Fyn (Snyder et al 2005b)

432 The trafficking of NMDARs induced by SFKs

Various publications have shown that SFKs have the ability to regulate NMDAR

trafficking For example in support of a role for tyrosine phosphorylation by SFKs in

NMDAR trafficking phosphorylation at the Y1472 site on GluN2B prevented the

interaction of GluN2B with clathrin adaptor protein AP-2 and suppressed the

internalization of NMDARs (Prybylowski et al 2005) In addition Y842 of GluN2A was

also phosphorylated and dephosphorylation of this residue may increased the interaction

of NMDAR with the AP-2 adaptor resulting in the endocytosis of NMDARs (Vissel et

al 2001)

Furthermore a number of GPCRs and RTKs regulate NMDAR trafficking via

SFKs Dopamine D1 receptor activation lead to the trafficking and increased surface

expression of GluN2BRs specifically In contrast inhibition of tyrosine phosphatases

115

enhanced trafficking of both GluN2ARs and GluN2BRs This interaction required the

Fyn kinase since in Fyn -- mice dopamine D1 receptor agonist failed to induce

subcellular redistribution of NMDARs (Dunah et al 2004 Hallett et al 2006)

Consistently the activation of dopamine D1 receptors significantly increased GluN2B

insertion into plasma membrane in cultured PFC neurons this movement required Fyn

kinase but not Src (Hu et al 2010) Moreover activation of neuregulin 1 was found to

promote rapid internalization of NMDARs from the cell surface by a clathrin-dependent

mechanism in prefrontal pyramidal neurons Neuregulin 1 was supposed to activate

ErbB4 resulting in the increase of Fyn activity and GluN2B tyrosince phosphorylation

(Bjarnadottir et al 2007)

A variety of studies have implicated elevated Aβ42 in the reduction of excitatory

synaptic transmission and reduced expression of AMPARs in the plasma membrane

(Hsieh et al 2006 Walsh et al 2002) Recently acute application of Aβ42 was also

demonstrated to reduce the surface expression of NMDAR This occurred via its binding

to α7-nicotinic acetylcholine receptors (α7AChRs) The enhancement of Ca2+ influx

through α7AChR activated PP2B which then dephosphorylated and activated STEP61

which dephosphorylated the GluN2B subunit at Y1472 directly or via the reduction of Fyn

activity (Braithwaite et al 2006 Hsieh et al 2006) and promoted internalization of

GluN2BRs (Snyder et al 2005b)

My results also implied that different SFKs might selectively modulate the

trafficking of NMDAR subtypes Src might increase GluN2AR trafficking while Fyn

selectively modulates GluN2BR trafficking

116

433 The role of the scaffolding proteins on the potentiation of NMDARs by SFKs

At the synapse the C terminus of GluN2 subunits interacts with MAGUKs

including PSD95 PSD93 SAP97 and SAP102 These scaffolding proteins bind to many

signaling proteins including SFKs (Kalia and Salter 2003) This may imply that these

scaffolding proteins are involved in the regulation of NMDARs by SFKs

Scaffolding proteins such as PSD95 can even inhibit the potentiation of NMDARs

by SFKs In Xenopus oocytes PSD95 reduced the Zn2+ inhibition of GluN2AR channels

and eliminated the potentiation of NMDAR currents by Src (Yamada et al 2002)

Another study showed that Src only interacted with amino acids 43ndash54 of PSD95 but not

other scaffolding protein such as PSD93 and SAP102 (Kalia and Salter 2003)

Furthermore this region of PSD95 inhibited the ability of Src to potentiate NMDARs

(Kalia et al 2006)

In contrast other studies proposed that these scaffolding proteins might promote

the potentiation of NMDARs by SFKs In 1999 Tezuka et al (Tezuka et al 1999)

demonstrated that in HEK293 cells PSD95 promoted Fyn-mediated tyrosine

phosphorylation of GluN2A by interacting with NMDARs Different regions of PSD95

associated with GluN2A and Fyn respectively (Tezuka et al 1999) Fyn not only

interacts with PSD95 but also PSD93 In PSD93 knockout (PSD93 --) mice the

phosphorylation of tyrosines of GluN2A and GluN2B was reduced Moreover deletion

of PSD93 blocked the SFKs-mediated increase in phosphorylated tyrosines of GluN2A

and GluN2B in cultured cortical neurons (Sato et al 2008)

Whether or not interaction with these scaffolding proteins modulates the ability of

SFKs to differentially regulate the subtypes of NMDARs requires further study In

117

addition the potential role of these scaffolding proteins in the trafficking of NMDARs by

SFKs remains poorly understood

434 The involvement of SFKs in synaptic plasticity in the hippocampus

Since SFKs can regulate NMDAR activity and trafficking it is not surprising that

SFKs are also involved in the synaptic plasticity LTD induced by group I mGluR

activation in CA1 neurons was accompanied by the reduction of both tyrosine

phosphorylation and surface expression of GluA2 of AMPARs (Huang and Hsu 2006b

Moult et al 2006) Kandelrsquos group (ODell et al 1991) showed that inhibitors of

tyrosine kinases blocked LTP induction without affecting normal synaptic transmission

but had no effect on established LTP (ODell et al 1991) Thus SFKs suppressed LTD

through tyrosine phosphorylation of GluA2 of AMPARs (Boxall et al 1996) In contrast

it has been shown that tyrosine phosphorylation of C-terminal tyrosine residues in GluA2

results in the internalization of GluA2 in cortical neuron (Hayashi and Huganir 2004)

indicating the induction of LTD

So far the involvement of Src in the induction of LTP has been well supported

(Huang et al 2001 Lu et al 1998 Pelkey et al 2002 Xu et al 2008) The role of Fyn

in synaptic plasticity has also been studied using Fyn transgenic mice because there were

no specific Fyn inhibitors previously available In Fyn -- mice LTP induction was

inhibited although basal synaptic transmission paired pulse facilitation (PPF) remained

unchanged This defect was unique because Src (Src --) Yes (Yes --) and Abl knockout

(Abl --) mice showed no change in LTP In addition Fyn -- mice show impaired spatial

learning in Morris water maze (Grant et al 1992) Although these findings seem to

118

exclude the involvement of Src in LTP induction it might be caused by functional

redundancy between Src and Fyn (Salter 1998 Yu and Salter 1999) In addition my

study demonstrated that Src and Fyn modulate GluN2ARs and GluN2BRs respectively

so in Src -- mice although the activity of GluN2ARs remains no change because of Src

deficiency GluN2BR activity can still be increased by Fyn resulting in the LTP

induction These findings also implicate that indeed both GluN2AR and GluN2BR have

ability to mediate LTP induction

Later in order to determine whether the impairment of LTP in Fyn -- mice was

caused directly by Fyn deficiency in adult hippocampal neurons or indirectly by the

impairment of neuronal development exogenous Fyn was introduced into the Fyn --

mouse (Kojima et al 1997) In these Fyn rescue mice the impairment of LTP was

restored although the morphology of their brains demonstrated some abnormalities

These results suggest that the Fyn has ability to modulate the threshold for LTP induction

directly (Kojima et al 1997) Consistently when LTP was induced both the activity of

Fyn and phosphorylation of Y1472 at GluN2B subunit were increased (Nakazawa et al

2001)

Additionally conditionally transgenic mice overexpressing either wild type Fyn

or the constitutively activated Fyn have also been generated (Lu et al 1999b) In the

hippocampal slices expressing constitutively activated Fyn PPF was reduced while basal

synaptic transmission was enhanced (Lu et al 1999b) A weak theta-burst stimulation

which could not induce LTP in control slices induced LTP in CA1 region of the slices

But the magnitude of LTP induced by strong stimulation in constitutively activated Fyn

slices was similar to that in control slices (Lu et al 1999b) By contrast the basal

119

synaptic transmission and the threshold for the induction of LTP were not altered in the

slices overexpressing wild type Fyn (Lu et al 1999b)

435 The specificity of Fyn inhibitory peptide Fyn (39-57)

In order to investigate if Gαs coupled receptors can signal through Fyn to

modulate NMDARs we designed a specific Fyn inhibitory peptide Fyn (39-57) based

on the fact that Src and Fyn are highly conserved except in the unique domain Src (40-58)

mimics a portion of the unique domain of Src and prevents its regulation of NMDARs

(Gingrich et al 2004) Using an analogous approach we synthesized a peptide Fyn (39-

57) which corresponds to a region of the unique domain of Fyn I demonstrated that Fyn

(39-57) but not Src (40-58) attenuated the effect of Fyn Importantly Fyn (39-57) did

not alter the potentiation by Src kinase In contrast Src (40-58) failed to block the

increase of NMDAR currents by Fyn In addition I showed that although both the

activation of VPAC receptors and dopamine D1 receptor enhanced NMDAR currents

Src (40-58) did not block this potentiation (Yang unpublished data) Instead the

inclusion of Fyn (39-57) in the patch pipette abolished the effect of these two GPCRs on

NMDARs So far all the studies we have performed indicate that Fyn (39-57) is a

selective inhibitor for Fyn over Src

My results have shown that Fyn (39-47) can interfere with the signaling events

targeting GluN2BRs but the mechanism remains unknown Similar to Src (40-58) Fyn

(39-57) might disrupt the interaction between Fyn and proteins which are important for

Fyn regulation of NMDAR

120

44 The function of PACAPVIP in the CNS

441 Mechanism of NMDAR modulation by VIP

Using acutely isolated hippocampal CA1 neurons I demonstrated that application

of the lower concentration of VIP (1 nM) enhanced NMDAR peak currents and it did so

by stimulating VPAC12 receptors as the effect was blocked by [Ac-Tyr1D-Phe2]GRF

(1-29) (a specific VPAC12 receptor versus PAC1 receptor antagonist) The enhancement

of NMDAR currents induced by the low concentration of VIP was also blocked by both

the selective cAMP inhibitor Rp-cAMPS and specific PKA inhibitor PKI14-22 but not by

the specific PKC inhibitor bisindolylmaleimide I nor by Src (40-58) Moreover the

VIP-induced enhancement of NMDA-evoked currents was accentuated by application of

a phosphodiesterase 4 inhibitor This regulation of NMDARs also required the

scaffolding protein AKAP since St-Ht31 a specific AKAP inhibitor also blocked the

VIP-induced potentiation These results are consistent with signaling via VPAC12

receptors and the cAMPPKA signal cascade The dependency of this response on Ca2+

buffering indicates that VPAC receptor signaling relies on the increase in intracellular

Ca2+

A low concentration of VIP (1 nM) likely activated both VPAC1 and VPAC2

receptor as an increase was also observed using either the VPAC1 receptor selective

agonist [Ala112228]VIP or the VPAC2 receptor selective agonist Bay55-9837 The VPAC

receptor antagonist [Ac-Tyr1 D-Phe2] GRF (1-29) (1 μM) inhibited the enhancement of

NMDA-evoked currents caused by VIP (1 nM) or by either of the VPAC receptor

selective agonists This provided evidence for the involvement of both VPAC1 and

121

VPAC2 receptors in the regulation of hippocampal synaptic transmission through

modulation of NMDARs

All PAC1 and VPAC12 receptors couple strongly to the Gαs and stimulate the

cAMPPKA signaling pathway The PAC1 receptor also strongly stimulates the PLC

pathway whereas VPAC1 and VPAC2 receptors activate PLC only weakly (McCulloch

et al 2002) Our studies showed that the activation of VPAC receptors by low

concentration of VIP (1 nM) increased evoked NMDAR currents via cAMPPKA

pathway whereas the activation of PAC1 receptor induced by low concentration of

PACAP (1 nM) induced PLCPKC signaling pathway to enhance NMDA-evoked

currents in hippocampal neurons (Macdonald et al 2005) While induction of cAMP

production is commonly reported after the activation of these receptors mobilization of

intracellular Ca2+ is also documented (Vaudry et al 2000 Vaudry et al 2009) VIP has

been shown to increase prolactin secretion in cultured rat pituitary cells (GH4C1)

involving a transient elevation of intracellular Ca2+ (Bjoro et al 1987) Also VIP was

found to increase cytoplasmic Ca2+ levels in leukemic myeloid cells isolated from

patients with myeloid leukaemia (Hayez et al 2004) VIP has been reported to increase

intracellular Ca2+ levels in hamster CHO ovary cells the effect being higher in VPAC1

than in VPAC2 receptor expressing cells (Langer et al 2001) The efficient coupling of

the VPAC1 receptor to [Ca2+]i increase has been attributed to a small sequence in its third

intracellular loop that probably interacts with Gαi and Gαq proteins (Langer et al 2002)

Our studies showed that the increase of NMDA-evoked current induced by VIP (1 nM)

also required the increase of [Ca2+]i in the acutely isolated hippcampal cells although

PKC was not showed to be involved

122

Despite the broad and varied substrates targeted by PKA local pools of cAMP

within the cell generate a high degree of specificity in PKA-mediated signaling cAMP

microdomains are controlled by adenylate cyclases that form cAMP as well as PDEs that

degrade cAMP AKAPs target PKA to specific substrates and distinct subcellular

compartments providing spatial and temporal specificity for mediation of biological

effects mediated by the cAMPPKA pathway Our study showed that a specific

phosphodiesterase 4 inhibitor accentuated the VIP-induced enhancement of NMDA-

evoked currents this implied that PDE4 was also involved in the synaptic plasticity

Many studies were consistent with our conclusions The selective PDE4 inhibitor

Rolipram improved long-term memory consolidation and facilitated LTP in aged mice

with memory deficits (Ghavami et al 2006) Another study also found an ameliorating

effect of Rolipram on learning and memory impairment in rodents (Imanishi et al 1997)

Rolipram reversed the impairment of either working or reference memory induced by the

muscarinic receptor antagonist scopolamine (Egawa et al 1997 Imanishi et al 1997

Zhang and ODonnell 2000) In addition Rolipram has been shown to reinforce an early

form of long-term potentiation to a long-lasting LTP (late LTP) (Navakkode et al 2004)

and early LTD could also be transformed into late LTD by the activation of cAMPPKA

pathway using rolipram (Navakkode et al 2005) Moreover theta-burst LTP selectively

required presynaptically anchored PKA whereas LTP induced by multiple high-

frequency trains required postsynaptically anchored PKA at CA1 synapses (Nie et al

2007) Our study also showed that the existence of AKAP was required for the regulation

of NMDARs by VIP suggesting that AKAP may play an important role in synaptic

plasticity Specificity in PKA signaling arises in part from the association of the enzyme

123

with AKAPs Synaptic anchoring of PKA through association with AKAPs played an

important role in the regulation of AMPAR surface expression and synaptic plasticity

(Snyder et al 2005a) PKA phosphorylation increased AMPAR channel open probability

and is necessary for synaptic stabilization of AMPARs recruited by LTP (Esteban et al

2003) PKA and NMDARs were also closely linked via an AKAP In this model

constitutive PP1 keep NMDAR channels in a dephosphorylated and low activity state

PKA was bound to the AKAP scaffolding protein yotiao With high levels of cAMP

PKA was released leading to a shift in the balance of the channel to a phosphorylated and

higher activity state (Westphal et al 1999) Infusion St-Ht31 to the amygdala also

impaired memory consolidation of fear conditioning (Moita et al 2002)

The involvement of Src or Fyn in the VIP (1 nM)-mediated increase of NMDA-

evoked currents was also investigated Intracellular application of Src (40-58) did not

block the effect of VIP on NMDAR currents (Yang et al 2009) In contrast in the

presence of Fyn (39-57) the potentiation of NMDAR by VIP (1 nM) was inhibited

Additionally the activation of VPAC receptors targeted GluN2BR to increase NMDAR

currents since the presence of the GluN2BR antagonist Ro 25-6981 in the bath totally

abolished VIP modulation of NMDAR currents

442 The regulation of synaptic transmission by PACAPVIP system

Since PACAPVIP can regulate AMPAR-mediated current it is not surprising to

see PACAPVIP can also modulate basal synaptic transmission in the hippocampus The

effect of PACAP on the basal synaptic transmission is quite complicated different

concentrations of PACAP may inhibit (Ciranna and Cavallaro 2003 Roberto et al 2001

124

Ster et al 2009) enhance (Michel et al 2006 Roberto et al 2001 Roberto and Brunelli

2000) or have a biphasic effect (Roberto et al 2001) on the basal synaptic transmission

in the CA1 region of the hippocampus In 1997 Kondo et al (Kondo et al 1997)

reported that very high concentrations of PACAP (1 microM) persistently reduced basal

synaptic transmission from CA3 to CA1 pyramidal neurons and this effect didnrsquot share

mechanisms with low frequency-induced LTD In addition neither NMDAR antagonist

nor PKA inhibitor could block it (Kondo et al 1997) Instead Epac was found to be

involved (Ster et al 2009) Another study also supported this conclusion (Roberto et al

2001) Recently it was discovered that even lower concentration of PACAP (10 nM)

could reduce the amplitude of evoked EPSCs but this effect was mediated by

cAMPPKA pathway and was reversed upon drug washout (Ciranna and Cavallaro 2003)

In contrast a relatively low concentration of PACAP (005 nM) enhanced field

EPSPs in the hippocampus CA1 region This enhancement was partially mediated by

NMDARs and shares a common mechanism with LTP (Roberto et al 2001)

Consistently endogenous PACAP was found to exert a tonic enhancement on CA3-CA1

synaptic transmission since the presence of the PAC1 receptor antagonist PACAP 6-38

significantly reduced basal synaptic transmission (Costa et al 2009) In the

suprachiasmatic nucleus PACAP (10 nM) also enhanced spontaneuous EPSC (Michel et

al 2006) this enhancement depended on both presynaptic and postsynaptic mechanisms

Surprisingly although high concentration of PACAP (1 microM) induced a long-lasting

depression of transmission at the Schaffer collateral-CA1 synapse in the hippocampus it

enhanced synaptic transmission at the perforant path-granule cell synapse in the dentate

125

gyrus However this effect was not mediated by NMDAR and cAMPPKA signaling

pathway (Kondo et al 1997)

These studies raise an important question How do different concentrations of

PACAP induce different effects on basal synaptic transmission As mentioned above

different doses of PACAP may act predominantly on different receptors to recruit

different signaling pathways and produce opposite effects On the contrary only

stimulatory effect on basal synaptic transmission by VIP was reported in the

hippocampus The application of VIP (10 nM) enhanced the amplitude of EPSCs and this

effect was completely abolished by cAMPPKA antagonist (Ciranna and Cavallaro

2003) But this VIP-induced enhancement of synaptic transmission was mainly mediated

by VPAC1 receptor activation since the effect of the VPAC1-selective agonist was nearly

as big as the effect of VIP In addition this effect could be blocked by VPAC1 receptor

antagonist (Cunha-Reis et al 2005) Recently VIP-induced facilitation of synaptic

transmission in the hippocampus was found to be dependent on both adenosine A1 and

A2A receptor activation by endogenous adenosine (Cunha-Reis et al 2007) In addition

the enhancement of synaptic transmission to CA1 pyramidal cells by VIP was also

dependent on GABAergic transmission This action occurred both through presynaptic

enhancement of GABA release and post-synaptic facilitation of GABAergic currents in

interneurones (Cunha-Reis et al 2004)

But our studies demonstrated that the application of low concentration of PACAP

(1 nM) had no effect on basal synaptic transmission The most possible explanation was

that the solution we used was different from that of Cunha-Reis et al they used high

concentration of K+ in the recording solution Instead we found that the application of

126

PACAP (1 nM) favoured LTP induction In addition endogenous PACAP was required

for the LTP induction by HFS since the PAC1 receptor antagonist M65 significantly

inhibited LTP induction by HFS (unpublished data)

443 The involvement of PACAPVIP system in learning and memory

Given the distribution of VIP PACAP and their cognate receptors in the

hippocampus in addition to their impacts on the synaptic transmission their important

roles in learning and memory are also demonstrated following the generation of

transgenic animals and selective ligands

Mutant mice with either complete or forebrain-specific inactivation of PAC1

receptor showed a deficit in contextual fear conditioning and an impairment of LTP at

mossy fiber-CA3 synapses In contrast water maze spatial memory was unaffected in

these PAC1 receptor mutant mice (Otto et al 2001) Additionally in Drosophila

melanogaster mutation in the PACAP-like neuropeptide gene amnesiac affected both

learning memory and sleep (Feany and Quinn 1995) In line with these observations

intra-cerebroventricular injection of very low doses of PACAP improved passive

avoidance memory in rat (Sacchetti et al 2001)

Furthermore in a mouse mutant with a 20 reduction in brain VIP expression

there were learning impairments including retardation in memory acquisition (Gozes et

al 1993) Consistent with these findings intra-cerebral administration of a VIP receptor

antagonist in the adult rats resulted in deficits in learning and memory in the Morris water

maze (Glowa et al 1992) Consistently treatment of AD model mice with daily injection

of Stearyl-Nle17-VIP (SNV) which exhibited a 100-fold greater potency for VPAC

127

receptors than VIP was associated with significant amelioration for memory deficit

(Gozes et al 1996)

444 The other functions of PACAPVIP system in the CNS

My study contributed to the growing body of evidence demonstrating a role for

the modulation of NMDAR activity by PACAPVIP system Both PACAPVIP system

and NMDA also share several other common roles

One role is development Recent studies have indicated that VIP had an important

role in the regulation of embryonic growth and development during the period of mouse

embryogenesis (Hill et al 2007) Treatment of pregnant mice using a VIP antagonist

during embryogenesis resulted in microcephaly and growth restriction of the fetus

(Gressens et al 1994) as well as developmental delays in newborn mice (Hill et al

2007) Blockage of VIP during development resulted in permanent damage to the brain

(Hill et al 2007) VIP-induced growth occured at least in part through the actions of

ADNF (activity-dependent neurotrophic factor) (Glazner et al 1999) and insulin-like

growth factor (IGF) which were important growth factors in embryonic development

(Baker et al 1993) VIP also regulated nerve growth factor in the mouse embryo (Hill et

al 2002) providing further evidence of the broad role of VIP in neural development In

addition VIP application to cultured hippocampal neurons caused dendritic elongation by

facilitating the outgrowth of microtubes (Henle et al 2006 Leemhuis et al 2007) VIP

has been implicated in several neurodevelopmental disorders too Cortical astrocytes

from the mouse model of Down syndrome Ts65Dn showed reduced responses to VIP

stimulation as well VPAC1 expression was increased in several brain regions of these

128

mice (Sahir et al 2006) Also elevated VIP concentrations have been found in the

umbilical cord blood of newborns with Down syndrome or autism (Nelson et al 2001)

providing a link between VIP and autism

Similarly PACAP is also required for the development of the CNS PACAP and

PAC1 receptor were up-regulated during embryonic development indicating the

importance of this peptide for the development (Jaworski and Proctor 2000 Vaudry et

al 2000 Vaudry et al 2009) PACAP also induced neuronal differentiation in several

cell lines this role exerted by PACAP was mainly mediated by cAMPPKA signaling

pathway (Gerdin and Eiden 2007 Monaghan et al 2008 Shi et al 2006 Shi et al

2010a) But recently several studies demonstrated that another cAMP effector Epac was

also involved in the neuronal differentiation induced by PACAP (Gerdin and Eiden 2007

Monaghan et al 2008 Shi et al 2006 Shi et al 2010a) Furthermore PACAP induced

astrocyte differentiation in cortical precursor cells by expressing glial fibrilary acidic

protein (GFAP) not only PKA but also Epac mediated the expression of GFAP by

PACAP (Lastres-Becker et al 2008)

The other common role of PACAPVIP system and NMDAs is neurotoxicity

Paradoxically both PACAP and VIP provide neuroprotection while NMDARs are often

associated with neurotoxicity Toxicity associated with TTX treatment of spinal cord

cultures was prevented by VIP (Brenneman and Eiden 1986) Recent studies have

indicated a unique role for VIP in neuroprotection from excitotoxicity in white matter

(Rangon et al 2005) In this model VPAC2 receptors mediated neuroprotection from

excitotoxicity elicited by ibotenate The evidence was provided by both the action of

pharmacological agents and the lack of VIP-mediated activity in VPAC2 knockout mice

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(VPAC2 --) (Rangon et al 2005) VIP administration reduced the size of ibotenate-

induced lesions in brains of neonatal mice (Gressens et al 1994) The activation of

VIPVPAC1 signaling cascade in the vicinity of the injury site was also found to

circumvent the synergizing degenerative effects of ibotenate and pro-inflammatory

cytokines (Favrais et al 2007) Neuroprotective activity of VIP seems to involve an

indirect mechanism requiring astrocytes VIP-stimulated astrocytes secreted

neuroprotective proteins including ADNF (Dejda et al 2005) Beside the release of

neurotrophic factors astrocytes actively contributed to neuroprotective processes through

the efficient clearance of extracellular glutamate A recent study showed that activation

of VIPVPAC2 receptor in astrocytes increased GLAST-mediated glutamate uptake this

effect required both PKA and PKC activation (Goursaud et al 2008)

PACAP also could protect cells from death in various models of toxicity

including transient middle cerebral artery occlusion (Reglodi et al 2002) and nitric oxide

activation induced by glutamate (Onoue et al 2002) PACAP could inhibit several

signaling pathways including Jun N-terminal kinase (JNK)stress-activated protein kinase

(SAPK) and p38 which induce apoptosis (Vaudry et al 2000 Vaudry et al 2009) In

addition PACAP played the neuroprotective roles via the expression of neurotrophic

factors as well For example PACAP could increase the expression of BDNF in both

astrocytes (Pellegri et al 1998) and in neurons (Pellegri et al 1998 Yaka et al 2003)

My work in the thesis provided strong evidence that Src and Fyn signaling

cascades activated by Gαq- versus Gαs-coupled receptors respectively differentially

45 Significance

130

enhance GluN2AR and GluN2BR activity The activation of the Gαq coupled receptors

selectively stimulates PKCSrc cascade and increases the tysrosine phosphorylation of

GluN2A subunits In contrast Gαs coupled receptor activation preferentially induces

PKAFyn pathway and the increase of tyrosine phosphorylation of GluN2B subunits

(Yang et al unpublished data) This study provides us with the means to selectively

enhance either GluN2ARs or GluN2BRs By this means we can investigate the role of

NMDAR subtypes in the direction of synaptic plasticity

In addition it is well accepted that hyperactivation of NMDAR is the most

compelling molecular explanation for the mechanism underlying AD Memantine a

NMDAR antagonist has been approved for treatment of moderate to severe AD (Kalia et

al 2008 Parsons et al 2007) Recently overactivation of GluN2BR activity has been

implicated in AD (Ittner et al 2010) Based on my work some interfering peptides and

drugs can be designed and used to selectively inhibit the activity of GluN2BRs by

interrupting Fyn mediated signaling cascade It will provide new candidate drugs for the

treatment of AD

My current work has provided strong evidence to propose that the subtypes of

NMDARs are differentially regulated by SFKs and GPCRs It also raises several

questions which have to be answered in the future

46 Future experiments

461 Is the trafficking of GluN2AR andor GluN2BR to the surface increased by Src and

Fyn activation respectively

131

Previous studies have shown that Fyn could regulate the trafficking of GluN2BR

surface expression (Hu et al 2010 Snyder et al 2005b) but if Src also had the same

ability to modulate the trafficking of NMDARs to the surface remains unknown Our lab

has demonstrated that PKC enhanced NMDAR currents via Src activation in

hippocampal CA1 neurons (Kotecha et al 2003 Lu et al 1999a Macdonald et al

2005) In addition PKC activation phosphorylated SNAP25 and increased the surface

insertion of GluN1 subunits (Lau et al 2010) These studies implicate that Src may be

involved in the regulation of NMDAR trafficking although there is limited evidence of

GluN1 tyrosine phosphorylation (Lau and Huganir 1995 Salter and Kalia 2004)

Additionally my current work provide strong evidence that in CA1 neurons the activity

of GluN2ARs and Glun2BRs are differentially regulated by discrete Src and Fyn

signaling cascades It implicates that Src and Fyn may also differentilly modulate the

trafficking of GluN2ARs and GluN2BRs to the membrane

We will determine if the activation of PAC1 receptors via endogenous Src leads

to a selective increase of GluN2AR over GluN2BR at the membrane surface of

hippocampal neurons In contrast we will also study if VPAC receptor activation

selectively enhances the surface expression of GluN2BR versus GluN2AR through Fyn

activation

462 Sites of Tyrosine phosphorylation of GluN2 subunits

Although I have shown that the activity of GluN2AR and GluN2BR can be

enhanced by Src and Fyn respectively the evidence that tyrosine phosphorylations of

GluN2A andor GluN2B subunits directly cause the enhancement of GluN2AR or

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GluN2BR activity is lacking In order to answer this question potential tyrosine

phosphorylation sites on GluN2 subunits have to been mutated and expressed in HEK293

cells or Xenopus oocytes then whether or not the potentiation of NMDAR by SFKs is

blocked is studied Howover this approach is complicated by the large number of

potential tyrosine phosphorylation sites on GluN2A and GluN2B subunits as well as by

the recognition that these receptors behave very differently in cell lines (Kalia et al 2006

Salter and Kalia 2004)

Recently one paper demonstrated that when tyrosine residue at 1325 on the

GluN2A subunit was mutated to Phenylalanine (Phe) Src failed to increase NMDAR

currents in HEK cells (Taniguchi et al 2009) In addition the potentiation of EPSCNMDAs

induced by Src was blocked in medium spiny neurons of these knockin Y1325F

transgenic mice (Taniguchi et al 2009) indicating that the phosphorylation of GluN2A

Y1325 mediates the potentiation of NMDARs by Src Although many papers implicated

that Y1472 on the GluN2B subunit was strongly phosphorylated by Fyn (Nakazawa et al

2001 Nakazawa et al 2006) whether or not the phosphorylation of this residue induced

the increase of NMDAR activity by Fyn requires further study

Firstly we will study whether Y1325 in GluN2A subunit and Y1472 in GluN2B

subunit are strongly phosphoyrlated by Src and Fyn respectively Then if tyrosine

phosphorylation of these sites underlies the effects of SKFs on NMDARs will also be

investigated Recently two knockin transgenic mice which blocked the phosphorylation

of Y1325 in the GluN2A subunit (Y1325F) and Y1472 in the GluN2B subunit (Y1472F)

respectively were generated (Nakazawa et al 2006 Taniguchi et al 2009) These

transgenic mice have less compensation compared to GluN2A -- and GluN2B -- mice

133

With the help of these knockin transgenic mice we will confirm that the potentiation of

NMDARs by the PAC1 receptor activation and Src is absent in acutely isolated CA1

neurons as well as confirm that the increase of EPSCNMDAs at CA1 synapses is lost in

Y1325F knockin mice Using Y1472F mice we will also determine if Fyn and VPAC

receptors upregulate GluN2BR activity

463 How does Fyn inhibitory peptide (Fyn (39-57)) inhibit the increased function of

GluN2B subunits by Fyn

My current work demonstrated that Fyn inhibitory peptide (Fyn (39-57))

specifically blocked the increase of NMDARs currents by Fyn but not Src We propose

that it does so by interfering with the binding of proteins to GluN2B subunit which is

required for the potentiation of NMDARs by Fyn

We will use yeast-two hybrid (Y2H) assay to identify the proteins which bind the

unique domain of Fyn Since Fyn (39-57) effectively uncouples GluN2BRs from Fyn-

mediated regulation binding of candidate proteins must be displaced by Fyn (39-57) In

addition candidate proteins should associate with GluN2BRs

464 Are scaffolding proteins involved in the differential regulation of NMDAR

subtypes by SFKs

So far several studies have demonstrated that among scaffolding proteins only

PSD95 interacted with Src (Kalia and Salter 2003) it blocked the regulation of

NMDARs by Src (Kalia et al 2006 Yamada et al 2002) possibly this effect was

mediated by GluN2ARs (Yamada et al 2002) In contrast although PSD95 and PSD93

134

have been shown to bind Fyn (Sato et al 2008 Tezuka et al 1999) whether or not other

scaffolding proteins including SAP102 and SAP97 requires further study

Firstly we will determine which scaffolding proteins interact with Fyn using co-IP

assay Secondly how these scaffolding proteins modulate the ability of Fyn to selectively

regulate GluN2BRs will be investigated Thirdly we will study the potential role of these

scaffolding proteins in the trafficking of GluN2BRs by Fyn

135

Section 5 Appendix

Project 3 Epac activated by Gαs coupled receptors also modulates NMDARs

136

Introduction

Although PKA is involved in most of cAMP-mediated cellular functions some

functions induced by cAMP are independent of PKA For example cAMP-induced

activation of the small GTPase

51 cAMP effector Epac

Rap1 was not blocked by PKA inhibitiors This mystery

was clarified when Epac1 was identified (Bos 2003 Bos 2006 Gloerich and Bos 2010)

Subsequent studies showed that this protein was a cAMP effector which stimulated Rap

upon activation (de et al 1998) Epac2 was a close relative of Epac1 but it contained

two cAMP-binding domains (CBD) at its N terminus (Borland et al 2009 Roscioni et

al 2008)

Epac1 and Epac2 had distinct expression patterns Epac1 was expressed

ubiquitously whereas Epac2 was predominantly expressed in the brain and endocrine

tissues (Kawasaki et al 1998) Epac2 exists as three different splicing variants including

Epac2A Epac2B and Epac2C which differ only at their N terminus Epac2A has the full

length of protein while Epac2B lacks the N terminal CBD which is only expressed in

adrenal glands Epac2C is only detected in the liver which lacks the N terminal CBD and

DEP (Dishevelled Egl-10 and Pleckstrin domain)

In addition Epac1 and Epac2 are also localized in different subcellular

compartments For Epac1 many studies showed that it was located in centrosomes the

nuclear pore complex mitochondria and plasma membrane Its different subcellular

localizations link Epac1 to specific cellular functions For example activation of Epac1

in Rat1a cells predominantly stimulated Rap1 at the peri-nuclear region since at the

plasma membrane RapGAP activity was high it inactivated Rap quickly (Ohba et al

137

2003) Additionally in the nucleus Epac1 regulated the DNA damagendashresponsive kinase

(DNA-PK) (Huston et al 2008) The target to the plasma membrane of Epac1 resulted

from cAMP induced conformational changes and depended on the integrity of its DEP

domain Furthermore this translocation was required for cAMP-induced Rap activation

at the plasma membrane (Ponsioen et al 2009) Epac1 was also targeted to microtubules

to regulate microtubule polymerization This targeting might be mediated by the

microtubule-associated protein (MAP1) In contrast Epac2 was distributed in the plasma

membrane Epac2 targeted to the plasma membrane via its Ras associating (RA) domain

(Li et al 2006) In addition N-terminus of Epac2 also helped its delivery to the plasma

membrane (Niimura et al 2009)

Although one study showed that the binding affinities of cAMP for PKA and

Epac were similar (Dao et al 2006) in vivo support for this observation is currently

lacking In addition several studies demonstrated that Epac had a lower sensitivity for

cAMP compared with PKA (Ponsioen et al 2004) Indeed cAMP sensors based on PKA

were more sensitive than that based on Epac (Ponsioen et al 2004) Although Epac

required high concentration of cAMP to be activated the intracellular concentration of

cAMP after receptor stimulation was sufficient to activate Epac and its downstream

targets

Epac is a multi-domain protein including an N-terminal regulatory region and a

C-terminal catalytic region The N-terminal regulatory domain contains a DEP domain

although its deletion did not affect the regulation of Epac1 by cAMP it resulted in a more

cytosolic localization of Epac1 (Ponsioen et al 2009) This suggested that this domain

was involved in the localization of Epac1 in the plasma membrane Another domain is

138

CBD-B Although this domain mainly interacts with cAMP it also acts as a protein-

interaction domain For example it was found to interact with the MAP1B - light chain 1

(LC1) (Borland et al 2006) The entire N-terminal region of Epac1 might also serve as a

protein-interaction domain because one report showed that this region directed Epac1 to

mitochondria (Qiao et al 2002) Additionally Epac2 contained a second low-affinity

CBD-A domain with unknown biological function (Bos 2003 Bos 2006) Although this

domain bound cAMP with a 20-fold lower affinity than the conserved CBD-B it was not

involved in the activation of Epac2 by cAMP (Rehmann et al 2003)

Between the regulatory and the catalytic regions is a Ras exchange motif (REM)

which stabilizes the GEF domain of Epac Epac also has a RA domain and this domain

has been found to interact with GTP-bound Ras With the help of RA domain Epac 2

was recruited to the plasma membrane (Li et al 2006) The last domain of Epac is

CDC25 homology domain (CDC25HD) which exhibits GEF activity for Rap (Bos 2003

Bos 2006)

In the inactive conformation of Epac the CBD-B domain interacts with the

CDC25HD domain and hinders the binding and activation of Rap Upon binding of

cAMP to CBD-B domain a subtle change within this domain allows the regulatory

region to move away from the catalytic region No significant differences between the

conformation of the CDC25-HD in the active and inactive conformations have been

observed indicating that cAMP regulates the activity of Epac by relieving the inhibition

by the regulatory doamin rather than by inducing an allosteric change in the GEF domain

(Bos 2006 Rehmann et al 2003)

139

The activation of Gαs coupled receptors increases the concentration of cAMP

activating PKA dependent signaling pathway Recently many studies demonstrated that

Epac could also be activated by many Gαs coupled receptors and mediate cellular

functions (Ster et al 2007 Ster et al 2009 Woolfrey et al 2009)

52 Epac and Gαs coupled receptors

So far no specific Epac antagonist is available there are only two indirect ways to

claim the involvement of Epac in Gαs coupled receptor mediated effects One is to

reproduce Gαs coupled receptor induced effects by Epac agonist 8-pCPT-2prime-O-Me-cAMP

For example PACAP was proposed to induce LTD via Epac since this PACAP induced

LTD was inhibited by the non-specific Epac inhibitor BFA In addition occlusion

experiments were also done to investigate if PACAP was upstream of Epac Saturated

Epac-LTD occluded PACAP-LTD and vice versa These results provided strong evidence

that high concentration of PACAP induced LTD through Epac (Ster et al 2009)

The other way is to investigate if the actions of Gαs coupled receptors can be

abolished by the down-regulation of Epac expression In order to investigate if Epac2

wass involved in the dopamine D1D5 receptor induced synaptic remodeling after Epac2

was knocked down using Epac2 siRNA synaptic remodeling by dopamine D1D5

receptor did not occur (Woolfrey et al 2009) This study indicated that dopamine D1D5

receptor activation induced synaptic changes via Epac2

Epac proteins were initially characterized as cAMP-activated GEFs for Rap (de et

al 1998 Kawasaki et al 1998) Later Epac proteins were found to stimulate many

53 Epac mediated signaling pathways

140

effectors and played important roles in various cellular functions Schmidt demonstrated

that Gαs coupled receptors stimulated Rap2PLCε dependent signaling pathway via Epac

Activation of PLCε resulted in the generation of IP3 and the increase of cellular Ca2+

(Evellin et al 2002 Schmidt et al 2001) In contrast Gαi coupled receptors inhibited

the Epac-Rap2-PLCε signaling pathway (Vom et al 2004) Additionally Epac1 also

directly bound and activated R-Ras The activation of R-Ras by Epac stimulated

phospholipase D (PLD) activity then PLD hydrolyzed phosphatidylcholine (PC) to

phosphatidic acid (PA) in the plasma membrane (Lopez de et al 2006)

Several studies demonstrated that Rap1 activated by Epac also modulated

mitogen-activated protein kinase (MAPK) activity including ERK12 and JNK

(Hochbaum et al 2003 Stork and Schmitt 2002) The activated Rap1 by Epac may

enhance or inhibit ERK12 depending on specific cell types Recently it was

demonstrated that Epac-triggered activation of ERK12 relied on the mode of Rap1

activation Rap1 had to be colocalized with Epac in the plasma membrane for the

activation of ERK12 (Wang et al 2006) In addition it has been shown that Epac

activated JNK as well surprisingly the activation of JNK by Epac was independent of its

GEF activity (Hochbaum et al 2003)

Furthermore Epac interacts with microtube-associated protein 1B (MAP1B) and

its GEF activity was controlled by this interaction (Gupta and Yarwood 2005) Moreover

Rap1 increased the GAP activity of ARAP3 and inhibited RhoA-dependent signaling

pathway (Krugmann et al 2004) Such signaling pathway may present a link between

Rap1 and RhoA Recently it demonstrated that Rap1 activated by Epac activated Rac

through a Tiam1Vav2-dependent pathway in human pulmonary artery endothelial cells

141

(Birukova et al 2007) In addition the secretion of the amyloid precursor protein (APP)

by Epac required Rap1Rac dependent signaling pathway in mouse cortical neurons

(Maillet et al 2003) Epac activated by PACAP also stimulated a small GTPase Rit to

mediate neuronal differentiation (Shi et al 2006 Shi et al 2010a) Recently several

studies demonstrated that Epac modulated protein kinase B (PKB)Akt activity Again

Epac activation can either stimulate or inhibit Akt activity depending on cell types (Hong

et al 2008 Huston et al 2008 Nijholt et al 2008)

Depending on their cellular localizations and binding partners Epac proteins

activate different downstream effectors Therefore the coupling of Epac to specific

signaling pathways is determined by its localization to subcellular compartments (Dao et

al 2006) It is well demonstrated that spatio-temporal cAMP signaling involved AKAP

family (Carnegie et al 2009 Scott and Santana 2010) and recently the interaction of

Epac with AKAP have been identified in the heart and neurons (Dodge-Kafka et al 2005

Nijholt et al 2008) In neonatal rat cardiomyocytes muscle specific AKAP (mAKAP)

interacted with PKA PDE4D3 and Epac1 and formed a multiprotein complex which was

regulated by different cAMP concentrations At high cAMP concentration Epac1 was

activated and resulted in the inhibition of ERK5 via Rap1 subsequently PDE4D3 was

activated and the concentration of cAMP was reduced Whereas at low cAMP

concentration PDE4D3 was inactivated by ERK5 and subsequent PKA signaling was

enhanced (Dodge-Kafka et al 2005) A recent study reported that AKAP79150 bound

to Epac2 as well as PKA in neuron Direct binding of PKA or Epac2 to AKAP79150

54 Compartmentalization of Epac signaling

142

exerted opposing effects on neuronal PKBAkt activity The activation of PKA inhibited

PKBAkt phosphorylation whereas the stimulation of Epac2 enhanced PKBAkt

phosphorylation (Nijholt et al 2008)

In addition there are several studies supporting that PDEs also interacted with

Epac directly and contributed to the specificity of Epac signaling (Dodge-Kafka et al

2005 Huston et al 2008 Raymond et al 2007) For example In HEK-B2 cells PDE4D

was found in the cytoplasm and excluded from the nucleus while PDE4B was located in

the nucleus only PDE4B activity specifically controlled the ability of nuclear Epac1 to

export DNA-PK out of the nucleus while cytosolic PDE4D regulated PKA-mediated

nuclear import of DNA-PK DNA-PK was an enzyme which is involved in DNA repair

systems (Huston et al 2008) In addition a recent study by Raymond demonstrated that

in HEK293T cells there were several distinct PKA- and Epac-based signaling complexes

which included several different PDEs Individual PKA- or Epac-containing complexes

could contain either PDE3B or PDE4D but they did not contain both of these PDEs

PDE3B was largely located in Epac-based complexes but PDE4D enzymes were only

found in PKA-based complexes (Raymond et al 2007) Although the interaction

between PDEs and Epac are well demonstrated its physiological function requires further

study

It is well known that cAMP not only activates PKA but also Epac In order to

investigate the role of Epac in physiological functions of the cell Epac selective agonist

is required With the development of a selective Epac agonist 8-pCPT-2prime-O-Me-cAMP

55 A selective Epac agonist 8-pCPT-2prime-O-Me-cAMP

143

the research on Epac has been well expanded For this agonist the 2primeOH group of cAMP

has been replaced with 2primeO -Me in order to increase the binding with Epac In addition

the substitution of 8-pCPT on 2prime -O-Me-cAMP not only enhanced its affinity and

selectivity with Epac but also increased its membrane permeability (Enserink et al

2002) In vitro this specific Epac agonist 8-pCPT-2prime-O-Me-cAMP has demonstrated more

than three-fold ability to stimulate Epac1 compare to cAMP (Enserink et al 2002)

Later this specific Epac agonist was found to be hydrolyzed by PDE in vivo and

its metabolites might interfer with some cellular functions (Holz et al 2008 Poppe et al

2008) Beavo et al demonstrated that 8-pCPT-2prime-O-Me-cAMP had an anti-proliferative

effect in cultures of the protozoan Trypanosoma brucei but this action was mediated by

its degradation product 8-pCPT-2prime-O-Me-adenosine (8-pCPT-2prime-O-Me-Ado) Since

another Epac agonist Sp-8-pCPT-2prime-O-Me-cAMPS which was resistant to the hydrolysis

of PDEs had no such anti-proliferative effect In addition the PDEs expressed in

Trypanosomes could hydrolyze 8-pCPT-2prime-O-Me-cAMP to its 5prime-AMP derivative in vitro

(Laxman et al 2006) Very recently another study showed that the induction of cortisol

synthesis in adrenocortical cells by 8-pCPT-2prime-O-Me-cAMP involved an Epac-

independent pathway (Enyeart and Enyeart 2009) For these reasons the actions of 8-

pCPT-2prime-O-Me-cAMP in living cells have to be reproduced by PDE-resistant Sp-8-

pCPT-2prime-O-Me-cAMPS thereby reducing the possibility that the measured effect is

mediated by the metabolites of 8-pCPT-2prime-O-Me-cAMP

8-pCPT-2prime-O-Me-cAMP is not only susceptible to be hydrolysed by PDEs but

also inhibits PDEs This action may raises the level of cAMP and activate PKA For

example when the applied concentration of 8-pCPT-2prime-O-Me-cAMP was higher than

144

100 μM it activated PKA in NIH3T3 cells (Enserink et al 2002) Recently in one study

using pancreatic β cells the potentiation of Ca2+ dependent exocytosis by 8-pCPT-2prime-O-

Me-cAMP (100 μM) was reduced by PKA inhibitor PKI indicating PKA would act in a

permissive manner to mediate Epac-regulated exocytosis (Chepurny et al 2010) In

addition it has been reported that 13 distinct cyclic nucleotide analogs widely used in

studing cellular signaling might result in elevation of cAMP upon inhibition of PDEs in

human platelets (Poppe et al 2008) Thus when investigating Epac-mediated actions

using 8-pCPT-2prime-O-Me-cAMP another control experiment should be done to

demonstrate that this action is resistant to PKA inhibitors

Recently in order to increase membrane permeability of 8-pCPT-2-O-Me-cAMP

an acetoxymethyl (AM)-ester was introduced to mask its negatively charged phosphate

group This new compound could enter cells quickly thereby being intracellularly

hydrolyzed into 8-pCPT-2-O-Me-cAMP by cytosolic esterases Importantly intracellular

8-pCPT-2-O-Me-cAMP produced by this AM compound still kept its selectivity for

Epac (Chepurny et al 2009 Chepurny et al 2010 Kelley et al 2009)

Although the regulation of ion channels by cAMP is well studied most studies

contribute its effects to activation of PKA Now the involvement of Epac in the cAMP-

dependent regulation of ion channel function emerges

56 Epac mediates the cAMP-dependent regulaton of ion channel function

For example in pancreatic β cells Epac was reported to interact with nucleotide

binding fold-1 (NBF-1) of SUR1 subunits of ATP-sensitive K+ channels (KATP channels)

and inhibited their activities (Kang et al 2006) Once Epac was activated its effector

145

Rap stimulated PLC-ε to hydrolyze phosphatidylinositol 45-bisphosphate (PIP2)

(Schmidt et al 2001) PIP2 enhanced the activity of KATP channels by reducing the

channels sensitivity to ATP (Baukrowitz et al 1998 Shyng and Nichols 1998) the

hydrolysis of PIP2 by Epac may mediate the inhibitory action of Epac on KATP channels

In rat pulmonary epithelial cells Epac also increased the activity of amiloride-

sensitive Na+ channels (ENaC) (Helms et al 2006) This stimulatory effect was not

mediated by PKA since the mutation of PKA motif in the cytosolic domain of ENaC did

not block this effect In contrast the mutation of ERK motif inhibited the action of Epac

(Yang et al 2006) Recently in rat hepatocytes glucagon was shown to stimulate Epac

which then regulates Clndash channel (Aromataris et al 2006) since the PKA-selective

cAMP analogue N6-Bnz-cAMP could not activate this Clndash channel

Epac regulates not only ion channels but also ion transporters In rodent renal

proximal tubules Epac inhibited Na+ndashH+ exchanger 3 (NHE3) activity and this effect

was not mediated by PKA (Honegger et al 2006) Additionally Epac regulated the

activation of ATP-dependent H+ndashK+ transporter activity in the Iα cells of rat renal

collecting ducts (Laroche-Joubert et al 2002)

Although Epac modulates many ion channels and transporters including

AMPARs (Woolfrey et al 2009) if it also regulates NMDARs remains unknown

Furthermore given the importance of cAMP signaling in the hippocampus it is possible

that activation of cAMP effector Epac may be also involved in the synaptic plasticity

Recently several studies have demonstrated this possibility Epac was involved in not

57 Hypothesis

146

only memory consolidation but also memory retrieval (Ma et al 2009 Ostroveanu et al

2009) In addition Epac induced LTD (Ster et al 2009 Woolfrey et al 2009) although

one study indicated that Epac enhanced the maintenance of various forms of LTP in area

CA1 of the hippocampus (Gelinas et al 2008) Furthermore a lot of Gαs coupled

receptors have the capacity to activate Epac but if Epac activated by Gαs coupled

receptors selectively modulated subtypes of NMDARs has not previously been explored

147

Results

In order to investigate if Epac can regulate NMDA evoked current in acutely

isolated hippocampal CA1 neurons a specific Epac agonist 8-pCPT-2prime-O-Me-cAMP (10

μM) was used This agonist incorporates a 2rsquo-O-methyl substitution on the ribose ring of

cAMP This modification impairs their ability to activate PKA while increasing their

ability to activate Epac In addition this substitution also increases its membrane

permeability (Enserink et al 2002) NMDAR currents were evoked once every 1 minute

using a 3 s exposure to NMDA (50 microM) and glycine (05 microM) Epac agonist 8-pCPT-2prime-

O-Me-cAMP (10 μM) was applied in the bath continuously for 5 minutes Application of

8-pCPT-2prime-O-Me-cAMP (10 μM) increased NMDA-evoked currents up to 316 plusmn 39

(N = 8) compared with baseline but NMDA-evoked currents in control cells were stable

over the recording period (18 plusmn 27 n = 5) (Fig 61) Recently one study showed that

PDE-catalysed hydrolysis of 8-pCPT-2prime-O-Me-cAMP could generate bioactive

derivatives of adenosine and alter cellular function independently of Epac (Laxman et al

2006) This metabolism could complicate the interpretation of studies using 8-pCPT-2prime-

O-Me-cAMP (Holz et al 2008) To validate that the stimulatory action of 8-pCPT-2prime-O-

Me-cAMP reported here did not result from its hydrolysis we applied PDE-resistant

Epac agonist Sp-8-pCPT-2prime-O-Me-cAMPS (10 microM) in the bath for 5 minutes In the

presence of Sp-8-pCPT-2prime-O-Me-cAMPS NMDA evoked current was increased up to

455 plusmn 46 (n = 5) (Fig 61) excluding the involvement of the degradation of 8-pCPT-

2prime-O-Me-cAMP on the potentiation of NMDAR currents in acutely isolated cells

The Epac selectivity of 8-pCPT-2prime-O-Me-cAMP was not absolute since

concentrations of the analog in excess of 100 μM also activated PKA in vitro (Enserink et

148

al 2002) In addition one study showed that 8-pCPT-2prime-O-Me-cAMP could also inhibit

all PDEs and increase cAMP concentration to activate PKA (Poppe et al 2008) Thus

when examining the action of 8-pCPT-2prime-O-Me-cAMP in living cells control

experiments have to be done to exclude the involvement of PKA It should be

demonstrated that treatment of cells with PKI14-22 or Rp-cAMPs fails to block the action

of 8-pCPT-2prime-O-Me-cAMP In order to confirm the potentiation of NMDARs induced by

8-pCPT-2prime-O-Me-cAMP here was mediated by Epac but not by PKA PKA inhibitor

PKI14-22 which binds to catalytic subunit and inhibits PKA kinase activity was added in

the patch pipette In the presence of PKI14-22 (03 μM) the application of 8-pCPT-2prime-O-

Me-cAMP (10 μM) still caused a robust increase in NMDA evoked current (364 plusmn 22

n = 6) Another PKA inhibitor Rp-cAMPs was also used it binds to regulatory subunit of

PKA and inhibits dissociation of the catalytic subunit from the regulatory subunit of PKA

The presence of Rp-cAMPs (500 μM) also could not block potentiation of NMDARs

caused by the application of 8-pCPT-2prime-O-Me-cAMP (10 μM) (313 plusmn 2 n = 5) (Fig

62)

Previous studies indicated that activation of the Gαs-coupled β2-adrenoceptor

expressed in HEK293 cells or the endogenous receptor for prostaglandin E1 in N2E-115

neuroblastoma cells induced PLC stimulation via Epac and Rap2B (Schmidt et al 2001)

In addition in IB4 (+) subpopulation of sensory neurons cAMP activated by β2-

adrenergic receptor also enhanced PLC activity through Epac (Hucho et al 2005) To

check for the involvement of PLC PLC inhibitor U73122 (10 microM) was added in the

patch pipette The incubation of Epac agonist 8-pCPT-2prime-O-Me-cAMP failed to

potentiate NMDARs in the presence of U73122 (U73122 -42 plusmn 23 n = 6 8-pCPT-

149

2prime-O-Me-cAMP plus U73122 6 plusmn 24 n = 5 8-pCPT-2prime-O-Me-cAMP 402 plusmn 58 n

= 6) (Fig 63) In contrast the inactive analog of PLC inhibitor U73122 U73343 (10

microM) could not block the increase of NMDA evoked current induced by 8-pCPT-2prime-O-

Me-cAMP (U73343 -07 plusmn 28 n = 5 8-pCPT-2prime-O-Me-cAMP plus U73343 511 plusmn

37 n = 6) (Fig 63) In addition U73122 (10 microM) or U73343 (10 microM) alone also

failed to impact on NMDAR currents

In addition PLC activated by Epac can signal through PKC to regulate

presynaptic transmitter release at excitatory central synapses (Gekel and Neher 2008)

This signal pathway was also involved in inflammatory pain (Hucho et al 2005) To

investigate if PKC was involved in the potentiation of NMDARs induced by 8-pCPT-2prime-

O-Me-cAMP we included PKC inhibitor bisindolylmaleimide I (bis) (500nM) in both

patch pipette and bath solution The presence of bis blocked the enhancement of NMDA

evoked current induced by 8-pCPT-2prime-O-Me-cAMP (8-pCPT-2prime-O-Me-cAMP plus Bis

52 plusmn 3 n = 7 8-pCPT-2prime-O-Me-cAMP 324 plusmn 65 n = 6) Bis alone had no effect

on NMDA evoked current (Fig 64)

Our lab previously showed that PKC activation induced by Gq protein coupled

receptors such as muscarine receptors and mGluR5 receptors enhance NMDA-evoked

currents through Src (Kotecha et al 2003 Lu et al 1999a) So next we studied if the

PKC activation induced by Epac also stimulated Src activity and if this increase of Src

activity is required for the potentiation of NMDARs induced by Epac Src inhibitory

peptide (Src (40-58)) (25 microg) was included in the patch pipette and results showed that

Src inhibitory peptide blocked the potentiation of NMDAR currents induced by Epac (Fig

64)

150

A growing body of evidence shows that Epac also regulated intracellular Ca2+

dynamics (Holz et al 2006) In pancreatic β cells there existed an Epac-mediated action

of 8-pCPT-2-O-Me-cAMP to mobilize Ca2+ from intracellular Ca2+ stores (Kang et al

2003 Kang et al 2006) Another study showed that after PLC was activated by Epac

PIP2 was hydrolyzed to generate IP3 and DAG Then IP3 bound to IP3 receptors and

released Ca2+ from the ER resulting in the increase the intracellular Ca2+ concentration

In order to investigate if Ca2+ elevation in the hippocampal CA1 cells was required for

the potentiation of NMDARs by Epac BAPTA (20 microM) was added to the patch pipette

In the presence of BAPTA 8-pCPT-2prime-O-Me-cAMP failed to increase NMDA evoked

currents (8-pCPT-2prime- O-Me-cAMP plus BAPTA -147 plusmn 41 n = 6 8-pCPT-2prime-O-Me-

cAMP 333 plusmn 123 n = 6) BAPTA alone did not change NMDA mediated currents

(Fig 65)

Next we started to study if Epac regulated presynaptic neurotransmitter release in

hippocampal slices Several studies which investigated the role of Epac in

neurotransmitter release have reported the inconsistent results (Gelinas et al 2008

Woolfrey et al 2009) PPF was used to measure the change in the probability of

transmitter release in the hippocampal slices PPF is a well known presynaptic form of

short-term plasticity (Zucker and Regehr 2002) I stimulated the Schaffer collateral

pathway at 005 Hz with 01 ms pulses to evoke synaptic response in the hippocampal

slices After reaching the maximal synaptic response the baseline was chosed to yield a

13 maximal response by adjusting the stimulation intensity In control slices baseline

should be stable for a minimum of 20 minutes before the stimulation In drug treated slice

baseline responses were stable for 10 minutes before the application of 8-pCPT-2prime-O-Me-

151

cAMP Drug treatment was continued for 10 minutes before the stimulation When I

measured PPF the hippocampal slices were stimulated using two stimulations with

different intervals Then the slope of field EPSP evoked by the second stimulation was

compared to that induced by the first stimulation After the application of Epac agonist 8-

pCPT-2prime-O-Me-cAMP (10 microM) for 10 minutes PPF was increased (Fig 66) indicating

that Epac reduced presynaptic neurotransmitter release

In addition whether or not Epac increased the amplitude of NMDAREPSCs in the

hippocampal slices was also studied Whole cell recording was done on Pyramidal

neurons and holding voltage was -60 mV Schaffer Collateral fibers were stimulated

using constant current pulses (50-100 micros) to induce NMDAREPSCs every 30 s

Surprisingly bath application of Epac agonist 8-pCPT-2prime-O-Me-cAMP (10 microM) slightly

reduced NMDAREPSCs In addition when we increased the concentration of this Epac

agonist to 100 microM the reduction of NMDAREPSCs became more obvious (Fig 67) In

order to exclude Epacrsquos effect on the presynaptic site we applied another Epac agonist 8-

OH-2prime-O-Me-cAMP (10 microM) in the patch pipette this Epac agonist is membrane

impermeable so if I add it to the patch pipette it will not reach the presynaptic site and

affect presynaptic neurotransmitter release Indeed in the presence of this membrane

impermeable Epac agonist NMDAREPSCs was significantly increased (Fig 68)

152

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pe

ak C

urre

nt

Time (minutes)

Control (N=5) 10uM Epac agonist (N=8) 10uM PDE resistant Epac agonist (N=5)

Figure 51 Application of an Epac selective agonist (10 microM) 8-pCPT-2prime-O-Me-cAMP

to acutely isolated CA1 pyramidal neurons increased NMDA-evoked peak currents

(316 plusmn 39 n = 8 data obtained at 30 min of recording) it lasted throughout the

recording period But NMDA-evoked currents in control cells were stable over the

recording period (18 plusmn 27 n = 5 data obtained at 30 min of recording) In addition in

the presence of Sp-8-pCPT-2prime-O-Me-cAMPS a PDE resistant Epac selective agonist

NMDAR currents were increased up to 455 plusmn 46 (n = 5)

153

0 5 10 15 20 25 30 3508

10

12

14

16

10uM Epac (N=6) 10uM Epac + PKI (N=6) 10uM Epac + RpCAMPS (N=5)

Norm

alize

d Pe

ak C

urre

nt

Time (minutes)

Figure 52 PKA was not involved in the potentiation of NMDARs by Epac

Intracellular administration Rp-cAMPs (500 μM) (a specific cAMP inhibitor) or PKI14-22

(03 microM) failed to block the effect of Epac (PKI14-22 plus 8-pCPT-2prime-O-Me-cAMP 364 plusmn

22 n = 6 Rp-cAMPs plus 8-pCPT-2prime-O-Me-cAMP 313 plusmn 2 n = 5 data obtained

at 30 min of recording)

154

0 5 10 15 20 25 30 3508

10

12

14

16

10uM Epac (N=6) PLC inhibitor alone (N=6) 10uM Epac + PLC inhibitor (N=5)

Norm

alize

d Pea

k Cur

rent

Time (minutes)

0 5 10 15 20 25 30 35

07080910111213141516171819

10uM Epac (N=6) 10uM Epac + PLC control U73343 (N=5) PLC control U73343 (N=6)

Norm

alize

d Pe

ak C

urre

nt

Time (minutes)

Figure 53 PLC was involved in the potentiation of NMDARs by Epac The

incubation of Epac agonist failed to potentiate NMDARs in the presence of U73122

(U73122 -42 plusmn 23 n = 6 8-pCPT-2prime-O-Me-cAMP plus U73122 6 plusmn 24 n = 5 8-

pCPT-2prime-O-Me-cAMP 402 plusmn 58 n = 6 data obtained at 30 min of recording) while

PLC alone had no effect on NMDA evoked current In contrast the inactive analog of

PLC inhibitor U73343 could not block the increase of NMDA evoked current induced

by Epac (U73343 -07 plusmn 28 n = 5 8-pCPT-2prime-O-Me-cAMP plus U73343 511 plusmn

37 n = 6 data obtained at 30 min of recording) In addition U73343 alone also failed

to impact on NMDAR currents

155

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15 10uM Epac (N=6) 10uM Epac + Bis (N=7)

Nor

mal

ized

Pea

k C

urre

nt

Time (minutes)

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pea

k Cur

rent

Time (minutes)

10uM Epac (N=7) 10uM Epac + Src inhibitory peptide (N=8) 10uM Epac + Scrambled Src inhibitory

Peptide (N=5)

Figure 54 PKCSrc dependent signaling pathway mediated the potentiation of

NMDARs by Epac A The presence of bis blocked the enhancement of NMDA evoked

current induced by 8-pCPT-2prime-O-Me-cAMP (8-pCPT-2prime-O-Me-cAMP plus Bis 52 plusmn 3

n = 7 8-pCPT-2prime-O-Me-cAMP 324 plusmn 65 n = 6 data obtained at 30 min of

recording) Bis alone had no effect on NMDA evoked current B Src inhibitory peptide

(Src (40-58)) inhibited Epac induced potentiation of NMDARs

156

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pe

ak C

urre

nt

Time (minutes)

10uM Epac (N=6) 10uM Epac and BAPTA (N=6)

Figure 55 The elevated Ca2+ concentration in the cytosol was required for the

potentiation of NMDAR currents by Epac In the presence of BAPTA 8-pCPT-2prime-O-

Me-cAMP failed to increase NMDA evoked currents (8-pCPT-2prime-O-Me-cAMP plus

BAPTA -147 plusmn 41 n = 6 8-pCPT-2prime-O-Me-cAMP 333 plusmn 123 n = 6 data

obtained at 30 min of recording) BAPTA alone could not change NMDA mediated

current

157

Figure 56 In the presence of Epac agonist 8-pCPT-2prime-O-Me-cAMP paired-pulse

facilitation was increased indicating that Epac reduced presynaptic transmitter release

0 50 100 150 200-02

00

02

04

06

08

F

acilit

atio

n

Paired-Pulse Interval (ms)

Control (N=9) 10uM Epac (N=9)

158

Figure 57 Bath application of Epac agonist 8-pCPT-2prime-O-Me-cAMP reduced

NMDAREPSCs Low concentration of this Epac agonist (10 microM) slightly reduced

NMDAREPSCs but in the presence of Epac agonist (100 microM) the reduction of

NMDAREPSCs was significantly reduced

0 5 10 15 20025

050

075

100

125

EPAC

Norm

alize

d NM

DARs

EPS

Cs

Time (min)

10 uM 100 uM

159

Figure 58 Intracellular application of a membrane impermeable Epac agonist 8-

OH-2prime-O-Me-cAMP increased NMDAREPSCs

0 5 10 15 20 25

05

10

15

20

25

30

35

401

2

01s

40pA

1

2

01s

50pA

EPSC

NM

DA (

of b

asel

ine)

Time (min)

Control Epac agonist

1 2


160

Discussion

In my study I demonstrated that a selective Epac agonist 8-pCPT-2prime-O-Me-cAMP

(10 microM) could enhance NMDA evoke currents in acutely isolated hippocampal CA1 cells

Furthermore PDE-resistant Epac agonist Sp-8-pCPT-2prime-O-Me-cAMPS also potentiated

NMDA mediated currents this result excluded the possibilities that the increase of

NMDA evoked current by Epac agonist 8-pCPT-2prime-O-Me-cAMP was mediated by its

degradation products of PDEs in vivo This potentiation of NMDARs by 8-pCPT-2prime-O-

Me-cAMP was also not mediated by PKA since it could not be blocked in the presence of

two PKA inhibitors PKI14-22 and Rp-cAMPs But the application of PLC inhibitor

U73122 abolished the increase of NMDA mediated currents induced by Epac In the

presence of either PKC inhibitor bisindolylmaleimide I or Ca2+ chelator BAPTA Epac

agonist pCPT-2prime-O-Me-cAMP also failed to potentiate NMDARs

58 The regulation of NMDARs by Epac

Our results showed that the increase of NMDA evoked currents by Epac was

blocked by PLC inhibitor U73122 in the hippocampal CA1 cells Several other studies

further supported this notion Schmidt et al (2001) demonstrated that two Gαs coupled

GPCRs the β2-adrenergic receptors and prostaglandin E1 receptors stimulated PLC-ε

through EpacRap2 signaling cascade Activation of PLC-ε by Epac and Rap2 then

generated IP3 and increased Ca2+ in the cytosol (Schmidt et al 2001) Evellin et al have

further reported that the M3 muscarinic acetylcholine receptor could also stimulate PLCε

by the activation of Epac and Rap2B (Evellin et al 2002) Later the same group

demonstrated that in contrast to Gαs-coupled receptor the activation of Gαi-coupled

receptor inhibited PLCε activity by suppressing Epac mediated Rap2B activation (Vom et

161

al 2004) Another group demonstrated that activation of Epac by its specific agonist

increased Ca2+ release in single mouse ventricular myocytes while this agonist had no

effect on Ca2+ release in myocytes isolated from PLCε knockout mice (PLCε --)

Moreover the introduction of exogenous PLCε to myocytes from PLCε -- mice

recovered the enhancement of Ca2+ release induced by Epac agonist (Oestreich et al

2007)

Previous research on GPCR signaling has identified several different pathways

resulting in the activation of PKC including G-proteins αq and βγ (Clapham and Neer

1997) and transactivation of growth factor receptors (Lee et al 2002) Recently several

studies showed that the Gαs coupled receptors might indeed activate PKC through Epac

(Gekel and Neher 2008 Hoque et al 2010 Hucho et al 2005 Hucho et al 2006

Parada et al 2005) Our data provided strong proof showing that the activation of PLC

induced by Epac could result in the hydrolysis of PIP2 and consequently activate PKC So

far a number of studies also supported these results One study demonstrated that Epac

stimulated PKCε and mediated a cAMP-to-PKCε signaling in inflammatory pain (Hucho

et al 2005) In addition estrogen interfered with the signaling pathway leading from

Epac to PKCε which was downstream of the β2-adrenergic receptors If estrogen was

applied before β2-adrenergic receptors or Epac stimulation estrogen abrogated the

activation of PKCε by Epac (Hucho et al 2006) Recently Epac1 was found to mediate

PKA-independent mechanism of forskolin-activated intestinal Cl- secretion via

EpacPKC signaling pathway (Hoque et al 2010) Epac to PKC signaling was also

involved in the regulation of presynaptic transmitter release at excitatory central synapse

One study demonstrated that the application of Epac agonist 8-pCPT-2prime-O-Me-cAMP

162

augmented the enhancement of EPSC amplitudes by phorbol ester (PDBu) which

activated PKC In addition this effect induced by PDBu was abolished if PKC activity

was inhibited (Gekel and Neher 2008)

Although my study provided strong evidences that Epac regulated NMDAR

currents through PLCPKC signaling pathway which subtype of NMDAR mediated its

effect requires further study In addition we will also investigate which Gαs coupled

receptors have ability to regulate NMDAR via Epac

My study has also shown that intracellular Ca2+ signaling was required for the

potentiation of NMDARs by Epac since BAPTA blocked the increase of NMDAR

currents induced by Epac activation There are three different mechanisms which can be

used to explain how Epac modulates Ca2+ dynamics inside the cells

59 A role for Epac in the regulation of intracellular Ca2+ signaling

Firstly Epac might interact directly with IP3 receptors and ryanodine receptors

(RyRs) thereby promoting their opening in response to the increase of Ca2+ or Ca2+-

mobilizing second messengers such as IP3 cADP-ribose (cADPR) and nicotinic acid

adenine dinucleotide phosphate (NAADP) (Dodge-Kafka et al 2005 Kang et al 2005)

In cardiac myocytes a macromolecular complex consisting of Epac1 mAKAP PKA

PDE and ryanodine receptor 2 existed cAMP could act via Epac to modulate Ca2+

dynamics (Dodge-Kafka et al 2005) In addition in mouse pancreatic β cells (Kang et

al 2005) and rat renal inner medullary collecting duct (IMCD) cells (Yip 2006) Epac

could act on ryanodine receptors directly and mobilize Ca2+ from the intracellular Ca2+

store

163

Secondly Epac might activate ERK and CaMKII to promote the PKA-

independent phosphorylation of IP3 receptors and ryanodine receptors thereby increasing

their sensitivity to Ca2+ or Ca2+-mobilizing second messengers (Pereira et al 2007)

Thirdly Epac might act via Rap to stimulate PLC-ε thereby hydrolyzing PIP2 and

generating IP3 Then IP3 binds to IP3 receptors and release Ca2+ from the ER resulting in

the increase of intracellular Ca2+ concentration (Oestreich et al 2007)

510 Epac reduces the presynaptic release

cAMP is one of the well known second messenger to facilitate transmitter release

cAMPPKA signaling enhances vesicle fusion at multiple levels including recruitment of

synaptic vesicles from the reserve pool to the plasma membrane and regulation of vesicle

fusion (Seino and Shibasaki 2005) In cerebellar and hippocampal synapses cAMPPKA

signaling enhanced synaptic transmission by increasing release probability (Chavis et al

1998 Chen and Regehr 1997) In addition PKA phosphorylated a number of the

proteins which are involved in the exocytosis of synaptic vesicles in neurons in vitro

(Beguin et al 2001 Chheda et al 2001)

Recently PKA-independent actions of cAMP which facilitate releases of

transmitters have been reported Epac was proposed to be involved (Hatakeyama et al

2007) A recent study investigated the differential effects of PKA and Epac on two types

of secretory vesicles large dense-core vesicles (LVs) and small vesicles (SVs) in mouse

pancreatic β-cells Epac and PKA selectively regulated exocytosis of SVs and LVs

respectively (Hatakeyama et al 2007) In addition using Epac2 knockout mice (Epac2 -

-) Epac2 was demonstrated to be required for the potentiation of the first phase of

164

insulin granule release probably it might controll granule density near the plasma

membrane (Shibasaki et al 2007)

In addition a number of papers demonstrated that Epac also enhanced

neurotransmitter release at glutamatergic synapses (Sakaba and Neher 2003) at the calyx

of Held (Kaneko and Takahashi 2004) cultured excitatory autaptic neurons (Gekel and

Neher 2008) and cortical neurons (Huang and Hsu 2006a) At the calyx of Held the

forskolin exerted a presynaptic action to facilitate evoked transmitter release which could

be mimicked by 8-Br-cAMP a cAMP analogue (Sakaba and Neher 2003) This action of

forskolin was Epac-mediated because it was reproduced by 8-pCPT-2prime-O-Me-cAMP In

addition it was insensitive to PKA inhibitors (Sakaba and Neher 2003) Additionally at

crayfish neuromuscular junctions the increase of cAMP concentration induced by

serotonin (5-HT) enhanced glutamate release resulting in the increase of synaptic

transmission (Zhong et al 2004 Zhong and Zucker 2004 Zhong and Zucker 2005)

This cAMP-dependent enhancement of transmission involved two direct targets the

hyperpolarization-activated cyclic nucleotide gated (HCN) channels and Epac (Zhong et

al 2004 Zhong and Zucker 2004 Zhong and Zucker 2005) Activation of the HCN

channels promoted integrity of the actin cytoskeleton while Epac facilitated

neurotransmission (Zhong et al 2004 Zhong and Zucker 2004 Zhong and Zucker

2005)

Although several studies claimed that the application of Epac agonist 8-pCPT-2prime-

O-Me-cAMP could not change the PPF in the CNS indicating no impact on the

presynaptic neurotransmitter release by Epac (Gelinas et al 2008 Woolfrey et al 2009)

But my data showed that even 10 min application of 8-pCPT-2prime-O-Me-cAMP (10 microM)

165

increased the PPF in the brain slices in the other word bath application of Epac agonist

reduced neurotransmitter release One recent report supported my result it demonstrated

that both the amplitude and frequency of miniature EPSC could be suppressed by the

activation of Epac2 and this Epac2 mediated reduction of miniature EPSC frequency was

not blocked by inhibiton of Epac2 expression at postsynaptic sites (Woolfrey et al 2009)

In addition the expression of Epac2 in the presynaptic site was also detected (Woolfrey

et al 2009) These data implied that Epac might reduce the presynaptic transmitter

release

Although my study has demonstrated that the activation of Epac reduced the

release of presynaptic transmitter which mechanism mediated this inhibition applied by

Epac requires further study

My study showed that similar to PKA Epac had ability to regulate the NMDARs

so it is not suprising that Epac is also involved in the synaptic plasticity and learning and

memory Recently the role of Epac-mediated signaling in learning and memory began to

emerge

511 Epac and learning and memory

Using pharmacologic and genetic approaches to manipulate cAMP and

downstream signaling it was demonstrated that both PKA and Epac were required for

memory retrieval (Ouyang et al 2008) When Rp-2prime-O-MB-cAMPS a cAMP inhibitor

was infused into the dorsal hippocampus (DH) of mice before contextual fear memory

examination memory retrieval was impaired (Ouyang et al 2008) consistently when

Sp-2prime-O-MB-cAMPS a cAMP activator was infused into the DH of dopamine β-

166

hydroxylase deficient mice (this mice showed the impairment in contextual fear memory

retrieval) memory retrieval was rescued (Ouyang et al 2008) indicating that cAMP was

required for the memory retrieval Next which cAMP effectors mediated this cAMP-

dependent memory retrieval was studied when PKA selective agonist Sp-6-Phe-cAMPS

was infused no rescue was observed In addition when Epac selective agonist 8-pCPT-

2prime-O-Me-cAMP was infused retrieval was also not rescued However when low doses of

both Epac-selective and PKA-selective agonists were infused together memory retrieval

was rescued (Ouyang et al 2008) These studies implicated both Epac and PKA

signaling were required for DH-dependent memory retrieval (Ouyang et al 2008)

Recently another study demonstrated that Epac activation alone could

significantly improve memory retrieval in contextual fear conditioning this enhancement

of memory retrieval was even stronger in a passive avoidance paradigm (Ostroveanu et

al 2009) When mice were injected with 8-pCPT-2prime-O-Me-cAMP 20 min before the test

a significant increase in freezing behavior was observed (Ostroveanu et al 2009) The

effect of Epac on memory retrieval was also examined in the passive avoidance task

Mice injected with 8-pCPT-2prime-O-Me-cAMP 20 min before the test showed a significantly

improvement These data demonstrated that Epac activation alone in the hippocampus

modulated the retrieval of contextual fear memory (Ostroveanu et al 2009) Additionally

downregulation of Epac expression by Epac siRNA completely abolished the 8-pCPT-2prime-

O-Me-cAMP induced enhancement of memory retrieval (Ostroveanu et al 2009)

Epac is not only involved in memory retrieval but also memory consolidation

The infusion of 8-pCPT-2prime-O-Me-cAMP into the hippocampus was found to enhance

memory consolidation (Ma et al 2009) Indirect evidence showed that Rap1 signaling

167

was involved since the infusion of 8-pCPT-2prime-O-Me-cAMP activated Rap1 in the

hippocampus (Ma et al 2009)

It is well known that synaptic plasticity is one of cellular mechanisms which

underlie learning and memory Since Epac is involved in both memory consolidation and

retrieval it is not surprising to find out that Epac also mediates synaptic plasticity in the

hippocampus Recently one study showed that 8-pCPT-2prime-O-Me-cAMP enhanced the

maintenance of several forms of LTP in hippocampal CA1 area while it had no effects

on basal synaptic transmission or LTP induction (Gelinas et al 2008) Usually one train

of HFS resulted in a short-lasting LTP which required no protein synthesis but in the

presence of Epac agonist 8-pCPT-2prime-O-Me-cAMP it induced a stable and protein

synthesis dependent LTP (Gelinas et al 2008) In addition both PKA inhibitor and

transcription inhibitors failed to block the enhancement of Epac induced LTP (Gelinas et

al 2008)

In contrast another study demonstrated that application of high concentration of

Epac agonist 8-pCPT-2prime-O-Me-cAMP (200 microM) induced LTD This kind of LTD was not

mediated by PKA since PKA inhibitor did not block this Epac mediated LTD (Ster et al

2009) Instead Epac was found to be involved because the pre-treatment of hippocampal

slices with brefeldin-A (BFA) an non-specific Epac inhibitor abolished this Epac-

mediated LTD (Ster et al 2009) Additionally this Epac-LTD was mediated by

Rapp38MAPK signaling pathway (Ster et al 2009) Consistently one recent study also

showed that in cortical neurons the application of Epac agonist 8-pCPT-2prime-O-Me-cAMP

resulted in the endocytosis of GluA23 subunits of AMPAR indicating LTD was induced

In addition both amplitude and frequency of AMPAR-mediated miniature EPSCs was

168

depressed (Woolfrey et al 2009) Furthurmore Epac2 was required for the endocytosis

of AMPARs induced by the activation of dopamine D1 receptor Incubation of neurons

with dopamine D1 agonist caused a reduction of the surface expression of AMPARs but

in the presence of Epac2 siRNA this effect was blocked (Woolfrey et al 2009)

So far the studies about the role of Epac in synaptic plasticity drew inconsistent

conclusions In the future we will also investigate if Epac activation has ability to change

the direction of synaptic plasticity and which mechanism mediates its effect on synaptic

plasticity

169

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ii



By

Kai Yang

Doctor of Philosophy

Department of Physiology University of Toronto

2011

The role of NMDAR subtypes in synaptic plasticity is very controversial partially

caused by the lack of specific GluN2A containing NMDA receptor (GluN2AR)

antagonists Here we took a novel approach to selectively modulate NMDAR subtype

activity and investigated its role in the induction of plasticity Whole cell recording in

both acutely isolated CA1 cells and hippocampal slices demonstrated that pituitary

adenylate cyclase activating peptide 1 receptors (PAC1 receptors) which are Gαq

coupled receptors selectively recruited Src kinase and enhanced currents mediated by

GluN2ARs In addition biochemical experiments showed that the activation of PAC1

receptors phosphorylated GluN2ARs specifically In contrast vasoactive intestinal

peptide receptors (VPAC receptors) which are Gαs coupled receptors selectively

stimulated Fyn kinase potentiated currents mediated by GluN2B containing NMDARs

(GluN2BRs) Furthermore dopamine D1 receptor activation (another Gαs coupled

receptor) specifically phosphorylated GluN2BRs Interestingly field recording

experiments showed that PAC1 receptor activation lowered the threshold for LTP whilst

iii







study







schizophrenia

iv

ACKNOWLEDGEMENTS












lab



v

TABLE OF CONTENTS











vi



311 Hypothesis 67










vii

NMDARs

1 Introduction


2 Results 147

3 Discussion



viii





CA1 neurons





CA1 cells




specifically



residues of GluN2A






ix








NMDAR currents


VPAC receptors







and GluN2BRs







x





NMDARs by Epac






NMDAREPSCs



xi





















cADPR - cADP-ribose











xii
































xiii
























NO - nitric oxide








xiv
































xv






























1

Section 1

Introduction

2






















111 AMPAR

3























4
















112 LTP and LTD







5




1121 LTP



















6



1122 LTD




















7






















8













1133 Drug addiction









9

















12 NMDARs





10


et al 2007)

131 GluN1 subunits

13 NMDAR subunits



















11












132 GluN2 subunits










12


Leszkiewicz 2004)












Leszkiewicz 2004)








13























14


2008)


















signaling


15























16









Sabatini 2010)














17


et al 2003)








Malinow 2005)














18

133 GluN3 subunits























19























20






















21























2006)

22























23




(Steinberg 2008)










al 2006)










24






















1997)

25






















26









influx of Ca2+







2004)






27



















phosphatises (PTPs)



28
























29




















(SHP12)

30




31























32


33




changing gating




















34
























35























36























37























38






















39














2001)










40























41














2009)








18 Metaplasticity

42












Zhao et al 2008)












43














191 PACAP and VIP

19 PACAPVIP system







44


(Fahrenkrug 2010)


















Vaudry et al 2009)




45


Vaudry et al 2009)









2010)







VPAC2 receptor





46
























47























48























49






















110 The Hippocampus

50



















model system




51


introduced here






















52























53























54









and some behaviours













2002)

55




















GαqPKCSrc pathway


56






57

10 min afterPACAP

Baseline

1s200pA

1a

A

091

1112131415161718

PACAPPeak

PACAPSS

Norm

alize

d Cu

rrent




B

58

Section 2


59
























60







61























62











City CA)













63























64








24 Animals














65





-NVHI)



groups

26 Statistics

66

Section 3 Results


LTP induction

67













NMDARs

311 Hypothesis








312 Results

68
























69























70


(23 + 35 n=6) (Fig 312)






















71








WT 718 + 151 n=6 GluN2A KO 34 + 43 n = 6) (Fig 314)
















72















designed









73
























74
























75









nM) (Fig 319)















76
























77






78










79






80









81








82








83








GluN2A KO mice

84

IP GluN2A

pTyr

GluN2A

Ctrl PACAP

Glu

N2A

pho

spho

ryla

tion

Ctrl PACAP

pTyr

GluN2B

IP GluN2B

A B

C D






indicates p lt 001

85

pSrcY416

Src

Ctrl PACAP





PACAP application

86




Ro25-6981 or by Fyn (39-57)

87

-40 -20 0 20 40 6005

10

15

20

25


Norm

alize

d fE

PSP

Slop

e

time (min)

-20 0 20 40 6005

10

15

20

25

Norm

alize

d fE

PSP

Slop

e

time (minutes)






88

-40 -30 -20 -10 0 10 20 30 40 50 60 70

06

07

08

09

10

11

12


rmali

zed

fEPS

P Sl

ope

time (min)





89

06

08

10

12

14

16

Nor

mal

ized

Fiel

d Am

plitu

de


1 10 20 50 100








90


91











321 Hypothesis












322 Results

92























93
























94
























95












(Fig 325)












96




(Figure 327)






97

0 5 10 15 20 25 30 35

09

10

11

12

13

14

15

16

VIP application


Norm

alized

Peak

Curre

nt

Time Course (min)

1nM VIP

2

1

200pA

1s

1nM VIP+GRF

2

1

200pA

1s

1nM VIP+M65

2

1

100pA











98

0 5 10 15 20 25 30 3508

10

12

14



Nor

mal

ized

Peak

Cur

rent

Time Course (min)

0 5 10 15 20 25 30 3508

10

12

14

16



Nor

mal

ized

Peak

Cur

rent

Time Course (min)









= 5)

99

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

VIP application


Norm

aliz

edPe

akC

urre

nt

Time Course (min)

0 5 10 15 20 25 30 35

09

10

11

12

13

14

15

16

VIP application

1nM VIP VIP+PKI PKI

Nor

mal

ized

Peak

Curre

nt

Time Course (min)








100

0 5 10 15 20 25 30 35

08

10

12

14

16

18

VIP application

1nM VIP Bis VIP+Bis

Norm

alize

dPe

akCu

rrent

Time Course (min)

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

VIP application


Norm

alize

dPe

akCu

rrent

Time Course (min)








101

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

16

17

VIP application


Norm

aliz

edPe

akC

urre

nt

Time Course (min)

0 5 10 15 20 25 30 35

08

09

10

11

12

13

14

15

16

17

VIP application


Norm

aliz

edPe

akC

urre

nt

Time (minutes)








recording)

102

0 5 10 15 20 25 30 35

08

09

10

11

12

13

14

15

16

17

VIP application


Nor

mal

ized

Peak

Cur

rent

Time Course (min)




103

0 5 10 15 20 25 30 35

08

10

12

14

16

18VIP

2 sec

500 p

A15

0 pA

21

21

Ro25-6981 control

norm

alized

I NMDA

time (min)

+ Ro2

5-698

1

+ Scra

mbled Ipe

p

+ Fyn(

39-57

)

VIP

08

10

12

14

16

18

B

A

norm

alized

I NMDA







104

Section 4

Discussion

105

Discussion







NMDAR currents
















106











GluN2ARs













107



















and Wagner 2008)




108
























109
























110


















had no change

111



112




113
























114





further study















al 2001)




115























116












(Kalia et al 2006)












117























118
















2001)








119






















120
















Ca2+







121
























122
























123























124























125
























126























127


(Gozes et al 1996)





















128
























129






















45 Significance

130















treatment of AD







131


















activation





132
























133

















subtypes by SFKs





134







135

Section 5 Appendix


136

Introduction










al 2008)














137


















targets






138














Bos 2006)









139























140
























141























142


















study





143
























144























145






















57 Hypothesis

146







147

Results























148















62)









149























64)

150












(Fig 65)












151



















152

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pe

ak C

urre

nt

Time (minutes)









153

0 5 10 15 20 25 30 3508

10

12

14

16


Norm

alize

d Pe

ak C

urre

nt

Time (minutes)






154

0 5 10 15 20 25 30 3508

10

12

14

16


Norm

alize

d Pea

k Cur

rent

Time (minutes)

0 5 10 15 20 25 30 35

07080910111213141516171819


Norm

alize

d Pe

ak C

urre

nt

Time (minutes)










155

0 5 10 15 20 25 30 3508

09

10

11

12

13

14


Nor

mal

ized

Pea

k C

urre

nt

Time (minutes)

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pea

k Cur

rent

Time (minutes)


Peptide (N=5)







156

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pe

ak C

urre

nt

Time (minutes)







current

157



0 50 100 150 200-02

00

02

04

06

08

F

acilit

atio

n



158





0 5 10 15 20025

050

075

100

125

EPAC

Norm

alize

d NM

DARs

EPS

Cs

Time (min)

10 uM 100 uM

159



0 5 10 15 20 25

05

10

15

20

25

30

35

401

2

01s

40pA

1

2

01s

50pA

EPSC

NM

DA (

of b

asel

ine)

Time (min)


1 2


160

Discussion























161






2007)


















162






















store

163























164



















2005)





165








release







emerge








166
























167













al 2008)











168








plasticity

169

Reference List












170












171













172














173














174














175













176













177














178














179













180













181













182













183












184













185













186













187














188













189













190















191













192












193














194













195













196














197














198














199














200














201













202













203













204













205













206













207














iii







study







schizophrenia

iv

ACKNOWLEDGEMENTS












lab



v

TABLE OF CONTENTS











vi



311 Hypothesis 67










vii

NMDARs

1 Introduction


2 Results 147

3 Discussion



viii





CA1 neurons





CA1 cells




specifically



residues of GluN2A






ix








NMDAR currents


VPAC receptors







and GluN2BRs







x





NMDARs by Epac






NMDAREPSCs



xi





















cADPR - cADP-ribose











xii
































xiii
























NO - nitric oxide








xiv
































xv






























1

Section 1

Introduction

2






















111 AMPAR

3























4
















112 LTP and LTD







5




1121 LTP



















6



1122 LTD




















7






















8













1133 Drug addiction









9

















12 NMDARs





10


et al 2007)

131 GluN1 subunits

13 NMDAR subunits



















11












132 GluN2 subunits










12


Leszkiewicz 2004)












Leszkiewicz 2004)








13























14


2008)


















signaling


15























16









Sabatini 2010)














17


et al 2003)








Malinow 2005)














18

133 GluN3 subunits























19























20






















21























2006)

22























23




(Steinberg 2008)










al 2006)










24






















1997)

25






















26









influx of Ca2+







2004)






27



















phosphatises (PTPs)



28
























29




















(SHP12)

30




31























32


33




changing gating




















34
























35























36























37























38






















39














2001)










40























41














2009)








18 Metaplasticity

42












Zhao et al 2008)












43














191 PACAP and VIP

19 PACAPVIP system







44


(Fahrenkrug 2010)


















Vaudry et al 2009)




45


Vaudry et al 2009)









2010)







VPAC2 receptor





46
























47























48























49






















110 The Hippocampus

50



















model system




51


introduced here






















52























53























54









and some behaviours













2002)

55




















GαqPKCSrc pathway


56






57

10 min afterPACAP

Baseline

1s200pA

1a

A

091

1112131415161718

PACAPPeak

PACAPSS

Norm

alize

d Cu

rrent




B

58

Section 2


59
























60







61























62











City CA)













63























64








24 Animals














65





-NVHI)



groups

26 Statistics

66

Section 3 Results


LTP induction

67













NMDARs

311 Hypothesis








312 Results

68
























69























70


(23 + 35 n=6) (Fig 312)






















71








WT 718 + 151 n=6 GluN2A KO 34 + 43 n = 6) (Fig 314)
















72















designed









73
























74
























75









nM) (Fig 319)















76
























77






78










79






80









81








82








83








GluN2A KO mice

84

IP GluN2A

pTyr

GluN2A

Ctrl PACAP

Glu

N2A

pho

spho

ryla

tion

Ctrl PACAP

pTyr

GluN2B

IP GluN2B

A B

C D






indicates p lt 001

85

pSrcY416

Src

Ctrl PACAP





PACAP application

86




Ro25-6981 or by Fyn (39-57)

87

-40 -20 0 20 40 6005

10

15

20

25


Norm

alize

d fE

PSP

Slop

e

time (min)

-20 0 20 40 6005

10

15

20

25

Norm

alize

d fE

PSP

Slop

e

time (minutes)






88

-40 -30 -20 -10 0 10 20 30 40 50 60 70

06

07

08

09

10

11

12


rmali

zed

fEPS

P Sl

ope

time (min)





89

06

08

10

12

14

16

Nor

mal

ized

Fiel

d Am

plitu

de


1 10 20 50 100








90


91











321 Hypothesis












322 Results

92























93
























94
























95












(Fig 325)












96




(Figure 327)






97

0 5 10 15 20 25 30 35

09

10

11

12

13

14

15

16

VIP application


Norm

alized

Peak

Curre

nt

Time Course (min)

1nM VIP

2

1

200pA

1s

1nM VIP+GRF

2

1

200pA

1s

1nM VIP+M65

2

1

100pA











98

0 5 10 15 20 25 30 3508

10

12

14



Nor

mal

ized

Peak

Cur

rent

Time Course (min)

0 5 10 15 20 25 30 3508

10

12

14

16



Nor

mal

ized

Peak

Cur

rent

Time Course (min)









= 5)

99

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

VIP application


Norm

aliz

edPe

akC

urre

nt

Time Course (min)

0 5 10 15 20 25 30 35

09

10

11

12

13

14

15

16

VIP application

1nM VIP VIP+PKI PKI

Nor

mal

ized

Peak

Curre

nt

Time Course (min)








100

0 5 10 15 20 25 30 35

08

10

12

14

16

18

VIP application

1nM VIP Bis VIP+Bis

Norm

alize

dPe

akCu

rrent

Time Course (min)

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

VIP application


Norm

alize

dPe

akCu

rrent

Time Course (min)








101

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

16

17

VIP application


Norm

aliz

edPe

akC

urre

nt

Time Course (min)

0 5 10 15 20 25 30 35

08

09

10

11

12

13

14

15

16

17

VIP application


Norm

aliz

edPe

akC

urre

nt

Time (minutes)








recording)

102

0 5 10 15 20 25 30 35

08

09

10

11

12

13

14

15

16

17

VIP application


Nor

mal

ized

Peak

Cur

rent

Time Course (min)




103

0 5 10 15 20 25 30 35

08

10

12

14

16

18VIP

2 sec

500 p

A15

0 pA

21

21

Ro25-6981 control

norm

alized

I NMDA

time (min)

+ Ro2

5-698

1

+ Scra

mbled Ipe

p

+ Fyn(

39-57

)

VIP

08

10

12

14

16

18

B

A

norm

alized

I NMDA







104

Section 4

Discussion

105

Discussion







NMDAR currents
















106











GluN2ARs













107



















and Wagner 2008)




108
























109
























110


















had no change

111



112




113
























114





further study















al 2001)




115























116












(Kalia et al 2006)












117























118
















2001)








119






















120
















Ca2+







121
























122
























123























124























125
























126























127


(Gozes et al 1996)





















128
























129






















45 Significance

130















treatment of AD







131


















activation





132
























133

















subtypes by SFKs





134







135

Section 5 Appendix


136

Introduction










al 2008)














137


















targets






138














Bos 2006)









139























140
























141























142


















study





143
























144























145






















57 Hypothesis

146







147

Results























148















62)









149























64)

150












(Fig 65)












151



















152

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pe

ak C

urre

nt

Time (minutes)









153

0 5 10 15 20 25 30 3508

10

12

14

16


Norm

alize

d Pe

ak C

urre

nt

Time (minutes)






154

0 5 10 15 20 25 30 3508

10

12

14

16


Norm

alize

d Pea

k Cur

rent

Time (minutes)

0 5 10 15 20 25 30 35

07080910111213141516171819


Norm

alize

d Pe

ak C

urre

nt

Time (minutes)










155

0 5 10 15 20 25 30 3508

09

10

11

12

13

14


Nor

mal

ized

Pea

k C

urre

nt

Time (minutes)

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pea

k Cur

rent

Time (minutes)


Peptide (N=5)







156

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pe

ak C

urre

nt

Time (minutes)







current

157



0 50 100 150 200-02

00

02

04

06

08

F

acilit

atio

n



158





0 5 10 15 20025

050

075

100

125

EPAC

Norm

alize

d NM

DARs

EPS

Cs

Time (min)

10 uM 100 uM

159



0 5 10 15 20 25

05

10

15

20

25

30

35

401

2

01s

40pA

1

2

01s

50pA

EPSC

NM

DA (

of b

asel

ine)

Time (min)


1 2


160

Discussion























161






2007)


















162






















store

163























164



















2005)





165








release







emerge








166
























167













al 2008)











168








plasticity

169

Reference List












170












171













172














173














174














175













176













177














178














179













180













181













182













183












184













185













186













187














188













189













190















191













192












193














194













195













196














197














198














199














200














201













202













203













204













205













206













207














iv

ACKNOWLEDGEMENTS












lab



v

TABLE OF CONTENTS











vi



311 Hypothesis 67










vii

NMDARs

1 Introduction


2 Results 147

3 Discussion



viii





CA1 neurons





CA1 cells




specifically



residues of GluN2A






ix








NMDAR currents


VPAC receptors







and GluN2BRs







x





NMDARs by Epac






NMDAREPSCs



xi





















cADPR - cADP-ribose











xii
































xiii
























NO - nitric oxide








xiv
































xv






























1

Section 1

Introduction

2






















111 AMPAR

3























4
















112 LTP and LTD







5




1121 LTP



















6



1122 LTD




















7






















8













1133 Drug addiction









9

















12 NMDARs





10


et al 2007)

131 GluN1 subunits

13 NMDAR subunits



















11












132 GluN2 subunits










12


Leszkiewicz 2004)












Leszkiewicz 2004)








13























14


2008)


















signaling


15























16









Sabatini 2010)














17


et al 2003)








Malinow 2005)














18

133 GluN3 subunits























19























20






















21























2006)

22























23




(Steinberg 2008)










al 2006)










24






















1997)

25






















26









influx of Ca2+







2004)






27



















phosphatises (PTPs)



28
























29




















(SHP12)

30




31























32


33




changing gating




















34
























35























36























37























38






















39














2001)










40























41














2009)








18 Metaplasticity

42












Zhao et al 2008)












43














191 PACAP and VIP

19 PACAPVIP system







44


(Fahrenkrug 2010)


















Vaudry et al 2009)




45


Vaudry et al 2009)









2010)







VPAC2 receptor





46
























47























48























49






















110 The Hippocampus

50



















model system




51


introduced here






















52























53























54









and some behaviours













2002)

55




















GαqPKCSrc pathway


56






57

10 min afterPACAP

Baseline

1s200pA

1a

A

091

1112131415161718

PACAPPeak

PACAPSS

Norm

alize

d Cu

rrent




B

58

Section 2


59
























60







61























62











City CA)













63























64








24 Animals














65





-NVHI)



groups

26 Statistics

66

Section 3 Results


LTP induction

67













NMDARs

311 Hypothesis








312 Results

68
























69























70


(23 + 35 n=6) (Fig 312)






















71








WT 718 + 151 n=6 GluN2A KO 34 + 43 n = 6) (Fig 314)
















72















designed









73
























74
























75









nM) (Fig 319)















76
























77






78










79






80









81








82








83








GluN2A KO mice

84

IP GluN2A

pTyr

GluN2A

Ctrl PACAP

Glu

N2A

pho

spho

ryla

tion

Ctrl PACAP

pTyr

GluN2B

IP GluN2B

A B

C D






indicates p lt 001

85

pSrcY416

Src

Ctrl PACAP





PACAP application

86




Ro25-6981 or by Fyn (39-57)

87

-40 -20 0 20 40 6005

10

15

20

25


Norm

alize

d fE

PSP

Slop

e

time (min)

-20 0 20 40 6005

10

15

20

25

Norm

alize

d fE

PSP

Slop

e

time (minutes)






88

-40 -30 -20 -10 0 10 20 30 40 50 60 70

06

07

08

09

10

11

12


rmali

zed

fEPS

P Sl

ope

time (min)





89

06

08

10

12

14

16

Nor

mal

ized

Fiel

d Am

plitu

de


1 10 20 50 100








90


91











321 Hypothesis












322 Results

92























93
























94
























95












(Fig 325)












96




(Figure 327)






97

0 5 10 15 20 25 30 35

09

10

11

12

13

14

15

16

VIP application


Norm

alized

Peak

Curre

nt

Time Course (min)

1nM VIP

2

1

200pA

1s

1nM VIP+GRF

2

1

200pA

1s

1nM VIP+M65

2

1

100pA











98

0 5 10 15 20 25 30 3508

10

12

14



Nor

mal

ized

Peak

Cur

rent

Time Course (min)

0 5 10 15 20 25 30 3508

10

12

14

16



Nor

mal

ized

Peak

Cur

rent

Time Course (min)









= 5)

99

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

VIP application


Norm

aliz

edPe

akC

urre

nt

Time Course (min)

0 5 10 15 20 25 30 35

09

10

11

12

13

14

15

16

VIP application

1nM VIP VIP+PKI PKI

Nor

mal

ized

Peak

Curre

nt

Time Course (min)








100

0 5 10 15 20 25 30 35

08

10

12

14

16

18

VIP application

1nM VIP Bis VIP+Bis

Norm

alize

dPe

akCu

rrent

Time Course (min)

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

VIP application


Norm

alize

dPe

akCu

rrent

Time Course (min)








101

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

16

17

VIP application


Norm

aliz

edPe

akC

urre

nt

Time Course (min)

0 5 10 15 20 25 30 35

08

09

10

11

12

13

14

15

16

17

VIP application


Norm

aliz

edPe

akC

urre

nt

Time (minutes)








recording)

102

0 5 10 15 20 25 30 35

08

09

10

11

12

13

14

15

16

17

VIP application


Nor

mal

ized

Peak

Cur

rent

Time Course (min)




103

0 5 10 15 20 25 30 35

08

10

12

14

16

18VIP

2 sec

500 p

A15

0 pA

21

21

Ro25-6981 control

norm

alized

I NMDA

time (min)

+ Ro2

5-698

1

+ Scra

mbled Ipe

p

+ Fyn(

39-57

)

VIP

08

10

12

14

16

18

B

A

norm

alized

I NMDA







104

Section 4

Discussion

105

Discussion







NMDAR currents
















106











GluN2ARs













107



















and Wagner 2008)




108
























109
























110


















had no change

111



112




113
























114





further study















al 2001)




115























116












(Kalia et al 2006)












117























118
















2001)








119






















120
















Ca2+







121
























122
























123























124























125
























126























127


(Gozes et al 1996)





















128
























129






















45 Significance

130















treatment of AD







131


















activation





132
























133

















subtypes by SFKs





134







135

Section 5 Appendix


136

Introduction










al 2008)














137


















targets






138














Bos 2006)









139























140
























141























142


















study





143
























144























145






















57 Hypothesis

146







147

Results























148















62)









149























64)

150












(Fig 65)












151



















152

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pe

ak C

urre

nt

Time (minutes)









153

0 5 10 15 20 25 30 3508

10

12

14

16


Norm

alize

d Pe

ak C

urre

nt

Time (minutes)






154

0 5 10 15 20 25 30 3508

10

12

14

16


Norm

alize

d Pea

k Cur

rent

Time (minutes)

0 5 10 15 20 25 30 35

07080910111213141516171819


Norm

alize

d Pe

ak C

urre

nt

Time (minutes)










155

0 5 10 15 20 25 30 3508

09

10

11

12

13

14


Nor

mal

ized

Pea

k C

urre

nt

Time (minutes)

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pea

k Cur

rent

Time (minutes)


Peptide (N=5)







156

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pe

ak C

urre

nt

Time (minutes)







current

157



0 50 100 150 200-02

00

02

04

06

08

F

acilit

atio

n



158





0 5 10 15 20025

050

075

100

125

EPAC

Norm

alize

d NM

DARs

EPS

Cs

Time (min)

10 uM 100 uM

159



0 5 10 15 20 25

05

10

15

20

25

30

35

401

2

01s

40pA

1

2

01s

50pA

EPSC

NM

DA (

of b

asel

ine)

Time (min)


1 2


160

Discussion























161






2007)


















162






















store

163























164



















2005)





165








release







emerge








166
























167













al 2008)











168








plasticity

169

Reference List












170












171













172














173














174














175













176













177














178














179













180













181













182













183












184













185













186













187














188













189













190















191













192












193














194













195













196














197














198














199














200














201













202













203













204













205













206













207














v

TABLE OF CONTENTS











vi



311 Hypothesis 67










vii

NMDARs

1 Introduction


2 Results 147

3 Discussion



viii





CA1 neurons





CA1 cells




specifically



residues of GluN2A






ix








NMDAR currents


VPAC receptors







and GluN2BRs







x





NMDARs by Epac






NMDAREPSCs



xi





















cADPR - cADP-ribose











xii
































xiii
























NO - nitric oxide








xiv
































xv






























1

Section 1

Introduction

2






















111 AMPAR

3























4
















112 LTP and LTD







5




1121 LTP



















6



1122 LTD




















7






















8













1133 Drug addiction









9

















12 NMDARs





10


et al 2007)

131 GluN1 subunits

13 NMDAR subunits



















11












132 GluN2 subunits










12


Leszkiewicz 2004)












Leszkiewicz 2004)








13























14


2008)


















signaling


15























16









Sabatini 2010)














17


et al 2003)








Malinow 2005)














18

133 GluN3 subunits























19























20






















21























2006)

22























23




(Steinberg 2008)










al 2006)










24






















1997)

25






















26









influx of Ca2+







2004)






27



















phosphatises (PTPs)



28
























29




















(SHP12)

30




31























32


33




changing gating




















34
























35























36























37























38






















39














2001)










40























41














2009)








18 Metaplasticity

42












Zhao et al 2008)












43














191 PACAP and VIP

19 PACAPVIP system







44


(Fahrenkrug 2010)


















Vaudry et al 2009)




45


Vaudry et al 2009)









2010)







VPAC2 receptor





46
























47























48























49






















110 The Hippocampus

50



















model system




51


introduced here






















52























53























54









and some behaviours













2002)

55




















GαqPKCSrc pathway


56






57

10 min afterPACAP

Baseline

1s200pA

1a

A

091

1112131415161718

PACAPPeak

PACAPSS

Norm

alize

d Cu

rrent




B

58

Section 2


59
























60







61























62











City CA)













63























64








24 Animals














65





-NVHI)



groups

26 Statistics

66

Section 3 Results


LTP induction

67













NMDARs

311 Hypothesis








312 Results

68
























69























70


(23 + 35 n=6) (Fig 312)






















71








WT 718 + 151 n=6 GluN2A KO 34 + 43 n = 6) (Fig 314)
















72















designed









73
























74
























75









nM) (Fig 319)















76
























77






78










79






80









81








82








83








GluN2A KO mice

84

IP GluN2A

pTyr

GluN2A

Ctrl PACAP

Glu

N2A

pho

spho

ryla

tion

Ctrl PACAP

pTyr

GluN2B

IP GluN2B

A B

C D






indicates p lt 001

85

pSrcY416

Src

Ctrl PACAP





PACAP application

86




Ro25-6981 or by Fyn (39-57)

87

-40 -20 0 20 40 6005

10

15

20

25


Norm

alize

d fE

PSP

Slop

e

time (min)

-20 0 20 40 6005

10

15

20

25

Norm

alize

d fE

PSP

Slop

e

time (minutes)






88

-40 -30 -20 -10 0 10 20 30 40 50 60 70

06

07

08

09

10

11

12


rmali

zed

fEPS

P Sl

ope

time (min)





89

06

08

10

12

14

16

Nor

mal

ized

Fiel

d Am

plitu

de


1 10 20 50 100








90


91











321 Hypothesis












322 Results

92























93
























94
























95












(Fig 325)












96




(Figure 327)






97

0 5 10 15 20 25 30 35

09

10

11

12

13

14

15

16

VIP application


Norm

alized

Peak

Curre

nt

Time Course (min)

1nM VIP

2

1

200pA

1s

1nM VIP+GRF

2

1

200pA

1s

1nM VIP+M65

2

1

100pA











98

0 5 10 15 20 25 30 3508

10

12

14



Nor

mal

ized

Peak

Cur

rent

Time Course (min)

0 5 10 15 20 25 30 3508

10

12

14

16



Nor

mal

ized

Peak

Cur

rent

Time Course (min)









= 5)

99

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

VIP application


Norm

aliz

edPe

akC

urre

nt

Time Course (min)

0 5 10 15 20 25 30 35

09

10

11

12

13

14

15

16

VIP application

1nM VIP VIP+PKI PKI

Nor

mal

ized

Peak

Curre

nt

Time Course (min)








100

0 5 10 15 20 25 30 35

08

10

12

14

16

18

VIP application

1nM VIP Bis VIP+Bis

Norm

alize

dPe

akCu

rrent

Time Course (min)

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

VIP application


Norm

alize

dPe

akCu

rrent

Time Course (min)








101

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

16

17

VIP application


Norm

aliz

edPe

akC

urre

nt

Time Course (min)

0 5 10 15 20 25 30 35

08

09

10

11

12

13

14

15

16

17

VIP application


Norm

aliz

edPe

akC

urre

nt

Time (minutes)








recording)

102

0 5 10 15 20 25 30 35

08

09

10

11

12

13

14

15

16

17

VIP application


Nor

mal

ized

Peak

Cur

rent

Time Course (min)




103

0 5 10 15 20 25 30 35

08

10

12

14

16

18VIP

2 sec

500 p

A15

0 pA

21

21

Ro25-6981 control

norm

alized

I NMDA

time (min)

+ Ro2

5-698

1

+ Scra

mbled Ipe

p

+ Fyn(

39-57

)

VIP

08

10

12

14

16

18

B

A

norm

alized

I NMDA







104

Section 4

Discussion

105

Discussion







NMDAR currents
















106











GluN2ARs













107



















and Wagner 2008)




108
























109
























110


















had no change

111



112




113
























114





further study















al 2001)




115























116












(Kalia et al 2006)












117























118
















2001)








119






















120
















Ca2+







121
























122
























123























124























125
























126























127


(Gozes et al 1996)





















128
























129






















45 Significance

130















treatment of AD







131


















activation





132
























133

















subtypes by SFKs





134







135

Section 5 Appendix


136

Introduction










al 2008)














137


















targets






138














Bos 2006)









139























140
























141























142


















study





143
























144























145






















57 Hypothesis

146







147

Results























148















62)









149























64)

150












(Fig 65)












151



















152

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pe

ak C

urre

nt

Time (minutes)









153

0 5 10 15 20 25 30 3508

10

12

14

16


Norm

alize

d Pe

ak C

urre

nt

Time (minutes)






154

0 5 10 15 20 25 30 3508

10

12

14

16


Norm

alize

d Pea

k Cur

rent

Time (minutes)

0 5 10 15 20 25 30 35

07080910111213141516171819


Norm

alize

d Pe

ak C

urre

nt

Time (minutes)










155

0 5 10 15 20 25 30 3508

09

10

11

12

13

14


Nor

mal

ized

Pea

k C

urre

nt

Time (minutes)

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pea

k Cur

rent

Time (minutes)


Peptide (N=5)







156

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pe

ak C

urre

nt

Time (minutes)







current

157



0 50 100 150 200-02

00

02

04

06

08

F

acilit

atio

n



158





0 5 10 15 20025

050

075

100

125

EPAC

Norm

alize

d NM

DARs

EPS

Cs

Time (min)

10 uM 100 uM

159



0 5 10 15 20 25

05

10

15

20

25

30

35

401

2

01s

40pA

1

2

01s

50pA

EPSC

NM

DA (

of b

asel

ine)

Time (min)


1 2


160

Discussion























161






2007)


















162






















store

163























164



















2005)





165








release







emerge








166
























167













al 2008)











168








plasticity

169

Reference List












170












171













172














173














174














175













176













177














178














179













180













181













182













183












184













185













186













187














188













189













190















191













192












193














194













195













196














197














198














199














200














201













202













203













204













205













206













207














vi



311 Hypothesis 67










vii

NMDARs

1 Introduction


2 Results 147

3 Discussion



viii





CA1 neurons





CA1 cells




specifically



residues of GluN2A






ix








NMDAR currents


VPAC receptors







and GluN2BRs







x





NMDARs by Epac






NMDAREPSCs



xi





















cADPR - cADP-ribose











xii
































xiii
























NO - nitric oxide








xiv
































xv






























1

Section 1

Introduction

2






















111 AMPAR

3























4
















112 LTP and LTD







5




1121 LTP



















6



1122 LTD




















7






















8













1133 Drug addiction









9

















12 NMDARs





10


et al 2007)

131 GluN1 subunits

13 NMDAR subunits



















11












132 GluN2 subunits










12


Leszkiewicz 2004)












Leszkiewicz 2004)








13























14


2008)


















signaling


15























16









Sabatini 2010)














17


et al 2003)








Malinow 2005)














18

133 GluN3 subunits























19























20






















21























2006)

22























23




(Steinberg 2008)










al 2006)










24






















1997)

25






















26









influx of Ca2+







2004)






27



















phosphatises (PTPs)



28
























29




















(SHP12)

30




31























32


33




changing gating




















34
























35























36























37























38






















39














2001)










40























41














2009)








18 Metaplasticity

42












Zhao et al 2008)












43














191 PACAP and VIP

19 PACAPVIP system







44


(Fahrenkrug 2010)


















Vaudry et al 2009)




45


Vaudry et al 2009)









2010)







VPAC2 receptor





46
























47























48























49






















110 The Hippocampus

50



















model system




51


introduced here






















52























53























54









and some behaviours













2002)

55




















GαqPKCSrc pathway


56






57

10 min afterPACAP

Baseline

1s200pA

1a

A

091

1112131415161718

PACAPPeak

PACAPSS

Norm

alize

d Cu

rrent




B

58

Section 2


59
























60







61























62











City CA)













63























64








24 Animals














65





-NVHI)



groups

26 Statistics

66

Section 3 Results


LTP induction

67













NMDARs

311 Hypothesis








312 Results

68
























69























70


(23 + 35 n=6) (Fig 312)






















71








WT 718 + 151 n=6 GluN2A KO 34 + 43 n = 6) (Fig 314)
















72















designed









73
























74
























75









nM) (Fig 319)















76
























77






78










79






80









81








82








83








GluN2A KO mice

84

IP GluN2A

pTyr

GluN2A

Ctrl PACAP

Glu

N2A

pho

spho

ryla

tion

Ctrl PACAP

pTyr

GluN2B

IP GluN2B

A B

C D






indicates p lt 001

85

pSrcY416

Src

Ctrl PACAP





PACAP application

86




Ro25-6981 or by Fyn (39-57)

87

-40 -20 0 20 40 6005

10

15

20

25


Norm

alize

d fE

PSP

Slop

e

time (min)

-20 0 20 40 6005

10

15

20

25

Norm

alize

d fE

PSP

Slop

e

time (minutes)






88

-40 -30 -20 -10 0 10 20 30 40 50 60 70

06

07

08

09

10

11

12


rmali

zed

fEPS

P Sl

ope

time (min)





89

06

08

10

12

14

16

Nor

mal

ized

Fiel

d Am

plitu

de


1 10 20 50 100








90


91











321 Hypothesis












322 Results

92























93
























94
























95












(Fig 325)












96




(Figure 327)






97

0 5 10 15 20 25 30 35

09

10

11

12

13

14

15

16

VIP application


Norm

alized

Peak

Curre

nt

Time Course (min)

1nM VIP

2

1

200pA

1s

1nM VIP+GRF

2

1

200pA

1s

1nM VIP+M65

2

1

100pA











98

0 5 10 15 20 25 30 3508

10

12

14



Nor

mal

ized

Peak

Cur

rent

Time Course (min)

0 5 10 15 20 25 30 3508

10

12

14

16



Nor

mal

ized

Peak

Cur

rent

Time Course (min)









= 5)

99

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

VIP application


Norm

aliz

edPe

akC

urre

nt

Time Course (min)

0 5 10 15 20 25 30 35

09

10

11

12

13

14

15

16

VIP application

1nM VIP VIP+PKI PKI

Nor

mal

ized

Peak

Curre

nt

Time Course (min)








100

0 5 10 15 20 25 30 35

08

10

12

14

16

18

VIP application

1nM VIP Bis VIP+Bis

Norm

alize

dPe

akCu

rrent

Time Course (min)

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

VIP application


Norm

alize

dPe

akCu

rrent

Time Course (min)








101

0 5 10 15 20 25 30 3508

09

10

11

12

13

14

15

16

17

VIP application


Norm

aliz

edPe

akC

urre

nt

Time Course (min)

0 5 10 15 20 25 30 35

08

09

10

11

12

13

14

15

16

17

VIP application


Norm

aliz

edPe

akC

urre

nt

Time (minutes)








recording)

102

0 5 10 15 20 25 30 35

08

09

10

11

12

13

14

15

16

17

VIP application


Nor

mal

ized

Peak

Cur

rent

Time Course (min)




103

0 5 10 15 20 25 30 35

08

10

12

14

16

18VIP

2 sec

500 p

A15

0 pA

21

21

Ro25-6981 control

norm

alized

I NMDA

time (min)

+ Ro2

5-698

1

+ Scra

mbled Ipe

p

+ Fyn(

39-57

)

VIP

08

10

12

14

16

18

B

A

norm

alized

I NMDA







104

Section 4

Discussion

105

Discussion







NMDAR currents
















106











GluN2ARs













107



















and Wagner 2008)




108
























109
























110


















had no change

111



112




113
























114





further study















al 2001)




115























116












(Kalia et al 2006)












117























118
















2001)








119






















120
















Ca2+







121
























122
























123























124























125
























126























127


(Gozes et al 1996)





















128
























129






















45 Significance

130















treatment of AD







131


















activation





132
























133

















subtypes by SFKs





134







135

Section 5 Appendix


136

Introduction










al 2008)














137


















targets






138














Bos 2006)









139























140
























141























142


















study





143
























144























145






















57 Hypothesis

146







147

Results























148















62)









149























64)

150












(Fig 65)












151



















152

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pe

ak C

urre

nt

Time (minutes)









153

0 5 10 15 20 25 30 3508

10

12

14

16


Norm

alize

d Pe

ak C

urre

nt

Time (minutes)






154

0 5 10 15 20 25 30 3508

10

12

14

16


Norm

alize

d Pea

k Cur

rent

Time (minutes)

0 5 10 15 20 25 30 35

07080910111213141516171819


Norm

alize

d Pe

ak C

urre

nt

Time (minutes)










155

0 5 10 15 20 25 30 3508

09

10

11

12

13

14


Nor

mal

ized

Pea

k C

urre

nt

Time (minutes)

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pea

k Cur

rent

Time (minutes)


Peptide (N=5)







156

0 5 10 15 20 25 30 3508

10

12

14

16

Norm

alize

d Pe

ak C

urre

nt

Time (minutes)







current

157



0 50 100 150 200-02

00

02

04

06

08

F

acilit

atio

n



158





0 5 10 15 20025

050

075

100

125

EPAC

Norm

alize

d NM

DARs

EPS

Cs

Time (min)

10 uM 100 uM

159



0 5 10 15 20 25

05

10

15

20

25

30

35

401

2

01s

40pA

1

2

01s

50pA

EPSC

NM

DA (

of b

asel

ine)

Time (min)


1 2


160

Discussion























161






2007)


















162






















store

163























164



















2005)





165








release







emerge








166
























167













al 2008)











168








plasticity

169

Reference List












170












171













172














173














174














175













176













177














178














179













180













181













182













183












184













185













186













187














188













189













190















191













192












193














194













195













196














197














198














199














200














201













202













203













204













205













206













207














kai yang - university of toronto

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