VOLTAGE GATED SODIUM CHANNEL REGULATION OF

NEURITE OUTGROWTH: ROLE OF NMDA AND

NEUROTROPHIN RECEPTOR SIGNALING PATHWAYS

___________________________________

By

SAIRAM JABBA

___________________________________

A DISSERTATION

Submitted to the faculty of the Graduate School of the Creighton University in Partial

Fulfillment of the Requirements for the degree of Doctor of Philosophy in the Department

of Pharmacology.

_________________________________

Omaha, NE

(7/6/2012)

II

III

ABSTRACT

Activity-dependent N-methyl D-aspartate receptor (NMDAR) signaling, gene

transcription and protein synthesis play major roles in brain functions that regulate

neuronal morphology. Inasmuch as neuronal activity-induced increments in cytoplasmic

sodium may augment NMDAR-mediated currents (Rose and Konnerth, 2001; Yu and

Salter, 1998; George et al., 2009), we reasoned that intracellular Na+ may function as a

signaling molecule and positively regulate neuronal development in immature

cerebrocortical neurons. The central hypothesis of this study is that sodium channel

activators stimulate neuronal development by elevating [Na+]i , augmenting NMDAR

function in presence of activated SFKs, enhancing BDNF release and activating the

downstream TrkB signaling. The specific objective of the proposed work is to elucidate

the signaling mechanisms by which sodium channel activators influence neuronal

morphology in immature cerebrocortical neurons. More specifically, to understand the

relationship between increases in [Na+]i and NMDAR, brain-derived neurotrophic factor

(BDNF)-TrkB mediated neuronal development.

For these studies, sodium channel activators that increase [Na+]i ,antillatoxin

(ATX) and veratridine (VRT) were used as pharmacological tools to determine their

potential to mimic neuronal activity. VGSCs activators robustly stimulated neurite

outgrowth in a hormetic concentration-response relationship and this enhancement was

sensitive to the VGSC antagonist, tetrodotoxin. To unambiguously demonstrate the

enhancement of NMDA receptor function by ATX, we recorded single-channel currents

from cell-attached patches. ATX was found to increase the open probability of NMDA

IV

receptors. Na+ dependent upregulation of NMDAR function has been shown to be

regulated by Src family kinase (SFK) (Yu and Salter, 1998). The Src kinase inhibitor PP2

abrogated ATX-enhanced neurite outgrowth suggesting a SFK involvement in this

response. ATX-enhanced neurite outgrowth was also inhibited by the NMDAR antagonist,

MK-801, and the calmodulin dependent kinase kinase (CaMKK) inhibitor, STO-609,

demonstrating the requirement for NMDAR activation with subsequent downstream

engagement of the Ca2+ dependent CaMKK pathway.

Activity-dependent neuronal development involves N-methyl D-aspartate receptor

(NMDAR) mediated calcium influx and brain-derived neurotrophic factor (BDNF)-TrkB

signaling. We tested the effect of the VGSC activators on BDNF synthesis and release and

TrkB activation in DIV1 cerebrocortical neurons. Inhibition of TrkB receptors and its

downstream effector pathways, PI3K, and PLCγ inhibited VRT-enhanced NOG. VRT

stimulated phosphorylation of TrkB and its downstream effectors Akt, mTOR, PLCγ1,

ERK1/2 and CREB. VRT increased BDNF synthesis and release in a concentration

dependent manner; however, VRT stimulation of TrkB phosphorylation displayed a

biphasic concentration-response curve. VRT stimulation of BDNF synthesis required

VGSCs and NMDARs.

Taken together, these data suggest that VGSC activators seem to be capable of

mimicking activity-dependent neuronal development and hence may represent a novel

pharmacological strategy to regulate neuronal development through NMDA and

neurotrophin receptor-dependent mechanisms.

V

VI

DEDICATION

I dedicate this dissertation to my respected parents, my lovely wife, my family, all my

teachers and all of my friends for their constant love and support, without which I would

not have completed this degree.

VII

ACKNOWLEDGEMENTS

I express my sincere gratitude to Dr. Thomas F. Murray for his invaluable support

and guidance during my Ph.D years. Not only was he a wonderful teacher, but also an

inspirational role model. I would like to thank him for his patience and commitment

towards making me a good researcher and a better person. I would not hesitate to say that

I would like to take him as a role model for training my students in future.

I would like to thank all my committee members, Dr. D. Roselyn, Cerutis, Dr.

Shashank Dravid and Dr. Yaping Tu for their kind assistance and critical evaluation of my

progress, which helped me in learning good science and in becoming a better researcher. I

thank them for their inputs and guidance into my projects. My special thanks to Dr. Dravid

who was not only a wonderful collaborator, but also was a great friend and badminton

partner. He and his lab conducted some critical electrophysiological experiments

discussed in chapter-III.

A special thank you to members of my laboratory who were very helpful and

always made me feel comfortable in the laboratory during all these years. I thoroughly

enjoyed the insightful scientific interactions with Joju George, Zhengyu Cao, Lakshmi

Kelamangalath and Suneet Mehrotra. Special thanks to Bridget Sefranek (nee Leuschen)

for perfectly organizing the research needs and requirements for my projects. I want to

express my gratitude to the faculty, fellow graduate students and staff of Department of

Pharmacology for their help during my years in the program.

I greatly thank my wonderful wife, Sujatha Nagulapally, for her unconditional love and

encouragement. She was my ‘rock of Gibraltar’ during my most stressful period of my

graduate career. I would like to extend my humblest and sincere gratefulness to my

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parents, Lakshmi Rajyam and Panduranga Rao Jabba, for instilling in me the value of

education in life. They were there for me at every step of my life providing unwavering

love and support. I thank my sisters Suma Jabba and Uma Peddireddy for being my role

models and for showering me with unflinching love. My wishes to my lovely niece

Ramyatha Sai, and nephews, Ravi and Hitesh Sai, who always brightened my mood. My

special thanks to my brother-in-laws Ratna Kishore and Gurunath and to my in-laws

Radha and Venkat Nagulapally, for being ever supportive of my ambitions. I would like to

extend my sincere gratitude to Venkateshwaralu Karanam, who was one of my earliest

mentor and true inspiration to take up science. I take this opportunity to thank all of my

friends for providing me with ever constant love, support, encouragement and above all

friendship. I greatly appreciate Chandra Sekhar Baliwada, Bhanu Telugu, Lokaranjan

Somaraju, Pradeep Gendapodi, Kishor Devalaraja, Kalyan Nannuru and Hima Bindu

Ramachandrareddy along with their spouses. My special thanks to Kiran Kotu for being a

very good friend. I truly appreciate the help and company of Pradeep Malreddy, Raja

Rachaktla, Nithya Raveendran, Niranjan Butchi, Rajkumari Sanginaboyina, Vijay and

Rebekah Golla, Satish and Chitra Medicetty, Keil Regehr, Raj Maganti, Casey Devore,

Prasanna Kankanala, Hyma Gajula, Saurabh Jauhari, Vijay Yajjala, Neha Jain, Laxmi

Fogueri, Srinivas Dannaram, Praveen Ramanan, Anantha Gollapudi, Praneet Bathena and

Vamsi Karuturi.

Finally, last but not the least, I offer my humblest gratitude to God for guiding me all

through my life.

IX

Table of Contents

1 CHAPTER 1 - INTRODUCTION .................................................................. 1-14

1.1 Background ................................................................................................... 1-17

1.2 Significance ................................................................................................... 1-22

2 CHAPTER 2 - LITERATURE REVIEW .................................................... 2-23

2.1 Voltage-Gated Sodium Channels .................................................................. 2-23

2.1.1 Introduction: ........................................................................................ 2-23

2.1.2 Primary structure of VGSCs ............................................................... 2-23

2.1.3 VGSCs: Diversity in expression and function .................................... 2-24

2.1.4 Structure of VGSCs at Atomic Resolution ......................................... 2-25

2.1.5 VGSCs function: Molecular perspective ............................................ 2-26

2.2 Voltage-gated sodium channels activators and gating modifiers .................. 2-29

2.2.1 Antillatoxin ......................................................................................... 2-30

2.2.2 Veratridine .......................................................................................... 2-33

2.3 N-methyl-D-aspartate receptors (NMDARs) ................................................ 2-36

2.3.2 Neurotrophins and their receptors, tropomyosin-related kinases

(TRKs): ........................................................................................................ 2-41

2.3.3 Regulation of BDNF-TrkB signaling pathway ................................... 2-47

2.3.4 Role of BDNF-TrkB signaling in neuronal development ................... 2-50

2.4 References ..................................................................................................... 2-56

X

3 CHAPTER 3 - Antillatoxin, a Novel Lipopeptide, Enhances Neurite

Outgrowth in Immature Cerebrocortical Neurons Through Activation of Voltage–

Gated Sodium Channels .............................................................................................. 3-74

3.1 Abstract ......................................................................................................... 3-75

3.2 Experimental Procedures ............................................................................... 3-79

3.3 Results ........................................................................................................... 3-87

3.4 Discussion ..................................................................................................... 3-96

3.5 References ................................................................................................... 3-116

4 CHAPTER 4 – Sodium channel activator-stimulated neuronal development

involves BDNF-TrkB signaling pathway. ................................................................ 4-121

4.1 Abstract ....................................................................................................... 4-122

4.2 Introduction ................................................................................................. 4-123

4.3 Materials and Methods ................................................................................ 4-126

4.4 Results ......................................................................................................... 4-133

4.5 Discussions .................................................................................................. 4-142

List of Figures

Figure 2-1: Subunit structure of VGSCs. ....................................................................... 2-54

Figure 2-2: A) Voltage dependent activation: outward movement of S4 Voltage sensors 2-

54

Figure 2-3: Intracellular NA+ may act as a signalinging molecule and upregulate

NMDARs. ....................................................................................................................... 2-55

XI

Figure 3-1: ATX increases intracellular sodium levels in DIV-1 cerebrocortical neurons. 3-

103

Figure 3-2: Effect of ATX on neurite outgrowth. ........................................................ 3-104

Figure 3-3. Effect of TTX on ATX-induced neurite outgrowth in immature cerebrocortical

neurons. ......................................................................................................................... 3-106

Figure 3-4:Pharmacological evaluation of signaling pathways involved in ATX-enhanced

neurite outgrowth. ......................................................................................................... 3-107

Figure 3-5: ATX-induced neurite extension involves a Src Family kinase. ................. 3-108

Figure 3-6: Quantification of ATX-induced increase of intracellular sodium levels in DIV-

1 cerebrocortical neurons. ............................................................................................. 3-109

Figure 3-7:ATX evoked change in membrane potential in DIV-1 cereobrocortical neurons.

....................................................................................................................................... 3-110

Figure 3-8: ATX-induced Ca2+ influx and pharmacological evaluation in DIV-1

cerebrocortical neurons. ................................................................................................ 3-112

Figure 3-9:. Increase in NMDA receptor channel open probability by ATX. .............. 3-114

Figure 3-10:Schematic diagram of the pathways involved in ATX-induced neurite

outgrowth ...................................................................................................................... 3-115

Figure 4-1: Veratridine stimulated neurite outgrowth and dendritic arborization in

immature cerebrocortical neurons................................................................................. 4-148

Figure 4-2: Veratridine (VRT) increases intracellular Na+ and Ca2+ in DIV1

cerebrocortical neurons and this Ca2+ influx is TTX-sensitive and NMDAR dependent . 4-

149

Figure 4-3:TrkB is essential for veratridine-induced neurite outgrowthVeratridine .... 4-150

XII

Figure 4-4: In situ BDNF ELISA: Veratridine enhances BDNF release in immature

cerebrocortical neurons ................................................................................................. 4-151

Figure 4-5:Veratridine enhances BDNF synthesis in immature cerebrocortical neurons and

this requires VGSCs and partially involves NMDARs................................................. 4-152

Figure 4-6: Veratridine enhances BDNF synthesis in immature cerebrocortical neurons

and this requires VGSCs and partially involves NMDARs .......................................... 4-153

Figure 4-7: - Veratridine-induced neurite outgrowth involves PI3-kinase activity ...... 4-154

Figure 4-8- : Veratridine stimulated Akt phosphorylation involves TrkB receptors .... 4-155

Figure 4-9 - : Veratridine-induced neurite outgrowth involves the PI3K-Akt-mTOR

pathway ......................................................................................................................... 4-156

Figure 4-10: - Veratridine-induced Ca2+ influx involves PLC mediated release of Ca2+

from intracellular .......................................................................................................... 4-157

Figure 4-11: Veratridine-induced neurite outgrowth requires phospholipase C (PLC) 4-158

Figure 4-12:MAPK pathway has a modest role in veratridine-induced neurite outgrowth. 4-

159

Figure 4-13:Pharmacological characterization of Akt activation by veratridine .......... 4-160

Figure 4-14: – Model ................................................................................................... 4-161

List of Tables

Table 1: Location and pharmacological properties of various VGSCs isoforms……..2-55

Table 2: Receptor sites on VGSCs (modified from Catterall et al., Pharmacol Rev. 2005)…………………………………………………………………………………..2-56

XIII

1-14

1 CHAPTER 1 - INTRODUCTION

Activity-dependent N-methyl D-aspartate receptor (NMDAR) signaling, gene

transcription and protein synthesis play major roles in brain functions that regulate

neuronal morphology. Various neurological disorders including several mental

retardation conditions, learning and memory deficit conditions and traumatic brain injury

may be attributed to loss of one or more of these processes. The mechanisms by which

neuronal activity translate into morphological changes are complex and not completely

understood. Understanding these mechanisms will help to determine potential drug

targets to modulate these pathways and develop therapeutic approaches to these

neurological disorders.

Recent studies have demonstrated that neuronal activity-mediated increases in [Na+]i

in neuronal structures augment NMDAR function and may contribute to activity-

dependent synaptic plasticity (Rose and Konnerth, 2001; Yu and Salter, 1998; George et

al., 2009). Inasmuch as neuronal activity-induced increments in cytoplasmic sodium may

augment NMDAR-mediated currents, we reasoned that intracellular Na+ may function as

a signaling molecule and positively regulate neuronal development in immature

cerebrocortical neurons. In this study, sodium channel activators that increase [Na+]i

antillatoxin(ATX) and veratridine (VRT) will be used as pharmacological tools to

determine their potential to mimic neuronal activity. The findings of this study will be

useful in developing novel therapeutic strategies for better management of neurological

diseases involving abnormalities of neuronal morphologies.

1-15

Our long term research goal is to identify novel compounds that can mimic neuronal

activity-dependent neuronal development, and thereby to develop new therapeutic

strategies. The specific objective of the proposed work is to elucidate the signaling

mechanisms by which sodium channel activators influence neuronal morphology in

immature cerebrocortical neurons. More specifically, to understand the relationship

between increases in [Na+]i and NMDAR and brain-derived neurotrophic factor

(BDNF)-TrkB mediated neuronal development. Our proposal is based on published

observations and preliminary data indicating that sodium channel activators promote

neurite outgrowth.

HYPOTHESIS:

The central hypothesis of this proposal is that sodium channel activators stimulate

neuronal development by elevating [Na+]i , augmenting NMDAR function in presence of

activated SFKs, enhancing BDNF release and activating the downstream TrkB-PI3K-

Akt-mTOR pathway.”

SPECIFIC AIM 1:

Evaluate the influence of [Na+]i on NMDAR signaling and investigate the role of src

family kinases (SFKs) in sodium channel activator-enhanced neurite outgrowth. Our

working hypothesis is, sodium channel activators enhance neurite outgrowth by elevating

[Na+]i and augmenting NMDAR function and this Na+ dependent up-regulation of

NMDAR function is regulated by SFKs. To test this hypothesis, up-regulation of

NMDAR function by antillatoxin will be assessed by recording NMDAR single channel

1-16

currents from cell attached patches. The role of NMDARs in ATX and VRT induced

Ca2+ influx will be investigated. Neurite outgrowth assay and Ca2+ influx assay in

presence of PP2, a specific inhibitor of SFKs will be performed to investigate the role of

SFKs. Also the effect of ATX on activation of Src will be assessed.

SPECIFIC AIM 2:

Determine the significance of BDNF-TrkB signaling and its downstream pathways of

TrkB-PI3K-PLCγ-MAPK in regulation of VGSC activator-induced neurite outgrowth,

spinogenesis and synaptogenesis. Our working hypothesis is that sodium channel

activators potentiate NMDAR function and thereby exert trophic effects through

increased gene expression and release of BDNF. The released BDNF regulates neuronal

development by activating its cognate receptor TrkB and the downstream TrkB signaling

pathways, mainly a combination of the PI3K, MAPK and PLCγ pathways. To test this

hypothesis, the involvement of BDNF-TrkB signaling in sodium channel activator-

stimulated neuroal development will be examined by 1) Overexpression of dominant

negative isoform of TrkB, the TrkB.T1 (truncated TrkB) in immature cerebrocortical

cultures, and study its effects on veratridine-mediated neurite outgrowth, spinogensis and

synaptogenesis . 2) Activation of of TrkB receptors and its major downstream pathways,

the PI3K-Akt, PLCγ, MAPK pathways. Enhanced release and increased gene expression

of BDNF under the influence of veratridine will be studied using ELISA and qRT-PCR

respectively.

1-17

1.1 Background

Antillatoxin is a novel VGSC activator

Antillatoxin (ATX) is a structurally novel lipopeptide with an exceptionally high degree

of methylation unlike any known natural product (Lee and Loh, 2006). Isolated from the

cyanobacterium Lyngbya majuscula, this compound is also distinguished by multiple

stereocenters (Orjala et al., 1995a). The essential role of the asymmetric carbon atoms in

ATX is reflected in the stereoselective effects of ATX enantomers (Li et al., 2001).

ATX is considered to be the second most potent ichthyotoxic compound obtained from

marine sources after only brevetoxin (PbTx)-1 (Orjala et al., 1995b). Exposure to L.

majuscula blooms are associated with adverse human health effects, including respiratory

irritation, eye inflammation, and severe contact dermatitis. Previous work has

demonstrated that ATX is a potent activator of voltage-gated sodium channels (VGSCs)

that elevates intracellular Na+ concentration ([Na+]i) in intact neurons (Li et al., 2001;

Cao et al., 2008). Moreover, ATX has been shown to be neurotoxic in cerebellar granule

cells through an indirect activation of N-methyl-d-aspartate receptors (NMDARs) as a

consequence of glutamate release (Li et al., 2001; Li et al., 2004). ATX precise

recognition site on the channel protein remains to be defined. The structure of ATX

includes asymmetric carbon atoms, and the (4R,5R)-isomer is the naturally occurring

compound. The (4R,5R)-isomer appears in profile as an “L” shape with a hydrophobic

interior and a cluster of hydrophilic groups on the exterior of the macrocycle (Li et al.,

2001). Thus, the (4R,5R)-configuration is important for creating a molecular topology

1-18

that is recognized by the acceptor site on the voltage-gated sodium channel α subunit.

Given the unique structure and mechanism of action of ATX, we sought to further

characterize its pharmacologic actions and the functional consequences in cerebrocortical

neurons.

Role of NMDAR in neuronal development and synaptic plasticity.

Neuronal activity has a major role in the development of dendritic complexity and

neuronal circuits. The mechanisms by which neuronal activity translate into

morphological changes are complex. Numerous studies have shown that activity-

dependent neuronal development involves ionotropic glutamate receptors (NMDAR)

mediated calcium influx (Ghosh and Greenberg, 1995; West et al., 2002). These increases

in [Ca2+]i activate signaling cascades that control the transcriptional regulation of

neuronal development. Increase in cytosolic Ca2+ also involves calcium release form

intracellular stores (ER) and is involved in local effects like dendrtic branching and

stabilization of dendritic structures. Intracellular calcium acts as a signaling molecule

largely through binding to calmodulin, a calcium-binding protein that engages

downstream Ca2+/calmodulin-dependent protein kinase (CaMK) and mitogen-activated

protein kinase (MAPK) signaling pathways (Ghosh and Greenberg, 1995; West et al.,

2002). CaMK kinase (CaMKK) has been demonstrated to be an upstream regulator of

both CaMK- and MAPK-signaling pathways. Moreover, previous studies have

demonstrated that activity-dependent neurite outgrowth (Wayman et al., 2006) and

synaptogenesis (Saneyoshi et al., 2008) are regulated by NMDAR-dependent

CaMKK/calmodulin kinase I-signaling cascades. Therefore, NMDARs play a critical role

1-19

in activity-dependent development and plasticity (Ghosh and Greenberg, 1995), dendritic

arborization (Wayman et al., 2006; Wong and Ghosh, 2002; Miller and Kaplan, 2003),

spine morphogenesis (Ultanir et al., 2007), and synapse formation (Saneyoshi et al.,

2008) by stimulating these calcium-dependent signaling pathways. Preliminary results

show the involvement of NMDAR in VGSC activators-stimulated.

Intracellular sodium as a signaling molecule and its involvement in neuronal development.

Regulation of [Na+]i plays a critical role in the nervous system, not only because Na+

influx through VGSCs is responsible for the initiation and propagation of action

potentials but also because various neuronal cell functions, such as intracellular pH, Ca2+

homeostasis, and reuptake of neurotransmitters, are dependent on the Na+ gradient.

Previous studies have further indicated that intracellular Na+ can also act as a signaling

molecule to modulate cell functions, such as cell proliferation, ion channel permeability,

G-protein function, and opioid ligand-receptor interactions (Yu, 2006). Moreover, recent

studies have demonstrated that neuronal activity-mediated increases in [Na+]i in

structures, including soma, dendrites, and spines, may act as a signaling molecule and

contribute to activity-dependent synaptic plasticity (Rose and Konnerth, 2001). In

cerebellar Purkinje neurons, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

receptor-mediated Na+ influx was shown to be required for induction of long-term

depression (Linden et al., 1993). In both hippocampal and immature cerebrocortical

neurons, an elevation in intracellular Na+ was found to increase NMDAR-mediated

whole-cell currents and NMDAR single-channel activity by increasing both channel open

probability and mean open time (Yu and Salter, 1998; George et al., 2009). Yu and Salter

1-20

have demonstrated that an increment of [Na+]i of 10 mM was sufficient to produce

significant increases in NMDA receptor single-channel activity. It has also been reported

that increments of [Na+]i greater than 5 mM represent a critical threshold required to

regulate NMDAR-mediated Ca2+ influx. They used veratridine, a VGSC modulator, to

demonstrate that Na+ influx through TTX-sensitive VGSC was sufficient to up-regulate

NMDAR activity. Moreover, this [Na+]i-mediated up-regulation of NMDAR function has

been shown to require Src kinase activation (Yu and Salter, 1998; George et al., 2009).

Src family kinases (SFKs) act as a crucial point of convergence for signaling pathways

that enhance NMDAR activity, and, by up-regulating the function of NMDARs, Src gates

the production of NMDAR-dependent synaptic potentiation and plasticity (Salter and

Kalia, 2004). Hence it is important to explore the regulatory influence of [Na+]i on

NMDAR function in activity-dependent neuronal development.

Regulation of activity dependent dendritic growth and branching by BDNF-TrkB

signaling.

Neurotrophins cooperate with neuronal activity to modulate neuronal morphology.

Studies in the past few years have shown convincing evidence for the involvement of

BDNF-TrkB signaling in activity-dependent dendritic growth, dendric arborization,

synaptogenesis and synaptic transmission (Reichardt, 2006; Huang and Reichardt, 2003;

Yoshii and Constantine-Paton, 2007). BDNF-TrkB mediates increase in dendritic

complexity by increasing the total dendritic/neurite length, dendritic branching

(arborization) and increase in number of primary neurites (Baker et al., 1998; McAllister

et al., 1996). Activation of NMDARs and intracellular Ca2+ rise is critical for the

1-21

expression and release of BDNF (Reichardt, 2006). The released BDNF binds to its

cognate receptors TrkB and acts in an autocrine/paracrine mode to regulate neuronal

development. The binding of BDNF to full length TrkB (FL-TrkB) induces

autophosphorylation of tyrosine residues in the intracellular kinase domains of TrkB,

which in turn, leads to phosphorylation of two tyrosine residues (Tyr 515, Tyr 816)

located outside the kinase activation domain of TrkB (Reichardt, 2006). Phosphorylation

of these two residues recruit and activate three major intracellular signaling pathways that

mediate BDNF-TrkB regulated neuronal morphology. They are: the PI3K-Akt pathway,

the Ras-MAPK pathway and PLCγ-Ca2+ pathway (Reichardt, 2006). Activation of TrkB

at Y816 recruits and activates PLCγ which in turn hydrolyzes PIP2 to DAG and IP3,

where IP3 releases Ca2+ from intracellular stores. The other TrkB phosphorylation site is

Tyr515 that activates downstream MEK-MAPK/Erk signaling, which promotes neuronal

differentiation and growth by influencing transcription events, such as activation of

CREB transcription factor. Tyr 516 indirectly activates PI3K pathway by interacting with

adaptor proteins that regulate PI3K pathway. Another pathway that is indirectly activated

by Phospho-TrkB (Y516) is the PI3K-Akt pathway. Once activated, Akt can

subsequently phosphorylate a number of substrates (GSK3β, MAP2, mTOR) that are

involved in cell survival, NOG/actin dynamics, dendritogenesis and synaptic plasticity

(Read and Gorman, 2009; Yoshii and Constantine-Paton, 2010). Preliminary results for

the present study show the involvement of TrkB in VGSC activators-stimulated NOG in

immature cerebrocortical cultures. This proposed research project will help us understand

1-22

the role and function of BDNF-TrkB signaling in sodium channel-activators mediated

neuronal development.

1.2 Significance

Inasmuch as activity-dependent N-methyl D-aspartate receptor (NMDAR) signaling,

gene transcription and protein synthesis play major roles in brain functions that regulate

neuronal morphology and synaptic plasticity, disruption of one or more of these

processes gives rise to dendritic abnormalities implicated in mental retardation and

autism-spectrum disorders. On the other hand, normal functioning of these mechanisms

are crucial for structural remodeling of dendritic arbor required in synaptogenisis,

synaptic plasticity, and learning and memory. Recently, there is increasing evidence for

mTOR-mediated protein synthesis being important in long term memory formation, and

misregulation of this protein synthesis playing an important role in autism and mental

retardation disorders. Given the increasingly critical role of NMDAR-BDNF-TrkB-PI3K-

mTOR signaling in synapse maturation, plasticity and neurological diseases, expanding

our knowledge of how these signaling pathways function in the brain is key to

understanding learning and memory, as well as developing novel therapeutic approaches

neurological disorders related to NMDAR dysfunction

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2 CHAPTER 2 - LITERATURE REVIEW

2.1 Voltage-Gated Sodium Channels

2.1.1 Introduction:

Voltage gated sodium channels (VGSCs) play an important role in the initiation

(the rising phase) and propagation of action potentials (AP) in neurons and other

electrically excitable cells like myocytes (especially cardiac myocytes) and

neuroendocrine cells. In response to local membrane depolarization they mediate the

rapid influx of the sodium ions into the excitable cell, generating an action potential.

These action potentials will initiate various physiological events like neuronal firing and

muscle contraction. Seminal work in this regard was performed by Hodgkin and Huxley

in 1952 and showed that electrical signals in nerves are initiated by voltage-dependent

activation of sodium current that carries Na+ inward. This sodium current is followed by

inactivation of VGSCs in a few milliseconds and subsequently terminated by activation

of voltage-gated potassium channels.

2.1.2 Primary structure of VGSCs

VGSCs consists of huge complex of four structurally homologous domains of α-

subunits (each 260 kDa), β1 (36 kDa), β2 (33 kDa) subunits (Hartshorne and Catterall,

1981). The ∝-subunit was sufficient enough for functional VGSC currents (Noda et al.,

1986) but the β subunits are necessary for modulation of kinetics and voltage dependence

2-24

of gating (Isom et al., 1995). All four α subunit domains consists of six helical

transmembrane (TM) segments (S1-S6). The voltage sensor for each of the domain is

located in the S4 which consists of positively charged amino acids at every 3 position of

the segment. The loop between S5-S6 re-enters and gets embedded in the TM region and

four such loops of all domains forms the narrow extracellular side of the ion-selective

pore of the channel. The S6 segments of all four domains make the wider intracellular

portion of the pore. Each of the TM segments is connected by short extracellular loops

except for the large one between S5-S6. The domains are connected to each other by

large intracellular loops. The C & N terminal ends are located intracellularly. The β

subunits are unique ion channel subunits with N-terminal extracellular immunoglobulin-

like fold, a single transmembrane segment and a small intracellular segment (Figure 1)

(Isom et al., 1992; Isom et al., 1995). The hydrophobic interactions hold the 2 beta sheets

like a sandwich to form immunoglobin like fold.

2.1.3 VGSCs: Diversity in expression and function

There are at least 10-12 different kinds of VGSCs expressed in various excitable

tissues with subtle amino acid changes among them. Nav1.1, 1.2, 1.3 and 1.6 are

primarily in central nevous system (CNS) and 1.7, 1.8 and 1.9 in peripheral nervous

system (PNS). Nav1.4 is the primary sodium channel in skeletal muscle and Nav1.5 in

heart. Physiological and pharmacological signatures for some of these VGSCs have been

established which clearly support that different VGSCs have different physiological

characteristics in various excitable tissues. Studies on neuronal electrogenic apparatus

plasticity have confirmed that VGSCs gene expression is dynamic, like in diseased and

2-25

normal states (Nav1.8&1.9 are down regulated in injured neurons-(Dib-Hajj et al., 1998),

different functional states (Nav1.2 & 1.6 are over expressed in fast bursting state

compared to quiescent state-(Tanaka et al., 1999), and also different developmental

states. In regard to Navβ subunits, a total of 4 Navβ subunits have been discovered. They

bind to α subunits either noncovalently (β1 and β3) or by disulfide bonds (β2 and β4).

These Navβ subunits resemble the family of cell adhesion molecules (Isom et al., 1995)

and are required for localization and immobilization of VGSCs in specific locations of

excitable cells (Patino et al., 2011).

Various studies have lead to discovery of various sub-type specific toxins and

some sub-type specific blockers which may further help in discovering therapeutic

alternatives for diseases involving excitable cells and tissues. Table 1 is a compilation of

various isoforms of VGSCs discovered till date and their expressed locations in the body

and their pharmacological significance.

2.1.4 Structure of VGSCs at Atomic Resolution

A high resolution crystal structure of the bacterial sodium channel NaVAb was

determined, (Payandeh et al., 2011) revealing abundant novel information about the

structural basis for sodium selectivity and conductance. It also shed new light on the

mechanism of VGSCs block by therapeutic drugs and mechanisms of voltage-

dependence of gating. A top view of NaVAb structure revealed that, the central pore is

formed by four pore-forming modules (S5-S6 segments) and the intervening pore loop.

The outer rim of the pore module was formed by four voltage- sensing modules (S1-S4

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segments). The transmembrane architecture demonstrates that the voltage-sensing

module of one subunit is closely associated with the pore-forming module of its adjacent

subunit, thereby enforcing a concerted gating of the four subunits.

2.1.5 VGSCs function: Molecular perspective

There are three important features that characterize the sodium channels: 1)

selective permeability for sodium conductance, 2) Voltage dependent activation, 3) rapid

inactivation (HODGKIN and HUXLEY, 1952a; HODGKIN and HUXLEY, 1952b;

HODGKIN and HUXLEY, 1952c).

2.1.5.1 selective permeability for sodium conductance

VGSCs pore and Ion-selectivity filter: Studies utilizing Na+ channel blockers tetradotoxin

(TTX) and saxitoxin (SXT) were responsible in identifying outer pore and selectivity

filter (Heinemann et al., 1992; Noda et al., 1989; Terlau et al., 1991). These initial studies

on pore structure were further confirmed by the crystal structure study of NavAb.

Glutamate 387 on S5-S6 inter segmental loop (membrane-reentrant loop) of domain I has

been revealed as the crucial binding amino acid (aa) for TTX and SXT. Two negatively

charged aa residues present at analogous positions on all 4 domains in S5-S6 loop form

the inner and outer rings for the TTX and SXT receptor binding site and also the

selectivity filter of the outer pore of the Na+ channel. Mutational studies of the residues

forming these rings have strongly supported the idea that these aa residues form the

selectivity filters (Schlief et al., 1996).

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Though all VGSCs have similar Na+ permeation, they have different affinity for

TTX. Nav1.1, 1.2, 1.3 and 1.7 have more affinity for TTX. They are called TTX

sensitive VGSCs and are broadly expressed in neurons. Nav1.5, 1.8, 1.9 have 200 times

lesser affinity for TTX and are called TTX resistant VGSCs. These are mostly expressed

in cardiac myocytes and dorsal root ganglion neurons. This difference is due to a single

aa change at couple of residues preceding the glutamate 387 of domain 1 selectivity filter

from tyrosine/ phenylalanine to cysteine. Similarly, for cadmium (which is a high

affinity cardiac VGSCs blocker), the presence of cysteine at the above said site in the

selectivity filter of heart VGSCs attributes for stronger affinity in cardiac VGSCs over

other VGSCs.

2.1.5.2 Voltage-dependent activation

Voltage dependent activation of VGSCs was first demonstrated by Hodgkin and Huxley

and predicted that it requires the outward movement of gating charges in response to

changes in the membrane electric media (HODGKIN and HUXLEY, 1952a; HODGKIN

and HUXLEY, 1952b; HODGKIN and HUXLEY, 1952c). The S4 is a unique segment

with repeat motifs consisting of positively charged aa residues (usually arginine)

followed by 2 hydrophobic residues, thereby creating a spiral of positive charge around

the helix (Figure 2). The negatively charged internal TM electric field will strongly

attract the positive charges into the plasma membrane. Depolarization of the membrane

will lead to movement of this positively charged S4 segment in a spirally outward

direction bringing a conformational change that makes the pore open. This model is

called the sliding helix or helical screw model of voltage sensing (Catterall et al., 1986;

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Catterall, 1986; DeCaen et al., 2008; DeCaen et al., 2009; DeCaen et al., 2011; Guy and

Seetharamulu, 1986; Kontis and Goldin, 1997; Kontis et al., 1997; Wang et al., 2011;

Yarov-Yarovoy et al., 2012; Zhang et al., 2011). This transmembrane position of the S4

segment was confirmed by mapping the receptor sites for scorpion toxins. β scorpion

toxin enhances the VGSCs activation by pushing the voltage dependence to much

negative membrane potentials. Upon activation the β-scorpion toxins trap the activated

S4 voltage sensor in its outward, activated position and thereby slowing down the

deactivation of the channel. Voltage sensor trapping is a common mechanism employed

by various neurotoxins like β and α scorpion toxins.

2.1.5.3 Inactivation:

VGSCs get inactivated within milliseconds of activation/depolarization. The short

conserved intracellular loop connecting III-IV domains of α subunit functions as the

inactivation gate. The aa triad of isoleucine, phenylalanine and methionine (IFM motif)

play an important role in the inactivation of the channel (West et al., 1992). The IFM

motif binds like a tether and blocks the pore by binding to the receptor site present

intracellular of the pore. (Figure 3)

2.1.5.4 Coupling of activation to inactivation:

S4 sensor outward movement (especially of domain IV) leads to activation of VGSCs

and changes in its voltage dependence leading to their inactivation. This inactivation

signal leads to closure of intracellular inactivation gate. Some toxins like α scorpion

toxin and sea anemone toxins would bind to the receptor site in such a way that it would

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allow only partial movement of the S4 voltage sensor and thereby activating it but will

lead to slow inactivation. This clearly shows that activation couples with inactivation in

the normal functionality of the channel.

2.2 Voltage-gated sodium channels activators and

gating modifiers

VGSCs channel function can be altered by neurotoxins binding to various

molecular targets called receptor sites located on the channels. These receptor

neurotoxins bind with high affinity and specificity and hence provide excellent tools to

study the structure, function, conductance and gating properties of VGSCs.

Pharmacological studies have revealed that neurotoxins act on six unique receptor sites

(Table 2) with the possibility of additional two more sites (pyrethroid and antillatoxin

binding sites). Upon binding, these neurotoxins alter the Na+ conductance and voltage-

dependent gating by bringing about conformational changes at the site of binding and

thereby changing the equilibrium between open and the closed/inactive state of the

VGSCs. Also, upon binding they can alter the affinity for other neurotoxins acting at a

different receptor site. VGSCs modifiers bring about their actions primarily by 1) slowing

the coupling of sodium channel activation to inactivation (reduce the rate of inactivation),

2) increasing the mean open time of the channel 3) inhibiting channel inactivation or 4)

shifting the activation potential to more negative values, thereby augmenting Na+ influx

through VGSCs.

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2.2.1 Antillatoxin

Antillatoxin (ATX) is a structurally novel lipopeptide with an exceptionally high

degree of methylation unlike any known natural product 28 Lee,K.C. 2006. Isolated

from the cyanobacterium Lyngbya majuscula, this compound is also distinguished by

multiple stereocenters 261 Orjala,J. 1995. The essential role of the asymmetric

carbon atoms in ATX is reflected in the stereoselective effects of ATX enantiomers 13

Li,W.I. 2004. ATX is considered to be the second most potent ichthyotoxic compound

obtained from marine sources after only brevetoxin (PbTx)-1 261 Orjala,J. 1995.

Exposure to L. majuscula blooms are associated with adverse human health effects,

including respiratory irritation, eye inflammation, and severe contact dermatitis. Previous

work has demonstrated that ATX is a potent activator of voltage-gated sodium channels

(VGSCs) that elevates intracellular Na+ concentration ([Na+]i) in intact neurons 24

Li,W.I. 2001; 7 Cao,Z. 2008; 296 Jabba,S.V. 2010. Moreover, ATX has been shown to

be neurotoxic in cerebellar granule cells through an indirect activation of N-methyl-d-

aspartate receptors (NMDARs) as a consequence of glutamate release 24 Li,W.I.

2001.

ATX is a novel activator of VGSC; however, its precise recognition site on the

channel protein remains to be defined. The structure of ATX includes asymmetric carbon

atoms, and the (4R,5R)-isomer is the naturally occurring compound. The (4R,5R)-isomer

appears in profile as an “L” shape with a hydrophobic interior and a cluster of

hydrophilic groups on the exterior of the macrocycle (Li et al., 2001). Thus, the (4R,5R)-

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configuration is important for creating a molecular topology that is recognized by the

acceptor site on the voltage-gated sodium channel α subunit. Previous studies from our

lab have shown that ATX allosterically enhances the specific binding of

[3H]batrachotoxin to intact cerebellar granule cells (Li et al., 2001). This effect of ATX

on [3H]batrachotoxin binding was synergistically augmented by brevetoxin. The strong

synergistic interaction of the ATX recognition site with neurotoxin site 5 suggests that

these sites may be topologically close and/or conformationally coupled. The results

obtained using [3H]batrachotoxin as a probe for sodium channel conformation allowed us

to exclude the interaction of ATX with neurotoxin sites 1, 2, 3, and 5 on VGSCs. Site 1

was ruled out because tetrodotoxin and saxitoxin bind to the outer vestibule of the pore of

the ion channel and allosterically inhibit the binding of [3H]batrachotoxin; this is an

effect that is antipodal to that of ATX. We were able to rule out sites 2 and 5 inasmuch as

these sites display positive allosteric coupling to the ATX site. Neurotoxin receptor site 3,

the target for α-scorpion toxins and sea-anemone toxins, was excluded because ATX

enhanced [3H]batrachotoxin binding in the presence of a maximally effective

concentration of sea-anemone toxin. Although we cannot exclude an interaction of ATX

with neurotoxin site 4, the target for β-scorpion toxin, it is reasonable to posit that ATX

binds to a novel recognition domain on the α-subunit of the VGSC. The relatively small

lipotripeptide structure of ATX would not be restricted to an extracellular target, as is the

case for the scorpion toxins, which are composed of 60 to 65 amino acids. Given the

unique structure and mechanism of action of ATX, it would be important to further

characterize its binding site on VGSCs.

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Most recent study on ATX has demonstrated that it displays a unique efficacy

with respect to stimulation of sodium influx in cells expressing rNav1.2, rNav1.4 and

rNav1.5 α-subunits (Cao et al., 2011). The efficacy of ATX was distinctive inasmuch as it

was not shared by activators of neurotoxin sites 2 and 5. It was also demonstrated that

the ATX binding site shares with neurotoxin sites 2 and 5 the phenomenon of partial

agonism. Finally, it was observed a reciprocal allosteric interaction between neurotoxin

site 5 and the ATX binding site. Collectively, these data indicate that ATX is a sodium

channel gating modifier with unique efficacy in cells heterologously expressing VGSC α-

subunits. Defining the molecular determinants and mechanisms of action of ATX may

provide further insight into the gating properties of sodium channels.

Our lab previously demonstrated that NMDA receptor function may be increased

through activation of VGSCs with attendant elevation of intracellular sodium in

cerebrocortical neurons (George et al., 2009). VGSC activators function as gating

modifiers that elevate [Na+]i in the absence of substantial depolarization of neurons (Cao

et al., 2008; George et al., 2009). These findings have been confirmed and extended for

this structurally novel lipopeptide, ATX. Jabba et al., found that ATX promoted

neuritogenesis by elevating [Na+]i, which in turn augmented NMDAR function leading to

Ca2+ influx and engagement of a CaMKK pathway (Jabba et al., 2010) which stimulated

neuritogenesis in DIV-1 cerebrocortical neurons. These data provide further support for

the hypothesis that sodium channel activators seem to be capable of mimicking activity-

dependent neuronal development through potentiation of NMDAR signaling pathways

that influence neuronal plasticity.

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2.2.2 Veratridine

Veratridine (VRT) is a lipid-soluble alkaloid extracted from plants of family Liliaceae

(suborder Melanthaceae) belonging to genus Veratrum (species album, viride etc.).

Veratrine, the alkaloid fraction of the seeds is a mixture that mostly consists of the ester

alkaloids, veratridine, and cevadine and of the alkamine veracevine or its isomer cevine.

Veratridine binds to receptor site 2 of VGSCs and preferentially to activated state of

VGSCs, thereby causing them to stay open (persistent activation via allosteric

mechanism) during a sustained membrane depolarization. This leads to abolishment of

VGSCs inactivation and shift of the voltage dependence of activation to more negative

potentials 395 Ulbricht,W. 1969; 384 Albuquerque,E.X. 1988; 394 Catterall,W.A.

1975; 393 Catterall,W.A. 1975; 392 Catterall,W.A. 1980; 391 Catterall,W.A. 1980; 389

Khodorov,B.I. 1985. This primarily leads to Na+ influx, with secondary effects like

increased Na+-K+ pump activity, increased Ca2+ influx and in turn exocytosis .

Veratridine is a partial agonist at site 2 with batrachotoxin being the full agonist. Most of

the experiments involving localization of receptor site-2 utilized batrachotoxin and

determined that site 2 neurotoxins bind to the S6 TM region of domain I 359

Trainer,V.L. 1996. It is thought that the abolishment of VGSCs inactivation by VRT

and other site 2 neurotoxins is due to its interaction with S6 TM of domain IV (IVS6) that

is required for fast inactivation. Also, site 2 neurotoxins binding to IVS6 may cause the

toxin to change the voltage-dependent movements of the adjacent IVS4 voltage-sensor

and thereby affect activation and coupling of activation to inactivation 351

Linford,N.J. 1998.

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2.2.2.1 Intracellular sodium as a signaling molecule and its involvement in neuronal

development.

The electrical signals of neurons are fundamentally dependent on Na+ influx

through VGSCs and are primarily responsible for the rising phase of action potential and

hence supply the current that drives the membrane potential to the peak of action

potential. Na+ is a major physiological ion present in the extracellular milieu. Apart from

action potential dependent Na+ influx, Na+ can enter into the cells via various routes

including influx through voltage and ligand-gated ion channels, uptake via membrane

exchangers and gradient-driven co-transporters 252 Nicholls,D. 1990. Recent studies

have indicated that changes in intracellular sodium concentration ([Na+]i) produced in the

soma and dendrites as a result of neuronal activity may act as a signaling molecule and

play a role in activity-dependent synaptic plasticity. It has been shown that short burst or

tectonic stimulation of afferents that induce synaptic LTP causes [Na+]i increments of 10

mM in dendrites and of up to 35-40 mM in dendritic spines 93 Rose,C.R. 1999; 91

Rose,C.R. 2001 . In cerebellar Purkinje neurons, α-amino-3-hydroxy-5-methyl-4-

isoxazolepropionic acid receptor-mediated Na+ influx was shown to be required for

induction of long-term depression 249 Linden,D.J. 1993. In both hippocampal and

immature cerebrocortical neurons, an elevation in intracellular Na+ was found to increase

NMDAR-mediated whole-cell currents and NMDAR single-channel activity by

increasing either channel open probability or mean open time or both (Figure 3) 96

Yu,X.M. 1998; 296 Jabba,S.V. 2010; 6 George,J. 2009. Yu and Salter have

demonstrated that an increment of [Na+]i of 10 mM was sufficient to produce significant

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increases in NMDA receptor single-channel activity. It has also been reported that

increments of [Na+]i greater than 5 mM represent a critical threshold required to regulate

NMDAR-mediated Ca2+ influx. They used veratridine, a VGSC modulator, to

demonstrate that Na+ influx through TTX-sensitive VGSC was sufficient to up-regulate

NMDAR activity. Moreover, this [Na+]i-mediated up-regulation of NMDAR function has

been shown to require Src kinase activation 6 George,J. 2009; 96 Yu,X.M. 1998. In

DIV 1 dorsal root ganglion (DRG) neurons obtained from E7 pups, Semaphorin3A-

induced facilitation of axonal guidance involved VGSCs activation and required changes

in [Na+]i 309 Yamane,M. 2012. [Na+]i downregulated the amiloride-sensitive

currents in epithelial sodium channels (ENaCs) 433 Cook,D.I. 2002; 434 Dinudom,A.

2001; 436 Komwatana,P. 1996. Further, studies have demonstrated [Na+]i may also

activate potassium channels 432 Bhattacharjee,A. 2005; 431 Dryer,S.E. 2003. Some

studies have found the role of [Na+]i in regulation of ligand-receptor and ligand-

transporters interactions 440 Pert,C.B. 1974; 439 Pert,C.B. 1974; 438 Bloch,R.J. 1986;

437 Werling,L.L. 1986; 435 Puttfarcken,P. 1986. Also, a significant increase in [Na+]i

is a characteristic feature of tissue injury and various studies have demonstrated the

neuro-protectiveness of blocking Na+ influx during tissue injury. The detailed mechanism

of action for the above listed affects of Na+ are not clearly evident and require further

studies are very important to elucidate them. One important possibility is the manifold

influx of Ca2+ into cytoplasm with subsequent activation of Ca2+-dependent activation.

Also, Na+ entry could change intracellular pH 443 Boonstra,J. 1983; 442

Moolenaar,W.H. 1983; 441 Moolenaar,W.H. 1986, thereby regulating enzymatic

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activity, neuronal growth and death. Hence, it would be critical to determine these

mechanisms and elucidate the potential Na+ binding target sites.

2.3 N-methyl-D-aspartate receptors (NMDARs)

NMDARs are located at excitatory glutamate synapses in the central nervous system

and play several important roles including, but not limited to, excitatory synaptic

transmission, neuronal plasticity, learning and memory formation and excitotoxicity.

They possess unique features like voltage-dependent block by extracellular Mg2+, high

Ca2+ permeability and slow activation/deactivation kinetics. Though NMDARs are

sensitive to various endogenous ligands, a critical requirement for their activation is

binding to the co-agonist glycine. Others that modify the receptor activity are

extracellular Zn2+, polyamines, interactions with scaffolding, anchoring and signaling

molecules associated with postsynaptic density (PSD). Numerous subtypes of NMDARs

have been identified in CNS, distinct in their channel properties, including, but not

limited to, ligand sensitivity, divalent ion block, kinetics, and interaction with

intracellular proteins. Understanding the role of various subtypes of NMDARs in normal

CNS functions like neuronal development, learning and memory and synaptic plasticity

will be important in developing therapeutic strategies for various pathophysiological

conditions involving NMDARs like epilepsy, ischemic brain damage, schizophrenia,

depression and, more speculatively, neurodegenerative disorders such as Parkinson’s and

Alzheimer’s diseases, Huntington’s chorea, and amyotrophic lateral sclerosis.

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2.3.1.1 NMDARs subunits and composition:

Electrophysiological, binding and cloning experiments provided the first evidence for

the presence of diversity in NMDARs and also regarding the heterologous composition of

the NMDARs in forming a functional NMDAR. Three families of NMDAR subunits

having significant sequence homology with other ionotrophic glutamate receptors were

discovered: GluN1, GluN2 (4 distinct GluN2’s-A, B, C and D) and GluN3 (2 different

GluN3’s) (previously called NR1, NR2 and NR3 respectively) 378 Moriyoshi,K. 1991;

368 Monyer,H. 1994; 363 Mori,H. 1995 with each having several splice variants.

NMDARs function as hetertetrameric assemblies, usually comprising of 2 obligatory

GluN1 subunits and 2 GluN2 subunits. GluN3 containing NMDARs contain

diheteromeric (GluN1/GluN3) or triheteromeric (GluN1/GluN2 / GluN3) complexes

430 Traynelis,S.F. 2010. The presence of such diverse NMDAR subunits allow for

various combinations of functionally distinct NMDAR subunit assembly.

The obligatory GluN1 subunit is ubiquitously expressed in the CNS, both in embryo

and adult, but with developmental and spatial variations in regard to GluN1 isoform

expressed. In contrast, GluN2 subunits have expression patterns that differ in

developmental time and space (one brain region to other) 369 Watanabe,M. 1992; 367

Akazawa,C. 1994; 368 Monyer,H. 1994; 373 Monyer,H. 1992. In embryonic CNS,

GLuN2B and GluN2D are preferentially expressed, with GluN2B being widely expressed

all over the brain and with GluN2D exclusively expressed in diencephalon and brain

stem. Postnatal, GluN2 subunit expression undergoes a drastic turnover, especially during

the first two weeks of birth, with gradual increase in GluN2A subunit expression in entire

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CNS. GluN2B expression reaches maximum at P7-10 but gets restricted more to the fore

brain regions (cortex, hippocampus, striatum, olfactory bulb) and remains high

throughout at these regions. GluN2A subunit expression increases steeply at around P7-

10. GluN2C subunit expression starts around P7, exclusively in the cerebellum granule

cells (CGC) and olfactory bulb neurons. GluN2D subunit expression is drastically

reduced postnatally, with little expression in the diencephalon and brainstem in adult life.

These developmental switches in GluN2 subunit expression patterns are important

functionally and bring about a change in the kinetics and sensitivity of NMDAR

mediated currents to various endogenous GluN ligands.

2.3.1.2 NMDAR Transmembrane topology:

NMDARs are integral membrane proteins, and as described earlier, form

heteroterameric channels with a central ion channel pore selective for Na+, Ca2+ and K+

cations. Each GluN subunit consists four distinct domains 81 Mayer,M.L. 1984; 325

Paoletti,P. 2007, 1)N-terminal domain (NTD): first 380 amino acids in the extracellular

region which forms a large clamshell-like domain, 2) Agonist binding domain (ABD): a

300 amino acids region that binds glycine in GluN1 and glutamate in GluN2 subunits, 3)

membrane domain: consists of 3 TM segments (M1, M3 and M4) and a short re-entrant

loop (pore or P-loop; M2) that forms the ion channel, and 4) An intracellular C-terminal

domain: highly variable in length (subunit dependent) and involved in scaffolding,

interaction with signaling molecules and receptor trafficking.

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2.3.1.3 NMDARs role in neuronal development:

Neuronal activity has a major role in the development of dendritic complexity and

neuronal circuits. The mechanisms by which neuronal activity translate into

morphological changes are complex. Numerous studies have shown that activity-

dependent neuronal development involves NMDARs mediated calcium influx 254

Ghosh,A. 1995; 32 West,A.E. 2002. Calcium influx through the NMDA receptor and

the subsequent initiation of signaling pathways have a well established role in activity-

dependent neuronal development and plasticity. NMDAR-mediated increase in cytosolic

Ca2+ also involves calcium release form intracellular stores (ER) and is involved in local

effects like dendrtic branching and stabilization of dendritic structures37 Redmond,L.

2002 . Intracellular calcium acts as a signaling molecule largely through binding to

calmodulin, a calcium-binding protein that engages downstream Ca2+/calmodulin-

dependent protein kinase (CaMK) and mitogen-activated protein kinase (MAPK)

signaling pathways 254 Ghosh,A. 1995; 32 West,A.E. 2002. Also, NMDAR-

mediated activity-dependent signaling to the nucleus may be influenced by the

developmental regulation of NMDAR subunit composition. Similarly, NMDAR-

dependent changes in synaptic function and calcium signaling can mediate the

developmental regulation of activity-dependent transcriptional programs 328

Majdan,M. 2006. Moreover, previous studies have demonstrated that activity-

dependent neurite outgrowth 99 Wayman,G.A. 2006 and synaptogenesis 98

Saneyoshi,T. 2008 are regulated by NMDAR-dependent CaMKK/calmodulin kinase I-

signaling cascades. Also, Epherin B (EphB) receptor tyrosine kinases regulates NMDARs

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function, thereby altering Ca2+ influx mediated gene expression, providing further

evidence for the role of NMDARs in neuronal development 344 Dalva,M.B. 2000.

Moreover, EphB interaction with GluN1 subunit of NMDARs during synaptic

development and plasticity plays an important role in NMDAR-dependent induction of

LTP and LTD 342 Grunwald,I.C. 2001; 341 Henderson,J.T. 2001. More

importantly, Src family tyrosine kinases phosphorylation of GluN2B subunit leads to

NMDARs function up-regulation, thereby increasing activity-dependent gene expression

340 Takasu,M.A. 2002. Moreover, NMDAR function is up-regulated due to changes

in [Na+]i and it has been shown that this requires Src kinase activation96 Yu,X.M.

1998; 6 George,J. 2009 . Src family kinases (SFKs) act as a crucial point of

convergence for signaling pathways that enhance NMDAR activity, and, by up-regulating

the function of NMDARs, Src gates the production of NMDAR-dependent synaptic

potentiation and plasticity97 Salter,M.W. 2004 .

Therefore, NMDARs play a critical role in activity-dependent development and

plasticity (Ghosh and Greenberg, 1995), dendritic arborization 99 Wayman,G.A. 2006;

1 Wong,R.O. 2002; 84 Miller,F.D. 2003, spine morphogenesis 36 Ultanir,S.K.

2007 and synapse formation 98 Saneyoshi,T. 2008 by stimulating these NMDAR-

mediated calcium-dependent signaling pathways.

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2.3.2 Neurotrophins and their receptors, tropomyosin-related kinases

(TRKs):

Neurotrophins play an important role in regulating neural survival, development,

function and plasticity. The discovery of neurotrophins and its cognate receptor marked a

breakthrough in developmental neurobiology. These biochemicals that are secreted in

small quantities regulate both local and global effects, for e.g., growth cone motility and

gene transcription respectively.

As of now, six neurotrophins are identified, out of which, four have been characterized.

1) Nerve growth factor (NGF), 2) Brain-derived neurotrophic factor (BDNF), 3)

Neurotrophin-3 (NT-3), and 4) Neurotrophin-4 (NT-4). All four neurotrophins have

similar sequence and structure and originate from a common ancestral gene. Their genes

share various similarities, including the presence of multiple promoters. The protein

product consists of three different sequences connected in a row. They are 1) signal

sequence 2) prodomain sequence, and 3) mature neurotrophin sequence. Hence, each

gene product has to undergo proteolysis to form a mature protein.

The neurotrophins interact with two different classes of receptors. Primarily, they

activate receptor tyrosine kinases called tropomyosin-related kinases (TRKs) 269

Huang,E.J. 2003 which in turn regulate signaling pathways involved in neuronal

survival, proliferation, neurite and axonal outgrowth, dendritic aroborization and

remodeling, cytoskeleton rearrangement, membrane trafficking and synaptogenesis and

synaptic plasticity. Trk receptors consist of three domains 1) extracellular domain, 2)

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transmembrane domain, and 3) cytoplasmic domain. The extracellular domain consists of

a) cysteine-rich cluster followed by b) three leucine-rich repeats, c) another cysteine-rich

cluster and d) two immunoglobulin-like domains. The cytoplasmic domain consists of a

tyrosine kinase domain and is surrounded by several tyrosines. These tyrosines upon

phosphorylation interact with several cytoplasmic adaptors and enzymes. Upon binding

with neurotrophins, Trk receptors dimerize, leading to the transphosphorylation

activation of the kinases present in the cytoplasmic domain. Three distinct TRKs have

been identified, TrkA, B, and C. Neurotrophins binds to their cognate receptor in an

autocrine or/and pracrine manner, subsequently activating them. NGF binds to TrkA and

activates its tyrosine kinase activity. Similarly BDNF and NT-4 activates TrkB, and NT-3

activates TrkC 380 Kaplan,D.R. 1991; 381 Klein,R. 1991; 379 Klein,R. 1991; 269

Huang,E.J. 2003; 345 Bibel,M. 2000. In addition, NT-3 can activate other Trk

receptors but with lower efficiency. Alternate splicing of the receptors will introduce

additional short amino sequences to the extracellular domain, thereby affecting their

ligand interactions 366 Clary,D.O. 1994; 376 Meakin,S.O. 1992; 358 Strohmaier,C.

1996. Similarly, splicing can also introduce changes in the intracellular catalytic

domain and thereby affecting signaling mechanisms initiated by neurotrophin binding.

TrkB and TrkC isoforms generated by alternate slicing include those which contain

comparatively short cytoplasmic motifs with no tyrosine kinase domains. Those isoforms

that lack the kinase domain inhibit productive dimerization with kinase-containing Trk

receptors, thereby inhibiting responses to neurotrophins 429 Eide,F.F. 1996. More

recent works, however, have demonstrated that these isoforms have a role to play in

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regulating various cellular signaling mechanisms. BDNF mediated activation of T1

isoform of TrkB controls release of Ca2+ from intracellular stores in a G-protein and IP3-

dependent pathway 336 Rose,C.R. 2003. Differential splicing also affects the

substrate specificity of one of the TrkC isoform, inhibiting its activation of several

substrates and thereby its ability to promote neuronal differentiation 364 Guiton,M.

1995; 356 Meakin,S.O. 1997.

Neurotrophins also interact with another family of receptor called the p75

Neurotrophin receptor (p75NTR). These are low affinity receptors, but bind to all

neurotrophins with almost same affinity 382 Rodriguez-Tebar,A. 1990; 353 Frade,J.M.

1998. p75NTR belongs to tumour necrosis receptor superfamily and its structure

consists of three domains. 1) an extracellular domain containing four cysteine-rich

motifs, 2)a transmembrane domain, and 3) cytoplasmic domain, that includes the ‘death’

domain 357 Liepinsh,E. 1997. Although, p75NTR do not have a catalytic domain, it

can interacts with several proteins that play an important role in neuronal survival and

development and synaptic plasticity 330 Makkerh,J.P. 2005; 346 Bentley,C.A. 2000;

347 Harrison,S.M. 2000.

2.3.2.1 Brain Derived Neurotrophic Factor (BDNF):

BDNF is one of the best characterized neurotrophin that plays a critical role in neuronal

development and plasticity. More recent studies in the field of neurotrophins have

associated BDNF deficits with various neurodevelomental, neurodegenerative and

psychiatric disorders. BDNF is widely expressed in most brain areas, with high

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expression in the hippocampus, amygdale, and cerebellum with principal glutamatergic

neurons being the main cell type expressing it 324 Aid,T. 2007; 418 Ernfors,P. 1990;

420 Ernfors,P. 1990; 424 Lindholm,D. 1992. An activity-regulated gene, Bdnf gene is

composed of at least nine exons and is transcribed by many unique promoters leading to

synthesis of distinct 5’untranslated regions (UTRs). Exons I to VIII have their own

distinct promoter and are associated by splicing mechanism to exon IX, which is the only

one being translated into a protein. Also, these mRNA synthesized have two distinct

3’UTRs, with differences due to the presence of two distinct polyadenylation sites,

leading to expression of Bdnf gene with 2 different 3’UTR lengths 324 Aid,T. 2007.

Due to a combination of several 5’UTRs, 10 different exons and 2 distinct 3’UTRs, there

is a possibility of at least 18 distinct pre-mRNA being transcribed, but all translated into

one BDNF protein. This transcription is highly regulated, in a developmental age wise,

tissue-specifically and neuronal activity-dependent manner 332 Rattiner,L.M. 2005;

324 Aid,T. 2007. For e.g., neuronal activity-dependent and Ca2+ influx mediated Bdnf

transcription is controlled by promoter I and IV 321 Kidane,A.H. 2009; 332

Rattiner,L.M. 2005. Bdnf gene with long 3’UTRs are transported to the dendrites and

locally translated to the BDNF protein 417 An,J.J. 2008, where as the short 3’UTR

containing Bdnf gene is localized to the soma. Though most genes that are locally

translated are tightly regulated by local synaptic activity, mechanisms of regulation of

Bdnf gene local translation in the dendrites is not well established. Also, other

mechanisms of transcriptional control of BDNF are not well understood, and given the

utmost biological significance of BDNF in neuronal development and plasticity, it will be

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very important to focus future studies on transcriptional regulation of Bdnf gene synthesis

in the nervous system.

The pre sequence of the pre-RNA directs the translation of the BDNF to endoplasmic

reticulum, giving rise to a dimeric pro-BDNF (29 kDa) that is involved in the sorting of

BDNF into appropriate pathway of secretion 333 Chen,Z.Y. 2005; 287 Lee,C.C.

2005. This pro-BDNF is later cleaved to give rise to mature BDNF 360 Seidah,N.G.

1996. Two different pathways are utilized in the secretion of BDNF 1) constitutive and

2) regulated pathways. In the constitutive pathway, BDNF is packaged into smaller sized

granules (Ø 50-100 nm) and released continuously, in a Ca2+-independent fashion. This

pathway occurs mostly at the soma and proximal parts of the neuron and does not involve

of any specific triggering stimulus 349 Mowla,S.J. 1999; 337 Lessmann,V. 2003; 331

Brigadski,T. 2005. In the regulated pathway, BDNF is packaged into more bigger

sized vesicles (Ø ~300 nm), and following an increase in [Ca2+]i these vesicles fuse to the

plasma membrane and release BDNF. This pathway occurs primarily at the distal parts of

the neuronal processes 331 Brigadski,T. 2005. As mentioned earlier, pro-BDNF is

converted into mature BDNF in the golgi and trans-golgi network and then sorted for

these two different pathways. Two different amino acid motifs, each present in the pro

and mature form are involved in this sorting process. The interaction of the motif

specifically present in the mature form with carboxypeptidase E (CPE) targets it to the

regulated secretory pathway and an absence of the CPE interaction leads to the BDNF

sorted to the constitutive pathway 416 Pang,P.T. 2004 . Similarly, interaction of the

motif specifically present in the pro form with sortilin targets it to the regulated secretory

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pathway. A single nucleotide polymorphism in the motif specifically present in the pre

form (val66met) will lead to the loss of interaction with sortilin, thereby impairing the

sorting mechanisms to the regulated secretory pathway and causing deficits in neuronal

development and cognition 339 Egan,M.F. 2003. Even though both pathways release

both pro and mature form, studies have shown that, comparatively more mature BDNF is

released by the regulated pathway than constitutive pathway. The possible reason is, in

regulated pathway, the time between synthesis and release of BDNF is longer, thereby

providing more time for the intracellular convertases to convert the pro form to mature

form. Vice versa, in constitutive pathway, more pro-BDNF is released due to the less

available time for the convertases to convert the pro form to mature form.

Several studies in the last decade have demonstrated that, BDNF release in regulated

pathway requires increase in [Ca2+]i. The route for increase in [Ca2+]i can be manifold.

Calcium influx through NMDARs or VGCCs or release of Ca2+ from intracellular

calcium stores or a combination of any of the above (depending on the kind of

stimulation or activation), can trigger the release of BDNF. Mechanistic studies on BDNF

release have demonstrated that the rise in the calcium activates the calcium calmodulin II

(CamKII) leading to the fusion of BDNF containing secretory granules with the plasma

membrane and slowly releasing BDNF into the extracellular milieu 331 Brigadski,T.

2005; 323 Kolarow,R. 2007. It has been also demonstrated that protein kinase A

(PKA) have a regulatory role in the release of BDNF through regulated pathway 323

Kolarow,R. 2007 in the dendritic neuronal processes.

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2.3.3 Regulation of BDNF-TrkB signaling pathway

Upon binding with neurotrophins, Trk receptors dimerize, leading to the

transphosphorylation activation of the kinases (activation loop tyrosines) present in the

cytoplasmic domain 415 Huang,E.J. 2003. Proteases and convertases that control the

processing of pro-BDNF to mature-BDNF regulate TrkB receptor responsiveness to

BDNF, as only mature form of BDNF and not the pro-forms of BDNF can activate TrkB

receptors 400 Lee,R. 2001. The TrkB receptor contain additional tyrosine residues in

the cytoplasmic domain that act as phosphorylation substrates for TrkB tyrosine kinase.

These phosphorylated tyrosines then interact with various scaffolding proteins and other

intermediary proteins that are part of intracellular signaling cascades. Y670, Y674 and

Y675 (human TrkA sequence) are present in the autoregulatory loop of the tyrosine kinse

domain. Phosphorylation of these tyrosines increases the Trk tyrosine kinase activity.

Phosphorylation of other tyrosine residues leads to the formation of binding sites for

proteins containing phosphor-tyrosine binding (PTB) or Src-homology-2 (SH2) domains.

Three major intracellular signaling pathways are activated by BDNF-TrkB. They are: 1)

PI3K-Akt pathway, 2) the Ras-MAPK pathway and 3) PLCγ-Ca2+ pathway (Reichardt,

2006). BDNF-TrkB signaling mediated functional changes can involve any one of these

or a combination of these signaling cascades.

2.3.3.1 Phospholipase-C signaling cascade

Activation of TrkB at Y816 recruits and activates PLCγ which in turn hydrolyzes

phosphotidylinositol 4,5-biphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-

2-48

triphosphate (IP3), where DAG activates protein kinase C (PKC) and IP3 releases Ca2+

from intracellular stores 415 Huang,E.J. 2003; 399 Reichardt,L.F. 2006. Together

these signaling molecules activate many intracellular enzymes, including most PKC

isoforms, Ca2+/CamKs and other Ca2+/Cam-regulated targets. Activation of PLCγ1

activates PKCδ leading to neurotrophin-mediated activation of MEK1 and Erk1/2 401

Corbit,K.C. 1999. Also, BDNF-TrkB-PLC γ1 signaling activates transient receptor

potential canonical subfamily of cation channels 3/6 (TRPC3/6) and contributes to

BDNF-induced Ca2+ elevations at growth cones and synapses 403 Li,Y. 2005; 402

Amaral,M.D. 2007. In nociceptive sensory neurons, the capsaicin receptor VR1, the

heat-activated TRP channel is repressed due to depletion of PIP2. Since activation of Trk-

PLC γ1 signaling leads to depletion of PIP2, Trk receptor activation leads to

hypersensitization of this channel to thermal and mechanical stimuli 404 Chuang,H.H.

2001; 405 Prescott,E.D. 2003. Also, some recent studies have implicated the role of

BDNF-TrkB-PLC γ1 signaling induced Ca2+ increase in synaptic plasticity 406

Nakata,H. 2007; 407 Shaywitz,A.J. 1999.

2.3.3.2 Mitogen-activated protein kinase pathway

The other TrkB phosphorylation site is Tyr515, which activates downstream MEK-

MAPK/Erk signaling and promotes neuronal differentiation and growth by influencing

transcription events, such as activation of CREB transcription factor. Phosphorylation of

Y515 on TrkB by BDNF recruits Shc to TrkB and phosphorylates it. This leads to Shc

interaction with Grb2, an adaptor protein that recruits and activates the guanine

nucleotide exchange factor (GEF) SOS. Subsequently SOS activates Ras which thereby

2-49

activates downstream kinases B-raf, MEK and MAPK/Erk 399 Reichardt,L.F. 2006;

415 Huang,E.J. 2003. MEK-MAPK/Erk signaling pathway activates transcription

factors, such as CREB 407 Shaywitz,A.J. 1999. MAPK/Erk plays a critical role in

protein synthesis dependent plasticity by increasing phosphorylation of eukaryotic

initiation factor 4E (eIF4E), the 4E-binding protein 1 (4E-BP1) and ribosomal protein S6

409 Kelleher,R.J.,3rd 2004; 408 Klann,E. 2004.

2.3.3.3 Phosphotidyl-Inositol-3-kinase pathway

Phosphorylation of Tyr 516 indirectly activates PI3K pathway by interacting with

adaptor proteins (Shc-Grb2-Ras) that regulate PI3K pathway 399 Reichardt,L.F.

2006. PI3K activation leads to the changes in inositol phospholipids composition in the

plasma membrane (cytoplasmic side), resultingin translocation of Akt/protein kinase B

(PKB) to the plasma membrane. Activated Akt/PKB is involved in a variety of functions

such as cell survival, proliferation, neuronal differentiation, neurite outgrowth and protein

translation 410 Read,D.E. 2009; 415 Huang,E.J. 2003; 399 Reichardt,L.F. 2006. The

TrkB-PI3K-Akt mediated protein translation is mediated by signaling cascade called

mammalian target of rapamycin (mTOR), a mjor regulator of protein synthesis 411

Sarbassov,D.D. 2005; 412 Sarbassov,D.D. 2005. Akt activation of mTOR pathway

involves, phosphorylation and inhibition of tuberous sclerosis complex proteins: hamartin

(TSC1) and tuberin (TSC2). TSC1/2 complex is a GTPase-activating protein for Ras

homolog enriched in brain (Rheb), immediate upstream activator of mTOR. PI3K-Akt

mediated inhibition of TSC1/2 complex increases activation of Rheb which subsequently

causes mTOR activation 413 Jaworski,J. 2006. Active mTOR phosphorylates p70S6

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and 4E-BP1 leading to mRNA translation. Also, The PI3K-Akt pathway plays an

important role in long-term maintenance of synaptic plasticity by regulating the

trafficking of synaptic proteins 414 Yoshii,A. 2007.

Activation of TrkB at Y816 recruits and activates PLCγ which in turn hydrolyzes

PIP2 to DAG and IP3, where IP3 releases Ca2+ from intracellular stores. The other TrkB

phosphorylation site is Tyr515 that activates downstream MEK-MAPK/Erk signaling,

which promotes neuronal differentiation and growth by influencing transcription events,

such as activation of CREB transcription factor. Tyr 516 indirectly activates PI3K

pathway by interacting with adaptor proteins that regulate PI3K pathway. Another

pathway that is indirectly activated by Phospho-TrkB (Y516) is the PI3K-Akt pathway.

2.3.4 Role of BDNF-TrkB signaling in neuronal development

Neurotrophins cooperate with neuronal activity to modulate neuronal morphology.

Studies in the past few years have shown convincing evidence for the involvement of

BDNF-TrkB signaling in activity-dependent dendritic growth, dendric arborization,

synaptogenesis and synaptic transmission 399 Reichardt,L.F. 2006415 Huang,E.J.

2003; 414 Yoshii,A. 2007. BDNF-TrkB mediates increase in dendritic complexity by

increasing the total dendritic/neurite length, dendritic branching (arborization) and

increase in number of primary neuritis 295 Baker,R.E. 1998; 275 McAllister,A.K.

1996. Activation of NMDARs and intracellular Ca2+ rise is critical for the expression

and release of BDNF399 Reichardt,L.F. 2006. The released BDNF binds to its

cognate receptors TrkB and acts in an autocrine/paracrine mode to regulate neuronal

2-51

development. Through BDNF-TrkB-PI3K-Akt pathway, Akt can subsequently

phosphorylate a number of substrates (GSK3β, MAP2, mTOR) that are involved in cell

survival, NOG/actin dynamics, dendritogenesis and synaptic plasticity 410 Read,D.E.

2009; 414 Yoshii,A. 2007.

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Table 1: Location and pharmacological properties of various VGSCs isoforms.

Channel name Location Pharmacological significance Nav1.1 Primarily in the cell

bodies of the central neurons; cardiac myocytes

Site of action for epileptic drugs; Site for local anesthetics that enter circulation and Cerebro spinal fluid (CSF) and thereby causing side effects.

Nav1.2 Primarily in the myelinated and premyelinated axons of central neurons of the central neurons

Site of action for epileptic drugs; Site for local anesthetics that enter circulation and CSF and thereby causing side effects.

Nav1.3 Cell bodies of central neurons in embryonic and prenatal life: Cardiac myocytes

Site of action for epileptic drugs; Site for local anesthetics that enter circulation and CSF and thereby causing side effects.

Nav1.4 Skeletal muscle Site for local anesthetics treating myotonia

Nav1.5 Cardiac myocytes; immature and denervated skeletal muscle; Some neurons in the brain

Site for antiarrhythmic drugs; Site for local anesthetics that enter circulation and thereby causing side effects.

Nav1.6 Cerebellum, cerebral cortex, brainstem, spinal cord; DRG; nodes of Ranvier in PNS and CNS.

Target for anti epileptic drugs and analgesics.

Nav1.7 DRG neurons, Schwann cells, sympathetic neurons; neuroendocrine cells

Site for local anesthetics in PNS.

Nav1.8 Small and medium sized DRG neurons and axons.

Target for Analgesic drugs

Nav1.9 Mostly in the nociceptive DRG neurons (c-type); trigeminal neurons

Target for Analgesic drugs

2-53

Table 2: Receptor sites on VGSCs (modified from Catterall et al., Pharmacol Rev. 2005)

Receptor Site Toxin or Drug Domains

Neurotoxin receptor site 1 Tetrodotoxin IS2–S6, IIS2–S6

Saxitoxin IIIS2–S6, IVS2–S6

µ-Conotoxin

Neurotoxin receptor site 2 Veratridine IS6, IVS6

Batrachotoxin

Grayanotoxin

Neurotoxin receptor site 3 α -Scorpion toxins IS5–IS6, IVS3–S4

Sea anemone toxins IVS5–S6

Neurotoxin receptor site 4 β-Scorpion toxins IIS1–S2, IIS3–S4

Neurotoxin receptor site 5 Brevetoxins IS6, IVS5

Ciguatoxins

Neurotoxin receptor site 6 δ -Conotoxins IVS3–S4

Local anesthetic receptor site Local anesthetic drugs IS6, IIIS6, IVS6

Antiarrhythmic drugs

Pyrethroid insecticide receptor site

Deltamethrin and other pyrethroids Unknown site

2-54

Figure 2-1: Subunit structure of VGSCs.

( Source: Yu & Catterall, 2003, Genome Biology 2003, 4:207)

Figure 2-2: A) Voltage dependent activation: outward movement of S4 Voltage sensors

(Source: Yu & Catterall, 2003, Genome Biology)

A

2-55

B) Voltage sensor trapping model of β scorpion toxin action. (Source: Catterall WA,

2002, Nov Found symp.)

Figure 2-3: Intracellular NA+ may act as a signalinging molecule and upregulate

NMDARs. (Courtesy Yu XM, 2006)

2-56

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3 CHAPTER 3 - Antillatoxin, a Novel Lipopeptide, Enhances

Neurite Outgrowth in Immature Cerebrocortical Neurons

Through Activation of Voltage–Gated Sodium Channels

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3.1 Abstract

Antillatoxin (ATX) is a structurally novel lipopeptide that activates voltage-gated

sodium channels (VGSC) leading to sodium influx in cerebellar granule neurons and DIV

8-9 cerebrocortical neurons (Cao et al., 2008; Li et al., 2001). The precise recognition site

for ATX on the VGSC, however, remains to be defined. Inasmuch as elevation of

cytoplasmic sodium may increase N-methyl-D-aspartate receptor (NMDAR) mediated

Ca2+ influx, intracellular sodium [Na+]i may function as a signaling molecule. Here we

hypothesized that ATX may enhance neurite outgrowth in cerebrocortical neurons by

elevating [Na+]i and augumenting NMDAR function. ATX concentrations of 30-100 nM

robustly stimulated neurite outgrowth and this enhancement was sensitive to the VGSC

antagonist, tetrodotoxin. To unambiguously demonstrate the enhancement of NMDA

receptor function by ATX, we recorded single-channel currents from cell-attached

patches. ATX was found to increase the open probability of NMDA receptors. Na+

dependent upregulation of NMDAR function has been shown to be regulated by Src

family kinase (SFK) (Yu and Salter, 1998). The Src kinase inhibitor PP2 abrogated

ATX-enhanced neurite outgrowth suggesting a SFK involvement in this response. ATX-

enhanced neurite outgrowth was also inhibited by the NMDAR antagonist, MK-801, and

the calmodulin dependent kinase kinase (CaMKK) inhibitor, STO-609, demonstrating the

requirement for NMDAR activation with subsequent downstream engagement of the Ca2+

dependent CaMKK pathway. These results with the structurally and mechanistically

novel natural product, ATX, confirm and generalize our earlier results with a neurotoxin

site 5 ligand. These data suggest that VGSC activators may represent a novel

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pharmacological strategy to regulate neuronal plasticity through NMDAR-dependent

mechanisms.

Antillatoxin (ATX) is a structurally novel lipopeptide with an exceptionally high

degree of methylation unlike any known natural product (Lee and Loh, 2006). Isolated

from the cyanobacterium Lyngbya majuscula, this compound is also distinguished by

multiple stereocenters (Orjala et al., 1995). The essential role of the asymmetric carbon

atoms in ATX is reflected in the stereoselective effects of ATX enantiomers (Li et al.,

2004). ATX is considered to be the second most potent ichthyotoxic compound obtained

from marine sources following only brevetoxin (PbTx-1) (Perez-Otano and Ehlers, 2005).

Exposure to L. majuscula blooms are associated with adverse human health effects,

including respiratory irritation, eye inflammation and severe contact dermatitis. Previous

work has further demonstrated that ATX is a potent activator of voltage-gated sodium

channels (VGSCs) that elevates intracellular Na+ concentration ([Na+]i) in intact neurons

(Cao et al., 2008; Li et al., 2001). ATX has moreover been shown to be neurotoxic in

cerebellar granule cells (CGCs) through an indirect activation of N-methyl-D-aspartate

receptors (NMDARs) as a consequence of glutamate release (Li et al., 2001; Li et al.,

2004).

Regulation of [Na+]i plays a critical role in the nervous system, not only because

Na+ influx through VGSCs is responsible for the initiation (the rising phase) and

propagation of action potentials, but also because various neuronal cell functions such as

intracellular pH, Ca2+ homeostasis and reuptake of neurotransmitters are dependent on

the Na+ gradient. Previous studies have further indicated that intracellular Na+ can also

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act as a signaling molecule to modulate cell functions including, but not restricted to, cell

proliferation, ion channel permeability, G-protein function and opioid ligand-receptor

interactions (Yu, 2006). Recent studies have moreover demonstrated that neuronal

activity mediated increases in [Na+]i in structures including soma, dendrites and spines

may act as a signaling molecule and contribute to activity-dependent synaptic plasticity

(Rose and Konnerth, 2001). In cerebellar Purkinje neurons, AMPA receptor mediated

Na+ influx was shown to be required for induction of LTD (Linden et al., 1993). In both

hippocampal and immature cerebrocortical neurons an elevation in intracellular Na+ was

found to increase NMDAR-mediated whole-cell currents and NMDAR single channel

activity by increasing both channel open probability and mean open time (George et al.,

2009; Yu and Salter, 1998). This [Na+]i mediated upregulation of NMDAR function has

been shown to require Src kinase activation (George et al., 2009; Yu and Salter, 1998).

Src family kinases act as a crucial point of convergence for signaling pathways that

enhance NMDAR activity, and, by upregulating the function of NMDARs, Src gates the

production of NMDAR-dependent synaptic potentiation and plasticity (Salter and Kalia,

2004).

Neuronal activity has a major role in the development of dendritic complexity and

neuronal circuits. The mechanisms by which neuronal activity translate into

morphological changes are complex. Numerous studies have shown that activity-

dependent neuronal development involves various calcium influx pathways mediated by

ionotropic glutamate receptors (mainly NMDAR) and voltage-gated Ca2+ channels

(VGCCs) (Ghosh and Greenberg, 1995; West et al., 2002). Intracellular calcium acts as a

signaling molecule largely through the binding to calmodulin, a calcium binding protein

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that engages downstream Ca2+/calmodulin dependent protein kinase (CaMK) and

mitogen activated protein kinase (MAPK) signaling pathways (Ghosh and Greenberg,

1995; West et al., 2002). CaMK kinase (CaMKK) has been demonstrated to be an

upstream regulator of both CaMK- and MAPK-signaling pathways. Moreover, previous

studies have demonstrated that activity-dependent neurite outgrowth (Wayman et al.,

2006) and synaptogenesis (Saneyoshi et al., 2008) are regulated by NMDAR-dependent

CaMKK/calmodulin kinase I-signaling cascades. NMDARs therefore play a critical role

in activity-dependent development and plasticity (Ghosh and Greenberg, 1995), dendritic

arborization (Miller and Kaplan, 2003; Wayman et al., 2006; Wong and Ghosh, 2002),

spine morphogenesis (Ultanir et al., 2007) and synapse formation (Saneyoshi et al., 2008)

by stimulating these calcium-dependent signaling pathways.

Inasmuch as neuronal activity induced increments in cytoplasmic sodium may

augment NMDAR mediated currents, we reasoned that intracellular Na+ may function as

a signaling molecule and regulate neuritogenesis in immature cerebrocortical neurons.

We have recently demonstrated that brevetoxin (PbTx-2), a VGSC activator, enhanced

NMDAR function and augumented neurite outgrowth (George et al., 2009). In the

present study we extend our earlier work to demonstrate that these pharmacologic actions

of the neurotoxin site 5 ligand brevetoxin generalize to the structurally and

mechanistically novel VGSC activator, ATX. We found that ATX promoted

neuritogenisis by elevating [Na+]i which in turn augumented NMDAR function leading to

Ca2+ influx and engagement of a CaMKK pathway. These data provide further support

for the hypothesis that sodium channel activators appear capable of mimicking activity-

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dependent neuronal development through potentiation of NMDAR signaling pathways

that influence neuronal plasticity.

3.2 Experimental Procedures

Cerebrocortical neuron culture- Primary cultures of cerebrocortical neurons were

harvested from Swiss–Webster mice on embryonic day 16 and cultured as described

previously (Cao et al., 2008). Cells were plated onto poly-L-lysine -coated (Sigma) 96-

well (9 mm), clear-bottomed, black-well culture plates (Costar) at a density of 1.8 x 106

cells per mL (150 µL per well) , 24-well (15.6 mm) culture plates at a density of 0.05 x

106 cells per ml (0.5 mL per well), or 6-well (35 mm) culture dishes at a density of 2.25 x

106 cell per ml (2 ml per well), respectively, and incubated at 37°C in a 5% CO2 and 95%

humidity atmosphere. All animal use protocols were approved by the Creighton

University Institutional Animal Care and Use Committee.

Immunocytochemistry and determination of total neurite length- Cells were plated

on poly-lysine coated 12 or 15 mm glass coverslips (Fisher Scientific), and placed inside

of 24-well culture plates at a low density of 0.05 x 106 cells per ml (0.5 mL per well).

To assess the influence of ATX on neuritogenesis, primary cultures of immature

cerebrocortical neurons were exposed to various concentrations of ATX ranging from 1-

1,000 nM for 24 h beginning three hours post plating and total neurite outgrowth was

measured. In some experiments these concentrations of ATX were coincubated with

tetrodotoxin (TTX) (1 µM) (Biomol), MK-801 (1 µM) (Sigma), nifedipine (1 µM)

(Sigma), 1,8-naphthoylene benzimidazole-3-carboxylic acid (STO-609) (2.6 µM)

(Calbiochem), 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3, 4-d] pyrimidine (PP2)

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or 4-amino-7-phenylpyrazol [3, 4-d] pyrimidine (PP3) (Calbiochem). At 24 h post

plating, cultures were fixed at room temperature for 20 min using 4% paraformaldehyde

in PBS. Post-fixation, neurons were blocked and permeabilized by incubating for 30 min

with PBS containing 2% fetal bovine serum (Atlanta Biologicals) and 0.15% Triton X-

100 (Sigma). The coverslips were incubated overnight at 4°C with protein gene product

9.5 (anti-PGP 9.5) primary antibody (AbD SeroTec). After washing 3X in blocking

buffer, coverslips were incubated with a secondary antibody [FITC (anti-rabbit IgG)]

(Jackson ImmunoResearch Laboratories) for 60 min at room temperature. Coverslips

were washed and mounted on microscope slides and analyzed by fluorescence

microscopy on an Olympus IX 71 inverted microscope with a Nikon camera. Digital

images of individual neurons were captured and total neurite length quantified using

Image Pro plus (Media Cybernetics). To reduce the effect of paracrine neurotrophic

factors on neurite growth, only those neurons that were separated from surrounding cells

by approximately 150 µm were digitally acquired and analysed. At least 25 randomly

chosen neurons from different cultures were evaluated for each treatment group.

Diolistic labeling- The Helios Gene Gun System (Bio-Rad, Hercules, CA) was used to

deliver DiI coated tungsten particles (1.3 µM) (Bio-Rad) into paraformaldehyde fixed

DIV-1 cerebrocortical neurons. Diolistic bullet preparation was based on the method of

O`Brien and Lummis (O'Brien and Lummis, 2006). Briefly, 2.5-3.5 mg of DiI

(Invitrogen) was suspended in 200 µl of dichloromethane (Sigma). The dissolved dye

was added over evenly spread 35 mg tungsten particles placed on a clean glass slide, and

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then allowed to dry. The dye coated particles were scraped onto another clean glass slide

and chopped to fine particles using a clean razor blade and later re-suspended in 3 ml

deionized water. This dye slurry was sonicated for 10 minutes and then vortex briefly to

form a uniform suspension. After adding 100 µl of polyvinylpyrrolidone (PVP) stock

solution (0.96 % PVP in ethanol) to the dye slurry, it was drawn into a PVP pre-coated

tefzel tubing mounted on a preparation station (Bio-Rad) using a 5-10 ml syringe. The

dye particles were allowed to settle for 20-30 minutes and then the supernatant water was

carefully withdrawn from tefzel tubing using a syringe. The tubing was rotated for 1-2

min to uniformly spread the particles. The tubing was then allowed to dry for 5 min

before cutting into bullets using a tube cutter. The DIV-1 cerebrocortical neurons grown

on cover slips were shot post-fixation (1.5 % paraformaldehyde) using DiI bullets loaded

onto a Helios gene gun at 140-160 psi of helium pressure from a distance of 2.5 cm. The

dye particles were allowed to spread across the neuronal membrane overnight and cover

slips were then mounted for imaging.

Intracellular sodium concentration ([Na+]i) measurement- [Na+]i measurement and

full in situ calibration of SBFI fluorescence ratio were performed as described previously

(Cao et al., 2008). Cells grown in 96-well plates were washed four times with Locke's

buffer (in mM: 8.6 HEPES, 5.6 KCl, 154 NaCl, 5.6 glucose, 1.0 MgCl2, 2.3 CaCl2, 0.1

glycine, pH 7.4) using an automated microplate washer (Bio-Tek Instruments). After

measuring the background fluorescence of each well, cells were incubated for 1 h at 37°C

with dye-loading buffer (100 µl/well) containing 10 µM SBFI-AM (Invitrogen) and

0.02% pluronic F-127 (Invitrogen). Cells were then washed 5X with Locke's buffer

leaving a final volume of 120 µl in each well. The plate was then transferred back to the

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incubator for 15 min to allow the cells to equilibrate following washing and then placed

in a FlexStation II (Molecular Devices) chamber to detect Na+- bound SBFI emission at

505 nm (cells were excited at 340 and 380 nm). Fluorescence readings were taken once

every 5 s for 60 s to establish the baseline, and then 40 µl ATX was added to each well

from the compound plate at a rate of 26 µl/s, yielding a final volume of 160 µl/well. After

correcting for background fluorescence, SBFI fluorescence ratios (340/380) versus time

were analyzed, and time- or concentration-response graphs were generated using

GraphPad Prism (GraphPad Software).

Full in situ calibration of the SBFI fluorescence ratio was performed using

calibration media containing the following (in mM): 0.6 MgCl2, 0.5 CaCl2, 10 HEPES,

Na+ and K+ such that [Na+] plus [K+] = 130, 100 gluconate, and 30 Cl- (titrated with 10

mol/l KOH to pH 7.4). Gramicidin D (5 µM) (Na+ ionophore), monensin (10 µM)

(Na+/H+ carrier), and ouabain (100 µM) (Na+/K+-ATPase inhibitor) to equilibrate the

intracellular and extracellular sodium concentration. After five washes, Locke's buffer

was replaced by 150 µl sodium-containing calibration solution (0-130 mM). The plate

was then loaded onto the FlexStation chamber for recording of emitted fluorescence

during excitation at 340 and 380 nm. Fluorescence data were converted to a ratio

(340/380) after background correction. To convert the fluorescence ratio of emitted SBFI

signals into a [Na+]i value, the following equation was used: [Na+] = βKd [(R – Rmin)/(Rmax

– R)] (equation 1), where β is the ratio of the fluorescence of the free (unbound) dye to

bound dye at the second excitation wavelength (380 nm), Kd is the apparent dissociation

constant of SBFI for Na+, R is the background-subtracted SBFI fluorescence ratio, and

Rmin and Rmax are, respectively, the minimum and maximum fluorescence values. Data

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relating [Na+]i to R were fitted by a three-parameter hyperbolic equation having the

following form: R = Rmin + [a ([Na+])/(b + [Na+])] (equation 2), where a and b are

constants and equal to Rmax – Rmin and βKd, respectively (2, 33). These data relating

[Na+]i to R (see Fig. 6B) were well described (r2 = 0.98) by Equation 2. The derived

parameters were Rmin = 1.47 ± 0.03, a = 3.541 ± 0.11, and b = 63.30 ± 4.93. The value for

Rmin obtained by this method was similar to the value of Rmin derived experimentally at

[Na+] = 0 mM. The corresponding values for Rmax and βKd were, therefore, Rmax = 5.01 ±

0.13 and βKd = 63.30 ± 4.93 mM. We compared the values of Rmax and βKd obtained from

a Hanes plot (Cao et al., 2008) to those derived from the three-parameter hyperbolic fit.

The equation was rearranged to generate a Hanes plot such that [Na+]/(R – Rmin) =

[Na+]/(Rmax – Rmin) + [βKd/(Rmax – Rmin)] (equation 3).

The plotting of [Na+]/(R – Rmin) versus [Na+]i as a Hanes function yielded a

straight line (r2 = 1) (data not shown). The slope 1/(Rmax – Rmin) of this regression

provides a means to estimate of Rmax, whereas the intercept on the abscissa is equal to –

βKd. The value for Rmin was obtained from the experimental data. The values of Rmax and

βKd calculated from Hanes plot were 4.97 ± 0.10 and 63.3 ± 1.9 mM, respectively, and

were therefore not significantly different from the values derived from the three-

parameter hyperbolic fit which were 5.01 ± 0.13 (Rmax) and 63.3 ± 4.93 mM (βKd).

Intracellular Ca2+ monitoring- DIV-1 cerebrocortical neurons grown in 96-well plates

were used for intracellular Ca2+ concentration ([Ca2+]i) measurements as described

previously (George et al., 2009). Briefly, the growth medium was removed and replaced

with dye-loading medium (100 µl per well) containing 8 µM fluo-3 AM (Invitrogen) and

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0.04% pluronic acid in Locke's buffer. After 1 h of incubation in dye-loading medium, the

neurons were washed four times in fresh Locke's buffer (200 µl per well, 22°C) using an

automated microplate washer (Bio-Tek Instruments) and transferred to a FlexStation II

benchtop scanning fluorometer chamber. The final volume of Locke's buffer in each well

was 120 µl. Fluorescence measurements were performed at 37°C. The neurons were

excited at 488 nm and Ca2+-bound fluo-3 emission was recorded at 538 nm at 1.2 s

intervals. After recording baseline fluorescence for 27 s, 40 µl of a 4X concentration of

ATX in the presence or absence of either PP2, PP3, nifedipine or MK 801 were added to

the cells at a rate of 26 µl/s yielding a final volume of 160 µl/well; the fluorescence was

monitored for an additional 220–270 s. The fluo-3 fluorescence was expressed as (Fmax –

Fmin)/Fmin where Fmax is the maximum, and Fmin the minimum fluorescence measured in

each well.

Western blotting- Western blot analysis was performed using cells grown in 6-well

plates. Three hours post platting, cells exposed to 30 nM ATX for time periods ranging

from 0 to 24 h at 37ºC. At the end of each time period cultures were transferred onto an

ice slurry to terminate drug exposure and washed 3X with ice-cold PBS. Cells were

lysed using ice-cold lysis buffer (50 mM Tris, 50 mM NaCl, 2 mM EDTA, 2 mM EGTA,

1% NP-40, 0.1% SDS, 2.5 mM sodium pyrophosphate, and 1 mM sodium

orthovanadate). Phenylmethylsulfonyl fluoride (1 mM) and 1X protease inhibitor

mixture (Sigma) were then added and the lysate incubated for 30 min at 4°C. Cell lysates

were sonicated and then centrifuged at 13,000 x g for 15 min at 4°C. The supernatant was

assayed by the Bradford method to determine protein content. Equal amounts of protein

were mixed with the Laemmli sample buffer and heated for 5 min at 75°C. The samples

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were loaded onto a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane and

immunoblotted with anti-phospho Src (416) and total Src antibodies (Cell Signaling

Technology). Blots were developed with ECL Plus kit (GE Healthcare) for 3 min. Blots

were subsequently stripped (63 mM Tris base, 70 mM SDS, 0.0007% 2-mercaptoethanol,

pH 6.8) and reprobed for further use. Western blot densitometry data were obtained using

MCID Basic 7.0 software (Imaging Research).

Membrane potential assay fluorescence monitoring- Membrane potential in the

cerebrocortical neuron cultures was determined using the FLIPR membrane potential

(FMP) assay (Molecular Devices) as previously described (George et al., 2009). FMP

blue dye was used to assess the membrane potential of neurons in culture. Quantification

of changes in membrane potential was derived using KCl as a reference. In preliminary

experiments we determined that, with cerebrocortical neurons in culture, the optimum dye

concentration was one-eighth of that suggested by the manufacturer. After removing the

culture medium, 180 µl of assay buffer was added to the neurons, and the plate was

incubated at 37°C in a 5% CO2 and 95% humidity atmosphere for 30 min. For KCl

calibration measurements, varying concentrations of 10X KCl standard solutions in assay

buffer were prepared. After 30 min equilibration incubation, the plate was transferred to a

FlexStation II chamber, and the fluorescence measurements were performed at 37°C.

Neurons were excited at 530 nm, and emission was recorded at 565 nm at 2 s intervals.

After recording the baseline for 60 s, either 20 µl KCl or ATX was added to a final

volume of 200 µl at a rate of 26 µl/s, and the fluorescence was monitored for an

additional 240 s.

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A linear regression analysis of the log [K+] versus FMP blue fluorescence change

(F-F0) was generated. We used the Goldman–Hodgkin–Katz equation to generate a

standard curve for the estimation of membrane potential (EM) at various concentrations of

extracellular K+:

, ,

[ ] [ ] [ ], ln( )

[ ] [ ] [ ]out out inNa K Cl

m K Na Clin in outNa K Cl

P Na P K P ClRTEF P Na P K P Cl

+ + −

+ + −

+ + −+

+ + −

+=

+ + (equation 4)

where EM is membrane potential, R is universal gas constant, T is temperature using the

Kelvin scale, and PK, PNa, and PCl are permeabilities for K+, Na+, and Cl–, respectively.

[K+]out, [Na+]out, and [Cl–]out, and [K+]in, [Na+]in, and [Cl–]in are the respective extracellular

and intracellular concentrations of K+, Na+, and Cl–. A 1 d in vitro (DIV-1) neuronal [Cl–

]in value of 140 mM was used for these calculations(35). The regression for the [K+]out

versus Δfluorescence and EM was used for estimating ATX-induced change in membrane

potential.

Electrophysiology- Single-channel currents were recorded at 23°C in the cell-attached

configuration (Hamill et al., 1981). Patch pipettes were pulled from borosilicate glass

capillaries (Warner Instruments), coated with Sylgard 184 (Dow Corning) and fire-

polished to a resistance of 10–15 MΩ when filled with the pipette solution. The external

recording solution consisted of Mg2+-free Locke's buffer with 20 µM EDTA to chelate

trace amounts of divalent cations. ATX was always bath-applied. The patch pipette

solution consisted of extracellular Locke's buffer without MgCl2 and with 100 µM

NMDA and 100 µM glycine. In some experiments, 10 µM strychnine, 10 µM bicuculline

methiodide and 10 µM DNQX were included in the external solution to block nonspecific

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components. All recordings were done from DIV-1 cerebrocortical neurons. Cell-attached

patch recordings were done using an Axopatch 200B amplifier (Molecular Devices),

filtered at 8 kHz (–3 dB, 8-pole Bessel), and digitized at 40 kHz digitized with Axon

pClamp 10.2 software. The pipette potential was +60 mV. Records were idealized with a

segmental k-means algorithm (Qin, 2004) using QUB software (www.qub.buffalo.edu).

All conductance levels were assumed to be equal for the analysis. Dwell-time histograms

were generated and fitted using Channelab (Synaptosoft) with an imposed dead time of 50

µs. The open probability (Po), mean open time, and amplitude were compared by paired t

test. For representation in figures, the Po and mean open time were normalized to the

average of respective control values. The corresponding ATX-treated values were

normalized to their paired control values.

3.3 Results

Antillatoxin is a VGSC activator in immature cerebrocortical neurons. In previous

reports we demonstrated that ATX is an activator of VGSCs in cerebellar granule

neurons (Li et al., 2001) and mature (DIV-9) cerebrocortical neurons (Cao et al., 2008).

We therefore sought to determine whether immature cerebrocortical neurons were also

sensitive to ATX-induced elevation of [Na+]i. We assessed ATX-induced elevation of

[Na+]i in DIV-1 cerebrocortical neurons loaded with SBFI. As shown in Figure 1A & B,

30 nM ATX elevated [Na+]i in DIV-1 cerebrocortical neurons. To confirm that the

observed Na+ influx was mediated by VGSCs, we tested the influence of TTX, a selective

antagonist of VGSCs, on the response to ATX. Pretreatment of DIV-1 cerebrocortical

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neurons with TTX (1 μM) abolished ATX-induced Na+ influx. These results indicate that

ATX is an activator of VGSCs in DIV-1 cerebrocortical neurons.

Antillatoxin enhances neurite outgrowth in immature cerebrocortical neurons. We next

wanted to determine the influence of ATX on neuritogenesis in immature cerebrocortical

neurons. Three hours post plating, primary cultures of immature cerebrocortical neurons

were exposed to various concentrations of ATX ranging from 1-1,000 nM for 24 h and

total neurite outgrowth was then assessed. Either immunostaining of PGP 9.5 or diolistic

labeling were used to visualize neurons and determine the influence of ATX on neurite

outgrowth (Fig. 2A). ATX significantly enhanced total neurite outgrowth in immature

cerebrocortical neurons with concentrations of 30 and 100 nM producing a robust >2-fold

increase in total neurite length. As previously observed with PbTx-2, the ATX

concentration-response profile was bidirectional, or hormetic (Fig 2B).

Antillatoxin-induced neurite outgrowth is mediated by VGSCs. Given that ATX is a

VGSC activator with the ability to augment neurite outgrowth, we wanted to confirm the

involvement of VGSCs in the latter functional response. DIV-1 cerebrocortical neurons

were coincubated in the presence or absence of TTX (1 µM) and 30 nM ATX for 24 h

and total neurite length was determined. Consistent with the involvement of VGSCs,

TTX completely abolished ATX- induced neurite outgrowth (control, 109.5 ± 22.5 μm;

TTX, 153.23 ± 8.4 μm; ATX, 236.65 ± 17.9 μm; TTX plus ATX, 115.3 ± 11.8 μm) (Fig

3A, B).

Antillatoxin-induced neurite outgrowth involves NMDARs, VGCCs and the Ca2+

dependent CaMKK pathway. Inasmuch as previous studies have indicated that activity-

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dependent neuritogenesis and neuronal development involve Ca2+ influx pathways

through NMDAR and voltage-gated calcium channels (VGCC) with subsequent

engagement of a CaMKK pathway (Konur and Ghosh, 2005; Wayman et al., 2006), we

assessed the role of this signaling cascade in ATX-induced neurite outgrowth. Co-

incubation of MK-801 (1 µM), an uncompetitive antagonist of NMDAR with 30 nM

ATX abrogated ATX-enhanced neurite outgrowth in immature cerebrocortical neurons

(Fig 4A,B) (control, 109.5 ± 22.5 µm; ATX, 236.65 ± 17.9 µm; ATX plus MK-801, 113.8

± 9.8 µm) demonstrating that ATX-enhanced neurite outgrowth involves NMDARs. To

investigate the role of VGCCs in the response to ATX, we used the L-type calcium

channel blocker, nifedipine (1 µM). Nifedipine pretreatment partially reduced ATX-

stimulated neurite outgrowth (Fig 4A,B) (control, 109.5 ± 22.5 µm; ATX, 236.65 ± 17.9

µm; ATX plus nifedipine, 166.3 ± 13.45 µm) suggesting that VGCCs may play a role in

the response to ATX. Next we investigated the involvement of a downstream Ca2+

dependent CaMKK in ATX-induced stimulation of neurite outgrowth. CaMKK is an

important upstream activator of essential signaling mediators of activity-dependent

neurite outgrowth such as CaMK1, CaMKIV, and MAPKs. STO-609 (2.6 µM), a

selective CaMKK inhibitor (Tokumitsu et al., 2002), eliminated the stimulatory effect of

ATX on neurite outgrowth in immature cerebrocortical neurons (Fig 4A,B) (control,

109.5 ± 22.5 µm; ATX, 236.65 ± 17.9 µm; ATX plus STO-609, 120.4 ± 9.02 µm). This

observation suggests that a Ca2+-dependent CaMKK pathway contributes to the

stimulatory effects of ATX on neuritogenesis.

Antillatoxin-induced neurite outgrowth is mediated by Src family tyrosine kinase

activation. Activity-dependent neurite outgrowth involves upregulation of NMDAR

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function. As earlier studies (Salter and Kalia, 2004; Yu et al., 1997; Yu and Salter, 1998)

have shown that [Na+]i and activated Src family kinases (SFKs) upregulate NMDAR

function, we reasoned that Src family kinases may participate in ATX-enhanced neurite

outgrowth. Exposure of neurons to the Src family kinase inhibitor PP2 (2 µM), but not its

inactive congener PP3 (2 µM), eliminated the stimulatory effect of ATX on neurite

outgrowth. These findings establish a role for Src family kinases in ATX-induced

stimulation of neurite outgrowth (Fig 5A,B) (control, 118.8 ± 12.9 µm; ATX, 194.4 ±

17.2 µm; ATX plus PP2, 83.9 ± 9.7 µm; ATX plus PP3, 181.3 ± 15.3 µm). The catalytic

activity of Src kinase is controlled by phosphorylation and dephosphorylation events,

primarly that of Y416. Intermolecular autophosphorylation of Y416 stimulates Src

kinase activity by permitting access to its substrates and ligands (Yu et al., 1997). To

assess the ability of ATX to activate Src, we determined the phosphorylation of the Y416

residue using an anti-phospho-Y416 Src antibody. Immature cerebrocortical neurons

were exposed to 30 nM of ATX and cell lysates were collected at various time periods for

western blot analysis. These results revealed that 30 nM ATX produced a robust

activation of Src kinase as reflected in the sustained increase in the phosphorylation of

tyrosine 416 (Fig 5B,C). These findings indicate that ATX exposure produces an

activation of Src kinase that is temporally correlated with the stimulation of neurite

outgrowth.

Antillatoxin increases intracellular sodium levels in immature cerebrocortical neurons.

Given that the earlier studies of Yu and Salter (Yu et al., 1997; Yu and Salter, 1998; Yu,

2006) demonstrated that [Na+]i is a regulator of NMDAR-mediated signaling, it was

important to quantify the magnitude of ATX-induced elevation of [Na+]i in immature

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cerebrocortical neurons. SBFI, a sodium-sensitive fluorescent indicator, was used to

determine the influence of ATX on [Na+]i in DIV-1 cerebrocortical neurons. Full in situ

calibration was performed in DIV-1 cerebrocortical neurons to determine the relationship

between the ratiometric SBFI signal and [Na+]i (Cao et al., 2008; George et al., 2009).

Cells loaded with the SBFI were excited at 340 and 380 and the emitted fluorescence was

recorded at 505. The 340/380 emission ratio was calculated after background correction

(Fig. 6A). A three-parameter hyperbolic function adequately fit the calibration data

relating SBFI fluorescence ratio to [Na+]i (Fig. 6B). ATX produced a concentration-

dependent increase in [Na+]i (Fig. 6C) with an EC50 value of 114.2 nM (70.8 to 184.2

nM, 95% CI). The in situ SBFI calibration showed that basal [Na+]i in DIV-1

cerebrocortical neurons was 17.3 ± 0.37 mM, and ATX produced a maximum elevation

of 78.6 ± 6.9 mM (Fig. 6D). Since a 30 nM concentration of ATX was sufficient to

produce a robust increase in neurite outgrowth, it was important to quantify the [Na+]i

increment associated with this treatment. The 30 nM ATX treatment produced a

maximum [Na+]i of 26.1 ± 0.4 mM, representing an increment of 8.8 mM over basal.

Previous reports in hippocampal neurons suggested that an increment of [Na+]i of 10 mM

was sufficient to produce significant increases in NMDAR channel activity (Yu and

Salter, 1998; Yu, 2006). It has moreover been reported that increments of [Na+]i of >5

mM may represent a critical threshold required to regulate NMDAR-mediated Ca2+ influx

in primary cultures of hippocampal neurons (Xin et al., 2005). Consistent with these

findings, the increment of [Na+]i detected in immature cerebrocortical neurons appears

sufficient to upregulate NMDAR function.

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Antillatoxin-evoked change in membrane potential is inadequate to relieve the Mg2+

blockade of NMDARs. Given the evidence in support of the involvement of NMDARs in

ATX-induced stimulation of neuritogenesis, we considered mechanisms apart from the

increment of [Na+]i. The ability of ATX to engage NMDARs could be a consequence of

either the elevation of [Na+]i or neuronal depolarization with attendant relief of the Mg2+

block of NMDAR. To ascertain the magnitude of ATX-induced membrane

depolarization, we assessed membrane potential changes in DIV-1 cerebrocortical

neurons using the membrane-potential sensitive fluorescence dye, FMP blue.

As previously reported (George et al., 2009), FMP blue behaved as a Nernstian

fluorescent indicator of membrane potential in DIV-1 cerebrocortical neurons. This was

demonstrated by assessing the relationship between extracellular K+ concentration and

fluorescence intensity. Extracellular K+ produced a concentration-dependent increase in

maximum FMP blue fluorescence consistent with a depolarization-induced redistribution

of the lipophillic anion dye and attendant increase in fluorescence quantum efficiency

(Fig. 7A). As depicted in Fig. 7B, the regression analysis for K+ concentration-dependent

changes FMP blue fluorescence showed marked linear correlation (r2= 0.99). For a

Nernstian fluorescent indicator of membrane potential, the ratio of fluorescence inside to

the outside of the cell should be related to the membrane potential as described by the

Nernst equation (Ehrenberg et al., 1988). This prediction is based on the principal that the

membrane potential of isolated neurons is largely the result of the K+ diffusion potential

(Hille, 1992). We therefore used the Goldman-Hodgkin-Katz equation to generate a

standard curve for the estimation of membrane potential (EM) at various concentrations of

extracellular K+. The membrane potential of cerebrocortical neurons was dependent on

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the external concentration of K+ (Hille, 1992). The concordance of the [K+]out versus

membrane fluorescence and [K+]out versus EM regressions indicates that changes in

cerebrocortical neuron FMP blue fluorescence can be used to estimate membrane

potential. Therefore, the relationship between fluorescence change and EM depicted in

Fig. 7B was generated to determine ATX-induced changes in membrane potential of

cerebrocortical neurons. The resting membrane potential of DIV-1 cerebrocortical

neurons was found to be –29.6 mV. This is consistent with previous demonstrations of a

relatively depolarized resting membrane potential of immature neurons that later becomes

more hyperpolarized as neurons mature (Kim et al., 1995; Ramoa and McCormick,

1994). As shown in Figure 7C, ATX produced a rapid and concentration-dependent

increment in FMP blue fluorescence in DIV-1 cerebrocortical neurons. Nonlinear

regression analysis of the ATX concentration–response relationship yielded an EC50

value of 92.3 nM (63.6–136.8 nM, 95% CI) (Fig. 7D). Because the 30 nM concentration

of ATX was sufficient to elevate [Na+]i, it was important to assess the membrane potential

changes associated with this treatment. The 30 nM ATX treatment produced a transient

increase in FMP blue fluorescence that was equivalent to the fluorescence change

produced by an extracellular K+ concentration of 7.6 mM. The corresponding membrane

potential change was accordingly found to be negligible, representing only a 0.9 mV

depolarization (from -29.6 ± 0.01 to 28.7 ± 0.15 mV). This change in membrane potential

would, therefore, not be sufficient to influence the voltage-dependent Mg2+ block of

NMDARs (Mayer et al., 1984).

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Antillatoxin increases intracellular calcium levels ([Ca2+]i) in DIV 1 cerebrocortical

neurons. Previous studies have suggested that activity-dependent neuritogenesis and

neuronal development involves Ca2+- dependent signaling pathways through NMDAR

and VGCCs. Due to the finding that ATX-induced neurite outgrowth involved NMDARs

and VGCCs, we hypothesized that ATX exposure would produce Ca2+ influx in these

immature cerebrocortical neurons. To investigate this, cells loaded with fluo-3 were

exposed to various concentrations of ATX and [Ca2+]i was monitored. ATX produced

rapid and concentration-dependent increases in [Ca2+]i with even 30 nM ATX producing

a significant increase in calcium influx (Fig 8A).

To delineate the Ca2+ influx pathways triggered by ATX, the role of VGSCs, NMDARs

and VGCCs in DIV-1 cerebrocortical neurons were investigated. A pharmacologic

evaluation of the [Ca2+]i response to 100 nM ATX was performed. Cells were pretreated

with specific antagonists: TTX (VGSCs), MK-801 (NMDARs) or nifedipine (VGCCs) to

evaluate the role of these channels in ATX induced Ca2+ influx. TTX (1 µM) completely

blocked the response to ATX (data not shown), while MK-801 (1 µM) and nifedipine (1

µM) both significantly reduced ATX-induced Ca2+ influx (Fig 8B,C). Given the

previously demonstrated role of SFK activation in the upregulation of NMDAR function

and in ATX-induced neurite outgrowth, we examined the role of SFKs in ATX-induced

Ca2+ influx. PP2 (2 µM), a specific SFK family inhibitor, but not PP3 (2 µM), blocked

ATX stimulation of Ca2+ influx consistent with the involvement of a SFK in this response

(Fig 8D,E).

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Antillatoxin increases NMDA receptor single-channel open probability but not the mean

open time. To gain insight into the effect of ATX on single-channel properties of NMDA

receptors, unitary currents were recorded from DIV-1 cerebrocortical neurons. Cell-

attached patch recording was performed with 100 µM NMDA and 100 µM glycine in the

patch pipette at a patch potential of +60 mV. Experiments were performed in the nominal

absence of extracellular Mg2+ in the recording buffer supplemented with 20 µM EDTA to

chelate trace amounts of divalent ions. In the majority of patches, only single openings

were observed with no apparent simultaneous double openings. The absence of double

openings can be presumably attributed to the supposedly low expression of NMDA

receptors in immature cerebrocortical neurons. Patches in which we observed

simultaneous double openings were not further analyzed. Single-channel recordings were

idealized using the QUB and analyzed using ChanneLab with an imposed resolution of

50 µs. Bath application of 100 nM ATX significantly increased the open probability (Po)

of NMDA receptors from 0.0053 ± 0.002 under control conditions to 0.012 ± 0.004 (206

± 46% of control) after ATX (n = 6, p < 0.05, paired t test) (Fig. 9A,B) The mean open

time was not affected by ATX (1.862 ± 0.38 ms without ATX; 1.90 ± 0.38 ms, with

ATX-101 ± 4.5% of control) (n = 8, p < 0.05, paired t test) (Fig. 9B). ATX similarly did

not affect the amplitude of single-channel currents (data not shown). The composite open

and shut dwell-time histograms were generated and fitted using Channelab. The open

time histogram could be fitted by the sum of three exponential components with time

constants of 0.127 (27%), 1.584 (53%) and 3.5 (20%). The time constants after ATX

application were 0.091 (19%), 1.409 (70%) and 4.67 (11%). The composite shut time

histograms could be fitted by sum of five exponential components with time constants of

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0.7 (34%), 0.095 (17%), 20.8 (14%), 240 (22%), and 1090 (12%). The time constants

were similar after ATX application 0.78 (35%), 0.169 (23%), 11.0 (10%), 84 (16%), and

580 (16%), except that the duration of the longer shut time constants were reduced (Fig.

9C).

3.4 Discussion

ATX is a novel activator of VGSC; however, its precise recognition site on the channel

protein remains to be defined. The structure of ATX includes asymmetric carbon atoms

and the (4R,5R)-isomer is the naturally occurring compound. The (4R,5R)-isomer appears

in profile as an “L” shape with a hydrophobic interior and a cluster of hydrophilic groups

on the exterior of the macrocycle (Li et al., 2004). Thus the (4R,5R)-configuration is

important for creating a molecular topology that is recognized by the acceptor site on the

voltage-gated sodium channel alpha subunit.

We have previously shown that ATX allosterically enhances the specific binding

of [3H]batrachotoxin to intact cerebellar granule cells (Li et al., 2001) This effect of ATX

on [3H]batrachotoxin binding was synergistically augmented by brevetoxin. The strong

synergistic interaction of the ATX recognition site with neurotoxin site 5 suggests that

these sites may be topologically close and/or conformationally coupled. The results

obtained using [3H]batrachotoxin as a probe for sodium channel conformation allowed us

to exclude the interaction of ATX with neurotoxin sites 1, 2, 3, and 5 on VGSCs. Site 1

was ruled out because tetrodotoxin and saxitoxin bind to the outer vestibule of the pore of

the ion channel and allosterically inhibit the binding of [3H]batrachotoxin; this is an

effect that is antipodal to that of ATX. We were able to rule out sites 2 and 5 inasmuch as

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these sites display positive allosteric coupling to the ATX site. Neurotoxin receptor site 3,

the target for α-scorpion toxins and sea-anemone toxins, was excluded because ATX

enhanced [3H]batrachotoxin binding in the presence of a maximally effective

concentration of sea-anemone toxin. Although we cannot exclude an interaction of ATX

with neurotoxin site 4, the target for β-scorpion toxin, it is reasonable to posit that ATX

binds to a novel recognition domain on the α-subunit of the VGSC. The relatively small

lipotripeptide structure of ATX would not be restricted to an extracellular target, as is the

case for the scorpion toxins, which are composed of 60-65 amino acids. Given the unique

structure and mechanism of action of ATX, we sought to further characterize its

pharmacologic actions in cerebrocortical neurons.

We have earlier demonstrated that NMDA receptor function may be increased

through activation of VGSCs with attendant elevation of intracellular sodium in

cerebrocortical neurons (George et al., 2009). VGSC activators function as gating

modifiers that elevate [Na+]i in the absence of substantial depolarization of neurons (Cao

et al., 2008; George et al., 2009). These findings have been confirmed and extended in

the present study by demonstrating that the structurally novel lipopeptide, ATX, elevates

intracellular Na+, increases NMDAR function and enhances neurite outgrowth in DIV-1

cerebrocortical neurons. These findings in DIV-1 murine cerebrocortical cultures provide

compelling evidence in support of a role for [Na+]i in activity-dependent processes of

neuronal development.

Antillatoxin enhances neurite outgrowth- Here we found that ATX enhanced

neurite outgrowth in DIV-1 cerebrocortical neurons as a result of elevation of

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cytoplasmic [Na+], potentiation of NMDAR function and stimulation of calcium influx.

ATX enhanced total neurite outgrowth in immature cerebrocortical neurons in a

bidirectional, or hormetic, concentration-response relationship with 30-100 nM producing

a robust increases of more than 2-fold (Fig. 2). Thus, the ability of ATX to augment

NMDAR channel activity translated into an enhancement of the trophic influence of

NMDAR on developing cerebrocortical neurons. Based on the premise that the effects of

neuronal activity on dendritic arbor growth and structural plasticity are primarily

mediated by engagement of NMDA receptors (Tolias et al., 2005), our results suggest

that ATX activation of sodium channels with attendant enhancement of NMDA receptor

signaling mimics the response to neuronal activity.

Antillatoxin concentration- response for neurite growth is an inverted-U- An

inverted-U model describes the relationship between NMDA receptor activity and

neuronal survival and growth (Lipton and Nakanishi, 1999). This inverted-U

concentration–response relationship has primarily, but not exclusively, been attributed to

[Ca2+]i regulation. An optimal window for [Ca2+]i is required for activity-dependent

neurite extension and branching, with lower levels stabilizing growth cones and higher

levels stalling them, in both cases preventing extension (Gomez and Spitzer, 2000; Hui et

al., 2007). Although the precise mechanism for the ATX bidirectional concentration-

response curve is not known, one plausible explanation is therefore related to the

involvement of NMDA receptors in the trophic response to ATX. Other potential

explanations for the inverted-U response include the possibility that high concentrations

of ATX might promote slow inactivation of VGSCs with attendant reduction in sodium

influx (Mitrovic et al., 2000). Alternatively, high concentrations of ATX could increase

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VGSC internalization, which has been shown to be a consequence of Na+ influx in

immature neuronal tissue. These results with ATX concur with those recently reported

for PbTx-2-induced stimulation of neuritogenesis in DIV-2 cerebrocortical neurons

(George et al., 2009). Although PbTx-2 is known to activate neurotoxin site 5 on VGSC

α-subunits, the molecular determinants for ATX on the VGSC remain to be defined (Li et

al., 2001).

ATX stimulated Ca2+ influx in cerebrocortical neurons through both NMDARs and

VGCCs. Ca2+-signaling pathways initiated by Ca2+ entry through L-type Ca2+ channels

and NMDA receptors have been shown to differ (Bading et al., 1993). Although MK-801

and nifedipine produced comparable reductions in ATX-induced Ca2+ influx, we found

that the NMDAR antagonist MK-801 completely blocked ATX-enhanced neurite

outgrowth whereas the L-type calcium channel blocker nifedipine produced only a partial

block of the stimulation of neurite outgrowth. These results suggest that Ca2+ which

enters neurons through NMDA receptors may have privileged access to the CaMKK and

CaMKI signaling elements that drive neuritogenesis.

In mature neurons a strong depolarizing stimulus (50 mM KCl) is required for the

engagement of L-type Ca2+ channels in dendritic growth and arborization, whereas a

smaller depolarizing stimulus (16 mM KCl) induced neurite outgrowth preferentially due

to Ca2+ influx through NMDARs (Redmond et al., 2002; Wayman et al., 2006). Our

observation that ATX concentrations of 30-100 nM provided a sufficient stimulus to

produce Ca2+ influx through VGCCs may be explained by the relatively depolarized

resting membrane potential of immature cerebrocortical neurons. The resting membrane

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potential of DIV-1 cerebrocortical neurons was found to be –29.6 mV and ATX (30-300

nM) produced modest changes of 1-5 mV. These modest changes in membrane potential

produced by ATX may however be sufficient to activate L-type Ca2+ channels given the

relatively depolarized resting membrane potential (Nowycky et al., 1985).

Regulatory influence of Na+ on NMDAR activity- Recent studies have shown that

intracellular Na+ might act as a signaling molecule. Based on the original work of

Hodgkin and Huxley (HODGKIN and HUXLEY, 1952) with squid axons, a single action

potential was calculated to minimally change the Na+ electrochemical gradient (Hille,

1992). The situation in mammalian neurons with fine axons, dendrites, and spines is,

however, much different, due to greater surface-to-volume ratios. Thus, a single action

potential may elevate [Na+]i substantially (Hille, 1992). Yu and Salter (Yu et al., 1997;

Yu and Salter, 1998) previously demonstrated that elevation of intracellular Na+ increases

NMDA receptor mediated whole-cell currents and NMDAR single channel current by

increasing the open probability and mean open time of the channel. An increment of

[Na+]i of 10 mM was sufficient to produce significant increases in NMDA receptor

single-channel activity. They used veratridine, a VGSC modulator to demonstrate that

Na+ influx through TTX-sensitive VGSC was sufficient to upregulate NMDAR activity.

Moreover, this Na+-dependent regulation of NMDA receptor function was shown to be

controlled by Src-induced phosphorylation of the receptor (Yu et al., 1997; Yu and Salter,

1998). These results were extended in the present study using the novel sodium channel

activator ATX as a probe to elevate intracellular Na+. Single channel current recording in

presence of ATX directly demonstrated the enhancement of NMDA receptor function. An

increase in intracellular Na+ and Src activation following exposure to ATX increased the

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open probability of the NMDAR. The shut time histogram with slow time constants

resemble NR2B-containing receptors (Erreger et al., 2005), consistent with the

expression of NR1/NR2B-containing receptors in immature neurons (Williams et al.,

1993). Given that the single-channel recordings were done in the absence of extracellular

Mg2+, these results additionally argue against relief of the voltage-dependent Mg2+ block

of the NMDAR in the actions of ATX. These data, therefore, confirm the regulatory

influence of Na+ on NMDAR channel activity in hippocampal neurons described

previously (Yu and Salter, 1998) and extend this relationship between [Na+]i and NMDA

receptor function to cerebrocortical neurons.

ATX represents a structurally and mechanistically novel activator of VGSCs whose

recognition domain on the α-subunit remains to be established (Li et al., 2001). Here we

found that ATX was capable of mimicking activity-dependent neuronal development by

upregulating NMDAR function. We propose a model for ATX-induced neuritogenisis

(Fig. 10) which involves direct activation of TTX-sensitive VGSCs, elevation of [Na+]i,

activation of a Src family kinase, potentiation of NMDAR function leading to Ca2+ influx

and engagement of a CaMKK pathway. We have recently reported another activator of

VGSCs, brevetoxin 2, is capable of upregulating NMDAR function and stimulating

neuritogenesis (George et al., 2009).

Structurally dissimilar sodium channel activators therefore appear capable of mimicking

activity-dependent structural plasticity by upregulating NMDA receptor signaling

pathways.

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Figure 3-1: ATX increases intracellular sodium levels in DIV-1 cerebrocortical neurons. A. Time-response curve for ATX stimulation of Na+ influx. This ATX-induced stimulation of Na+ influx was prevented by coapplication of 1 µM TTX. B. Histogram representing SBFI fluorescence ratio (340/380) values following the indicated treatments. Data shown are from an experiment performed in octuplicates. ***p < 0.001, unpaired t test.

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Figure 3-2: Effect of ATX on neurite outgrowth. A.Representative images of DiI-loaded immature cerebrocortical neurons at 24 h post plating (scale bar, 10 µm). Various concentrations of ATX were added to the culture medium at 3 h after plating. Depicted neurons were visualized by diolistic loading with DiI. B. Quantification of concentration-response effects of ATX on neurite outgrowth at 24 h post plating. ATX-enhanced neurite outgrowth displayed a hormetic concentration-response relationship with maximum enhancement seen at 30-100 nM ATX. Quantification of total neurite length was performed with Image Pro Plus.

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Experiment was performed twice and each point represents the mean value derived from analysis of 25-30 neurons. ***p < 0.001, unpaired t test.

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Figure 3-3. Effect of TTX on ATX-induced neurite outgrowth in immature cerebrocortical neurons. A. Representative images (scale bar, 10 µm) and (B) quantification of the effects of TTX on ATX-enhanced neurite outgrowth at 24 h post plating. Neurons were treated with 30 nM ATX in the presence and absence of 1 µM TTX beginning at 3 h post plating. Experiment was repeated twice and 25-30 neurons were quantified for each exposure condition. *p < 0.05, unpaired t test.

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Figure 3-4:Pharmacological evaluation of signaling pathways involved in ATX-enhanced neurite outgrowth. A. Representative images (scale bar, 10 µm) and (B) quantification of neurite extension at 24 h. The 30 nM ATX exposure was examined in the presence or absence of MK-801 (1 µM), nifedipine (1 µM) or STO-609 (2.6 µM) beginning at 3 h after plating. Experiment was repeated twice and 25-30 neurons were quantified for each exposure condition. ***p < 0.001, *p < 0.05, unpaired t test.

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Figure 3-5: ATX-induced neurite extension involves a Src Family kinase.

A. Representative images (scale bar, 10 µm) and (B) quantification of neurite extension at 24 h. Cerebrocortical neurons were treated with 30 nM ATX in the presence or absence of either 2 µM PP2 or PP3 beginning at 3 h after plating. Experiment was repeated four times and 20-30 neurons neurons were quantified for each exposure condition. ***p < 0.001, *p < 0.05, unpaired t test. C.Tyrosine phosphorylation (Y416) of Src kinase determined by immunoblotting. Cerebrocortical neurons were treated with 30 nM ATX beginning at 3 h post plating and P-Src (Y416) assessed at the indicated times. A representative blot is shown. The experiment was performed twice with independent cultures. Also depicted is the quantitative analysis of the relative band densities of immunoblots. Each bar represents mean ± SEM of two values.

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Figure 3-6: Quantification of ATX-induced increase of intracellular sodium levels in DIV-1 cerebrocortical neurons.

A. In situ calibration of SBFI fluorescence ratio (340/380). Time-response data show stepwise changes in SBFI fluorescence ratio values evoked by successive increments in extracellular sodium concentration. B. Three parameter hyperbolic fit adequately describes calibration data. C. Nonlinear regression analysis of the ATX concentration-response data (EC50 =114.2 nM; 70.8-184.1 nM 95% CI). Data represent the mean ± SEM of 2 separate experiments each with 2-5 replicates. The scale on the left ordinate represents the SBFI fluorescence ratio, while that on the right ordinate depicts the [Na+]i determined from the calibration curve shown in C. Addition of 30 nM ATX produced an 8.8 mM increment in [Na+]i over basal.

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Figure 3-7:ATX evoked change in membrane potential in DIV-1 cereobrocortical neurons.

A. Concentration-response profile for KCl-evoked FMP blue fluorescence change as a function of time. Each point represents the mean ± SEM of 3-9 values. B. The integrated time-response data for the increment in FMP blue fluorescence (Fmax-F0) plotted as a function of K+ concentration. The displayed regression and correlation coefficient (r2 = 0.995) were derived from linear regression analysis. The right ordinate scale shows membrane potential for each [K+] which was calculated using the Goldman-Hodgkin-Katz equation as described in materials and methods. The resting membrane potential was -29.6 mV. C. Time-response profiles for ATX-

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induced changes in membrane potential as determined by changes in FMP blue fluorescence. D. Nonlinear regression analysis of the integrated time-response data for the increment in FMP blue fluorescence (Fmax-F0) as a function of ATX concentration. The membrane potential values were determined by performing K+ calibration regressions in the same culture plate. The membrane potential change evoked by 30 nM ATX was 0.9 mV.

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Figure 3-8: ATX-induced Ca2+ influx and pharmacological evaluation in DIV-1 cerebrocortical neurons. A. Time-response profile of ATX-induced Ca2+ influx in fluo-3 loaded cerebrocortical neurons. Data shown are from a representative experiment performed with 2-5 replicates per point and repeated twice. ATX (30 nM) produced a significant increase in Ca2+

influx in these DIV-1 cerebrocortical neurons (inset). B. Pharmacological evaluation of ATX (100 nM) induced Ca2+ influx. Data are from representative experiment performed in triplicate and repeated twice. Cerebrocortical neurons were treated with either 1 µM MK-801 or 1 µM nifedipine before the addition of 100 nM ATX. C. Histogram representing quantification of the data shown in B. MK-801 and nifedipine significantly blocked ATX-induced calcium influx. D. Involvement of Src family kinase in ATX-induced Ca2+ influx. Data are from representative experiment performed in triplicate and repeated twice. Cerebrocortical neurons were treated with

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either 2 µM PP2 or 2 µM PP3 before the addition of 100 nM ATX. E, Histogram representing quantification of the data shown in D. **p < 0.005, *p < 0.05, unpaired t test

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Figure 3-9:. Increase in NMDA receptor channel open probability by ATX. A. Cell-attached patch recording from DIV-1 cerebrocortical neurons. NMDA receptor unitary currents were evoked by100 µM NMDA and 100 µM glycine in the patch pipette (pipette potential = +60 m V, filtered at 5 kHz for representation, digitized at 40 kHz). Enhancement of NMDA receptor activity by bath application of 100 nM ATX. B. Bath application of 100 nM ATX increased NMDA receptor channel open probability (Po), but not the mean open time (MOT) (n=6, *p<0.05, paired t test). C. Pooled dwell-time histograms were fitted using Channel lab. The open time histogram was fitted by the sum of three Gaussian components, and the shut time histogram was fitted by the sum of five Gaussian components. The time constants and area are described in Results.

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Figure 3-10:Schematic diagram of the pathways involved in ATX-induced neurite outgrowth

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4 CHAPTER 4 – Sodium channel

activator-stimulated neuronal

development involves BDNF-TrkB

signaling pathway.

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4.1 Abstract

N-methyl D-aspartate receptor (NMDAR) activation directly stimulates calcium

influx and leads to an increase in brain-derived neurotrophic factor (BDNF) – TrkB

signaling. This pathway is implicated in activity-dependent neuronal development and

synaptic plasticity. Voltage-gated sodium channel (VGSC) activators promote neuronal

development by increasing [Na+]i and upregulating NMDAR function. Here we tested the

effect of the VGSC activator veratridine (VRT) on neurite outgrowth (NOG), synthesis

and release of BDNF and TrkB activation in DIV1 cerebrocortical neurons. Primary

cultures of murine cerebrocortical neurons were prepared from E16-17 Swiss-Webster

mice. Diolistic loading of DiI and confocal imaging were performed to visualize and

image neurons respectively to assess total neurite outgrowth was determined. BDNF

ELISA was performed to determine BDNF synthesis and release (BDNF ELISA in situ).

Activation of various signaling molecules was determined by western blotting. VRT

enhanced NOG in a hormetic concentration-response manner, and this response was

dependent on NMDAR and TrkB signaling. Inhibitors of NMDAR, TrkB, PI3K, and PLC

inhibited VRT-enhanced NOG. Acute treatment with VRT stimulated phosphorylation

of TrkB and its downstream effectors Akt, mTOR, PLC, ERK1/2 and CREB. VRT

increased BDNF synthesis and release in a concentration dependent manner; however,

VRT stimulation of TrkB phosphorylation displayed a biphasic concentration-response

curve. VRT stimulation of BDNF synthesis required VGSCs and NMDARs. These data

suggest the influence of VGSC activators on neurite outgrowth may involve BDBF

synthesis and release.

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4.2 Introduction

Events that occur during neuronal development play a critical role in establishing

the morphological diversity and neuronal connectivity in brain. Dendritic arbor shape

determines the extent of neuronal connectivity and integration of synaptic signals: both

essential to the proper formation of neural circuits and proper function of nervous system.

In addition to intrinsic genetic programs, neuronal activity signals regulate neuronal

developmental events including neurogenesis, neurite outgrowth, dendritic arborization,

spinogenesis and synaptogenesis 452 Ben-Ari,Y. 2001; 206 McAllister,A.K. 2000; 1

Wong,R.O. 2002. Activity-dependent control of neuronal development primarily

involves calcium-dependent signaling and neurotrophins signaling. Activity-dependent

calcium signaling involves various calcium influx pathways including ionotropic

glutamate receptors (NMDAR) and voltage-gated Ca2+ channels (VGCCs) 254

Ghosh,A. 1995; 446 West,A.E. 2001; 32 West,A.E. 2002. Intracellular calcium acts as

a signaling molecule largely through the binding to calmodulin, a calcium-binding

protein that engages downstream Ca2+/calmodulin-dependent protein kinase (CaMK).

One such important CaMK is CaMKII, an important downstream regulator of dendritic

remodeling and synaptic activity 467 Fink,C.C. 2003; 468 Vaillant,A.R. 2002; 471

Zou,D.J. 1999; 469 Shen,K. 1998; 470 Wu,G.Y. 1998. Moreover, previous studies

have demonstrated that activity-dependent neurite outgrowth 99 Wayman,G.A. 2006

and synaptogenesis 98 Saneyoshi,T. 2008 are regulated by NMDAR-dependent

CaMKK/calmodulin kinase I-signaling cascades. Therefore, NMDARs play a critical role

in activity-dependent development and plasticity 254 Ghosh,A. 1995, dendritic

arborization 1 Wong,R.O. 2002; 84 Miller,F.D. 2003; 99 Wayman,G.A. 2006, spine

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morphogenesis 36 Ultanir,S.K. 2007, and synapse formation 98 Saneyoshi,T.

2008 by stimulating these calcium-dependent signaling pathways.

In addition to neuronal activity, numerous studies have implicated a role for

neurotrophins in dendritic development. Brain-derived neurotrophic factor (BDNF)

increase dendritic complexity of cortical pyramidal neurons by increasing total dendritic

length, the number of branch points and the number of primary dendrites 460

McAllister,A.K. 1995; 461 Dijkhuizen,P.A. 2005. Also, activity-dependent increases

in [Ca2+]i trigger a release of BDNF through the regulated pathway of BDNF release

456 Hartmann,M. 2001; 455 Goodman,L.J. 1996; 457 Nakajima,T. 2008; 331

Brigadski,T. 2005; 323 Kolarow,R. 2007; 458 Balkowiec,A. 2002 and mediates

activity-dependent dendritic development and synaptic plasticity455 Goodman,L.J.

1996; 456 Hartmann,M. 2001; 464 Ghosh,A. 1994; 463 Kohara,K. 2001; 462 Kohara,K.

2003. Changes in neuronal [Ca2+]i are due to influx either through NMDAR or VGCCs

or due to release from intracellular Ca2+ stores. The calcium activates CamKII leading to

the fusion of BDNF containing secretory granules with the plasma membrane and slow

release of BDNF into the extracellular milieu 331 Brigadski,T. 2005; 323 Kolarow,R.

2007. The effects of BDNF on dendritic morphology are due to the activation of

signaling mechanisms downstream of TrkB that influence neuronal development. Three

major intracellular signaling pathways are activated by BDNF binding to TrkB receptor.

They are: 1) the PI3K-Akt pathway, 2) the Ras-MAPK pathway and 3) the PLCγ-Ca2+

pathway 267 Reichardt,L.F. 2006.

Recent studies have demonstrated that neuronal activity-mediated increases in neuronal

[Na+]i augment NMDAR function and may contribute to activity-dependent synaptic

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plasticity 91 Rose,C.R. 2001; 96 Yu,X.M. 1998. Inasmuch as neuronal activity-

induced increments in cytoplasmic sodium may augment NMDAR-mediated currents, we

reasoned that intracellular Na+ may function as a signaling molecule to positively

regulate neuronal development in immature cerebrocortical neurons. In the present study,

we used veratridine (VRT), a VGSC gating modifier to manipulate [Na+]i in immature

cerebrocortical neurons. We have previously demonstrated that in cerebrocortical neurons

VGSC activators, brevetoxin (PbTx-2) and antillatoxin (ATX) elevated [Na+]i and

augmented NMDAR function 296 Jabba,S.V. 2010; 6 George,J. 2009. These VGSC

activators also enhanced neurite outgrowth in a hormetic concentration-relationship. The

inverted-U response to ATX and PbTx-2 on neurite outgrowth is similar to that of

NMDA (unpublished). Moreover, BDNF also display an inverted-U concentration-

response for retinal ganglion survival following optic nerve transaction 466 Klocker,N.

1998, and in promoting serotonergic axonal growth and remodeling in the adult brain

447 Mamounas,L.A. 2000

We therefore hypothesized that sodium channel activators stimulate neuronal

development by elevating [Na+]i , augmenting NMDAR function and enhancing BDNF

release with activation of downstream BDNF-TrkB signaling pathways. Here, we

demonstrate that VRT enhances NOG with a hormetic concentration-response

relationship, and this response is dependent on TrkB receptor signaling. We also show

that acute exposure to VRT caused increases in [Na+]i and [Ca2+]i with the [Na+]i

increment being sufficient to upregulate NMDAR function. Further, inhibition of TrkB

receptors and its downstream signaling molecules, PI3K, and PLC inhibited VRT-

enhanced NOG. Acute treatment with VRT stimulated phosphorylation of TrkB and its

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downstream effectors Akt, mTOR, PLC, ERK1/2 and CREB. More importantly, VRT

increased BDNF synthesis and release in a concentration dependent manner. Veratridine

stimulation of TrkB phosphorylation displayed a biphasic concentration-response curve.

Taken together, these data suggest that VRT activates VGSCs and elevates [Na+]i , which

in turn augments NMDAR function leading to increased BDNF synthesis and release.

Released BDNF binds to the TrkB receptor causing activation of BDNF-TrkB receptor

signaling, thereby mediating VGSC activator-enhanced neurite outgrowth. These data

provide further support for the hypothesis that sodium channel activators are capable of

mimicking activity-dependent neuronal development through potentiation of NMDAR

and neurotrophin signaling pathways.

4.3 Materials and Methods

Cerebrocortical Neuron Culture.

Primary cultures of cerebrocortical neurons were harvested from Swiss Webster mice on

embryonic day 16 and cultured as described previously (Cao et al., 2008). Cells were

plated onto poly-l-lysine-coated (Sigma-Aldrich, St. Louis, MO) 96-well (9 mm), clear-

bottomed, black-well culture plates (Corning Life Sciences, Lowell, MA) at a density of

1.8 × 106 cells/ml (150 μl/well), 24-well (15.6 mm) culture plates at a density of 0.05 ×

106 cells/ml (0.5 ml/well), 12-well (22 mm) culture lates at a density of 1.8 × 106 cells/ml

(1.0 ml/well), or 6-well (35 mm) culture dishes at a density of 2.25 × 106 cells/ml (2

ml/well), respectively, and incubated at 37°C in a 5% CO2 and 95% humid atmosphere.

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All animal use protocols were approved by the Creighton University Institutional Animal

Care and Use Committee.

Determination of Total Neurite Length and Diolistic Labeling.

Cells were plated on poly-lysine-coated 12- or 15-mm glass coverslips (Thermo Fisher

Scientific, Waltham, MA) and placed inside of 24-well culture plates at a low density of

0.05 × 106 cells/ml (0.5 ml/well). To assess the influence of VRT on neuritogenesis,

primary cultures of immature cerebrocortical neurons were exposed to various

concentrations of VRT ranging from 10 to 10000 nM for 24 h beginning 3 h after plating,

and total neurite outgrowth was measured. In some experiments, these concentrations of

VRT were coincubated with MK-801 (1 μM) (Sigma-Aldrich), LY29002 (10 μM)

(Sigma-Aldrich), U37122 (2 μM) (Calbiochem)K-252a (200 nM) (Calbiochem, San

Diego, CA), U0126 (10 μM) (Calbiochem) Rapamycin (1 μM) (Calbiochem). At 24 h

after plating, cultures were fixed at room temperature for 20 min using 1.5%

paraformaldehyde in phosphate-buffered saline (PBS). After fixation, neurons were

diolistically labeled with DiI. The Helios Gene Gun System (Bio-Rad Laboratories,

Hercules, CA) was used to deliver DiI-coated tungsten particles (1.3 μM) (Bio-Rad

Laboratories) into paraformaldehyde-fixed cerebrocortical neurons 1 day in vitro (DIV).

Diolistic bullet preparation was based on the method of O’Brien and Lummis (2006). In

brief, 2.5 to 3.5 mg of DiI (Invitrogen, Carlsbad, CA) was suspended in 200 μl of

dichloromethane (Sigma-Aldrich). The dissolved dye was added over evenly spread

tungsten particles (35 mg) placed on a clean glass slide and then allowed to dry. The dye-

coated particles were scraped onto another clean glass slide and chopped to fine particles

using a clean razor blade and later resuspended in 3 ml of deionized water. This dye

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slurry was sonicated for 10 min and then vortex briefly to form a uniform suspension.

After adding 100 μl of polyvinylpyrrolidone (PVP) (Bio-Rad Laboratories) stock solution

(0.96% PVP in ethanol) to the dye slurry, it was drawn into a PVP-precoated Tefzel

tubing mounted on a preparation station (Bio-Rad Laboratories) using a 5- to 10-ml

syringe. The dye particles were allowed to settle for 20 to 30 min, and then the

supernatant water was carefully withdrawn from Tefzel tubing (Bio-Rad Laboratories)

using a syringe. The tubing was rotated for 1 to 2 min to uniformly spread the particles.

The tubing was then allowed to dry for 5 min before cutting into bullets using a tube

cutter. The DIV-1 cerebrocortical neurons grown on coverslips were shot postfixation

(1.5% paraformaldehyde) using DiI bullets loaded onto a Helios gene gun at 140 to 160

psi of helium pressure from a distance of 2.5 cm. The dye particles were allowed to

spread across the neuronal membrane overnight, and coverslips were then mounted for

imaging on an Olympus IX 71 inverted microscope with a Himamatsu camera. Digital

images of individual neurons were captured, and total neurite length was quantified. To

reduce the effect of paracrine neurotrophic factors on neurite growth, only those neurons

that were separated from surrounding cells by approximately 150 μm were digitally

acquired and analyzed. Digital images of individual neurons were captured and exported

as 16-bit images. All neurites in a single neuron including those from secondary branches

were semi-automatically traced, and the length was measured by using the using

FilamentTracer module of Imaris 6.4.0 software. At least 25 randomly chosen neurons

from two different cultures were evaluated for each treatment group.

Intracellular Sodium Concentration Measurement.

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[Na+]i measurement and full in situ calibration of sodium-binding benzofuran

isophthalate (SBFI) fluorescence ratio were performed as described previously (Cao et

al., 2008). Cells grown in 96-well plates were washed four times with Locke's buffer (8.6

mM HEPES, 5.6 mM KCl, 154 mM NaCl, 5.6 mM glucose, 1.0 mM MgCl2, 2.3 mM

CaCl2, 0.1 mM glycine, pH 7.4) using an automated microplate washer (BioTek

Instruments, Winooski, VT). After measuring the background fluorescence of each well,

cells were incubated for 1 h at 37°C with dye-loading buffer (100 μl/well) containing 10

μM SBFI-AM (Invitrogen) and 0.02% Pluronic F-127 (Invitrogen). Cells were then

washed five times with Locke's buffer, leaving a final volume of 120 μl in each well. The

plate was then transferred back to the incubator for 15 min to allow the cells to

equilibrate after washing and then placed in a FlexStation II (Molecular Devices,

Sunnyvale, CA) chamber to detect Na+-bound SBFI emission at 505 nm (cells were

excited at 340 and 380 nm). Fluorescence readings were taken once every 5 s for 60 s to

establish the baseline, and then 40 μl of VRT was added to each well from the compound

plate at a rate of 26 μl/s, yielding a final volume of 160 μl/well. After correcting for

background fluorescence, SBFI fluorescence ratios (340/380) versus time were analyzed,

and time- or concentration-response graphs were generated using GraphPad Prism

(GraphPad Software Inc., San Diego, CA). Full in situ calibration of the SBFI

fluorescence ratio was performed as described previously (Cao et al., 2008, Jabba et al.,

2010)

Intracellular Ca2+ Monitoring.

DIV-1 cerebrocortical neurons grown in 96-well plates were used for intracellular Ca2+

concentration ([Ca2+]i) measurements as described previously (George et al., 2009, Cao et

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al., 2008, Jabba et al., 2010. In brief, the growth medium was removed and replaced with

dye-loading medium (100 μl/well) containing 8 μM fluo-3 AM (Invitrogen) and 0.04%

Pluronic acid in Locke's buffer. After 1-h incubation in dye-loading medium, the neurons

were washed four times in fresh Locke's buffer (200 μl/well) using an automated

microplate washer (BioTek Instruments) and transferred to a FlexStation II benchtop

scanning fluorometer chamber. The final volume of Locke's buffer in each well was 120

μl. Fluorescence measurements were performed at 37°C. The neurons were excited at 488

nm, and Ca2+-bound fluo-3 emission was recorded at 538 nm at 2-s intervals. After

recording baseline fluorescence for 27 s, 40 μl of a 4× concentration of VRT in the

presence or absence of specific agonists like TTX or MK-801 were added to the cells at a

rate of 26 μl/s, yielding a final volume of 160 μl/well; the fluorescence was monitored for

an additional 220 to 270 s. The fluo-3 fluorescence was expressed as (Fmax – F0)/F0,

where Fmax is the maximum and F0 is the fluorescence measured in each well at time

zero. In some experiments the fluo-3 fluorescence was expressed as area under the curve

(AUC).

Western Blotting.

Western blot analysis was performed by using cells grown in either six-well or 12-well

plates. DIV-1 cells were exposed to 1000 nM VRT for various time periods at 37°C. For

pharmacological experiments, along with 1000 nM VRT, cultures were co-incubated

either in the presence or absence of specific antagonists. At the end of each time period,

cultures were transferred onto an ice slurry to terminate drug exposure and washed three

times with ice-cold PBS. Cells were lysed using ice-cold lysis buffer (50 mM Tris, 50

mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% Nonidet P40, 0.1% SDS, 2.5 mM sodium

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pyrophosphate, and 1 mM sodium orthovanadate). Phenylmethylsulfonyl fluoride (1

mM) and 1× protease inhibitor mixture (Sigma-Aldrich) were then added, and the lysate

was incubated for 30 min at 4°C. Cell lysates were sonicated and then centrifuged at

13,000g for 15 min at 4°C. The supernatant was assayed by the Bradford method

(Bradford, 1976) to determine protein content. Equal amounts of protein were mixed with

the Laemmli sample buffer and heated for 5 min at 75°C. The samples were loaded onto

a 10% SDS-polyacrylamide gel electrophoresis gel and transferred to a nitrocellulose

membrane and immunoblotted with specific antibodies. Blots were developed with ECL

Plus kit (GE Healthcare, Chalfont St. Giles, UK) for 3 min. Blots were subsequently

stripped (63 mM Tris base, 70 mM SDS, 0.0007% 2-mercaptoethanol, pH 6.8) and

reprobed for further use. Western blot densitometry data were obtained by using MCID

Basic 7.0 software (Imaging Research, St. Catharines, ON, Canada).

4.3.1.1 BDNF immunoassays.

Sandwich BDNF ELISA and BDNF ELISA in situ (Balkoweic and Katz, 2000

J.Neurosci.) were performed to measure BDNF protein using a BDNF Emax immunoassay

System (Promega, Madison, WI) according to manufacturer specifications, except that

the concentrations of the anti-BDNF monoclonal antibody and anti-human BDNF

polyclonal antibody were two-fold of the recommended concentrations for BDNF ELISA

in situ. Also, the dilution of the anti IgY-HRP antibody was 1:50 in BDNF ELISA in situ.

Sandwich BDNF ELISA: Protein lysates were made from primary neuronal cultures

grown in 6 or 12-well plates. Cultures were treated with 1000 nM VRT for specific times,

either in presence or absence of various antagonists and changes in total BDNF protein

was measured by sandwich BDNF ELISA. Breifly, 96-well ELISA plates (Nunc

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maxisorp) were incubated overnight at 40C with anti-BDNF monoclonal antibody diluted

in carbonate coating buffer. The plates were washed 1X with TBST and blocked for 1h at

RT. Protein lysates were applied along with BDNF standards and incubated with shaking

(400 rpm) at RT for 2h. Subsequently plates were washed 5X and anti-human BDNF

polyclonal antibody was added and incubated with shaking at RT for 2h. After washing

5X, anti IgY-HRP antibody was added and with shaking at RT for 1h. The plate is

washed 5X and incubated with TMB solution for color development. After 10 minutes

reactions are stopped using1N HCl. Absorbance values were measured at 450 nm in a

plate reader (name the instrument).

BDNF ELISA in situ: This method was performed according to Balkoweic and Katz,

2000 J.Neurosci. Breifly, 96-well ELISA plates (Nunc maxisorp) were UV-sterilized for

30 minutes and then incubated overnight at 40C with anti-BDNF monoclonal antibody

diluted in carbonate coating buffer. Next, plates were blocked for 1h and then washed 2X

with plating media. Dissociated immature cerebrocortical neurons were plated and

cultured for 4 days. 3h post plating cells were treated to various concentrations of VRT

and 50 mM KCl. BDNF standards were also added to the plate at the start of the culture.

After 3-4d, the plates were washed vigorously with TBST to remove the cells and debris.

Subsequently anti-human BDNF polyclonal antibody was added and incubated with

shaking at RT for 2h. The remaining of the procedure was similar to that is described

above.

Plasmids and Nucleofection: FL-TrkB and DN TrkB (truncated TrkB) in pBluescript

sk-/- vector were a generous gift from Tony Hunter (Salk Institute, San Diego).

Dissociated cortical neurons obtained from E16-17 pups were re-suspended in

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Nucleofector solution (Mouse Neuron Kit, Amaxa Biosystems) and transfected according

to the manufacturer's directions with plasmids containing the genes of interest, using an

Amaxa Nucleofector (program O-005). Five million and 1 million neurons were used per

reaction for western blot and neurite outgrowth experiments, respectively. One

microgram of plasmid containing the gene of interest was used per nucleofection

reaction. Control neurons were transfected with the empty vector. Also, all nucleofection

reactions contained GFP plasmid (1 µg) bringing the total amount of plasmids to 2 µg per

reaction. Transfected neurons were plated at 2 X 106 neurons/well and 0.5 X 105

neurons/well for western blot experiments and neurite outgrowth assays, respectively. In

order to give more time for the expression of genes of interest, DIV-2 neurons were

utilized in experiments involving nucleofection.

4.4 Results

Veratridine Enhances Neurite Outgrowth in Immature Cerebrocortical Neurons.

In previous reports, we demonstrated that the VGSC activators brevetoxin (PbTx-2) and

antillatoxin (ATX) stimulated neurite out growth. Here, we wanted to determine whether

these transactions generalized to the VGSC site 2ligand, veratridine(VRT) . Three hours

after plating, primary cultures of immature cerebrocortical neurons were exposed to

various concentrations of VRT ranging from 10 to 10,000 nM for 24 h, and total neurite

outgrowth and dendritic branch points were assessed. Diolistic labeling of DiI was used

to visualize neurons and determine the influence of VRT on neurite outgrowth (Fig. 1A)

and dendritic branch points. Veratridine significantly enhanced total neurite outgrowth in

immature cerebrocortical neurons with concentrations of 300 and 1,000 nM producing a

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robust >2-fold increase in total neurite length (***, p < 0.001) (Fig 1B). As previously

observed with PbTx-2 and ATX, the VRT concentration-response profile was

bidirectional, or hormetic (Fig 1B). Veratridine also significantly enhanced dendritic

branch points in these neurons with concentrations of 300 and 1,000 nM producing a

robust 4- (***, p < 0.001) and 3-fold (**, p < 0.01) increases respectively.

Veratridine Increases Intracellular sodium ([Na+]i) Levels in Immature Cerebrocortical

Neurons.

Given that the earlier studies (Yu et al., 1997, Yu and Salter 1998, Yu et al 2006, George

et al 2009) demonstrated that [Na+]i is a regulator of NMDAR-mediated signaling, it was

important to quantify the magnitude of VRT-induced elevation of [Na+]i in immature

cerebrocortical neurons. SBFI, a sodium-sensitive fluorescent indicator, was used to

determine the influence of VRT on [Na+]i in DIV-1 cerebrocortical neurons. Full in situ

calibration was performed in DIV-1 cerebrocortical neurons to determine the relationship

between the ratiometric SBFI fluorescence signal and [Na+]i (Cao et al 2008, George et al

2009, Jabba et al 2010). VRT produced a concentration-dependent increase in [Na+]i (Fig

2 A,B) with an EC50 value of 3.12 µM (1.42-6.88 µM, 95% CI). The in situ SBFI

calibration showed that basal [Na+]i in DIV-1 cerebrocortical neurons was 16.1 ± 0.51

mM. Inasmuch as the VRT concentrations of 300 and 1,000 nM were sufficient to

produce a robust increase in neurite outgrowth, it was important to quantify the [Na+]i

increment associated with these treatments. The 300 nM VRT treatment produced a

maximal [Na+]i of 23.1± 0.3 mM and 1,000 nM produced 26.5 ± 0.36 mM, representing

increments of 7.0 and 10.4 mM over basal. Previous reports in hippocampal neurons

suggested that an increment of [Na+]i of 10 mM was sufficient to produce significant

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increases in NMDAR channel activity (Yi and Salter 1998, Yan et al 2006). Moreover, it

has been reported that increments of [Na+]i >5 mM represent a critical threshold required

to regulate NMDAR-mediated Ca2+ influx in primary cultures of hippocampal neurons

(Xin et al., 2005). Consistent with these findings, the increment of [Na+]i detected in

immature cerebrocortical neurons appears sufficient to up-regulate NMDAR function.

Veratridine Increases Intracellular Calcium Levels ([Ca2+]i) in DIV-1 Cerebrocortical Neurons.

Activity-dependent neuritogenesis and neuronal development involve Ca2+-influx and to

a large extent this Ca2+-influx is NMDAR dependent. Previous studies have also

suggested that sodium channel activator-induced neurite outgrowth and Ca2+-influx

involves NMDARs and VGSCs. Hence, we hypothesized that VRT exposure would

produce Ca2+ influx in these immature cerebrocortical neurons. To investigate this theory,

cells loaded with fluo-3 were exposed to various concentrations of VRT, and [Ca2+]i was

monitored. VRT produced rapid and concentration-dependent increases in [Ca2+]i, with

an EC50 value of 2.56 µM (1.19-16.56 µM, 95% CI).

To investigate the role of VGSCs and NMDARs in VRT-induced Ca2+-influx in DIV-1

cerebrocortical neurons, a pharmacological evaluation was performed using TTX

(VGSCs) or MK-801 (NMDARs), prior to 1,000 nM VRT exposure and changes in

[Ca2+]i were monitored. TTX (1 μM) completely blocked the response to VRT (Fig 2E,

F), whereas MK-801 (1 μM) significantly reduced VRT-induced Ca2+ influx (**, p <

0.01) (Fig 2G, H).

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Veratridine-induced neurite outgrowth involves TrkB receptors.

Inasmuch as previous studies have indicated that activity-dependent neuritogenesis and

neuronal development involve Ca2+ influx through NMDAR with subsequent

engagement of BDNF-TrkB signaling, we assessed the role of TrkB receptor in sodium

channel activator-induced neurite outgrowth. Coincubation of K-252a (200 nM), a TrkB

inhibitor, with 300 nM VRT inhibited VRT-stimulated neurite outgrowth in immature

cerebrocortical neurons (***, p < 0.001) (Fig. 3A, B and C), demonstrating the

requirement for TrkB receptor activation. These pharmacological data were confirmed

with a genetic approach in which DIV-1 neurons were transfected with either dominant

negative isoform of TrkB (truncated TrkB), the full length isoform of TrkB (TrkB.FL) or

the empty vector (back bone). Consistent with the involvement of TrkB receptors,

dominant negative TrkB completely abolished VRT-induced neurite outgrowth, whereas

neurons expressing TrkB.FL showed robust increase in neurite length similar to those

expressing empty back bone vector when exposed to VRT (Fig.3D and E).

Veratridine enhances BDNF release and synthesis in immature cerebrocortical neurons

Neurotrophins are expressed and released from neurons in an activity-dependent manner

and act in an autocrine/paracrine mode to induce morphological and functional changes

in neurons. Activation of NMDARs and elevation of [Ca2+]i are critical for the expression

and release of neurotrophins. Inasmuch as sodium channel activators mimic activity-

dependent neuronal development with attendant stimulation of NMDARs and increase in

intracellular Ca2+, we predicted that sodium channel activation will increase the synthesis

and release of BDNF with attendent activation of TrkB receptors. The influence of VRT

on release of endogenous BDNF was quantified using an in situ BDNF ELISA (Figure

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4A). Three hour post-plating, neurons were exposed to various concentrations of VRT

ranging from 100 to 10,000 nM for 72 h. As a positive control, neurons were exposed to

50 mM KCl. VRT produced a concentration-dependent increase in BDNF release with 10

µM VRT producing comparable increases in BDNF release to that of 50 mM KCl (Figure

4 B). VRT in concentrations of 1 (*, p < 0.05), 3 (**, p < 0.01) and 10 µM (***, p <

0.001) produced significant increases in BDNF release compared to control. We further

investigated BDNF synthesis in immature cerebrocortical neurons following VRT

exposure. Neurons were exposed to 1,000 nM VRT and cell lysates were collected for

various time points starting at 3 h post plating to 24 h (corresponding to the time of

neurite outgrowth assay). Results revealed that 1,000 nM VRT exposure increased BDNF

synthesis at 1, 6 and 12 h of exposure but showed reduced amount of BDNF after 24 h

VRT exposure. (Figure 5A, B). We also characterized for the increase in BDNF synthesis

on VRT exposure using BDNF sandwich ELISA (Figure 5C). Three hours post plating

neurons were exposed to 1000 nM VRT either in the presence or absence of various

specific antagonists for 12 h. Cell lysates (using ELISA lysate buffer) were collected and

stored for BDNF sandwich ELISA assay. These results revealed that TTX, a VGSC

blocker, completely inhibited VRT-enhanced BDNF synthesis. BDNF synthesis was also

partially blocked (75 %) by MK 801, a NMDAR blocker. The VGCC blocker nifedipine

(1 µM) and PLCγ blocker U73122 were without effect on VRT-stimulated BDNF

synthesis. These results demonstrate that VRT enhances BDNF synthesis in immature

cerebrocortical neurons and this requires VGSC and NMDARs activation (Figure 5C).

We also demonstrated using BDNF sandwich ELISA assay and western blot analysis,

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that VRT increased BDNF synthesis in a concentration- dependent manner.(Figure 6A,

B, C)

Veratridine-induced neurite outgrowth involves PI3-kinase activity

The PI3K-Akt signaling pathway is downstream of BDNF-TrkB receptor and plays an

important in neurotrophin-stimulated neurite outgrowth. Inasmuch as VRT stimulated the

synthesis and release of BDNF, we determined whether PI3K is involved in VRT-

induced neurite outgrowth. Coincubation of LY29002 (10 µM), a PI3K inhibitor, with

300 nM VRT completely inhibited VRT-stimulated neurite outgrowth in immature

cerebrocortical neurons (***, p < 0.001) (Figure 7 A, B and C), suggesting a PI3K

involvement in VRT-enhanced neurite outgrowth. Similarly, wortmanin, another PI3K

blocker, also inhibited VRT-stimulated neurite outgrowth (data not shown). The major

affecter downstream of PI3K that modulates neurite outgrowth is Akt. To assess the

ability of VRT to activate Akt, we determined the phosphorylation of the Ser473 residue

on Akt by using an anti-phospho-Ser473 Akt antibody. DIV-1 cerebrocortical neurons

were exposed to 1,000 nM VRT, and cell lysates were collected at various time periods

for western blot analysis. The results revealed that 1,000 nM VRT produced a robust

activation of Akt as reflected by the increase in the phosphorylation of Ser473 at 5

minutes after exposure (~2 fold). Akt activation peaked at 10-15 minutes of VRT

exposure (~3 fold) (Figure 7 C, D). Prior incubation with VGSC blocker TTX for 15

minutes attenuated VRT-stimulated Akt activation indicating the requirement for VGSC

activation (Figure 7 E,F). These findings establish a role for PI3K-Akt signaling in the

stimulatory effect of VRT on neurite outgrowth.

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Veratridine stimulated Akt phosphorylation involves TrkB receptors

To determine the involvement of TrkB receptors in Veratridine stimulated Akt

phosphorylation, DIV-1 cultures were \ pre-incubated with K252a (200 nM) for 30

minutes and then exposed to 1,000 nM VRT for 5 minutes. Cell lysates were collected

and investigated for activation of Akt (Ser473) by immunoblotting. Western blot analysis

revealed that K252a blockade completely inhibited VRT-stimulated Akt activation

indicating the requirement of TrkB receptors (Figure 8C, D). To further confirm these

pharmacologic results, we utilized a genetic approach by nucleofecting the neurons with

either DN-TrkB or FL-TrkB. Neurons nucleofected with an empty back-bone vector were

used as a control. All nucleofected neurons were exposed to 1,000 nM VRT for 5

minutes. Subsequently cell lysates were collected and investigated for activation of Akt

(Ser473) by immunoblotting. Nucleofection with DN TrkB completely blocked the VRT-

stimulated Akt activation, but neither the FL-TrkB (~4 fold) nor the empty vector (~6

fold) had any effect on VRT-stimulated Akt activation (Figure 8A,B). The basal Akt

activity was also higher in neurons nucleofected with FL TrkB. These data suggest the

involvement of TrkB receptors in Veratridine stimulated Akt phosphorylation.

Veratridine-induced neurite outgrowth involves the PI3K-Akt-mTOR pathway

Downstream of TrkB is the mTOR signaling complex which is critical for protein

synthesis in dendrites and participates in activity–dependent dendritic arborization. PI3K-

Akt signaling acting through, or in coordination with mTOR, is involved in the regulation

of dendritic morphogenesis and synaptic plasticity. To determine the involvement of

mTOR signaling in VRT-stimulated neurite outgrowth, cerebrocortical neurons were co-

incubated with VRT in the presence or absence of rapamycin, specific inhibitor of

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mTOR, for 24 h and total neurite length was determined. Consistent with the involvement

of mTOR, rapamycin inhibited VRT-stimulated neurite outgrowth (Figure 9 A, B, C). To

assess the ability of VRT to activate mTOR, we determined the phosphorylation of the

Ser2448 residue on mTOR using an anti-phospho-Ser2488 mTOR antibody. DIV-1

cerebrocortical neurons were exposed to 1,000 nM VRT, and cell lysates were collected

at various time periods for western blot analysis. The results revealed that 1,000 nM VRT

produced a ~2 fold activation of mTOR as reflected by an increase in the phosphorylation

of Ser2448 at 15 minutes post exposure. (Figure 9 D). Taken together, these results

indicate that mTOR activation participates in sodium channel activator-stimulated

neuronal neurite outgrowth.

Veratridine-induced Ca2+ influx and neurite outgrowth involves PLC mediated

release of Ca2+ from intracellular stores

Another important signaling pathway downstream of neurotrophin-TrkB pathway is the

PLCγ pathway. BDNF activation of TrkB receptors at Y816 recruits and activates PLCγ

which in turn hydrolyzes PIP2 to DAG and IP3, with IP3 triggering the release of Ca2+

from intracellular stores. Release of Ca2+ from intracellular calcium stores leads to release

of secretory growth factors vesicles. Here we explored the involvement of PLCγ in VRT-

induced changes in [Ca2+]i using the PLCγ inhibitor U73102. We also determined for the

role of PLCγ in VRT- induced neurite outgrowth pharmacologically using U73102. Fluo-

3 loaded DIV-1 neurons were pretreated with the PLCγ specific antagonist-U73102, prior

to 1,000 nM VRT exposure and changes in [Ca2+]i were monitored. U73102 (2 μM)

significantly reduced VRT-induced Ca2+ influx (**, p < 0.01) (Figure 10 A, B). We then

determined whether the attenuation in Ca2+ response was due to inhibition of release of

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Ca2+ from intracellular Ca2+ stores. We pretreated neurons with 10 μM thapsigargin for

40 minutes to deplete the ER of calcium and then treated with 1,000 nM VRT. VRT-

induced change in intracellular was significantly attenuated in presence of thapsigargin,

suggesting that PLC mediated release of Ca2+ from intracellular stores contributes to

VRT-induced elevation of Ca2+ (Figure 10 C, D). Next, we investigated whether PLC is

involved in VRT-induced neurite outgrowth. Coincubation of U73102 (2 µM) with 300

nM VRT completely inhibited VRT-stimulated neurite outgrowth in immature

cerebrocortical neurons (***, p < 0.001) (Figure 11 A), indicating that VRT-enhanced

neurite outgrowth involves PLC signaling. We next determined whether phosphorylation

of the Y816 residue on TrkB following VRT exposure was involved in the recruitment of

downstream PLCγ signaling machinery to TrkB receptor. DIV-1 cerebrocortical neurons

were exposed to 1,000 nM VRT, and cell lysates were collected at various time periods

for western blot analysis. The results revealed that 1,000 nM VRT produced a robust

activation of TrkB Y816 residue as reflected by the increase in the phosphorylation at 2

minutes post exposure. (Figure 11 B) We also examined the ability of VRT to activate

PLCγ by determining the phophorylation of Y783 residue on PLCγ1. We found the Y783

residue on PLCγ1 is activated by VRT exposure at 2-5 minutes. (Figure 11 C). Taken

together these data indicate the involvement of a TrkB- PLCγ signaling in VRT-

stimulated neurite outgrowth.

MAPK pathway has a modest role in veratridine-induced neurite outgrowth.

The third signaling pathway downstream of neurotrophin-TrkB pathway is the MAPK

pathway. BDNF phosphorylation of the Tyr515 site on TrkB receptors activates

downstream MEK-MAPK/Erk signaling, which promotes neuronal differentiation and

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growth. We investigated for involvement of the MAPK pathway in VRT-induced neurite

outgrowth using the MEK inhibitor U0126 (20 µM). Coincubation of U0126 with 300

nM VRT partially inhibited VRT-stimulated neurite outgrowth in immature

cerebrocortical neurons (*, p < 0.05) (Fig.12 A, B and C), indicating that VRT-enhanced

neurite outgrowth partially involves MEK-MAPK pathway. We further demonstrated

phosphorylation of ERK1/2 (T202/Y204) and CREB (S133) following VRT exposure.

DIV-1 cerebrocortical neurons were exposed to 1,000 nM VRT, and cell lysates were

collected at various time periods for western blot analysis. The results revealed that 1,000

nM VRT produced a robust activation of ERK1/2 ((T202/Y204) and CREB (S133) in

immature cerebrocortical neurons. (Figure 12 A, B and C).

4.5 Discussions

The electrical signals of neurons are fundamentally dependent on Na+ influx through

VGSCs. Sodium channels are primarily responsible for the rising phase of action

potential and hence supply the current that drives the membrane potential to peak

depolarization (Hille B, 2001). VGSCs activity has been shown to regulate

neurotransmitter release in developing cortex and also to mediate neuronal firing

dependent synaptic plasticity (Platel JC, 2005, Cantrell AR, 2001). In immature

cerebrocortical neurons, VGSCs activators enhanced neurite outgrowth through

potentiation of NMDAR signaling pathways that influence neuronal morphology 296

Jabba,S.V. 2010; 6 George,J. 2009, indicating that sodium channel activators appear

capable of mimicking activity-dependent neuronal development. Neuronal activity also

cooperates with neurotrophins to influence neuronal development and plasticity 1

Wong,R.O. 2002; 262 Yoshii,A. 2010; 267 Reichardt,L.F. 2006. Although activity-

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dependent neurotrophins influences on neuronal development are well studied, little is

known regarding the influence of VGSCs activation on neurotrophin signaling. Towards

achieving this goal, we used a VGSC gating modifier and determined the effects of

VGSCs activation on BDNF synthesis, secretion and BDNF receptor (TrkB) dependent

signaling pathways, and their role in neurite outgrowth. Our findings provide one of the

first reports of the influence a VGSC gating modifier on neurotrophin signaling. We

found that VGSCs activator increased BDNF release, synthesis and activated BDNF-

TrkB signaling pathways, including the PI3K-Akt cascasde, the PLCγ pathway, and the

MAPK/Erk1/2 pathway. An interesting feature of our results is that the influence of VRT

on TrkB activation followed a bidirectional pattern similar to that of VRTon neurite

outgrowth.

VRT enhanced total neurite outgrowth and dendritic arborization in immature

cerebrocortical neurons with a bidirectional, or hormetic, concentration-response

relationship with 300-1,000 nM producing robust increases of more than 2-fold. (Figure

1). This result is in accordance with previous reports using such as other VGSCs

activators such as PbTx-2 and ATX 6 George,J. 2009; 296 Jabba,S.V. 2010. PbTx-2

and ATX enhanced neurite outgrowth in DIV-1 cerebrocortical neurons as a result of

elevation of cytoplasmic [Na+], potentiation of NMDAR function and stimulation of

calcium influx. Yu and Salter 96 Yu,X.M. 1998first suggested that [Na+]i may act as

signaling molecule and augments NMDAR fuction. They used VRT to demonstrate that

Na+ influx through TTX-sensitive VGSC was sufficient to upregulate NMDAR activity

in hippocampal neurons. Similarly, previous reports 296 Jabba,S.V. 2010; 6 George,J.

2009 using single-channel current recordings from cell-attached patches

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unambiguously confirmed that VGSCs activators like PbTx-2, ATX augmented NMDAR

function by increasing open probability or mean open time (or both) of NMDARs. Yu

and Salter have also determined that an increment in [Na+]i of 10 mM was sufficient to

potentiate NMDAR single-channel activity. Moreover, this Na+-dependent regulation of

NMDA receptor function was shown to be controlled by Src-induced phosphorylation of

the receptor. These results were extended in the present study using sodium channel

activator VRT as a probe to elevate intracellular Na+ in cerebrocortical neurons. VRT

treatment increased the release of glutamate and enhanced NMDA-induced Ca2+ influx in

immature cerebrocortical neurons (data not shown). Veratridine exposure also led to the

activation of Src (data not shown) 95 Yu,X.M. 1997. We have previously shown that

an increase in intracellular Na+ and Src activation following exposure to ATX increased

the open probability of the NMDAR. These data, therefore, confirm the regulatory

influence of Na+ on NMDAR channel activity in hippocampal neurons described

previously 96 Yu,X.M. 1998 and extend this relationship between [Na+]i and NMDA

receptor function to cerebrocortical neurons.

NMDAR activation promotes neurite growth and dendritic arborization, whereas

pharmacological blockade of NMDARs reduces it 43 Rajan,I. 1998. Stimulation of

NMDAR function also activates Ca2+ signaling pathways that regulate neurite outgrowth

and dendritic arborization 38 Konur,S. 2005. Consistent with these reports, we found

that exposure of immature cerebrocortical neurons to VRT increased [Ca2+]i with

significant dependence on NMDARs. Thus, the ability of VRT to augment NMDAR

channel activity translated into an enhancement of the trophic influence of NMDAR on

developing cerebrocortical neurons. Based on the premise that the effects of neuronal

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activity on dendritic arbor growth and structural plasticity are primarily mediated by

engagement of NMDA receptors 106 Tolias,K.F. 2005, our results suggest that VRT

activation of sodium channels with attendant enhancement of NMDA receptor signaling

mimics the response to neuronal activity.

Another important finding of the study is that a VGSC activator increased BDNF

synthesis, release and activated BDNF-TrkB signaling pathways. BDNF plays an

important role in regulating neural survival, development, function and plasticity.

Veratridine-enhanced BDNF synthesis was TTX-sensitive, and pharmacological

blockade of NMDARs significantly attenuated the response. Nifedipine and U73122, a

VGCC and PLC inhibitor, respectively, were without effect on VRT-enhanced BDNF

synthesis (Figure 5). These data, therefore demonstrate that VGCCs and intracellular

Ca2+ stores are not required for VRT-induced BDNF synthesis. Transcription of Bdnf

from its promoters (I & IV) is highly regulated by neuronal activity-dependent Ca2+

influx 321 Kidane,A.H. 2009; 332 Rattiner,L.M. 2005, and is regulated by the route

of Ca2+ influx into the cell and by the pattern of phosphorylation induced on the

transcription factor CREB 446 West,A.E. 2001. In rat cerebrocortical cultures,

Ghosh et al., 445 Ghosh,A. 1994 demonstrated that transcription of Bdnf is

preferentially driven by Ca2+ influx through L-VGCCs, whereas it is poorly induced by

calcium coming through NMDARs. In that study they demonstrated that glutamate

increased Bdnf gene expression transiently, with peak expression around 1 h and with

reduced expression thereafter. The stimulation of Bdnf gene expression by depolarizing

levels of KCl was more delayed and peaked at approximately 3 h with a subsequent

sustained elevation. This difference could be due to inability of VRT to alter membrane

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potential in cerebrocortical neurons, which would not provide the stimulus for VGCC

activation. Also, similar to glutamate increased Bdnf gene expression, VRT increased

BDNF protein expression peaked at approximately 1 h and was sustained for 12 h.

Exposure to VRT in immature cerebrocortical neurons moreover increased BDNF release

(secretion) in a concentration-dependent manner (0.1-10 µM VRT). The released BDNF

binds to its cognate receptor TrkB in an autocrine/paracrine fashion to activate

downstream signaling pathways and thereby influence neuronal development. Similarly,

a 12 h VRT exposure increased BDNF synthesis in a concentration-dependent manner

(0.1-10 µM VRT). Interestingly, the influence of VRT on TrkB activation (Y816)

followed a bidirectional concentration-relationship, similar to that of VRT-mediated

neurite outgrowth. Marini et al., 450 Marini,A.M. 1998 have demonstrated that

NMDA exertes a neuroprotective activity by increasing BDNF release (acute effect, 2-5

‘) and synthesis (chronic effect, ~3 h), leading to activation of TrkB signaling. They

proposed that there is an integral relationship between NMDAR activation and BDNF-

TrkB signaling in a bidirectional manner, consonant with the bidirectional pattern shown

by NMDA and VRT on neurite outgrowth. An inverted-U model describes the

relationship between NMDA receptor activity and neuronal survival and growth 80

Lipton,S.A. 1999. In both cases, this inverted-U concentration–response relationship

has primarily, but not exclusively, been attributed to [Ca2+]i regulation. An optimal

window for [Ca2+]i is required for activity-dependent neurite extension and branching,

with lower levels stabilizing growth cones and higher levels stalling them, in both cases

preventing extension 50 Gomez,T.M. 2000; 74 Hui,K. 2007. In regard to NMDAR-

BDNF-TrkB signaling, one possible explaination could be that , the TrkB receptor may

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be desensitized at higher concentrations of VRT exposure, due to higher amounts of

BDNF being synthesized and released. Conversely, at lower concentrations of VRT

exposure the neurons are not able to synthesize and release sufficient BDNF to activate

TrkB receptors. The trophic role of BDNF in promoting serotonergic neuron axonal

growth and remodeling in the adult brain also displayed an inverted U-concentration-

response relationship 447 Mamounas,L.A. 2000 . In this study, cortical infusions of

various BDNF concentrations activated TrkB signaling locally, leading to highly

localized seratonin sprouting response, in which both the seratonin sprouting and Trk

receptor signaling displayed maximal responses at intermediate BDNF doses and are

depressed at higher doses. BDNF is a key mediator of bidirectional responses to exercise

and anti-depressants 448 Gomez-Pinilla,F. 2008. Although higher levels of BDNF

are generally beneficial to neuronal survival, excessive activation of its receptor, TrkB

can have adverse affects on neuronal survival and plasticity.

Sodium channels gating modifiers such as VRT appear capable of mimicking

neuronal activity and neurotrophin-dependent neurite outgrowth and hence may represent

a novel pharmacological strategy to regulate neuronal development through NMDAR-

BDNF-TrkB-dependent mechanisms. Recent studies have shown that neurotrophin

signaling plays an important role in various neurodevelopmental, neurodegenerative and

psychiatric disorders, and hence it is feasible that VGSC gating modifiers that augment

neuritogenesis and neurotrophin signaling may help in recovering from such disorders.

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Figure 4-1: Veratridine stimulated neurite outgrowth and dendritic arborization in immature cerebrocortical neurons

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Figure 4-2: Veratridine (VRT) increases intracellular Na+ and Ca2+ in DIV1 cerebrocortical neurons and this Ca2+ influx is TTX-sensitive and NMDAR dependent

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Figure 4-3:TrkB is essential for veratridine-induced neurite outgrowthVeratridine

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Figure 4-4: In situ BDNF ELISA: Veratridine enhances BDNF release in immature cerebrocortical neurons

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Figure 4-5:Veratridine enhances BDNF synthesis in immature cerebrocortical neurons and this requires VGSCs and partially involves NMDARs

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Figure 4-6: Veratridine enhances BDNF synthesis in immature cerebrocortical neurons and this requires VGSCs and partially involves NMDARs

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Figure 4-7: - Veratridine-induced neurite outgrowth involves PI3-kinase activity

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Figure 4-8- : Veratridine stimulated Akt phosphorylation involves TrkB receptors

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Figure 4-9 - : Veratridine-induced neurite outgrowth involves the PI3K-Akt-mTOR pathway

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Figure 4-10: - Veratridine-induced Ca2+ influx involves PLC mediated release of Ca2+ from intracellular

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Figure 4-11: Veratridine-induced neurite outgrowth requires phospholipase C (PLC)

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Figure 4-12:MAPK pathway has a modest role in veratridine-induced neurite outgrowth.

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Figure 4-13:Pharmacological characterization of Akt activation by veratridine

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Figure 4-14: Model 12

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