Declaration of Conflict of Interest or RelationshipDeclaration of Conflict of Interest or Relationship

Speaker Name: Xin Yu Kai-Hsiang Chuang

I have no conflicts of interest to disclose with regard to the subject matter ofthis presentation.

Functional MRI of the Mouse BrainFunctional MRI of the Mouse Brain

Kai-Hsiang ChuangKai-Hsiang Chuang

Laboratory of Molecular ImagingLaboratory of Molecular ImagingSingapore Bioimaging ConsortiumSingapore Bioimaging Consortium

Agency for Science, Technology and ResearchAgency for Science, Technology and ResearchSingaporeSingapore

Xin YuXin Yu

Laboratory of Functional and Molecular ImagingLaboratory of Functional and Molecular ImagingNational Institute of Neurological Disorders and StrokeNational Institute of Neurological Disorders and Stroke

National Institues of HealthNational Institues of Health

OutlineOutline• Issues of hemodynamic-based functional Issues of hemodynamic-based functional

MRI in miceMRI in mice

• Functional brain imaging by manganese Functional brain imaging by manganese enhanced MRI (MEMRI)enhanced MRI (MEMRI)

• MEMRI applications in the olfactory and MEMRI applications in the olfactory and auditory system in transgenic mouse auditory system in transgenic mouse modelsmodels

BOLD-fMRI for functional brain mapping BOLD-fMRI for functional brain mapping

“…. increases in glucose use and blood flow that are much greater than those in oxygen consumption. As a result there is an increase in the oxygen level in those areas (supply exceeds demand). PET is usually used to monitor blood flow. fMRI detects the changes in oxygen availability as a local change in the magnetic field. The resulting fMRI signal is a ‘blood-oxygen-level-dependent’ (BOLD) Signal…”

Raichle, Raichle, NatureNature 2001 2001

Neurovascular coupling factors Neurovascular coupling factors • Blood Oxygenation Level Dependent Blood Oxygenation Level Dependent

(BOLD) fMRI(BOLD) fMRI

– Blood flow/volume↑ >> O2 & glucose↑

• HbO2/Hb (activated) > HbO2/Hb (rest)

• T2*↑ MRI signal↑

Examples of hemodynamic fMRI in mice Examples of hemodynamic fMRI in mice

– Mouse brain : ~1 cm long; Cortex : ~ 1.2 mm think– Resolution requirement : 200 m in-plane, 0.5 mm

slice thickness– Need dedicated surface receive coil– Data averaging to get a reasonable SNR– Use CBV fMRI by i.v. injecting iron oxide particle (eg,

MION, Feridex)

Nair & Duong, MRM 2004 Mueggler, et al, MRM 2001

CBV, bicucullineBOLD, hindpaw

Issues – anesthesia: delivery & controlIssues – anesthesia: delivery & control• -chloralose-chloralose

– i.p. & intubation (Ahrens & Dubowitz, NMR Biomed 2001)– Less impact to metabolic/hemodynamic responses– Toxic, terminal experiment

• UrethaneUrethane– i.p. & free-breathing (Xu et al, PNAS 2003)– Carcinogenic, terminal experiment

• IsofluraneIsoflurane– Intubation with muscle relaxant (Mueggler, et al. MRM 2001; Bosshard,

ISMRM 2009))– Free-breathing (Nair & Duong, MRM 2004)– Vasodilator, increase baseline CBF, suppression of neural

activity• Medetomidine Medetomidine

– s.c. & free-breathing (Adamczak, et al, ISMRM 2009, available for longitudinal studies)

– α2-adrenoreceptor agonist– Sedative & analgesia, stable ~90 min

Issues – physiology under anesthesiaIssues – physiology under anesthesia• Anesthesia altersAnesthesia alters

– Cardiopulmonary regulation– Neural activity– Neural connectivity/pathway– Metabolic rate– Blood glucose, etc

Rat blood glucose under isoflurane

0

50

100

150

200

250

300

350

400

450

0 10 20 30 40 50 60 70 80 90 100 110

[min]

Glc

[m

g/d

L]

1.5% isofl

3.0% isofl

0.9% isofl,intubated

isofl off

isofl off

Matsumura, et al, NeuroImage 2003

Rat brain FDGConscious Propofol Isoflurane

Chuang’s Laboratory

Issues – maintain physiologyIssues – maintain physiology• Challenging to monitor pCO2, BP, etcChallenging to monitor pCO2, BP, etc

– Fine artery and vein, difficult to perform surgery – Not enough blood for withdraw– Noninvasive monitoring by noninvasive methods (e.g.,

transcutaneous pCO2)

• Temperature controlTemperature control– Heated air may cause larger fluctuation due to coil heat

up by hot air– Carefully designed water bath is more stable

Limitation of fMRI in generalLimitation of fMRI in general• Contrast mechanismContrast mechanism

– Complicated relationship with electrophysiology– Neural-vascular coupling changes with disease, drug

• Spatial resolution Spatial resolution – Small mouse brain needs high spatial resolution to achieve

anatomical details

• Ways of delivering stimulationWays of delivering stimulation– In magnet, under anesthesia– So far, sensory only

In general, BOLD-fMRI on mice is still at technique In general, BOLD-fMRI on mice is still at technique developing phase. developing phase.

Manganese Enhanced MRIManganese Enhanced MRI

Manganese (MnManganese (Mn2+2+))• CaCa2+2+ analog analog

– Enter excitable cells thru voltage-gated Ca2+ channel– eg, neuron, cardiac muscle, pancreatic β-cell

• Transport along axon and cross synapsesTransport along axon and cross synapses– via microtubule– Anterograde, trans-synaptic– Fast axonal transport

• MRI visibleMRI visible– High T1 relaxivity

3 major applications of MEMRI3 major applications of MEMRI• Neural activationNeural activation

• Neuronal tract tracingNeuronal tract tracing

• Neural cytoarchitectureNeural cytoarchitecture

Lin and Koretsky, Magn Reson Med 1997Aoki et al, Magn Reson Med 2002

Silva et al, J Neurosci Meth 2008

Pautler et al, Magn Reson Med 1998

Advantages of MEMRIAdvantages of MEMRI• Signal related to CaSignal related to Ca2+2+ channel activity, axonal channel activity, axonal

transport, synaptic uptake, etctransport, synaptic uptake, etc– Better reflect underlying neural activity/connectivity– Potentially better localizationlocalization

• Intra-cellularIntra-cellular– Stay in cell at least several days– Allow experiments in awakeawake animal outsideoutside magnet

– Imaged by high-resolutionhigh-resolution T1-weighted MRI (no EPI artifacts, hardware requirement)

• Anesthesia during scanning has less influenceAnesthesia during scanning has less influence

Issues of MEMRIIssues of MEMRI• MnMn2+2+ doesn’t cross Blood-Brain Barrier easily doesn’t cross Blood-Brain Barrier easily

– Limited ways of delivering Mn2+ to targeted regione.g., stereotaxic injection or break down BBB

• ToxicityToxicity– Not suitable for human subjects

• Long clearance timeLong clearance time– Only one stimulation event per subject – Can be repeated when Mn2+ cleared after 2-3 weeks(one longitudinal study was done in a 24hr time interval with second Mn injection,

Yu et al., 2005)

• Mechanism not fully understoodMechanism not fully understood

Functional map without breaking BBBFunctional map without breaking BBB• Brain regions without BBBBrain regions without BBB

– Appetite related region in hypothalamic nuclei (Kuo, et al, NMR Biomed 2006; Kuo, et al, J Neurosci 2007, Just et al. ISMRM 2009; Zeeni et al., ISMRM 2009)

• Systemic uptakeSystemic uptake– Mn2+ slowly distribute in whole brain via ventricular

brain junction after systemic infusion– Auditory mapping with long lasting stimulation

(Yu, et al, Nat Neurosci 2005; Watanabe, et al, MRM 2008; Lee et al., 2007)

• Activity dependent tracingActivity dependent tracing– Olfactory mapping etc.

MEMRI application in the MEMRI application in the olfactory systemolfactory system

Activity dependent tracingActivity dependent tracing• Deliver MnDeliver Mn2+2+ to let to let activityactivity enhance uptake and enhance uptake and

watch watch tracing tracing into higher order regions– Activated pathway– Odor induced activation in olfactory bulb

Pautler and Koretsky, NeuroImage 2002

No odor Amyl acetate

Functional organization olfactory bulbFunctional organization olfactory bulb

Graeme Lowe, http://flavor.monell.org/~loweg/OlfactoryBulb.htm Matt Valley, http://wikipedia.org

Methods for odor mappingMethods for odor mapping

• Current methodsCurrent methods– HRP tracing, monoclonal antibody,

receptor gene-labeled projection– 2-Deoxyglucose (2-DG)– C-Fos mRNA expression– Optical imaging of intrinsic signal or dyee– BOLD fMRI

• ProblemsProblems– Invasive– Penetration depth, field-of-view– Resolution (BOLD-fMRI)

Johnson, J Comp Neurol, 1999

Xu, PNAS, 2003

Rubin, Neuron, 1999

2DG

Intrinsic signalIntrinsic signal

fMRI

ProcedureProcedure• Lightly anesthetize the mouse by isofluraneLightly anesthetize the mouse by isoflurane• Quickly inject low dose MnQuickly inject low dose Mn2+2+ into nostrils into nostrils

– Mouse wake up in 30 sec

• Expose to an odor for 20 min in a chamberExpose to an odor for 20 min in a chamber• Anesthetize again by isofluraneAnesthetize again by isoflurane• Continuous 3D T1w MRI scan for 2-3 hrContinuous 3D T1w MRI scan for 2-3 hr

5% isoflurane

7uL 10mM Mn2+

dilute odor20 min

5% isoflurane

MRI scan

olfactometer

ON

Gl

Mi

MEMRI: individual analysisMEMRI: individual analysis

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6000

60 80 100 120 140 160

Time (min)

Sig

nal i

nten

sity

(A

.U.)

1 mm

Chuang et al, NeuroImage 2009

No odor

Odor induced Mn enhancementOdor induced Mn enhancement

high

low

OctanalAcetophenone Carvone

Chuang et al, NeuroImage 2009

Post-processingPost-processing• Co-registrationCo-registration• SegmentationSegmentation• Cortical layer flatteningCortical layer flattening

– Flat odor map in the glomerular and mitral cell layers

LL

VentralVentral

Later

Later

alal M

edi

Med

ialal

DorsalDorsal

LateralLateral

VentralVentral

MedialMedial

VentralVentral

DorsalDorsal

LateralLateral

VentralVentral

MedialMedial

VentralVentral

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teriA

nteri

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or

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steriP

osteri

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GlGl

MiMi

Group odor mapsGroup odor maps

OctanalAcetophenone Carvone

• Group t-test Group t-test vsvs no odor control ( no odor control (NN = 5 – 8) = 5 – 8)

anterior

lateral

medial

p = 0.005

t-score

0

5.0

2.5

Chuang et al, NeuroImage 2009

Detect individual glomeruliDetect individual glomeruli• RI7 transgenic mice (Bozza et al., 2002)RI7 transgenic mice (Bozza et al., 2002)

– Replace mouse M71 receptor by rat I7 (rI7) receptor, which responses to octanal, and with GFP

• Sensitivity: 80%Sensitivity: 80%

MEMRI GFP

1 mm

Chuang et al, NeuroImage 2009

MEMRI application in the MEMRI application in the auditory systemauditory system

The central auditory system (CAS) The central auditory system (CAS)

CochleaCN: Cochlear Nucleus

SoC: Superior Olive Complex

LL: Lateral Leminiscus

IC: Inferior colliculus

MGN: Medial Geniculate Nuclues

AC: Auditory cortex

VIII Nerve

CN SoC

LL

IC

MGN

AC

2mm

Dorsal

Ventral

IC

Low Frequency < 1 kHz

High Frequency 60kHz

The tonotopic organization of the mouse The tonotopic organization of the mouse inferior colliculus (IC)inferior colliculus (IC)

16 kHz

32 kHz

40 kHz

Romand and Ehret, 1990

Dorsal

Ventral

Mn Inj. 0.4mmol/kg

Mn Inj. 0.2mmol/kg

20-50 kHz 40 kHzNo Stimulation

Clearance

Time

Post 24 hr Post 48 hr Post 72 hr

0 255

Longitudinal imaging studies over 3 days in 24 h Longitudinal imaging studies over 3 days in 24 h time intervalstime intervals

n=4

0 255

MEMRI detected pure tone stimulated neuronal MEMRI detected pure tone stimulated neuronal activity in the mouse ICactivity in the mouse IC

16 kHz

40 kHz

Coronal IC image

IC Tonotopic Map(Electrophysiology)

16kHz

40kHz

n=8 for each group

40 kHz

0 255

Rostral

Caudal

16 kHz

2D coronal IC slices along the caudal-rostral 2D coronal IC slices along the caudal-rostral axisaxis

16 kHz

40 kHz

P<0.05

3D contour of frequency specific activity patterns 3D contour of frequency specific activity patterns by voxel-wise t statistic analysis by voxel-wise t statistic analysis

16 kHz 40 kHz

n8 for each group

Fibroblast growth factor (Fgf) 17 knockout mice Fibroblast growth factor (Fgf) 17 knockout mice can survive to adulthoodcan survive to adulthood

• Fgf8 and Fgf17 are morphogens to regulate the mid-hindbrain formation, Fgf8 and Fgf17 are morphogens to regulate the mid-hindbrain formation, expressing at the mid-hind brain border.expressing at the mid-hind brain border.

Fgf8Fgf17

Mid-hind brain border

Embryonic mouse brain

•Fgf8 mutation is lethal•Fgf17 knockout mice can survive to adulthood

Anatomical midbrain phenotype of Anatomical midbrain phenotype of Fgf17Fgf17 mutant micemutant mice

Cyt

och

rom

e

O

xyd

ase

ME

MR

I

IC Cb

Fgf17+/- Fgf17-/-

Histology from Anamaria SudarovHistology from Anamaria Sudarov

• Phenotype of Phenotype of Fgf17 Fgf17 mutantmutant mice– Smaller IC in Fgf17-/- mice– Fgf17+/- mice are similar to wild type

• Normal peripheral auditory systemNormal peripheral auditory system– Inner ear morphology – Auditory brainstem response (ABR)

Longitudinal studies of 16 and 40 kHz pure tone Longitudinal studies of 16 and 40 kHz pure tone stimulation in stimulation in Fgf17Fgf17 mutant mice mutant mice

Mn Inj. 0.4mmol/kg

Mn Inj. 0.2mmol/kg

40 kHz 16 kHzNo Stimulation

Clearance

Time

P21 Two days

P23

P24

P20

Altered tonotopic organization of the IC in Altered tonotopic organization of the IC in Fgf17Fgf17-/--/- mice mice

Fgf17+/- Fgf17-/-16 kHz40 kHz

SIthreshold=Mean+1.5*SD

n=7 n=10

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16 40 16 40Fgf17+/- Fgf17-/-

*

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* P<0.01* P<0.01

Maximal intensity maps

Summary of MEMRI activity mapping in Summary of MEMRI activity mapping in transgenic mouse modelstransgenic mouse models

• Dynamic MEMRI enables mapping odorant Dynamic MEMRI enables mapping odorant information flow at the level of information flow at the level of single glomerulisingle glomeruli in in the mouse olfactory bulbthe mouse olfactory bulb

• MEMRI can characterize altered anatomical MEMRI can characterize altered anatomical structure and functional architecture of the inferior structure and functional architecture of the inferior colliculus in colliculus in Fgf17Fgf17-/--/- mice mice

• Non-invasive protocol allows repeated Non-invasive protocol allows repeated experiments in the same mouseexperiments in the same mouse

AcknowledgementsAcknowledgements• Laboratory of Functional and Laboratory of Functional and

Molecular Imaging, Molecular Imaging, NINDSNINDS, , NIH, USANIH, USA– Alan P Koretsky– Steve J Dodd– Hellmut Merkle– Afonso C Silva

• Singapore Bioimaging Singapore Bioimaging ConsortiumConsortium– Conny Schmidt– Bingwen Zheng– Way Cherng Chen

• Developmental Neural Developmental Neural Plasticity Unit, NINDS, NIH, Plasticity Unit, NINDS, NIH, USAUSA– Leonardo Belluscio– Carolyn Marks

• New York UniversityNew York University– Daniel Turnbull– Dan Sanes– Youssef Zaim Wadghiri

• Sloan Kettering InstituteSloan Kettering Institute– Alexandra Joyner

• Samsung Medical Center, Samsung Medical Center, Seoul, KoreaSeoul, Korea– Jung Hee Lee

• National Institute of National Institute of Radiological Science, Chiba, Radiological Science, Chiba, JapanJapan– Ichio Aoki