1.
Declaration 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 of this presentation.

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Functional MRI of the Mouse Brain Kai-Hsiang Chuang L aboratory of Molecular Imaging Singapore Bioimaging Consortium Agency for Science, Technology and Research Singapore Xin Yu Laboratory of Functional and Molecular Imaging National Institute of Neurological Disorders and Stroke National Institues of Health

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Outline <ul><li>Issues of hemodynamic-based functional MRI in m ice </li></ul><ul><li>Functional brain imaging by manganese enhanced MRI (MEMRI) </li></ul><ul><li>MEMRI applications in the olfactory and auditory system in transgenic mouse models </li></ul>

4.
BOLD-fMRI for functional brain mapping <ul><li>“… . 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) S ignal …” </li></ul>Raichle, Nature 2001

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Neurovascular coupling factors <ul><li>Blood Oxygenation Level Dependent (BOLD) fMRI </li></ul><ul><ul><li>Blood flow/volume↑ >> O 2 & glucose↑ </li></ul></ul><ul><ul><ul><li>HbO 2 /Hb (activated) > HbO 2 /Hb (rest) </li></ul></ul></ul><ul><ul><ul><li>T 2 *↑  MRI signal↑ </li></ul></ul></ul>

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Examples of hemodynamic fMRI in mice <ul><ul><li>Mouse brain : ~1 cm long ; Cortex : ~ 1.2 mm think </li></ul></ul><ul><ul><li>Resolution requirement : 200  m in-plane, 0.5 mm slice thickness </li></ul></ul><ul><ul><li>Need dedicated surface receive coil </li></ul></ul><ul><ul><li>Data averaging to get a reasonable SNR </li></ul></ul><ul><ul><li>Use CBV fMRI by i.v. injecting iron oxide particle (eg, MION, Feridex) </li></ul></ul>Nair & Duong, MRM 2004 Mueggler, et al, MRM 2001 CBV, bicuculline BOLD, hindpaw

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Issues – anesthesia: delivery & control <ul><li> -chloralose </li></ul><ul><ul><li>i.p. & intubation (Ahrens & Dubowitz, NMR Biomed 2001) </li></ul></ul><ul><ul><li>Less impact to metabolic/hemodynamic responses </li></ul></ul><ul><ul><li>Toxic, terminal experiment </li></ul></ul><ul><li>Urethane </li></ul><ul><ul><li>i.p. & free-breathing (Xu et al, PNAS 2003) </li></ul></ul><ul><ul><li>Carcinogenic, terminal experiment </li></ul></ul><ul><li>Isoflurane </li></ul><ul><ul><li>Intubation with muscle relaxant (Mueggler, et al. MRM 2001 ; Bosshard, ISMRM 2009) ) </li></ul></ul><ul><ul><li>Free-breathing (Nair & Duong, MRM 2004) </li></ul></ul><ul><ul><li>Vasodilator, increase baseline CBF, suppress ion of neural activity </li></ul></ul><ul><li>Medetomidine </li></ul><ul><ul><li>s.c. & free-breathing (Adamczak, et al, ISMRM 2009 , available for longitudinal studies ) </li></ul></ul><ul><ul><li>α 2 -adrenoreceptor agonist </li></ul></ul><ul><ul><li>Sedative & analgesia, stable ~90 min </li></ul></ul>

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Issues – physiology under anesthesia <ul><li>Anesthesia alters </li></ul><ul><ul><li>Cardiopulmonary regulation </li></ul></ul><ul><ul><li>Neural activity </li></ul></ul><ul><ul><li>Neural connectivity/pathway </li></ul></ul><ul><ul><li>Metabolic rate </li></ul></ul><ul><ul><li>Blood glucose, etc </li></ul></ul>isofl off isofl off Matsumura, et al, NeuroImage 2003 Chuang’s Laboratory Rat brain FDG Conscious Propofol Isoflurane

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Issues – maintain physiology <ul><li>Challenging to monitor pCO2, BP, etc </li></ul><ul><ul><li>Fine artery and vein, difficult to perform surgery </li></ul></ul><ul><ul><li>Not enough blood for withdraw </li></ul></ul><ul><ul><li>Noninvasive monitoring by noninvasive methods (e . g . , transcutaneous pCO2) </li></ul></ul><ul><li>Temperature control </li></ul><ul><ul><li>Heated air may cause larger fluctuation due to coil heat up by hot air </li></ul></ul><ul><ul><li>Carefully designed water bath is more stable </li></ul></ul>

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Limitation of fMRI in general <ul><li>Contrast mechanism </li></ul><ul><ul><li>Complicated relationship with electrophysiology </li></ul></ul><ul><ul><li>Neural-vascular coupling changes with disease, drug </li></ul></ul><ul><li>Spatial resolution </li></ul><ul><ul><li>Small mouse brain needs high spatial resolution to achieve anatomical details </li></ul></ul><ul><li>Ways of delivering stimulation </li></ul><ul><ul><li>In magnet, under anesthesia </li></ul></ul><ul><ul><li>So far, sensory only </li></ul></ul><ul><li>In general, BOLD-fMRI on mice is still at technique developing phase. </li></ul>

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Manganese Enhanced MRI

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Manganese (Mn 2+ ) <ul><li>Ca 2+ analog </li></ul><ul><ul><li>Enter excitable cells thru voltage-gated Ca 2+ channel </li></ul></ul><ul><ul><li>eg, neuron, cardiac muscle, pancreatic β -cell </li></ul></ul><ul><li>Transport along axon and cross synapses </li></ul><ul><ul><li>via microtubule </li></ul></ul><ul><ul><li>Anterograde, trans-synaptic </li></ul></ul><ul><ul><li>Fast axonal transport </li></ul></ul><ul><li>MRI visible </li></ul><ul><ul><li>High T 1 relaxivity </li></ul></ul>

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3 major applications of MEMRI <ul><li>Neural activation </li></ul><ul><li>Neuronal tract tracing </li></ul><ul><li>Neural cytoarchitecture </li></ul>Lin and Koretsky, Magn Reson Med 1997 Aoki et al, Magn Reson Med 2002 Silva et al, J Neurosci Meth 2008 Pautler et al, Magn Reson Med 1998

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Advantages of MEMRI <ul><li>Signal related to Ca 2+ channel activity, axonal transport, synaptic uptake, etc </li></ul><ul><ul><li>Better reflect underlying neural activity/connectivity </li></ul></ul><ul><ul><li>Potentially better localization </li></ul></ul><ul><li>Intra-cellular </li></ul><ul><ul><li>Stay in cell at least several days </li></ul></ul><ul><ul><li>Allow experiments in awake animal outside magnet </li></ul></ul><ul><ul><li>Imaged by high-resolution T 1 -weighted MRI (no EPI artifacts, hardware requirement) </li></ul></ul><ul><li>Anesthesia during scanning has less influence </li></ul>

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Issues of MEMRI <ul><li>Mn 2+ doesn’t cross Blood-Brain Barrier easily </li></ul><ul><ul><li>Limited ways of delivering Mn 2+ to targeted region </li></ul></ul><ul><ul><li>e . g . , stereotaxic injection or break down BBB </li></ul></ul><ul><li>Toxicity </li></ul><ul><ul><li>Not suitable for human subjects </li></ul></ul><ul><li>Long clearance time </li></ul><ul><ul><li>Only one stimul ation event per subject </li></ul></ul><ul><ul><li>Can be repeated when Mn 2+ cleared after 2-3 weeks </li></ul></ul><ul><ul><li>( one longitudinal study was done in a 24hr time interval with second Mn injection, Yu et al., 2005 ) </li></ul></ul><ul><li>Mechanism not fully understood </li></ul>

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Functional map without breaking BBB <ul><li>Brain regions without BBB </li></ul><ul><ul><li>Appetite related region in hypothalamic nuclei </li></ul></ul><ul><ul><li>(Kuo, et al, NMR Biomed 2006; Kuo, et al, J Neurosci 2007 , Just et al. ISMRM 2009; Zeeni et al., ISMRM 2009 ) </li></ul></ul><ul><li>Systemic uptake </li></ul><ul><ul><li>Mn 2+ slowly distribute in whole brain via ventricular brain junction after systemic infusion </li></ul></ul><ul><ul><li>Auditory mapping with long lasting stimulation </li></ul></ul><ul><ul><li>(Yu, et al, Nat Neurosci 2005; Watanabe, et al, MRM 2008 ; Lee et al., 2007 ) </li></ul></ul><ul><li>Activity dependent tracing </li></ul><ul><ul><li>Olfactory mapping etc. </li></ul></ul>

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MEMRI application in the olfactory system

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Activity dependent tracing <ul><li>Deliver Mn 2+ to let activity enhance uptake and watch tracing into higher order regions </li></ul><ul><ul><li>Activated pathway </li></ul></ul><ul><ul><li>Odor induced activation in olfactory bulb </li></ul></ul>Pautler and Koretsky, NeuroImage 2002 No odor Amyl acetate

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

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Methods for odor mapping <ul><li>Current methods </li></ul><ul><ul><li>HRP tracing, monoclonal antibody, receptor gene-labeled projection </li></ul></ul><ul><ul><li>2-Deoxyglucose (2-DG) </li></ul></ul><ul><ul><li>C-Fos mRNA expression </li></ul></ul><ul><ul><li>Optical imaging of intrinsic signal or dy e </li></ul></ul><ul><ul><li>BOLD fMRI </li></ul></ul><ul><li>Problems </li></ul><ul><ul><li>Invasive </li></ul></ul><ul><ul><li>Penetration depth, field-of-view </li></ul></ul><ul><ul><li>Resolution (BOLD-fMRI) </li></ul></ul>Johnson, J Comp Neurol , 1999 Xu, PNAS , 2003 Rubin, Neuron , 1999 2DG Intrinsic signal fMRI

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Procedure <ul><li>Lightly anesthetize the mouse by isoflurane </li></ul><ul><li>Quickly inject low dose Mn 2+ into nostrils </li></ul><ul><ul><li>Mouse wake up in 30 sec </li></ul></ul><ul><li>Expose to an odor for 20 min in a chamber </li></ul><ul><li>Anesthetize again by isoflurane </li></ul><ul><li>Continuous 3D T1w MRI scan for 2-3 hr </li></ul>5% isoflurane 7uL 10mM Mn 2+ dilute odor 20 min 5% isoflurane MRI scan olfactometer

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MEMRI: individual analysis 1 mm Chuang et al, NeuroImage 2009 ON Gl Mi No odor

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Odor induced Mn enhancement Octanal Acetophenone Carvone Chuang et al, NeuroImage 2009 high low

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Post-processing <ul><li>Co-registration </li></ul><ul><li>Segmentation </li></ul><ul><li>Cortical layer flattening </li></ul><ul><ul><li>Flat odor map in the glomerular and mitral cell layers </li></ul></ul>Gl Mi L Ventral Lateral Medial Dorsal Lateral Ventral Medial Ventral Dorsal Lateral Ventral Medial Ventral Anterior Posterior

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Group odor maps <ul><li>Group t-test vs no odor control ( N = 5 – 8) </li></ul>Octanal Acetophenone Carvone anterior lateral medial p = 0.005 Chuang et al, NeuroImage 2009 t-score 0 5.0 2.5

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Detect individual glomeruli <ul><li>RI7 transgenic mice (Bozza et al., 2002) </li></ul><ul><ul><li>Replace mouse M71 receptor by rat I7 (rI7) receptor, which responses to octanal, and with GFP </li></ul></ul><ul><li>Sensitivity: 80% </li></ul>MEMRI GFP 1 mm Chuang et al, NeuroImage 2009

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MEMRI application in the auditory system

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The central auditory system (CAS) Cochlea CN: Cochlear Nucleus SoC: Superior Olive Complex LL: Lateral Leminiscus IC: Inferior colliculus MGN: Medial Geniculate Nuclues AC: Auditory cortex 2mm VIII Nerve CN SoC LL IC MGN AC

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Dorsal Ventral IC Low Frequency < 1 kHz High Frequency  60kHz The tonotopic organization of the mouse inferior colliculus (IC) 16 kHz 32 kHz 40 kHz Romand and Ehret, 1990 Dorsal Ventral

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Longitudinal imaging studies over 3 days in 24 h time intervals Mn Inj. 0.4mmol/kg Mn Inj. 0.2mmol/kg 20-50 kHz 40 kHz No Stimulation Clearance Time Post 24 hr Post 48 hr Post 72 hr 0 255 n=4

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MEMRI detected pure tone stimulated neuronal activity in the mouse IC 16 kHz 40 kHz Coronal IC image IC Tonotopic Map (Electrophysiology) 16kHz 40kHz n=8 for each group 0 255

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2D coronal IC slices along the caudal-rostral axis 40 kHz 0 255 Rostral Caudal 16 kHz

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3D contour of frequency specific activity patterns by voxel-wise t statistic analysis 16 kHz 40 kHz P<0.05 16 kHz 40 kHz n  8 for each group

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Fibroblast growth factor (Fgf) 17 knockout mice can survive to adulthood <ul><li>Fgf8 and Fgf17 are morphogens to regulate the mid-hindbrain formation, expressing at the mid-hind brain border. </li></ul>Mid-hind brain border Embryonic mouse brain <ul><li>Fgf8 mutation is lethal </li></ul><ul><li>Fgf17 knockout mice can survive to adulthood </li></ul>Fgf8 Fgf17

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Anatomical midbrain phenotype of Fgf17 mutant mice Cytochrome Oxydase MEMRI IC Cb Fgf17 +/- Fgf17 -/- Histology from Anamaria Sudarov <ul><li>Phenotype of Fgf17 mutant mice </li></ul><ul><ul><li>Smaller IC in Fgf17 -/- mice </li></ul></ul><ul><ul><li>Fgf17 +/- mice are similar to wild type </li></ul></ul><ul><li>Normal peripheral auditory system </li></ul><ul><ul><li>Inner ear morphology </li></ul></ul><ul><ul><li>Auditory brainstem response (ABR) </li></ul></ul>

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Longitudinal studies of 16 and 40 kHz pure tone stimulation in Fgf17 mutant mice Mn Inj. 0.4mmol/kg Mn Inj. 0.2mmol/kg 40 kHz 16 kHz No Stimulation Clearance Time P21 Two days P23 P24 P20

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Altered tonotopic organization of the IC in Fgf17 -/- mice Fgf17 +/- Fgf17 -/- 16 kHz 40 kHz SI threshold =Mean+1.5*SD n=7 n=10 Maximal intensity maps 0 200 400 600 800 16 40 16 40 Fgf17 +/- Fgf17 -/- * Activity center to IC center distance (  m) Fgf17 +/- Fgf17 -/- 0 200 300 400 500 100 16 & 40 kHz activity center distance (  m) * P<0.01 * P<0.01

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Summary of MEMRI activity mapping in transgenic mouse models <ul><li>Dynamic MEMRI enables mapping odorant information flow at the level of single glomeruli in the mouse olfactory bulb </li></ul><ul><li>MEMRI can characterize a ltered anatomical structure and functional architecture of the inferior colliculus in Fgf17 -/- mice </li></ul><ul><li>Non-invasive protocol allows repeated experiments in the same mouse </li></ul>

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Acknowledgement s <ul><li>Laboratory of Functional and Molecular Imaging, NINDS , NIH, USA </li></ul><ul><ul><li>Alan P Koretsky </li></ul></ul><ul><ul><li>Steve J Dodd </li></ul></ul><ul><ul><li>Hellmut Merkle </li></ul></ul><ul><ul><li>Afonso C Silva </li></ul></ul><ul><li>Singapore Bioimaging Consortium </li></ul><ul><ul><li>Conny Schmidt </li></ul></ul><ul><ul><li>Bingwen Zheng </li></ul></ul><ul><ul><li>Way Cherng Chen </li></ul></ul><ul><li>Developmental Neural Plasticity Unit, NINDS, NIH, USA </li></ul><ul><ul><li>Leonardo Belluscio </li></ul></ul><ul><ul><li>Carolyn Marks </li></ul></ul><ul><li>New York University </li></ul><ul><ul><li>Daniel Turnbull </li></ul></ul><ul><ul><li>Dan Sanes </li></ul></ul><ul><ul><li>Youssef Zaim Wadghiri </li></ul></ul><ul><li>Sloan Kettering Institute </li></ul><ul><ul><li>Alexandra Joyner </li></ul></ul><ul><li>Samsung Medical Center, Seoul, Korea </li></ul><ul><ul><li>Jung Hee Lee </li></ul></ul><ul><li>National Institute of Radiological Science, Chiba, Japan </li></ul><ul><ul><li>Ichio Aoki </li></ul></ul>