This work was supported by the LabEx TRAIL grant, reference ANR-10-LABX-57, and a French-Swiss ANR-FNS grant reference ANR-15- CE37-0012. The authors thank Aur&#233;lien Trotier for his technical support.

All animal procedures were conducted in accordance with the Animal Experimentation Guidelines of the European Communities Council Directive of November 24, 1986 (86/609/EEC). The protocol met the ethical guidelines of the French Ministry of Agriculture and Forests and was approved by the local ethics committees (Comit&#233; d&#39;&#233;thique pour L&#39;exp&#233;rimentation Animale Bordeaux n&#176;50112090-A).
NOTE: During the MR measurements, an adequate level of anesthesia and physiological monitoring (body temperature, respiratory rate) are indispensable requirements.
1. Animals
<ol>
	<li>Use male Wistar rats weighing between 350 and 450 g.</li>
	<li>Keep them on a 12:12 h light:dark cycle and provide food and water ad libitum.</li>
</ol>
2. Anesthesia
<ol>
	<li>Prepare the equipment needed for anesthesia (Figure 1A,B, see Table of Materials): a 5 mL syringe containing medetomidine in physiological saline solution (240 &#181;g/kg/h, with a perfusion rate of 20 &#181;L/min), a 0.5 mL syringe containing atipamezole (20 &#956;L, in 0.5 mL of saline solution), and eye ointment. 
	NOTE: Keep all equipment under the extractor hood, except the 5 mL syringe containing medetomidine, which is placed in the syringe pump near the magnet for anesthesia during MR acquisitions.</li>
	<li>Place the rat in the induction chamber, start the anesthesia by delivering 4% isoflurane, and adjust the oxygen flow rate to 1.5 L/min.</li>
	<li>Evaluate the depth of anesthesia by assessing the withdrawal of paw reflexes.</li>
	<li>When the rat does not respond to stimulation, take it out of the anesthesia box, place it on the bench with its nose in the isoflurane mask, and maintain anesthesia by delivering 2.5% in oxygen at 1.5 L/min.</li>
	<li>Gently massage the tail and place the tourniquet (Figure 1C). 
	NOTE: The massage can be performed in warm water, with a temperature between 38 and 42 &#176;C, to obtain better vasodilation of the veins.</li>
	<li>Insert the peripheral intravenous catheter (22 G), previously heparinized, in the left or right tail vein. Note that a venous return is observed (a drop of blood is visible at the distal part of the needle) when the catheter is correctly inserted (Figure 1D).</li>
	<li>Blow out any air bubbles present in the catheter dead space volume using the 2 mL syringe containing physiological saline solution and heparin.</li>
	<li>Apply the eye ointment and prepare the syringe containing atipamezole (17 &#181;g/mL) to awaken the rat at the end of the experiment.</li>
</ol>
3. Rat Placement in Magnet for Whisker Stimulation
<ol>
	<li>Place a breathing sensor on the magnet bed and then transfer the rat from the bench to the magnet bed. Place it in the prone position with its nose in the isoflurane mask, with the breathing sensor located between the ribcage and the magnet bed. 
	NOTE: All equipment that enters the MRI room should be MRI-safe.</li>
	<li>Decrease the isoflurane (from 4% to 1.5%&#8211;2%) during rat placement and switch the anesthesia to medetomidine at the end of this procedure. Ensure that the right whiskers are free, having cut the right edge at the front of the rat MRI bed beforehand to allow movement of the whiskers.</li>
	<li>Hold the rat in position with tape and monitor its breathing which must be between 60 and 80 breaths per minute.</li>
	<li>Make a sail that traps all right whiskers in the paper tape (Figure 2). Align the flexible outlet pipe of the air-puff system along the rat MRI bed so that the part exiting the tube is perpendicular and at around 1.5 cm from the sail. Fix it with paper tape.</li>
</ol>
4. Whisker Stimulation
<ol>
	<li>Connect the flexible inlet pipe from a compressed air source (1 bar) to a solenoid control valve input and the outlet pipe to the solenoid control valve output (Figure 3). Ensure that the solenoid control valve stays outside the magnet room.</li>
	<li>Plug the pulsing device into the solenoid valve and into the magnet using the transistor-transistor logic (TTL)-port. Configure it so that the pulsing frequency = 8 Hz, the pulsing time = 20 s, and the resting time = 10 s. 
	NOTE: These parameters, visualized on the small liquid crystal display (LCD) screen, are adjustable via the three dedicated analogical potentiometers. The electronic pulsing device, which controls the paradigm, must be composed of high-quality electronic components to avoid any drift in time parameters (for correct postprocessing).</li>
</ol>
5. BOLD fMRI Acquisition
<ol>
	<li>Place the rat brain so that it is in an upright position and use the ear bars to maintain it. Place the volume array coil above the rat&#8217;s head (Figure 4A) and fix it using tape. Check that the sail is moving correctly (anteroposterior movement, no rotation, and no friction of sail) when the air-puff system is turned On; then, switch it Off.</li>
	<li>Insert the bed and the coil in the center of the magnet. Check that the sail is still moving correctly once the bed is inside the magnet when the air-puff system is On; then, switch it Off. Switch completely from isoflurane to medetomidine (perfusion rate: 20 &#181;L/min).</li>
	<li>Check that the rat is well located using a localization sequence (TE = 2.5 ms; TR = 100 ms; average = 1; repetition = 1; slice = 1 mm; image size = 256 x 256; field of view (FOV) = 50 x 50 mm; scan time = 12 s 800 ms). Drag the Localizer sequence tab into the Instruction name and click on Continue.</li>
	<li>Drag the T2_Star_FID_EPI sequence tab into the Instruction name, center the FOV on the middle of the brain, and click on the Adjustment platform tab to open the edited scan instruction. Record a B0 map and proceed to a scan shim. 
	NOTE: For a B0 map, use the following parameters: first echo time = 1.65 ms; TR = 20 ms, average = 1; flip angle = 30&#176;; echo spacing = 3.805 ms; slice = 58 mm; image size = 64 x 64 x 64; FOV = 58 x 58 x 58 mm; scan time = 1 m 24 s 920 ms. For scan shim, use the following parameters: voxel selective excitation = STEAM Gaussian pulse; TE = 5 ms; mixing time = 10 ms; acquisition duration = 204.8 ms; bandwidth = 10,000 Hz; dwell time = 50 &#956;s).</li>
	<li>Start the T2 Star_FID_EPI sequence (TE = 16.096 ms; TR = 500 ms; average = 1; repetition = 600; slice = 1 mm; four consecutive slices; image size = 128 x 128; FOV = 20 x 20 mm; bandwidth = 333,333.3 Hz; scan time = 5 min 00 s). 
	NOTE: Due to the TTL port, an external trigger signal will start the air-puff system at the same time. The paradigm = [20 s activation + 10 s rest] x 10, for the total duration of the 600 scans, 500 ms per scan. The slices are centered on the middle of the barrel field area.</li>
	<li>Acquire another localization sequence (same as the one described in step 5.3) to compare with the first one and check whether the rat has moved during the T2_Star_FID_EPI sequence.</li>
	<li>Bring the bed to its initial position, remove the volume array coil, and connect the surface coil.</li>
</ol>
6. BOLD Processing
<ol>
	<li>Open the T2 Star_FID_EPI file and read the T2 Star_FID_EPI image in Image Display. Open the start-up window of the functional controller, called FunController.</li>
	<li>In this Processing tab, select the functional imaging window and define the stimulation protocol (duration and alternation of On/Off periods, corresponding to the paradigm used).</li>
	<li>Select the protocol window (dataset with 600 frames) and insert the value of 40 in the On Period tab and 20 in the Off period tab. Click on the Invert Attribution tab and drag the Stimulation States slider to the left to select the value 1.</li>
	<li>In the preprocessing window, click on the Median filter in plane for preprocessing and on the Median filter (2D, 3D) for postprocessing.</li>
	<li>Click on the Execute tab and drag the cursors to adjust the overlay lookup table. Visualize the activated brain area (Figure 4B).</li>
</ol>
7. Proton MRS Acquisitions
<ol>
	<li>To correctly position the surface coil, modify the position of the rat head. Rotate the head (approximately 30&#176; clockwise) so that the surface coil (Figure 5A) can be placed just above the left barrel cortex while being horizontal and located at the magnet center when inside the magnet.</li>
	<li>Place&#160;the surface coil, fix it on the rat brain using tape (Figure 5B), and check that the sail is moving correctly (anteroposterior movement, no rotation, and no friction of the sail) when the air-puff system is turned On; then, switch it Off at the main switch.</li>
	<li>Check that the sail is moving correctly once the bed is inside the magnet when the air-puff system is On. Then, switch it Off.</li>
	<li>Check the rat is positioned correctly using a localization sequence. Set parameters as follows: TE = 2.5 ms; TR = 100 ms; average = 1; repetition = 1; slice = 1 mm; image size = 256 x 256; FOV = 50 x 50 mm; scan time = 12 s 800 ms.
	<ol>
		<li>Drag the Localizer sequence tab into the Instruction name window and click on the Continue tab to execute the scan program.</li>
	</ol>
	</li>
	<li>When the brain localization is correct, drag the T2_TurboRARE sequence tab in the Instruction name window and click on Continue to execute the scan program. These anatomical images, together with the previous BOLD fMRI acquisition, will allow the correct localization of the voxel in the S1BF for MRS. 
	NOTE: The T2_TurboRARE parameters are 14 slices, 2 mm per slice, FOV = 2.5 x 2.5 cm, TE = 100 ms, TR = 5,000 ms, matrix = 128 x 128, sequence time = 2 min 40 s.</li>
	<li>Drag the LASER sequence tab into the Instruction name window, place the voxel (2 mm high, 2.5 mm long, 3 mm deep) at the center of the S1BF area.
	<ol>
		<li>Use a rat brain atlas and the BOLD fMRI enhancement to localize the zone on the T2 images (Figure 6). Click on the Adjustment platform tab to open the edited scan instruction. Click on the Wobble tab and change the impedance (electronic loading) of the receive coil slightly to tune it. Click on the Apply tab when the tuning is finished to close the instruction editor and apply the changes in the edited instruction.</li>
	</ol>
	</li>
	<li>Record a B0 map and proceed to scan shim and, then, perform a local shim. 
	NOTE: For the B0 map, use the following parameters: first echo time = 1.65 ms; TR = 20 ms, average = 1; flip angle = 30&#176;; echo spacing = 3.805 ms; slice = 58 mm; image size = 64 x 64 x 64; FOV = 58 x 58 x 58 mm; scan time = 1 m 24 s 920 ms. For the scan shim, use the following parameters: voxel selective excitation STEAM Gaussian pulse; TE = 5 ms; mixing time = 10 ms; acquisition duration = 204.8 ms; bandwidth = 10,000 Hz; dwell time = 50 &#956;s. For the local shim, use the following parameters: water suppression, VAPOR acquisition duration = 1,363.15 ms; points = 4,096; bandwidth in Hz = 3,004.81 Hz; bandwidth in ppm = 10 ppm; dwell time = 166.40 &#956;s; spectral resolution = 0.37 Hz/points. The LASER parameters are: echo time = 19.26 ms; TR = 2,500 ms; averages = 128 or 32; scan time = 5 min 20 s or 1 min 20 s; acquisition points = 4,096.</li>
	<li>Perform 1H-MRS.
	<ol>
		<li>Start the 1H-MRS acquisition first during a resting period (4 x 32 LASER scans + 128 LASER scans; 2,500 ms per scan).</li>
		<li>Acquire another localization sequence (same as the one described in step 5.3) to compare with the first one recorded and ensure that the rat has not moved during the LASER acquisition.</li>
		<li>Perform 1H-MRS during whisker activation using the LASER sequence (4 x 32 LASER scans + 128 LASER scans; 2,500 ms per scan) with the air-puff system On (paradigm = 20 s of activation and 10 s of rest).</li>
		<li>Once again, perform a localization sequence to check whether the rat has moved. 
		NOTE: The number of scans and resting/activated periods can be adapted and modified, but always ensure that the rat is not moving by regularly performing a localization sequence.</li>
	</ol>
	</li>
	<li>Bring the bed to its initial position, remove the surface coil, and move the rat back to the bench. Inject atipamezole into a skin fold made in the rat&#8217;s back to reverse the anesthesia and awaken it.</li>
</ol>
8. Proton MRS Processing
<ol>
	<li>Open the LCModel software and click on the appropriate tab to select the right data type ( Free Induction Decay file) and choose the right file. Click on the OK tab when this is done.</li>
	<li>Optimize the quantification control parameters step by step.
	<ol>
		<li>In the Title section, manually enter a title and define an adequate ppm range (e.g., 0.2 to 4.0 ppm) by manually typing in the necessary value in the respective fields.</li>
		<li>In the Basis file section, select and download the required file to fit the macromolecule baseline correctly (it can be provided by the software provider).</li>
		<li>Define and load the input control parameters. Prepare the save process of all useful file types beforehand (TABLE = compact tables; PS = necessary PostScript output; CSV = format for spreadsheets; COORD = coordinates for plots). Click on the RunLCModel tab to start the LCModel quantification.</li>
	</ol>
	</li>
	<li>Define selected metabolites to generate statistics. 
	NOTE: LCModel provides metabolite quantification and estimates errors by a value termed Cram&#233;r-Rao lower bound (CRLB). A value with a CRLB &#60; 15 is considered as an optimal quantification. A CRLB &#62; 25 indicates an unreliable value.</li>
</ol>

<ol>
	<li>Pellerin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+regulation+of+energy+metabolism+by+astrocytes:+an+update.">Activity-dependent regulation of energy metabolism by astrocytes: an update.</a> Glia. 55, 1251-1262 (2007).</li><li>Pellerin, L., Magistretti, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+uptake+into+astrocytes+stimulates+aerobic+glycolysis:+a+mechanism+coupling+neuronal+activity+to+glucose+utilization.">Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization.</a> Proceedings of the National Academy of Sciences of the United States of America. 91, 10625-10629 (1994).</li><li>Cholet, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+injection+of+antisense+oligonucleotides+targeted+to+the+glial+glutamate+transporter+GLAST+decreases+the+metabolic+response+to+somatosensory+activation.">Local injection of antisense oligonucleotides targeted to the glial glutamate transporter GLAST decreases the metabolic response to somatosensory activation.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 21, 404-412 (2001).</li><li>Voutsinos-Porche, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+Glutamate+Transporters+Mediate+a+Functional+Metabolic+Crosstalk+between+Neurons+and+Astrocytes+in+the+Mouse+Developing+Cortex.">Glial Glutamate Transporters Mediate a Functional Metabolic Crosstalk between Neurons and Astrocytes in the Mouse Developing Cortex.</a> Neuron. 37, 275-286 (2003).</li><li>Zimmer, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[18F]FDG+PET+signal+is+driven+by+astroglial+glutamate+transport.">[18F]FDG PET signal is driven by astroglial glutamate transport.</a> Nature Neuroscience. 20 (3), 393-395 (2017).</li><li>Haiss, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+in+vivo+two-photon+imaging+after+blood+replacement+by+perfluorocarbon.">Improved in vivo two-photon imaging after blood replacement by perfluorocarbon.</a> The Journal of Physiology. , (2009).</li><li>Mullins, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+theory+of+functional+magnetic+resonance+spectroscopy+(fMRS):+A+meta-analysis+and+discussion+of+using+MRS+to+measure+changes+in+neurotransmitters+in+real+time.">Towards a theory of functional magnetic resonance spectroscopy (fMRS): A meta-analysis and discussion of using MRS to measure changes in neurotransmitters in real time.</a> Scandinvian Journal of Psychology. 59, 91-103 (2018).</li><li>Wong-Riley, M. T., Welt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+changes+in+cytochrome+oxidase+of+cortical+barrels+after+vibrissal+removal+in+neonatal+and+adult+mice.">Histochemical changes in cytochrome oxidase of cortical barrels after vibrissal removal in neonatal and adult mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 77, 2333-2337 (1980).</li><li>Petersen, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+organization+of+the+barrel+cortex.">The functional organization of the barrel cortex.</a> Neuron. 56, 339-355 (2007).</li><li>Cox, S. B., Woolsey, T. A., Rovainen, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+dynamic+changes+in+cortical+blood+flow+with+whisker+stimulation+corresponds+to+matched+vascular+and+neuronal+architecture+of+rat+barrels.">Localized dynamic changes in cortical blood flow with whisker stimulation corresponds to matched vascular and neuronal architecture of rat barrels.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 13, 899-913 (1993).</li><li>Feldmeyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitatory+neuronal+connectivity+in+the+barrel+cortex.">Excitatory neuronal connectivity in the barrel cortex.</a> Frontiers in Neuroanatomy. 6, 24 (2012).</li><li>Boussida, S., Traore, A. S., Durif, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+the+brain+hemodynamic+responses+to+sensorimotor+stimulation+in+a+rodent+model:+A+BOLD+fMRI+study.">Mapping of the brain hemodynamic responses to sensorimotor stimulation in a rodent model: A BOLD fMRI study.</a> PLoS One. 12, e0176512 (2017).</li><li>Heinke, W., Koelsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+anesthetics+on+brain+activity+and+cognitive+function.">The effects of anesthetics on brain activity and cognitive function.</a> Current Opinion in Anesthesiology. 18, 625-631 (2005).</li><li>Horn, T., Klein, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactate+levels+in+the+brain+are+elevated+upon+exposure+to+volatile+anesthetics:+a+microdialysis+study.">Lactate levels in the brain are elevated upon exposure to volatile anesthetics: a microdialysis study.</a> Neurochemistry International. 57, 940-947 (2010).</li><li>Boretius, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Halogenated+volatile+anesthetics+alter+brain+metabolism+as+revealed+by+proton+magnetic+resonance+spectroscopy+of+mice+in+vivo.">Halogenated volatile anesthetics alter brain metabolism as revealed by proton magnetic resonance spectroscopy of mice in vivo.</a> Neuroimage. 69, 244-255 (2013).</li><li>Sinclair, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+physiological+effects+of+alpha2-agonists+related+to+the+clinical+use+of+medetomidine+in+small+animal+practice.">A review of the physiological effects of alpha2-agonists related to the clinical use of medetomidine in small animal practice.</a> Canadian Veterinary Journal. 44, 885-897 (2003).</li><li>Weber, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fully+noninvasive+and+robust+experimental+protocol+for+longitudinal+fMRI+studies+in+the+rat.">A fully noninvasive and robust experimental protocol for longitudinal fMRI studies in the rat.</a> Neuroimage. 29, 1303-1310 (2006).</li><li>Hartmann, M. J., Johnson, N. J., Towal, R. B., Assad, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+characteristics+of+rat+vibrissae:+resonant+frequencies+and+damping+in+isolated+whiskers+and+in+the+awake+behaving+animal.">Mechanical characteristics of rat vibrissae: resonant frequencies and damping in isolated whiskers and in the awake behaving animal.</a> The Journal of Neuroscience. 23, 6510-6519 (2003).</li><li>Prichard, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactate+rise+detected+by+1H+NMR+in+human+visual+cortex+during+physiologic+stimulation.">Lactate rise detected by 1H NMR in human visual cortex during physiologic stimulation.</a> Proceedings of the National Academy of Sciences of the United States of America. 88, 5829-5831 (1991).</li><li>Sappey-Marinier, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+photic+stimulation+on+human+visual+cortex+lactate+and+phosphates+using+1H+and+31P+magnetic+resonance+spectroscopy.">Effect of photic stimulation on human visual cortex lactate and phosphates using 1H and 31P magnetic resonance spectroscopy.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 12, 584-592 (1992).</li><li>Mazuel, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neuronal+MCT2+knockdown+in+the+rat+somatosensory+cortex+reduces+both+the+NMR+lactate+signal+and+the+BOLD+response+during+whisker+stimulation.">A neuronal MCT2 knockdown in the rat somatosensory cortex reduces both the NMR lactate signal and the BOLD response during whisker stimulation.</a> PLoS One. 12, e0174990 (2017).</li><li>Castellano, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NAA+and+NAAG+variation+in+neuronal+activation+during+visual+stimulation.">NAA and NAAG variation in neuronal activation during visual stimulation.</a> Brazilian Journal of Medical and Biological Research. 45, 1031-1036 (2012).</li><li>Sarchielli, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+1H-MRS+findings+in+migraine+patients+with+and+without+aura+assessed+interictally.">Functional 1H-MRS findings in migraine patients with and without aura assessed interictally.</a> Neuroimage. 24, 1025-1031 (2005).</li><li>Baslow, M. H., Hrabe, J., Guilfoyle, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+relationship+between+neurostimulation+and+N-acetylaspartate+metabolism+in+the+human+visual+cortex:+evidence+that+NAA+functions+as+a+molecular+water+pump+during+visual+stimulation.">Dynamic relationship between neurostimulation and N-acetylaspartate metabolism in the human visual cortex: evidence that NAA functions as a molecular water pump during visual stimulation.</a> Journal of Molecular Neuroscience. 32, 235-245 (2007).</li><li>Mangia, S., Tkac, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+relationship+between+neurostimulation+and+N-acetylaspartate+metabolism+in+the+human+visual+cortex:+evidence+that+NAA+functions+as+a+molecular+water+pump+during+visual+stimulation.">Dynamic relationship between neurostimulation and N-acetylaspartate metabolism in the human visual cortex: evidence that NAA functions as a molecular water pump during visual stimulation.</a> Journal of Molecular Neuroscience. 35, 245-248 (2008).</li><li>Baslow, M. H., Hrabal, R., Guilfoyle, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+the+authors+to+the+Letter+by+Silvia+Mangia+and+Ivan+Tkac.">Response of the authors to the Letter by Silvia Mangia and Ivan Tkac.</a> Journal of Molecular Neuroscience. 35, 247-248 (2008).</li><li>Barros, L. F., Weber, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CrossTalk+proposal:+an+important+astrocyte-to-neuron+lactate+shuttle+couples+neuronal+activity+to+glucose+utilisation+in+the+brain.">CrossTalk proposal: an important astrocyte-to-neuron lactate shuttle couples neuronal activity to glucose utilisation in the brain.</a> The Journal of Physiology. 596, 347-350 (2018).</li><li>Bak, L. K., Walls, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CrossTalk+opposing+view:+lack+of+evidence+supporting+an+astrocyte-to-neuron+lactate+shuttle+coupling+neuronal+activity+to+glucose+utilisation+in+the+brain.">CrossTalk opposing view: lack of evidence supporting an astrocyte-to-neuron lactate shuttle coupling neuronal activity to glucose utilisation in the brain.</a> The Journal of Physiology. 596, 351-353 (2018).</li></ol>

The authors have nothing to disclose.

The barrel cortex, also called S1BF for the somatosensory cortex or barrel field, is a region within the cortical layer IV that can be observed using cytochrome c oxidase staining9, and its organization is well known since it has been largely described10,11. One vibrissa is connected to one barrel, in which around 19,000 neurons are organized in a column12. The whisker-to-barrel cortex pathway has several advantages...

The brain possesses intrinsic mechanisms that allow the regulation of its major substrate (i.e., glucose), both for its contribution and its utilization, depending on variations in local cerebral activity. Although glucose is the main energy substrate for the brain, experiments performed in recent years have shown that lactate, which is produced by the astrocytes, could be an efficient energy substrate for the neurons. This raises the hypothesis of a lactate shuttle between astrocytes and neurons1. Known as ANLS, for astrocyte-neuron lactate shuttle2, the theory is still highly debated but has led to the proposal that glucose, rather than going directly into neurons, may enter the astrocytes, where it is metabolized into lactate, a metabolite that is, then, transferred to the neurons, which use it as efficient energy substrate. If such a shuttle exists in vivo, it would have several important consequences, both for the understanding of basic techniques in functional cerebral imaging (positron emission tomography [PET]) and for deciphering the metabolic alterations observed in brain pathologies.
To study brain metabolism and, particularly, metabolic interactions between neurons and astrocytes, four main techniques are available (not including micro-/nanosensors): autoradiography, PET, two-photon fluorescent confocal microscopy, and MRS. Autoradiography was one of the first methods proposed and provides images of the regional accumulation of radioactive 14C-2-deoxyglucose in brain slices, while PET yields in vivo images of the regional uptake of radioactive 18F-deoxyglucose. They both have the disadvantage of using irradiative molecules while producing low-spatial resolution images. Two-photon microscopy provides cellular resolution of fluorescent probes, but light scattering by tissue limits the imaging depth. These three techniques have been used previously to study neuroenergetics in rodents during whisker stimulation3,4,5,6. In vivo MRS has the dual advantage of being noninvasive and nonradioactive, and any brain structure can be explored. Moreover, MRS can be performed during neuronal activation, a technique called functional MRS (fMRS), which has been developed very recently in rodents7. Therefore, a protocol to monitor brain metabolism during cerebral activity by 1H-MRS in vivo and noninvasively is proposed. The procedure is described in adult healthy rats with brain activation obtained by an air-puff whisker stimulation performed directly in a 7 T magnetic resonance (MR) imager but may be adapted in genetically modified animals, as well as in any pathological condition.

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			<td>0.5 mL syringe with needle</td>
			<td>Becton, Dickinson and Company, USA</td>
			<td>2020-10</td>
			<td>0.33 mm (29 G) x 12.7 mm</td>
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			<td>1H spectroscopy surface coil</td>
			<td>Bruker, Ettlingen, Germany</td>
			<td>T116344</td>
			<td></td>
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			<td>7T Bruker Biospec system</td>
			<td>Bruker, Ettlingen, Germany</td>
			<td>70/20 USR</td>
			<td></td>
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			<td>Arduino Uno based pulsing device</td>
			<td>custom made</td>
			<td></td>
			<td></td>
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			<td>Atipamezole</td>
			<td>V&#233;toquinol, S.A., France</td>
			<td>V8335602</td>
			<td>Antisedan, 4.28 mg</td>
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			<td>Breathing mask</td>
			<td>custom made</td>
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			<td>TVM laboratoire, France</td>
			<td>40365</td>
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			<td>custom made</td>
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			<td>30x17x15 cm</td>
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			<td>Inlet flexible pipe</td>
			<td>Gardena, Germany</td>
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			<td>4.6-mm diameter, 3m long</td>
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			<td>Isoflurane pump, Model 100 series vaporizer, classic T3</td>
			<td>Surgivet, Harvard Apparatus</td>
			<td>WWV90TT</td>
			<td>from OH 43017, U.S.A</td>
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			<td>Isoflurane, liquid for inhalation</td>
			<td>Vertflurane, Virbac, France</td>
			<td>QN01AB06</td>
			<td>1000 mg/mL</td>
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			<td>KD Scientific syringe pump</td>
			<td>KD sientific, Holliston, USA</td>
			<td>Legato 110</td>
			<td></td>
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			<td>LCModel software</td>
			<td>LCModel Inc., Ontario, Canada</td>
			<td>6.2</td>
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			<td>Medetomidine hydrochloride</td>
			<td>V&#233;toquinol, S.A., France</td>
			<td>QN05CM91</td>
			<td>Domitor, 1 mg/mL</td>
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			<td>Micropore roll of adhesive plaster</td>
			<td>3M micropore, Minnesota, United States</td>
			<td>MI912</td>
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			<td>Micropore roll of adhesive plaster</td>
			<td>3M micropore, Minnesota, United States</td>
			<td>MI925</td>
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			<td>Monitoring system of physiologic parameter</td>
			<td>SA Instruments, Inc, Stony Brook, NY, USA</td>
			<td>Model 1025</td>
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			<td>NaCl</td>
			<td>Fresenius Kabi, Germany</td>
			<td>B05XA03</td>
			<td>0.9 % 250 mL</td>
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			<td>Outlet flexible pipe</td>
			<td>Gardena, Germany</td>
			<td>1348-20</td>
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			<td>Paravision software</td>
			<td>Bruker, Ettlingen, Germany</td>
			<td>6.0.1</td>
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			<td>Peripheral intravenous catheter</td>
			<td>Terumo, Shibuya, Tokyo, Japon</td>
			<td>SP500930S</td>
			<td>22 G x 1&#34;, 0.85x25 mm, 35 mL/min</td>
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			<td>Rat head coil</td>
			<td>Bruker, Ettlingen, Germany</td>
			<td></td>
			<td></td>
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			<td>Sodic heparin, injectable solution</td>
			<td>Choai, Sanofi, Paris, France</td>
			<td>B01AB01</td>
			<td>5000 IU/mL</td>
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			<td>Solenoid control valves, plunger valve 2/2 way direct-acting</td>
			<td>Burkert, Germany</td>
			<td>3099939</td>
			<td>Model type 6013</td>
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		<tr>
			<td>Terumo 2 ml syringe</td>
			<td>Terumo, Shibuya, Tokyo, Japon</td>
			<td>SY243</td>
			<td>with 21 g x 5/8&#34; needle</td>
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		<tr>
			<td>Terumo 5 mL syringe</td>
			<td>Terumo, Shibuya, Tokyo, Japon</td>
			<td>05SE1</td>
			<td></td>
		</tr>
		<tr>
			<td>Wistar RJ-Han rats</td>
			<td>Janvier Laboratories, France</td>
			<td></td>
			<td></td>
		</tr>
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functional magnetic resonance spectroscopy at 7 t in the rat barrel cortex during whisker activation

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to technological developments over the last decade and the increase in magnetic field strength, it is now possible to obtain good resolution spectra in vivo in the rat brain. Neuroenergetics (i.e., the study of brain metabolism) and, especially, metabolic interactions between the different cell types have attracted more and more interest in recent years. Among these metabolic interactions, the existence of a lactate shuttle between neurons and astrocytes is still debated. It is, thus, of great interest to perform functional proton magnetic resonance spectroscopy (1H-MRS) in a rat model of brain activation and monitor lactate. However, the methyl lactate peak overlaps lipid resonance peaks and is difficult to quantify. The protocol described below allows metabolic and lactate fluctuations to be monitored in an activated brain area. Cerebral activation is obtained by whisker stimulation and 1H-MRS is performed in the corresponding activated barrel cortex, whose area is detected using blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI). All steps are fully described: the choice of anesthetics, coils, and sequences, achieving efficient whisker stimulation directly in the magnet, and data processing.

This protocol allows the quantification of metabolite fluctuations during cerebral activation, which is obtained by right whisker stimulation directly in the magnet.
In this study, the overall goal of BOLD fMRI was to check that the whisker stimulation was efficient, to visualize the activated S1BF area, and to correctly locate the voxel for 1H-fMRS. The device built for whisker activation is efficient. Indeed, when r...

Watch this Scientific Journal Video about Functional Magnetic Resonance Spectroscopy at 7 T in the Rat Barrel Cortex During Whisker Activation at JoVE.com

Functional Magnetic Resonance Spectroscopy at 7 T in the Rat Barrel Cortex During Whisker Activation

After checking by blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) that the corresponding somatosensory barrel field cortex area (called S1BF) is correctly activated, the main goal of this study is to quantify lactate content fluctuations in the activated rat brains by localized proton magnetic resonance spectroscopy (1H-MRS) at 7 T.

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to ...

functional-magnetic-resonance-spectroscopy-at-7-t-rat-barrel-cortex

Research

JoVE Journal

Neuroscience

8.6K Views.  Centre National de la Recherche Scientifique (CNRS)/Université Bordeaux. After checking by blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) that the corresponding somatosensory barrel field cortex area (called S1BF) is correctly activated, the main goal of this study is to quantify lactate content fluctuations in the activated rat brains by localized proton magnetic resonance spectroscopy (1H-MRS) at 7 T.

Video: Functional Magnetic Resonance Spectroscopy at 7 T in the Rat Barrel Cortex During Whisker Activation

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral line-widths and allows high-resolution spectra to be obtained from extracts, intact cells, cell cultures, and more importantly intact tissue to investigate relationships between metabolites and cellular processes. In vivo HRMAS 1H-MRS studies have yet to be reported in the live fruit fly Drosophila melanogaster. Drosophila, as a simpler genetic organism, allows the multiple biological functions and various evolutionarily conserved signaling pathways to be examined at the whole organism level and it is a useful model for investigating genetics and physiology. To this end, we developed and implemented an in vivo HRMAS 1H-MRS method to investigate live Drosophila at 14.1 T. Here, we outline an HRMAS 1H-MRS protocol for the molecular characterization of Drosophila with a conventional MR spectrometer equipped with an HRMAS probe. This technique is a novel, in vivo, non-destructive Drosophila metabolite measurement approach, which enables the identification of disease biomarkers and thus may contribute to novel therapeutic development.

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

This technique enables the use of high-resolution magic angle spinning proton MR spectroscopy (HRMAS 1H-MRS) for molecular characterization of live Drosophila melanogaster with a conventional 14.1 tesla spectrometer equipped with an HRMAS probe.

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral ...

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as &#947;-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.

This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic field pulses. The initiation of cortical activation or its modulation depends on the background activation of the neurons of the cortical region activated, the characteristics of the coil, its position and its orientation with respect to the head. TMS combined with simultaneous electrocephalography (EEG) and neuronavigation (nTMS-EEG) allows for the assessment of cortico-cortical excitability and connectivity in almost all cortical areas in a reproducible manner. This advance makes nTMS-EEG a powerful tool that can accurately assess brain dynamics and neurophysiology in test-retest paradigms that are required for clinical trials. Limitations of this method include artifacts that cover the initial brain reactivity to stimulation. Thus, the process of removing artifacts may also extract valuable information. Moreover, the optimal parameters for dorsolateral prefrontal (DLPFC) stimulation are not fully known and current protocols utilize variations from the motor cortex (M1) stimulation paradigms. However, evolving nTMS-EEG designs hope to address these issues. The protocol presented here introduces some standard practices for assessing neurophysiological functioning from stimulation to the DLPFC that can be applied in patients with treatment resistant psychiatric disorders that receive treatment such as transcranial direct current stimulation (tDCS), repetitive transcranial magnetic stimulation (rTMS), magnetic seizure therapy (MST) or electroconvulsive therapy (ECT).

The protocol presented here is for TMS-EEG studies utilizing intracortical excitability test-retest design paradigms. The intent of the protocol is to produce reliable and reproducible cortical excitability measures for assessing neurophysiological functioning related to therapeutic interventions in the treatment of neuropsychiatric diseases such as major depression.

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

Acquisition of Resting-State Functional Magnetic Resonance Imaging Data in the Rat

Resting-state functional magnetic resonance imaging (rs-fMRI) has become an increasingly popular method to study brain function in a resting, non-task state. This protocol describes a preclinical survival method for obtaining rs-fMRI data. Combining low dose isoflurane with continuous infusion of the &#945;2 adrenergic receptor agonist dexmedetomidine provides a robust option for stable, high-quality data acquisition while preserving brain network function. Furthermore, this procedure allows for spontaneous breathing and near-normal physiology in the rat. Additional imaging sequences can be combined with resting-state acquisition creating experimental protocols with anesthetic stability of up to 5 h using this method. This protocol describes the setup of equipment, monitoring of rat physiology during four distinct phases of anesthesia, acquisition of resting-state scans, quality assessment of data, recovery of the animal, and a brief discussion of post-processing data analysis. This protocol can be used across a wide variety of preclinical rodent models to help reveal the resulting brain network changes that occur at rest.

This protocol describes a method for obtaining stable resting-state functional magnetic resonance imaging (rs-fMRI) data from a rat using low dose isoflurane in combination with low dose dexmedetomidine.

Resting-state functional magnetic resonance imaging (rs-fMRI) has become an increasingly popular method to study brain function in a resting, non-task ...

Group Synchronization During Collaborative Drawing Using Functional Near-Infrared Spectroscopy

Functional near-infrared spectroscopy (fNIRS) is a noninvasive method particularly suitable for measuring cerebral cortex activation in multiple subjects, which is relevant for studying group interpersonal interactions in ecological settings. Although many fNIRS systems technically offer the possibility to monitor more than two individuals simultaneously, establishing easy-to-implement setup procedures and reliable paradigms to track hemodynamic and behavioral responses in group interaction is still required. The present protocol combines fNIRS and video-based observation to measure interpersonal synchronization in quartets during a cooperative task.This protocol provides practical recommendations for data acquisition and paradigm design, as well as guiding principles for an illustrative data analysis example. The procedure is designed to assess differences in brain and behavior interpersonal responses between social and non-social conditions inspired by a well-known ice-breaker activity, the Collaborative Face Drawing Task. The described procedures can guide future studies to adapt group naturalistic social interaction activities to the fNIRS environment.

The present protocol combines functional near-infrared spectroscopy (fNIRS) and video-based observationto measure interpersonal synchronization in quartets during a collaborative drawing task.

Functional near-infrared spectroscopy (fNIRS) is a noninvasive method particularly suitable for measuring cerebral cortex activation in multiple subjects, ...

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei.
Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases.
We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...