We thank Dr. D. Chen and Dr. C. Yen for sharing the AFNI script to set up the real-time fMRI for PV 5 and the AFNI team for the software support. This research was supported by NIH Brain Initiative funding (RF1NS113278-01, R01 MH111438-01), and the S10 instrument grant (S10 RR023009-01) to Martinos Center, German Research Foundation (DFG) Yu215/3-1, BMBF 01GQ1702, and the internal funding from Max Planck Society.

This study was performed in accordance with the German Animal Welfare Act (TierSchG) and Animal Welfare Laboratory Animal Ordinance (TierSchVersV). The experimental protocol described here was reviewed by the ethics commission (&#167;15 TierSchG) and approved by the state authority (Regierungspr&#228;sidium, T&#252;bingen, Baden-W&#252;rttemberg, Germany).
1. Preparing the BOLD-fMRI experimental set-up for small animal study
<ol>
	<li>Turn on the console software to control imaging parameters and acquire MRI data. 
	NOTE: The proposed real-time fMRI set-up is implemented utilizing macro-functions of the console software (version 6) in parallel with the image processing functions of AFNI.</li>
	<li>Find MR sequences (i.e., Position, Localizer, Rapid Acquisition with Relaxation Enhancement (RARE), and 3D echo-planar imaging (EPI) with the workspace explorer, and then drag and append them in the scan list. 
	NOTE: Position and Localizer sequences are used to identify a region of interest (ROI) in a brain. A RARE sequence is used for an anatomy scan. A 3D EPI sequence is used to measure dynamic BOLD responses.</li>
	<li>Place the predefined macro scripts, &#8220;Setup_rt3DEPI&#8221; and &#8220;Feed2AFNI_rt3DEPI&#8221; in the macro script path (e.g., &#8220;/opt/(PV version)/prog/curdir/(user name)/ParaVision/macros&#8221;). Activate the 3D EPI reconstruction options, &#8220;Pre Image Series Activities&#8221; and &#8220;Execute Macro&#8221; in the &#8220;Data Reconstruction&#8221; user interface menu, and then link the predefined macro script, &#8220;Setup_rt3DEPI&#8221;, before clicking the &#8220;Scan&#8221; button. 
	NOTE: The macro scripts are included in the Supplementary files.</li>
	<li>Install the AFNI software for the real-time BOLD-fMRI analysis and visualization.</li>
</ol>
2. Catheterization and ventilation surgery
<ol>
	<li>Set up a ventilator and physiological status monitoring systems such as thermometer, blood pressure and respiration recording as shown in Figure 1. Set a constant frequency of 60 &#177; 1 breath/min with the ventilator and a temperature of 37 &#176;C using an MR-compatible heating pad with a feedback control set.</li>
	<li>Anesthetize an adult male Sprague-Dawley rat (300-600 g) in a chamber with 5% isoflurane for induction and deliver 2-2.5% isoflurane for surgery from a vaporizer. Check the depth of anesthesia by pinching the hindpaw and confirming the lack of a withdrawal response.</li>
	<li>Intubate the animal with a 14 G plastic cannula for ventilation (60 &#177; 1 breath/min with a mixture of 70% air and 30% oxygen). Adjust end-tidal carbon dioxide (CO2) to be in the range of 25 &#177; 5 mmHg29. 
	NOTE: The intubation is critical for maintaining proper CO2 levels through fMRI experiments.</li>
	<li>Place the animal in a supine position on a surgery table and shave a thigh with an electric razor. And then, make an incision on the shaved skin with surgical scissors. 
	NOTE: The length of the incision is around 1-2 cm in a longitudinal direction.</li>
	<li>Find a femoral artery and vein under the incised region for catheterization and separate the individual femoral artery and vein from the surrounding tissues.</li>
	<li>Fasten one side of the separated femoral artery with a surgical suture and hold the other side with micro bulldog forceps. Then, make a small incision between the tied regions on the femoral artery.</li>
	<li>Insert a catheter into the femoral artery through the small incision and tie the catheter and the artery together with surgical sutures. Monitor the arterial blood pressure constantly with the physiological monitoring system to be in the range of 80-120 mmHg and measure the arterial blood gas regularly to maintain pO2 of minimum 90 mmHg and pCO2 of 30-45 mmHg during scanning. 
	NOTE: This catheterization is critical for monitoring the arterial blood pressure during fMRI experiments.</li>
	<li>Fasten both ends of the femoral vein with silk braided surgical sutures. Then, make a small incision between the tied regions on the femoral vein. Use forceps to perform the suturing. 
	NOTE: The size of the suture is around 1-2 cm.</li>
	<li>Insert a catheter into the femoral vein. Tie the catheter and the vein together with surgical sutures. 
	NOTE: This catheterization is critical for administrating alpha-chloralose through the vein and adjusting the anesthetic levels during fMRI experiments. If the animal is not well anesthetized, it will start to breathe spontaneously. In this case, more alpha-chloralose must be administrated to avoid respiratory motion artifacts.</li>
	<li>Suture the surgical incision on the shaved skin. Once the surgical procedures are completed, keep the animal anesthetized by infusing a bolus of alpha-chloralose with the dosage of ~80 mg/kg through the catheter connected to the femoral vein and stop isoflurane administration at the same time.</li>
</ol>
3. Placing the animal inside the MRI scanner
<ol>
	<li>Transfer the anesthetized animal to the MRI scanner as soon as 2.10 step is done and secure it on a custom-made cradle.</li>
	<li>Insert a real-time feedback rectal thermometer on the animal to monitor the animal&#8217;s temperature. Place a heating pad under the animal&#8217;s torso to control the temperature. Maintain the body temperature at 37.0 &#177; 0.5 &#176;C during MRI scans.</li>
	<li>Deliver alpha-chloralose with ~25 mg/kg/h solution in a mixture of pancuronium (~2 mg/kg/h), a muscle relaxer, continuously while keeping the animal anesthetized and reducing motion artifacts in fMRI images. Monitor the blood pressure and respiration by adjusting the amount of drug and the rate of ventilation according to the physiological status.</li>
	<li>Administer ophthalmic ointment on the eyes of the animal to prevent dryness during fMRI experiments. Fix the animal&#8217;s head safely with two ear bars to avoid head motion artifacts.</li>
	<li>Fix a transceiver surface coil on the head. Tune and match the coil to the Larmor frequency (e.g., 599 MHz on 14.1 T) on the head before MRI measurements. 
	NOTE: Here, 22 mm diameter coil is used to cover the whole brain of a rat.</li>
	<li>Insert a pair of needle electrodes into the skin of the forepaw between digits 1 and 4 and fix them with surgical tape. And then, confirm that the stimulation works properly after connecting a stimulation input cable to these electrodes30.</li>
	<li>Insert the animal into the MRI bore and place it at the iso-center approximately.</li>
</ol>
4. Measuring anatomical MR images
<ol>
	<li>Click the calibration menu button in the main user interface. Perform the calibrations of the MRI system clicking the following items in the Adjustment Platform user interface (see Help menu in the console software): Find the basic resonance frequency, Calibrate the RF pulse power, Set the optimal receiver gain, Measure the B0 map in the animal for shimming, Run global linear shims based on non-localized free induction decay (FID) integral. 
	NOTE: This step takes less than 2 min.</li>
	<li>Run a Position sequence by clicking the &#8220;Scan&#8221; button to find the head location of the animal inside the MRI bore. If the head is not located at the iso-center, adjust the head location while moving the cradle back and forth until the head is located at the iso-center.</li>
	<li>Run a Localizer sequence by clicking the &#8220;Scan&#8221; button to identify an ROI in the head. Select Map Shim and define the ROI of the shim volume to cover the whole brain in the localizer image and then, run a high order (e.g., 2nd or 3rd order) shimming using the &#8220;Shim up to&#8221; option to reduce the main magnetic field (B0) inhomogeneities at the ROI. 
	NOTE: The high order shimming is a critical step to improve the quality of BOLD-fMRI data when EPI sequences are used.</li>
	<li>Run a T2-weighted RARE sequence by clicking the &#8220;Scan&#8221; button to acquire anatomical images covering the whole brain in a coronal view (e.g., the following sequence parameters are used: repetition time (TR) 4000 ms, effective echo time (TE) 36.1 ms, matrix 128 x 128, field of view (FOV) 19.2x19.2 mm2, number of slices 32, slice thickness 0.3 mm, RARE factor 8). 
	NOTE: In the following real-time fMRI visualization step, the anatomical images are used to register 3D EPI images as a template.</li>
</ol>
5. Real-Time fMRI software set-up and fMRI response visualization
<ol>
	<li>Open a terminal window and go to the real-time AFNI plugin path using the following command: 
	cd /home/(user name)/rt_afni 
	NOTE: The AFNI plugin script, &#8220;afni_rt&#8221; is included in the Supplementary files.</li>
	<li>Execute AFNI software with the real-time plugin using the command and options below. 
	afni -rt 
	-yestplugouts 
	-DAFNI_REALTIME_MP_HOST_PORT=localhost:(port number) 
	-DAFNI_REALTIME_Graph=Realtime 
	-DAFNI_FIM_IDEAL=(Paradigm) 
	NOTE: In the first case, the code allows external programs to exchange data with AFNI while in the second case the real-time plugin will attempt to open a TCP socket to the user-defined localhost and port. In the third and fourth cases, the codes will plot the time course of fMRI data in real-time and plot the time course of the user-defined paradigm in the fMRI time course respectively when real-time fMRI data are acquired. For further details, check https://afni.nimh.nih.gov/pub/dist/doc/program_help/README.environment.html.</li>
	<li>Monitor upcoming AFNI BRIK files defined by using the command &#8220;Dimon&#8221; as shown in Figure 2 with the following options: 
	Dimon -tr (TR of EPI) -nt (NRepetitions of EPI) 
	-rt -quit 
	-infile_pattern realtime*.BRIK 
	-file_type AFNI 
	NOTE: &#8220;Dimon&#8221; is a command to monitor the real-time acquisition of AFNI image files using the following options: &#8220;-rt&#8221; which executes the real-time plugin and &#8220;-infile_pattern (data name).BRIK -file_type AFNI&#8221; which allows the plugin to read the specific BRIK files and to send them into AFNI for display and formatting. For further details, check https://afni.nimh.nih.gov/pub/dist/doc/program_help/Dimon.html.</li>
	<li>Use &#8220;pvcmd&#8221; command with the following options: 
	pvcmd -a JMacroManager JMMExecuteMacro -category $USER -macro Feed2AFNI_rt3DEPI 
	NOTE: This code exists in the macro script, &#8220;Setup_rt3DEPI&#8221;, to run the background macro script, &#8220;Feed2AFNI_rt3DEPI&#8221;, right after clicking &#8220;Scan&#8221; button for EPI acquisition.</li>
	<li>Use &#8220;exec pvcmd&#8221; command with the following options to get EPI acquisition parameters. 
	exec pvcmd -a ParxServer -r ParamGetValue -psid $ParSpaceId -param (PVM parameters of EPI) -id 10 -args $AcqKey $ParSpaceId $ProcnoPath</li>
	<li>Use &#8220;exec to3d&#8221; command with the following options to convert EPI raw data to AFNI files in real-time in the background macro script, &#8220;Feed2AFNI_rt3DEPI&#8221;. 
	exec to3d -omri -xFOV $FOV_X -yFOV $FOV_Y -zFOV $FOV_Z -prefix $LastVolName $ImgFormat$Path2dseq</li>
	<li>Make sure that EPI geometrical information is consistent with the anatomy orientation. 
	NOTE: The &#8220;to3d&#8221; AFNI command will run automatically with the geometrical information such as the field of view (FOV) and matrix size to convert the fMRI raw data into one AFNI BRIK data whenever each 3D volume data is stored after every single TR as shown in Figure 2. The image orientation can be changed with the geometrical information parameters of &#8220;to3d&#8221;. For further details, check https://afni.nimh.nih.gov/pub/dist/doc/program_help/to3d.html.</li>
	<li>Turn on an electrical stimulus isolator and perform electrical forepaw stimulation for one evoked fMRI study (e.g., 3Hz, 4s pulse width 300us, 2.5mA) using stimulation blocks. 
	NOTE: Here, the block-design paradigm consists of 10 pre-stimulation scans, 3 stimulation scans and 12 inter-stimulation scans (15 scans per epoch).</li>
	<li>Run a T2*-weighted 3D EPI sequence by clicking the &#8220;Scan&#8221; button for the BOLD-fMRI study (e.g., the following parameters are used: TR/TE 1500/14 ms, matrix 64 x 64 x 32, FOV 19.2 x 19.2 x 9.6 mm3, and resolution 300 x 300 x 300 &#181;m3). 
	NOTE: As soon as clicking the &#8220;Scan&#8221; button, monitoring, and processing raw data will be done by using the predefined macro scripts in real-time. Once one AFNI BRIK dataset is converted, voxel-wise time course graphs for 3D EPI images are displayed in the AFNI software and automatically updated for every single TR.</li>
	<li>To overlay the EPI images on top of the anatomical RARE images, convert the RARE images to an AFNI BRIK dataset using the command &#8220;to3d&#8221; as in step 5.6, then register the EPI images to the anatomical images using the &#8220;align_epi_anat.py&#8221; AFNI script with the following options: 
	align_epi_anat.py -anat anatomy_template_al+orig -epi epi.$(epi data number)+orig -epi_base 1 -suffix _volreg -rat_align -cost lpa -epi2anat 
	NOTE: For further details, check https://afni.nimh.nih.gov/pub/dist/doc/program_help/ align_epi_anat.py.html.</li>
	<li>To process functional maps of the BOLD responses, calculate the deconvolution of 3D+time dataset with a specific stimulus time series using the &#8220;3dDeconvolve&#8221; command with the following options: 
	3dDeconvolve -input (input file name)+orig. -nfirst 0 -polort 3 -num_stimts 1 -stim_times 1 (stimulation paradigm file name) &#39;BLOCK(4,1)&#39; -stim_label 1 forepaw -tout -fout -rout 
	NOTE: Image processing steps such as spatial smoothing or temporal filtering have been incorporated into a customized AFNI data processing script. For further details, check https://afni.nimh.nih.gov/afni/doc/help/3dDeconvolve.html.</li>
	<li>To visualize functional maps of the BOLD signals, use an interactive clustering in the AFNI software. Open the &#8220;Define Overlay&#8221; option and use the &#8220;Clusters&#8221; function from the AFNI user interface menu.</li>
	<li>After the last fMRI scan, take the animal out of the MRI scanner and euthanize it according to the approved protocols. 
	NOTE: Image processing functions of AFNI and macro-functions in the latest console software were used to process the real-time fMRI data. Detailed information and descriptions of macro-functions can be found from the help menu in the console software. The AFNI software is a freeware, that can be directly downloaded through the NIMH-AFNI website. The related scripts to build the linkage between AFNI and the console system are attached.</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFNI:+Software+for+analysis+and+visualization+of+functional+magnetic+resonance+neuroimages.">AFNI: Software for analysis and visualization of functional magnetic resonance neuroimages.</a> Computers and Biomedical Research. 29 (3), 162-173 (1996).</li><li>Liou, W. W., Goshgarian, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+the+effect+of+chronic+phrenicotomy+on+the+induction+of+the+crossed+phrenic+phenomenon.">Quantitative assessment of the effect of chronic phrenicotomy on the induction of the crossed phrenic phenomenon.</a> Experimental Neurology. 127 (1), 145-153 (1994).</li><li>Shih, Y. Y., 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=Ultra+high-resolution+fMRI+and+electrophysiology+of+the+rat+primary+somatosensory+cortex.">Ultra high-resolution fMRI and electrophysiology of the rat primary somatosensory cortex.</a> Neuroimage. 73, 113-120 (2013).</li><li>Masamoto, K., Kim, T., Fukuda, M., Wang, P., Kim, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+neural,+vascular,+and+BOLD+signals+in+isoflurane-anesthetized+rat+somatosensory+cortex.">Relationship between neural, vascular, and BOLD signals in isoflurane-anesthetized rat somatosensory cortex.</a> Cerebral Cortex. 17 (4), 942-950 (2007).</li><li>van Alst, T. M., 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=Anesthesia+differentially+modulates+neuronal+and+vascular+contributions+to+the+BOLD+signal.">Anesthesia differentially modulates neuronal and vascular contributions to the BOLD signal.</a> Neuroimage. 195, 89-103 (2019).</li><li>Wu, T. 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=Effects+of+isoflurane+anesthesia+on+resting-state+fMRI+signals+and+functional+connectivity+within+primary+somatosensory+cortex+of+monkeys.">Effects of isoflurane anesthesia on resting-state fMRI signals and functional connectivity within primary somatosensory cortex of monkeys.</a> Brain and Behavior. 6 (12), 00591 (2016).</li><li>Liu, X., Zhu, X. H., Zhang, Y., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+change+of+functional+connectivity+specificity+in+rats+under+various+anesthesia+levels+and+its+neural+origin.">The change of functional connectivity specificity in rats under various anesthesia levels and its neural origin.</a> Brain Topography. 26 (3), 363-377 (2013).</li><li>Liu, X. 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=Multiphasic+modification+of+intrinsic+functional+connectivity+of+the+rat+brain+during+increasing+levels+of+propofol.">Multiphasic modification of intrinsic functional connectivity of the rat brain during increasing levels of propofol.</a> Neuroimage. 83, 581-592 (2013).</li><li>Hutchison, R. M., Hutchison, M., Manning, K. Y., Menon, R. S., Everling, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+induces+dose-dependent+alterations+in+the+cortical+connectivity+profiles+and+dynamic+properties+of+the+brain's+functional+architecture.">Isoflurane induces dose-dependent alterations in the cortical connectivity profiles and dynamic properties of the brain's functional architecture.</a> Human Brain Mapping. 35 (12), 5754-5775 (2014).</li><li>He, Y., 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=Ultra-Slow+Single-Vessel+BOLD+and+CBV-Based+fMRI+Spatiotemporal+Dynamics+and+Their+Correlation+with+Neuronal+Intracellular+Calcium+Signals.">Ultra-Slow Single-Vessel BOLD and CBV-Based fMRI Spatiotemporal Dynamics and Their Correlation with Neuronal Intracellular Calcium Signals.</a> Neuron. 97 (4), 925-939 (2018).</li><li>Yoshida, K., 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=Physiological+effects+of+a+habituation+procedure+for+functional+MRI+in+awake+mice+using+a+cryogenic+radiofrequency+probe.">Physiological effects of a habituation procedure for functional MRI in awake mice using a cryogenic radiofrequency probe.</a> Journal of Neuroscience Methods. 274, 38-48 (2016).</li></ol>

Sascha K&#246;hler is an employee at Bruker BioSpin MRI GmbH.

Real-time monitoring of the fMRI signal helps experimenters adjust the physiology of animals to optimize functional mapping. Motion artifacts in awake animals, as well as the anesthetic effect, are major factors that mediate the variability of fMRI signals, confounding the biological interpretation of the signal by itself31,32,33,34,35,<sup class="x...

Functional Magnetic Resonance Imaging (fMRI) is a non-invasive method to measure the hemodynamic responses1,2,3,4,5,6,7,8,9, e.g., the blood-oxygen-level-dependent (BOLD), cerebral blood volume and flow signal, associated with neural activity in the brain. In animal studies, hemodynamic signals can be affected by anesthesia10, the stress level of awake animals11, as well as the potential non-physiological artifacts, e.g., cardiac pulsation and respiratory motions12,13,14,15. Although many post-processing methods have been developed to provide a retrospective analysis of the fMRI signal for the task-related and resting-state functional dynamics and connectivity mapping16,17,18,19, there are few techniques to provide a real-time brain function mapping solution and instantaneous readouts in the animal brain20 (most of which are mainly used for human brain mapping21,22,23,24,25,26,27). In particular, this kind of real-time fMRI mapping method is lacking in animal studies. It is necessary to set up an fMRI platform to enable the investigation of real-time brain state-dependent physiological stages and to provides real-time biofeedback stimulation paradigm for animal brain functional studies.
In the present work, we illustrate a real-time fMRI experimental set-up with the customized macro-functions of the MRI console software, demonstrating real-time monitoring of the evoked BOLD-fMRI responses in the primary forepaw somatosensory cortex (FP-S1) of the anesthetized rats. This real-time set-up allows for the visualization of the ongoing brain activation in functional maps, as well as individual time courses in a voxel-wise manner, using the existing neuroimage analysis software, Analysis of Functional NeuroImages (AFNI)28. The preparation of the real-time fMRI experimental set-up for the animal study is described in the protocol. Besides the animal set-up, we provide detailed procedures to set up the visualization and analysis of the real-time fMRI signals using the latest console software in parallel with the image processing scripts. In summary, the proposed real-time fMRI set-up for animal studies is a powerful tool for monitoring the dynamic fMRI signals in the animal brain using the MRI console system.

<table><tbody><tr><td>14.1T Bruker MRI system</td><td>Bruker BioSpin MRI GmbH</td><td>N/A</td><td></td></tr><tr><td>A365 Stimulus Isolator</td><td>World Precision Instruments</td><td>N/A</td><td></td></tr><tr><td>AcqKnowledge Software</td><td>Biopac</td><td>RRID:SCR_014279, http://www.biopac.com/product/acqknowledge-software/</td><td></td></tr><tr><td>AFNI</td><td>Cox, 1996</td><td>RRID:SCR_005927, http://afni.nimh.nih.gov</td><td></td></tr><tr><td>CO2SMO (ETCO2/SpO2 Monitor), Model 7100</td><td>Novametrix Medical Systems Inc</td><td>N/A</td><td></td></tr><tr><td>Isoflurane</td><td>CP-Pharma</td><td>Cat# 1214</td><td></td></tr><tr><td>Master-9</td><td>A.M.P.I</td><td>N/A</td><td></td></tr><tr><td>Nanoliter Injector</td><td>World Precision Instruments</td><td>Cat# NANOFIL</td><td></td></tr><tr><td>Pancuronium Bromide</td><td>Inresa Arzneimittel</td><td>Cat# 34409.00.00</td><td></td></tr><tr><td>ParaVision 6</td><td>Bruker BioSpin MRI GmbH</td><td>RRID:SCR_001964</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>Cat# 10010-023</td><td></td></tr><tr><td>Rat: Sprague Dawley rat</td><td>Charles River Laboratories</td><td>Crl:CD(SD)</td><td></td></tr><tr><td>SAR-830/AP Ventilator</td><td>CWE</td><td>N/A</td><td></td></tr><tr><td>&amp;alpha;-chloralose</td><td>Sigma-Aldrich</td><td>Cat# C0128-25G;RRID</td><td></td></tr></tbody></table>

real-time fmri brain mapping in animals

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a real-time fMRI platform to guide experimenters to monitor fMRI responses instantaneously during acquisition, which can be used to modify the physiology of animals to achieve desired hemodynamic responses in animal brains. The real-time fMRI set-up is based on a 14.1T preclinical MRI system, enabling the real-time mapping of dynamic fMRI responses in the primary forepaw somatosensory cortex (FP-S1) of anesthetized rats. Instead of a retrospective analysis to investigate confounding sources leading to the variability of fMRI signals, the real-time fMRI platform provides a more effective scheme to identify dynamic fMRI responses using customized macro-functions and a common neuroimage analysis software in the MRI system. Also, it provides immediate troubleshooting feasibility and a real-time biofeedback stimulation paradigm for brain functional studies in animals.

Figure 3 and Figure 4 show a representative real-time voxel-wise BOLD-fMRI time course and functional maps with electrical forepaw stimulation (3 Hz, 4 s, pulse width 300 us, 2.5 mA). The fMRI design paradigm comprises 10 pre-stimulation scans, 3 stimulation scans, and 12 inter-stimulation scans with a total of 8 epochs (130 scans). The total scan time is 3 min 15 sec (195 sec). Figure 3 shows the voxel-wise time course (black line)...

Watch this Scientific Journal Video about Real-Time fMRI Brain Mapping in Animals at JoVE.com

Real-Time fMRI Brain Mapping in Animals

Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal fMRI.

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a ...

Research

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Neuroscience

3.5K Views.  Max Planck Institute for Biological Cybernetics. Functionele mapping van dierlijke hersenen kan profiteren van de real-time functionele magnetische resonantie beeldvorming (fMRI) experimentele opzet. Met behulp van de nieuwste software die is geïmplementeerd in het MRI-systeem voor dieren, hebben we een real-time monitoringplatform voor fMRI voor kleine dieren opgezet.

Video: Real-Time fMRI Brain Mapping in Animals

Functional Magnetic Resonance Imaging (fMRI) with Auditory Stimulation in Songbirds

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and molecular approaches allow the investigation of either different stimuli on few neurons, or one stimulus in large parts of the brain, blood oxygenation level dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) allows combining both advantages, i.e. compare the neural activation induced by different stimuli in the entire brain at once. fMRI in songbirds is challenging because of the small size of their brains and because their bones and especially their skull comprise numerous air cavities, inducing important susceptibility artifacts. Gradient-echo (GE) BOLD fMRI has been successfully applied to songbirds 1-5 (for a review, see 6). These studies focused on the primary and secondary auditory brain areas, which are regions free of susceptibility artifacts. However, because processes of interest may occur beyond these regions, whole brain BOLD fMRI is required using an MRI sequence less susceptible to these artifacts. This can be achieved by using spin-echo (SE) BOLD fMRI 7,8 . In this article, we describe how to use this technique in zebra finches (Taeniopygia guttata), which are small songbirds with a bodyweight of 15-25 g extensively studied in behavioral neurosciences of birdsong. The main topic of fMRI studies on songbirds is song perception and song learning. The auditory nature of the stimuli combined with the weak BOLD sensitivity of SE (compared to GE) based fMRI sequences makes the implementation of this technique very challenging.

Functional Magnetic Resonance Imaging fMRI with Auditory Stimulation in Songbirds

This article shows an optimized procedure for imaging of the neural substrates of auditory stimulation in the songbird brain using functional Magnetic Resonance Imaging (fMRI). It describes the preparation of the sound stimuli, the positioning of the subject and the acquisition and subsequent analysis of the fMRI data.

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and ...

Behavior

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Optogenetic Functional MRI

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional magnetic resonance imaging (ofMRI), which is a novel technique that combines the relatively high spatial resolution of high-field fMRI with the precision of optogenetic stimulation. Fiber optics that enable delivery of specific wavelengths of light deep into the brain in vivo are implanted into regions of interest in order to specifically stimulate targeted cell types that have been genetically induced to express light-sensitive trans-membrane conductance channels, called opsins. fMRI is used to provide a non-invasive method of determining the brain's global dynamic response to optogenetic stimulation of specific neural circuits through measurement of the blood-oxygen-level-dependent (BOLD) signal, which provides an indirect measurement of neuronal activity. This protocol describes the construction of fiber optic implants, the implantation surgeries, the imaging with photostimulation and the data analysis required to successfully perform ofMRI. In summary, the precise stimulation and whole-brain monitoring ability of ofMRI are crucial factors in making ofMRI a powerful tool for the study of the connectomics of the brain in both healthy and diseased states.

This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Immunology and Infection

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

This protocol describes the quantification of volumetric cerebral hemodynamic variations in the mouse brain using functional ultrasound (fUS). Procedures for 3D functional activation map following sensory stimulation as well as resting-state functional connectivity are provided as illustrative examples, in anesthetized and awake mice.

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ...

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 ...

In vivo Positron Emission Tomography to Reveal Activity Patterns Induced by Deep Brain Stimulation in Rats

Deep brain stimulation (DBS) is an invasive neurosurgical technique based on the application of electrical pulses to brain structures involved in the patient&#39;s pathophysiology. Despite the long history of DBS, its mechanism of action and appropriate protocols remain unclear, highlighting the need for research aiming to solve these enigmas. In this sense, evaluating the in vivo effects of DBS using functional imaging techniques represents a powerful strategy to determine the impact of stimulation on brain dynamics. Here, an experimental protocol for preclinical models (Wistar rats), combined with a longitudinal study [18F]-fluorodeoxyclucose positron emission tomography (FDG-PET), to assess the acute consequences of DBS on brain metabolism is described. First, animals underwent stereotactic surgery for bilateral implantation of electrodes into the prefrontal cortex. A post-surgical computerized tomography (CT) scan of each animal was acquired to verify electrode placement. After one week of recovery, a first static FDG-PET of each operated animal without stimulation (D1) was acquired, and two days later (D2), a second FDG-PET was acquired while animals were stimulated. For that, the electrodes were connected to an isolated stimulator after administering FDG to the animals. Thus, animals were stimulated during the FDG uptake period (45 min), recording the acute effects of DBS on brain metabolism. Given the exploratory nature of this study, FDG-PET images were analyzed by a voxel-wise approach based on a paired T-test between D1 and D2 studies. Overall, the combination of DBS and imaging studies allows describing the neuromodulation consequences on neural networks, ultimately helping to unravel the conundrums surrounding DBS.

In vivo Positron Emission Tomography to Reveal Activity Patterns Induced by Deep Brain Stimulation in Rats

We describe a preclinical experimental method to evaluate metabolic neuromodulation induced by acute deep brain stimulation with in vivo FDG-PET. This manuscript includes all experimental steps, from stereotaxic surgery to the application of the stimulation treatment and the acquisition, processing, and analysis of PET images.

Deep brain stimulation (DBS) is an invasive neurosurgical technique based on the application of electrical pulses to brain structures involved in the ...

Real-Time fMRI Brain Mapping in Animals

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