This work was supported by funding from the National Institute of Health (NIH)&#39;s National Institute on Drug Abuse (NIDA) [DJW, EDKS, and EMB were supported by Grant R21DA044501 awarded to Alan I. Green and DJW was supported by Grant T32DA037202 to Alan J. Budney] and the National Institute on Alcohol Abuse and Alcoholism (NIAAA) [Grant F31AA028413 to Emily D. K. Sullivan]. Additional support was provided through Alan I. Green&#39;s endowed fund as the Raymond Sobel Professor of Psychiatry at Dartmouth.
Hanbing Lu is supported by the National Institute on Drug Abuse Intramural Research Program, NIH.
The authors wish to acknowledge and thank the late Alan I. Green. His unwavering dedication to the field of co-occurring disorders helped to establish collaboration among the authors. We thank him for his mentorship and guidance, which will be greatly missed.

All experiments were performed on a 9.4 T MRI scanner, and were approved by the Institutional Animal Care and Use Committee at Dartmouth College. Additional approval was obtained to record and show the animals used in the video and figures below.
1. Preparations before scanning
<ol>
	<li>Subcutaneous infusion line
	<ol>
		<li>Partially remove a 23 G needle from its package so that the needle point remains sterile.</li>
		<li>Securely hold the hub of the needle and use a razor blade to score ...

<ol>
	<li>Smitha, K. A., 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=Resting+state+fMRI:+A+review+on+methods+in+resting+state+connectivity+analysis+and+resting+state+networks.">Resting state fMRI: A review on methods in resting state connectivity analysis and resting state networks.</a> The Neuroradiology Journal. 30 (4), 305-317 (2017).</li><li>Gorges, 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=Functional+connectivity+mapping+in+the+animal+model:+Principles+and+applications+of+resting-state+fMRI.">Functional connectivity mapping in the animal model: Principles and applications of resting-state fMRI.</a> Frontiers in Neurology. 8, (2017).</li><li>Paasonen, J., Stenroos, P., Salo, R. A., Kiviniemi, V., Gr&#246;hn, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connectivity+under+six+anesthesia+protocols+and+the+awake+condition+in+rat+brain.">Functional connectivity under six anesthesia protocols and the awake condition in rat brain.</a> NeuroImage. 172, 9-20 (2018).</li><li>Pawela, C. 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=A+protocol+for+use+of+medetomidine+anesthesia+in+rats+for+extended+studies+using+task-induced+BOLD+contrast+and+resting-state+functional+connectivity.">A protocol for use of medetomidine anesthesia in rats for extended studies using task-induced BOLD contrast and resting-state functional connectivity.</a> NeuroImage. 46 (4), 1137-1147 (2009).</li><li>Jonckers, E., 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=Different+anesthesia+regimes+modulate+the+functional+connectivity+outcome+in+mice.">Different anesthesia regimes modulate the functional connectivity outcome in mice.</a> Magnetic Resonance in Medicine. 72 (4), 1103-1112 (2014).</li><li>Williams, K. A., 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=Comparison+of+alpha-chloralose,+medetomidine+and+isoflurane+anesthesia+for+functional+connectivity+mapping+in+the+rat.">Comparison of alpha-chloralose, medetomidine and isoflurane anesthesia for functional connectivity mapping in the rat.</a> Magnetic Resonance Imaging. 28 (7), 995-1003 (2010).</li><li>Zhurakovskaya, E., 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=Global+functional+connectivity+differences+between+sleep-like+states+in+urethane+anesthetized+rats+measured+by+fMRI.">Global functional connectivity differences between sleep-like states in urethane anesthetized rats measured by fMRI.</a> PloS One. 11 (5), 0155343 (2016).</li><li>Fukuda, M., Vazquez, A. L., Zong, X., 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=Effects+of+the+&#945;2-adrenergic+receptor+agonist+dexmedetomidine+on+neural,+vascular+and+BOLD+fMRI+responses+in+the+somatosensory+cortex.">Effects of the &#945;2-adrenergic receptor agonist dexmedetomidine on neural, vascular and BOLD fMRI responses in the somatosensory cortex.</a> The European Journal of Neuroscience. 37 (1), 80-95 (2013).</li><li>Brynildsen, J. 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+characterization+of+a+robust+survival+rodent+fMRI+method.">Physiological characterization of a robust survival rodent fMRI method.</a> Magnetic Resonance Imaging. 35, 54-60 (2017).</li><li>Lu, H., 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=Rat+brains+also+have+a+default+mode+network.">Rat brains also have a default mode network.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (10), 3979-3984 (2012).</li><li>Lu, H., 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=Low-+but+not+high-frequency+LFP+correlates+with+spontaneous+BOLD+fluctuations+in+rat+whisker+barrel+cortex.">Low- but not high-frequency LFP correlates with spontaneous BOLD fluctuations in rat whisker barrel cortex.</a> Cerebral Cortex. 26 (2), 683-694 (2016).</li><li>Tsai, P. -. 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=Converging+structural+and+functional+evidence+for+a+rat+salience+network.">Converging structural and functional evidence for a rat salience network.</a> Biological Psychiatry. 88 (11), 867-878 (2020).</li><li>Murphy, K., Bodurka, J., Bandettini, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+long+to+scan?+The+relationship+between+fMRI+temporal+signal+to+noise+ratio+and+necessary+scan+duration.">How long to scan? The relationship between fMRI temporal signal to noise ratio and necessary scan duration.</a> NeuroImage. 34 (2), 565-574 (2007).</li><li>Birn, R. 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=The+effect+of+scan+length+on+the+reliability+of+resting-state+fMRI+connectivity+estimates.">The effect of scan length on the reliability of resting-state fMRI connectivity estimates.</a> NeuroImage. 83, 550-558 (2013).</li><li>Lu, H., 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=Synchronized+delta+oscillations+correlate+with+the+resting-state+functional+MRI+signal.">Synchronized delta oscillations correlate with the resting-state functional MRI signal.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (46), 18265-18269 (2007).</li><li>Lu, H., 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=Registering+and+analyzing+rat+fMRI+data+in+the+stereotaxic+framework+by+exploiting+intrinsic+anatomical+features.">Registering and analyzing rat fMRI data in the stereotaxic framework by exploiting intrinsic anatomical features.</a> Magnetic Resonance Imaging. 28 (1), 146-152 (2010).</li><li>Cox, R. 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>Ash, J. A., 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+connectivity+with+the+retrosplenial+cortex+predicts+cognitive+aging+in+rats.">Functional connectivity with the retrosplenial cortex predicts cognitive aging in rats.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (43), 12286-12291 (2016).</li><li>Hsu, L. -. 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=Intrinsic+insular-frontal+networks+predict+future+nicotine+dependence+severity.">Intrinsic insular-frontal networks predict future nicotine dependence severity.</a> The Journal of Neuroscience. 39 (25), 5028-5037 (2019).</li><li>Li, Q., 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=Resting-state+functional+MRI+reveals+altered+brain+connectivity+and+its+correlation+with+motor+dysfunction+in+a+mouse+model+of+Huntington's+disease.">Resting-state functional MRI reveals altered brain connectivity and its correlation with motor dysfunction in a mouse model of Huntington's disease.</a> Scientific Reports. 7, (2017).</li><li>Lu, H., 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=Abstinence+from+cocaine+and+sucrose+self-administration+reveals+altered+mesocorticolimbic+circuit+connectivity+by+resting+state+MRI.">Abstinence from cocaine and sucrose self-administration reveals altered mesocorticolimbic circuit connectivity by resting state MRI.</a> Brain Connectivity. 4 (7), 499-510 (2014).</li><li>Seewoo, B. J., Joos, A. C., Feindel, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analytical+workflow+for+seed-based+correlation+and+independent+component+analysis+in+interventional+resting-state+fMRI+studies.">An analytical workflow for seed-based correlation and independent component analysis in interventional resting-state fMRI studies.</a> Neuroscience Research. 165, 26-37 (2021).</li><li>Broadwater, M. A., 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=Adolescent+alcohol+exposure+decreases+frontostriatal+resting-state+functional+connectivity+in+adulthood.">Adolescent alcohol exposure decreases frontostriatal resting-state functional connectivity in adulthood.</a> Addiction Biology. 23 (2), 810-823 (2018).</li><li>Jaime, S., Cavazos, J. E., Yang, Y., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+observations+using+simultaneous+fMRI,+multiple+channel+electrophysiology+recording,+and+chemical+microiontophoresis+in+the+rat+brain.">Longitudinal observations using simultaneous fMRI, multiple channel electrophysiology recording, and chemical microiontophoresis in the rat brain.</a> Journal of Neuroscience Methods. 306, 68-76 (2018).</li></ol>

Stability of the animal, both physically and physiologically, is key to obtaining high-quality resting-state data. This protocol achieves stability by moving through four distinct phases of anesthesia. It is imperative that the animal has met the set physiological thresholds before moving to the next phase of anesthesia; since this method relies on physiological autoregulatory mechanisms, individual animals may require slightly different amounts of time at each anesthesia phase. It is our experience that taking more time...

Resting-state functional magnetic resonance imaging (rs-fMRI) is a measure of the blood-oxygen-level-dependent (BOLD) signal when the brain is at rest and not engaged in any particular task. These signals can be used to measure correlations between brain regions to determine the functional connectivity within neural networks. rs-fMRI is widely used in clinical studies due to its non-invasiveness and the low amount of effort required of patients (as compared to task-based fMRI) making it optimal for diverse patient populations1.
Technological advances have allowed rs-fMRI to be adapted for use in rodent models to uncover mechanisms underlying disease states (see reference2 for review). Preclinical animal models, including disease or knockout models, allow a wide range of experimental manipulations not applicable in humans, and studies can also make use of post-mortem samples to further enhance experiments2. Nevertheless, due to the difficulty in both limiting motion and mitigating stress, MRI acquisition in rodents is traditionally performed under anesthesia. Anesthetic agents, depending on their pharmacokinetics, pharmacodynamics, and molecular targets, influence brain blood flow, brain metabolism, and potentially affect neurovascular coupling pathways.
There have been numerous efforts to develop anesthetic protocols that preserve neurovascular coupling and brain network function3,4,5,6,7,8. We previously reported an anesthetic regime that applied a low dose of isoflurane along with a low dose of the &#945;2 adrenergic receptor agonist dexmedetomidine9. Rats under this method of anesthesia exhibited robust BOLD responses to whisker stimulation in regions consistent with established projection pathways (ventrolateral and ventromedial thalamic nuclei, primary and secondary somatosensory cortex); large-scale resting-state brain networks, including the default mode network10,11 and salience network12 have also been consistently detected. Furthermore, this anesthetic protocol allows for repeated imaging on the same animal, which is important for monitoring the disease progression and the effect of experimental manipulations longitudinally.
In the present study, we detail the experimental setup, animal preparation, and physiological monitoring procedures involved. In particular, we describe the specific anesthetic phases and acquisition of scans during each phase. Data quality is assessed following each resting-state scan. A brief summary of post-scan analysis is also included in the discussion. Laboratories interested in uncovering the potential of using rs-fMRI in rats will find this protocol useful.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>9.4T MRI</td>
			<td>Varian/Bruker</td>
			<td></td>
			<td>Varian upgraded with Bruker console running Paravision 6.0.1 software</td>
		</tr>
		<tr>
			<td>Air-Oxygen Mixer</td>
			<td>Sechrist</td>
			<td>Model 3500CP-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Analysis of Functional NeuroImages (AFNI)</td>
			<td>NIMH/NIH</td>
			<td>Version AFNI_18.3.03</td>
			<td>Freely available at: https://afni.nimh.nih.gov/</td>
		</tr>
		<tr>
			<td>Animal Cradle</td>
			<td>RAPID Biomedical</td>
			<td>LHRXGS-00563</td>
			<td>rat holder with bite bar, nose cone and ear bars</td>
		</tr>
		<tr>
			<td>Animal Physiology Monitoring &#38; Gating System</td>
			<td>SAII</td>
			<td>Model 1025</td>
			<td>MR-compatible system including oxygen saturation, temperature, respiration and fiber optic pulse oximetry add-on</td>
		</tr>
		<tr>
			<td>Antisedan (atipamezole hydrochloride)</td>
			<td>Patterson Veterinary</td>
			<td>07-867-7097</td>
			<td>Zoetis, Manufacturer Item #10000449</td>
		</tr>
		<tr>
			<td>Ceramic MRI-Safe Scissors</td>
			<td>MRIequip.com</td>
			<td>MT-6003</td>
			<td></td>
		</tr>
		<tr>
			<td>Clippers</td>
			<td>Patterson Veterinary</td>
			<td>07-882-1032</td>
			<td>Wahl touch-up trimmer combo kit, Manufacturer Item #09990-1201</td>
		</tr>
		<tr>
			<td>Dexmedesed (dexmedetomidine hydrochloride)</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1801</td>
			<td>Dechra Veterinary Products, Manufacturer Item#17033-005-10</td>
		</tr>
		<tr>
			<td>Digital Rectal Thermometer Covers</td>
			<td>Medline</td>
			<td>MDS9608</td>
			<td></td>
		</tr>
		<tr>
			<td>FMRIB Software Library</td>
			<td>FMRIB</td>
			<td>MELODIC Version 3.15</td>
			<td>Freely available at: https://fsl.fmrib.ox.ac.uk/fsl/fslwiki</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>Cara Inc.</td>
			<td>Model 50</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostat forceps, straight</td>
			<td>Kent Scientific</td>
			<td>INS750451-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td>Patterson Private Label, Manufacturer Item #14043-0704-06</td>
		</tr>
		<tr>
			<td>Isoflurane Vaporizer</td>
			<td>VetEquip Inc.</td>
			<td>911103</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Tape, 3/4&#34;</td>
			<td>VWR International</td>
			<td>89097-990</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles, 23 gauge</td>
			<td>BD</td>
			<td>305145</td>
			<td>plastic hub removed</td>
		</tr>
		<tr>
			<td>Parafilm Laboratory Film</td>
			<td>Patterson Veterinary</td>
			<td>07-893-0260</td>
			<td>Medline Industries Inc., Manufacturer Item #HSFHS234526A</td>
		</tr>
		<tr>
			<td>Planar Surface Coil</td>
			<td>Bruker</td>
			<td>T12609</td>
			<td>2cm</td>
		</tr>
		<tr>
			<td>Polyethylene Tubing</td>
			<td>Braintree Scientific</td>
			<td>PE50 50FT</td>
			<td>0.023&#34; (inner diameter), 0.038&#34; (outer diameter)</td>
		</tr>
		<tr>
			<td>Puralube Ophthalmic Ointment</td>
			<td>Patterson Veterinary</td>
			<td>07-888-2572</td>
			<td>Dechra Veterinary Products, Manufacturer Item #211-38</td>
		</tr>
		<tr>
			<td>Sprague Dawley Rats</td>
			<td>Charles River</td>
			<td>400 SAS SD</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 0.9% Saline Solution</td>
			<td>Patterson Veterinary</td>
			<td>07-892-4348</td>
			<td>Aspen Vet, Manufacturer Item #14208186</td>
		</tr>
		<tr>
			<td>Sterile Alcohol Prep Pads</td>
			<td>Medline</td>
			<td>MDS090735</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Tape, 1&#34; (3M Durapore)</td>
			<td>Medline</td>
			<td>MMM15381Z</td>
			<td>3M Healthcare, &#34;wide medical tape&#34;</td>
		</tr>
		<tr>
			<td>Surgical White Paper Tape, 1/2&#34; (3M Micropore)</td>
			<td>Medline</td>
			<td>MMM15300</td>
			<td>3M Healthcare</td>
		</tr>
		<tr>
			<td>Syringes, 1 mL w/ 25 gauge needle</td>
			<td>BD</td>
			<td>309626</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes, 3 mL</td>
			<td>BD</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>Vented induction and scavenging system</td>
			<td>VetEquip Inc.</td>
			<td>942102</td>
			<td>2 liter induction chamber with active scavenging</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>411724</td>
			<td>omega flowmeter</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>931600</td>
			<td>scavenging cube, &#34;vacuum&#34;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>921616</td>
			<td>nose cone, non-rebreathing</td>
		</tr>
	</tbody>
</table>

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.

Following each resting-state scan, stability is assessed using an independent component analysis (ICA; example script included in Supplementary Files). Figure 6 shows examples of component outputs from resting-state scans. Figure 6a shows a signal component from a scan with high stability. Note that spatially, the component has high regionality. Within the time course below the spatial component, the signal is stable and not predictable, indicat...

Watch this Scientific Journal Video about Acquisition of Resting-State Functional Magnetic Resonance Imaging Data in the Rat at JoVE.com

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

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 ...

Resting-state functional magnetic resonance imaging, or resting-state fMRI, has become an increasingly popular method to study brain function in a resting non-task state. Technological advances have allowed resting-state fMRI to be adapted for use in rodent models, and can be used to uncover mechanisms underlying disease states. The protocol described here combines low-dose isoflurane with low dose dexmedetomidine, allowing for high quality data acquisition and preservation of brain network function.This method also allows for spontaneous breathing and near-normal physiology for up to five hours. In this video, we cover monitoring of the rat's physiology during four distinct phases of anesthesia, animal induction and preparation, animal setup, anatomical scan acquisition, and resting-state scan acquisition followed by quality assessment of the data. Using a system to deliver inhaled anesthetic as well as scavenge waste gases from all needed areas, induce anesthesia with 2.5%isoflurane in 30%oxygen-enriched air.Once the animal is anesthetized, remove it from the chamber, weigh the animal, and place it in the nose cone on the heating pad in the preparation space, remaining at 2.5%isoflurane. Apply ophthalmic lubricating ointment to each eye to prevent drying. Confirm the depth of anesthesia by a lack of toe pinch response, Use clippers to shave a two-inch by two-inch square area on the lower lumbar region of the animal's back directly above the tail.Administer 0.015 milligrams per kilogram of the dexmedetomidine solution with an intraperitoneal injection into the lower-right quadrant of the abdomen using a 25-gauge needle. Switch isoflurane flow from the preparation space to the animal cradle. Move the animal into the animal cradle.Place the rat's front teeth securely over and into the bite bar. Push the nose cone over the nose to ensure a tight fit. If the nose cone does not cover the lower jaw, Parafilm can be used to gently hold the jaw closed while also sealing around the nose cone.Position the respiration pad under the animal's abdomen below the rib cage and reposition it until the respiration waveform shows a deep trough centered on each breath. Move to the next phase of anesthesia when respiration is less than 40 breaths-per-minute. Insert that ear bars into the ear canal to stabilize the rat's head in the animal cradle.Once positioned, pull forward on the bite bar and confirm the head does not move. Readjust the nose cone and Parafilm as needed. Insert the temperature probe into a pre-lubricated disposable probe cover.Gently insert the temperature probe approximately 1/2 inch into the rectum and tape it to the base of the tail with medical tape. Place the pulse oximeter clip onto the metatarsal area of the hind foot, ensuring the light source is on the bottom of the foot. Rotation of the clip can affect the signal, thus, creating a holder to keep the paw and clip upright will lead to greater stability.Calculate the infusion rate for 0.015 milligrams per kilogram per hour of dexmedetomidine. Set the drug pump to the calculated infusion rate and fill the infusion line. Using an alcohol wipe, clean the shaved area to remove any stray hair.Pinch the skin approximately two finger widths above the base of the tail. Insert 1/3 of the infusion line needle into the tented skin. Secure the needle to the skin with a three-inch piece of wide medical tape.Place a second piece of wide medical tape over the first across the rat and attached to both sides of the animal cradle. It is critically important that the ferromagnetic needle is well-secured to prevent movement during the scan. Begin the infusion of subcutaneous dexmedetomidine.Place a piece of gauze on the bridge of the rat's nose to create a level surface for the coil. Use paper tape, which does not interfere with the MRI signal, to secure the coil to the rat's head, centering it over the brain. Place paper towels over the animal, securing them to the animal cradle with laboratory tape.If using an air heating system, wrap a plastic sheet around the entire cradle to contain the warm air. Move the animal into the bore and tune the magnet. Reduce isoflurane to 1.5%resulting in a steady increase in respiration to approximately 45 to 50 breaths-per-minute.Remain at this level for the duration of the anatomical scanning. Use the FLASH localizer scan to ensure the brain is aligned with the magnet isocenter. Reposition the animal and repeat if necessary.Run the higher resolution RARE localizer scan, and use this scan output to align 15 sagittal slices centered across the brain. Using the middle sagittal slice, align the center axial slice to the decussation of the anterior commissure, which appears as a dark spot. Note the slice offset to use later in the resting-state scans.Acquire 23 slices using both the FLASH and RARE axial protocols to aid in registration to a common space during post-scan analysis. Shim across the whole brain using the PRESS sequence. After completing anatomical scans, reduce isoflurane to 0.5 to 0.75%adjusting so that the animal's respiration is 60 to 65 breaths-per-minute.Remain at this level for at least 10 minutes before beginning resting-state scanning to ensure stability. When physiology is stable, acquire a 15 slice EPI scan using the same slice offset as the anatomical axial series. After each resting-state scan is complete, check the quality using an independent component analysis to decompose the data into spatial and temporal components.Obtain at least three high-quality resting-state scans. When scanning is complete, increase isoflurane to 2%and stop the subcutaneous dexmedetomidine infusion. Remove the animal cradle from the magnet bore and return the animal to the preparation space.Inject 0.015 milligrams per kilogram of the diluted atipamezole solution into the rat's hind leg muscle using a 25-gauge needle. Place the rat back in the home cage on top of a heating pad, and monitor until the animal is ambulatory. Using an independent component analysis, assess stability following each resting-state scan.Shown here is an example of very high stability. Note that, spatially, the components have high regionality. Within the time course shown in the middle panel, the signal is stable and not predictable, which is indicative of true brain activity.The power spectrum at the bottom shows predominantly low frequencies. Shown here is a noise component from the same scan. Note the non-regionality, high-frequency time course, and single high-frequency peak in the power spectrum.Noise components should be de-noised during post-scan analysis. Finally, this component is from a scan where anesthesia was not stable. The time course displayed is variable and irregular.When this happens, improvements are needed to the anesthetic protocol, commonly to the ceiling of the nose cone and the scavenging of waste gases. Stability of the animal, both physically and physiologically, is key to obtaining high-quality resting-state data. It is imperative that the animal meets the physiological thresholds established before moving to the next phase of anesthesia.Following this survival technique, resting state fMRI can be used in longitudinal studies, in conjunction with experimental manipulations, and to explore disease models. The combination of low dose isoflurane with dexmedetomidine utilized in this protocol allows for a wide variety of preclinical studies for investigators interested in imaging the rodent brain in its resting state.

acquisition-resting-state-functional-magnetic-resonance-imaging-data

Research

JoVE Journal

Neuroscience

4.0K vistas.  Dartmouth-Hitchcock Medical Center. La resonancia magnética funcional en estado de reposo, o fMRI en estado de reposo, se ha convertido en un método cada vez más popular para estudiar la función cerebral en un estado de reposo sin tareas. Los avances tecnológicos han permitido que la resonancia magnética funcional en estado de reposo se adapte para su uso en modelos de roedores, y se puede utilizar para descubrir mecanismos subyacentes a los estados de enfermedad. El protocolo descrito aquí combina dosis bajas de isoflur...