We acknowledge Clara Jones, James Stirwalt, Linh Hoang, Young Joon Ha, and Amirsoheil Zareh for their help during training of the rats and preparation of the slings. Funding was provided by the National Institutes of Health (NIH, Grant Number: NS119852) and Leducq Foundation (Grant Number:15CVD02).

All procedures were compliant with National Institute of Health guidelines and approved by the University of California, Irvine Animal Care and Use Committee. Seven males and one female rat (Sprague-Dawley, weight: 185-350 g) were used in this study. After study completion, the rats were sacrificed using carbon dioxide overdose.
1. Design of different components
<ol>
	<li>Design of the head implant:
	<ol>
		<li>Make the head implant using CAD software (Figure 1C) and design it to image the area posterior to the bregma and adjacent to the midline centered on the somatosensory cortex. Ensure that the head implant covers an area of 0.9 mm to 1.9 mm on the skull away from the imaging area.</li>
		<li>Use only three screws to anchor the head implant on the rat&#39;s skull. Design all the screw holes so that they remain on the opposite side of the midline in the contralateral hemisphere of the imaged hemisphere.</li>
		<li>Place a bar, hollowed from the inside, in the upper part of the head implant to allow wires to fix the head cap to the head implant as shown in Figure 1D.</li>
	</ol>
	</li>
	<li>Design of the head cap:
	<ol>
		<li>Ensure that the head cap covers the imaging area completely and protects it from any sort of trauma as shown in Figure 1A, B. Add a curvature to the head cap so that it aligns to the shape of the head without causing difficulty to the animal&#39;s daily activities in the standard enriched cages.</li>
		<li>Cut the inner side of the head cap in a wider rectangular shape so that the upper part of the head implant can fit into it as shown in Figure 1E. Perpendicular to this rectangle, cut two other rectangular regions to anchor the head cap to the head implant.</li>
		<li>Pass one wire through the upper hollowed bar of the head implant for fixation of the head cap on the rat head as shown in Figure 1E-G. Pass the second wire in the same way. 
		NOTE: These wires can be easily removed using pliers or forceps. The 3D printing files are provided (file format: STL) as Supplemental File 1 and Supplemental File 2.</li>
	</ol>
	</li>
	<li>Design of the head frame:
	<ol>
		<li>Design the head frame in a way that one cut part can move through the upper bar of the head implant and is fixed using a clamp.</li>
		<li>Angle the other cut part to provide extra strength for keeping the rat head-fixed to make the contralateral side completely accessible for imaging. For the purpose of this study, cut the steel plate with tin snips to produce the head frame (Figure 1H, I). 
		NOTE: This part can be 3D printed as well.</li>
	</ol>
	</li>
</ol>
2. Initial rat training
<ol>
	<li>Allow rats to acclimate to the vivarium environment in their cages for 2-3 days.</li>
	<li>Start handling the rat in a quiet room. Open the cage and have the experimenter put their hand inside the cage near the rat for 15-20 min to let the rat get habituated.</li>
	<li>Once the rat displays calmness by not getting startled or running away from the experimenter&#39;s hands, gently pick the rat up for handling. Handle the rat for 30-45 min each day before sling training.</li>
</ol>
3. Sling training
<ol>
	<li>Train the rats for at least 2-3 days in the slings before the surgical implantation of the head implant and head cap.</li>
	<li>Arrange the sling setup as shown in Figure 2A. Clean the sling setup using ethanol wipes. 
	NOTE: All the slings are hand-sewn and made of a netting material either on the bottom or on both sides as shown in Figure 2A, B.</li>
	<li>For sling training, anesthetize the rats using 4% isoflurane for induction and 1% for maintenance until there is no hind paw pinch reflex.</li>
	<li>Under isoflurane anesthesia, place the rats on a flexible plastic sheet measuring 20 cm x 8 cm (length x width), where 10 cm x 8 cm of the plastic sheet is fully covered with the softer part of the Velcro. 
	NOTE: Anesthetizing the rats for sling training is an optional step, primarily used to reduce stress and anxiety.</li>
	<li>For the first 2 days of the training, put the rat snuggly into a baby sock (size 0-3 months) with the head out through a small hole incised at the end of the sock.</li>
	<li>Wrap a small piece of absorbent pad around the lower body part to keep the rat dry and collect excrements.</li>
	<li>Wrap the rat in a breathable cotton cloth (size: 25 cm x 25 cm). Place the rat on a plastic sheet that has Velcro strips glued to it.</li>
	<li>Further secure the rat to the plastic sheet using 0.5 cm wide Velcro strips at a distance of 3-6 mm from each other.</li>
	<li>Secure the rat in the sling. Remove the gas anesthesia. Allow the rat to recover from gas anesthesia in the sling.</li>
	<li>When the rat starts whisking, offer a few drops of 10% sucrose solution as a reward every 10-15 min.</li>
	<li>Randomly present the rat with the sensory stimuli that will be used during imaging (here whisker stimulation, every 15-25 min) to make it accustomed to sensory stimuli. Manually stimulate the whiskers at random intervals.</li>
	<li>Train the rat in the sling for 1 h on day 1, 2 h on day 2, and 3 h on day 3 as shown in Figure 2C.</li>
</ol>
4. Presurgical preparation
<ol>
	<li>Print the head implant and head cap using the 3D printer (Figure 1).</li>
	<li>Sterilize all the surgical instruments and headpieces (implants and caps) by immersing the equipment in the Metricide28&#160;germicide for 10 hours. Rinse tools thoroughly with sterile water just before surgery.</li>
	<li>Expose the rat to 4% isoflurane and then maintain at 1%-2% isoflurane until there is no hind paw pinch reflex.&#160;This surgery can be performed under many types of anesthesia, such as isoflurane, sodium pentobarbital, and ketamine-xylazine.</li>
	<li>Inject atropine (0.05 mg/kg) intramuscularly to reduce mucous secretions to help in breathing.</li>
	<li>Shave the head of the rat 5 mm centered around the midline using a hair trimmer starting from between the eyes to the back of the ears.</li>
	<li>Monitor the partial oxygen saturation and heart rate through a pulse oximeter and heart rate monitor probe secured to the hind leg of the rat.</li>
	<li>Wipe the rat&#8217;s head and the surrounding area three times with alternating rounds of betadine and 70% alcohol wipes.</li>
	<li>Fix the rat in a stereotaxic system.</li>
	<li>Insert a petroleum jelly-lubricated rectal probe to measure the rat&#39;s body temperature and maintain it through the heating blanket&#39;s feedback system to avoid hypothermia after anesthetic administration.</li>
	<li>Administer local anesthetic lidocaine hydrochloride at a concentration of 20 mg/ml, 0.07 mg/kg +/-0.2 body weight subcutaneously at the surgical site.</li>
	<li>Apply ophthalmic ointment to both eyes to prevent drying.</li>
	<li>Administer 2% local anesthetic subcutaneously over the surgical site.</li>
	<li>Inject 3 mL of lactated ringer&#39;s solution at room temperature subcutaneously to prevent dehydration and provide nourishment during surgery.</li>
</ol>
5. Surgery
<ol>
	<li>Remove the part of the skin over the surgical site (4 mm diameter centered around the midline and center of the head) using sharp surgical scissors. Dissect and remove part of the skin (~2 mm diameter, over the left somatosensory cortex) between the ear and eye on the temporal part of the head.</li>
	<li>Remove, using a scalpel, the underlying skin (pericranium) tissue to expose the skull. Clean the skull using sterilized cotton gauze.</li>
	<li>Retract/resect temporal muscle to expose desired size for imaging area [7.5 mm by 7.5 mm for this study].&#160;</li>
	<li>Expose the skull on the contralateral hemisphere for the head implant. Place the head implant on the skull to ascertain the location of anchoring screws for the implant as shown in Figure 2D-F.</li>
	<li>Mark the skull for drilling the screws using India Ink with drill bit 1. Drill the burr holes for the screws using dental drill bit 3. Screw the head implant in place.</li>
	<li>Dry the skull using sterile gauze. Apply a thin layer of tissue adhesive around and beneath the head implant to glue it to the skull. Apply a layer of dental cement to further support the head implant in place and let the cement dry for 2-3 min. 
	NOTE: The use of tissue adhesive in addition to dental cement ensures a strong hold8.</li>
	<li>Using dental drill bit 3, thin a 7.5 mm x 7.5 mm area on the left side of skull just posterior to the bregma and lateral to the midline. Thin the skull to ~50 &#181;m as shown in Figure 3A.</li>
	<li>Apply topical antibiotic ointment over the surgical site and then cover it with a thin layer of silicone rubber to protect the thinned skull as shown in Figure 3B. Cover the surgical site using the head cap as shown in Figure 3C. Fix it in place with the two small pieces of wires going through both the head implant and head cap as shown in Figure 3D, E. Apply silicone rubber to cover the head cap and skull to stabilize the head cap further on the rat&#39;s head as shown in Figure 3F. 
	NOTE: Silicon rubber provides additional protection to thinned skull.</li>
	<li>Inject the rat with flunixin meglumine (2.5 mg/kg) subcutaneously for pain and inflammation management.&#160;To prevent infection, inject Enrosite antibiotic enrofloxacin (22.7mg/ml, 10mg/kg +/-.01), intraperitoneally.</li>
	<li>Move the rat to the recovery chamber to help maintain its body temperature with a warming blanket and a heat lamp. Monitor the rat continuously until it regains consciousness and can maintain sternal recumbency.</li>
	<li>Return the rat to its separate cage once it fully recovers.</li>
	<li>For the next 3 days, administer flunixin and buprenorphine to alleviate inflammation and pain and enrosite to prevent infection twice daily.</li>
</ol>
6. Awake imaging
<ol>
	<li>Anesthetize the rat with 4% isoflurane for induction and 1% for maintenance when there is no hind paw pinch reflex. Inject acepromazine (0.3-0.5 mg/kg) subcutaneously. 
	NOTE: This concentration of acepromazine is below mild sedation levels and only helps keep the rats calm throughout the imaging process.</li>
	<li>Using customized strips of Velcro, fix the rat on the plastic sheet used during the training procedures. Wrap the lower body part using an absorption pad and place the rat snugly in the sling.</li>
	<li>Remove the silicon rubber. Remove the head cap by removing the fixation wires. Fix the headframe in the head implant as shown in Figure 2G.</li>
	<li>Lock the headframe in clamps as shown in Figure 2H, I.</li>
	<li>Remove gas anesthesia. Flush the imaging area with saline 3x and clean with wet gauze. Dry the imaging area and make a well, using petroleum jelly, around the imaging area. Fill the well with sterilized saline and cover with a glass slide (Figure 2E).</li>
	<li>Refer to the acquisition procedures for intrinsic signal optical imaging, the whisker stimulation protocol, and data analysis and presentation, which have been discussed in detail previously6,7.</li>
	<li>Throughout the experiment, monitor the rats for signs of agitation and restlessness, which can be further reduced by covering the eyes of the rats with a soft cloth or gauze (optional).</li>
</ol>

<ol><li>Cicero, L., Fazzotta, S., Palumbo, V. D., Cassata, G., Lo Monte, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anesthesia+protocols+in+laboratory+animals+used+for+scientific+purposes%5BTitle%5D)+AND+%22Acta+Biomedica%22%5BJournal%5D)">Anesthesia protocols in laboratory animals used for scientific purposes.</a> Acta Biomedica. 89 (3), 337-342 (2018).</li>
<li>Lythgoe, M. F., Sibson, N. R., Harris, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuroimaging+of+animal+models+of+brain+disease%5BTitle%5D)+AND+%22British+Medical+Bulletin%22%5BJournal%5D)">Neuroimaging of animal models of brain disease.</a> British Medical Bulletin. 65, 235-257 (2003).</li>
<li>Albrecht, M., Henke, J., Tacke, S., Markert, M., Guth, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+repeated+anaesthesia+on+physiological+parameters+in+male+Wistar+rats%3A+A+telemetric+study+about+isoflurane%2C+ketamine-xylazine+and+a+combination+of+medetomidine%2C+midazolam+and+fentanyl%5BTitle%5D)+AND+%22BMC+Veterinary+Research%22%5BJournal%5D)">Influence of repeated anaesthesia on physiological parameters in male Wistar rats: A telemetric study about isoflurane, ketamine-xylazine and a combination of medetomidine, midazolam and fentanyl.</a> BMC Veterinary Research. 10, 310(2014).</li>
<li>Uhlig, C., Krause, H., Koch, T., Gama de Abreu, M., Spieth, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anesthesia+and+monitoring+in+small+laboratory+mammals+used+in+anesthesiology%2C+respiratory+and+critical+care+research%3A+A+systematic+review+on+the+current+reporting+in+top-10+impact+factor+ranked+journals%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Anesthesia and monitoring in small laboratory mammals used in anesthesiology, respiratory and critical care research: A systematic review on the current reporting in top-10 impact factor ranked journals.</a> PLoS One. 10 (8), 0134205(2015).</li>
<li>Chen-Bee, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+and+quantifying+evoked+cortical+activity+assessed+with+intrinsic+signal+imaging%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">Visualizing and quantifying evoked cortical activity assessed with intrinsic signal imaging.</a> Journal of Neuroscience Methods. 97 (2), 157-173 (2000).</li>
<li>Chen-Bee, C. H., Agoncillo, T., Xiong, Y., Frostig, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+triphasic+intrinsic+signal%3A+Implications+for+functional+imaging%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">The triphasic intrinsic signal: Implications for functional imaging.</a> The Journal of Neuroscience. 27 (17), 4572-4586 (2007).</li>
<li>Chen-Bee, C. H., Agoncillo, T., Lay, C. C., Frostig, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intrinsic+signal+optical+imaging+of+brain+function+using+short+stimulus+delivery+intervals%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">Intrinsic signal optical imaging of brain function using short stimulus delivery intervals.</a> Journal of Neuroscience Methods. 187 (2), 171-182 (2010).</li>
<li>Scott, B. B., Brody, C. D., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+Resolution+Functional+Imaging+in+Behaving+Rats+Using+Voluntary+Head+Restraint%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Cellular Resolution Functional Imaging in Behaving Rats Using Voluntary Head Restraint.</a> Neuron. 80 (2), 371-384 (2013).</li>
<li>Frostig, R. D., Lieke, E. E., Ts'o, D. Y., Grinvald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortical+functional+architecture+and+local+coupling+between+neuronal+activity+and+the+microcirculation+revealed+by+in+vivo+high-resolution+optical+imaging+of+intrinsic+signals%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Cortical functional architecture and local coupling between neuronal activity and the microcirculation revealed by in vivo high-resolution optical imaging of intrinsic signals.</a> Proceedings of the National Academy of Sciences of the United States of America. 87 (16), 6082-6086 (1990).</li>
<li>Chang, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+method+for+functional+brain+imaging+in+awake+minimally+restrained+rats%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">Novel method for functional brain imaging in awake minimally restrained rats.</a> Journal of Neurophysiology. 116 (1), 61-80 (2016).</li>
<li>Stenroos, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Awake+rat+brain+functional+magnetic+resonance+imaging+using+standard+radio+frequency+coils+and+a+3D+printed+restraint+kit%5BTitle%5D)+AND+%22Frontiers+in+Neuroscience%22%5BJournal%5D)">Awake rat brain functional magnetic resonance imaging using standard radio frequency coils and a 3D printed restraint kit.</a> Frontiers in Neuroscience. 12, 548(2018).</li>
<li>Vogler, G. A. Chapter 19 - Anesthesia and Analgesia (Second Edition). <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aVogler%2C+GA+%22The+Laboratory+Rat%22">The Laboratory Rat</a>. Suckow, M. A., Weisbroth, S. H., Franklin, C. L. , Academic Press. Cambridge, MA. 627-664 (2006).</li>
<li>Schwarz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+head-fixed+behaving+rat--Procedures+and+pitfalls%5BTitle%5D)+AND+%22Somatosensory+and+Mot+Research%22%5BJournal%5D)">The head-fixed behaving rat--Procedures and pitfalls.</a> Somatosensory and Mot Research. 27 (4), 131-148 (2010).</li>
<li>Roh, M., Lee, K., Jang, I. S., Suk, K., Lee, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acrylic+resin+molding+based+head+fixation+technique+in+rodents%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Acrylic resin molding based head fixation technique in rodents.</a> Journal of Visualized Experiments. (107), e53064(2016).</li>
<li>Ferris, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applications+in+awake+animal+magnetic+resonance+imaging%5BTitle%5D)+AND+%22Frontiers+in+Neuroscience%22%5BJournal%5D)">Applications in awake animal magnetic resonance imaging.</a> Frontiers in Neuroscience. 16, 854377(2022).</li>
<li>Tiran, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcranial+functional+ultrasound+imaging+in+freely+moving+awake+mice+and+anesthetized+young+rats+without+contrast+agent%5BTitle%5D)+AND+%22Ultrasound+in+Medicine+and+Biology%22%5BJournal%5D)">Transcranial functional ultrasound imaging in freely moving awake mice and anesthetized young rats without contrast agent.</a> Ultrasound in Medicine and Biology. 43 (8), 1679-1689 (2017).</li>
<li>Desjardins, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Awake+mouse+imaging%3A+From+two-photon+microscopy+to+blood+oxygen+level-dependent+functional+magnetic+resonance+imaging%5BTitle%5D)+AND+%22Biological+Psychiatry%3A+Cognitive+Neuroscience+and+Neuroimaging%22%5BJournal%5D)">Awake mouse imaging: From two-photon microscopy to blood oxygen level-dependent functional magnetic resonance imaging.</a> Biological Psychiatry: Cognitive Neuroscience and Neuroimaging. 4 (6), 533-542 (2019).</li>
<li>Koletar, M. M., Dorr, A., Brown, M. E., McLaurin, J., Stefanovic, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Refinement+of+a+chronic+cranial+window+implant+in+the+rat+for+longitudinal+in+vivo+two-photon+fluorescence+microscopy+of+neurovascular+function%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Refinement of a chronic cranial window implant in the rat for longitudinal in vivo two-photon fluorescence microscopy of neurovascular function.</a> Scientific Reports. 9, 5499(2019).</li>
<li>Drew, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+optical+access+through+a+polished+and+reinforced+thinned+skull%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Chronic optical access through a polished and reinforced thinned skull.</a> Nature Methods. 7 (12), 981-984 (2010).</li>
<li>Cao, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+and+oxygen-metabolic+photoacoustic+microscopy+of+the+awake+mouse+brain%5BTitle%5D)+AND+%22Neuroimage%22%5BJournal%5D)">Functional and oxygen-metabolic photoacoustic microscopy of the awake mouse brain.</a> Neuroimage. 150, 77-87 (2017).</li>
<li>Grinvald, A., Frostig, R. D., Siegel, R. M., Bartfeld, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+optical+imaging+of+functional+brain+architecture+in+the+awake+monkey%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">High-resolution optical imaging of functional brain architecture in the awake monkey.</a> Proceedings of the National Academy of Sciences of the United States of America. 88 (24), 11559-11563 (1991).</li>
<li>Roe, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+optical+imaging+of+intrinsic+signals+in+anesthetized+and+awake+monkeys%5BTitle%5D)+AND+%22Applied+Optics%22%5BJournal%5D)">Long-term optical imaging of intrinsic signals in anesthetized and awake monkeys.</a> Applied Optics. 46 (10), 1872-1880 (2007).</li>
<li>Polley, D., Kvašňák, E., Frostig, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Naturalistic+experience+transforms+sensory+maps+in+the+adult+cortex+of+caged+animals%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Naturalistic experience transforms sensory maps in the adult cortex of caged animals.</a> Nature. 429 (6987), 67-71 (2004).</li>
</ol>

The authors have no conflicts of interest to disclose.

The use of awake, head-fixed rat imaging offers many advantages in terms of ease and customization. The custom-designed slings allow the rats to be wrapped through breathable netting material, eliminating the need to enclose animals in closed, plastic restraining chambers for extended periods of time10,11. Rats are kept calm and stress-free throughout the long durations of successive imaging sessions using a very low dose of acepromazine below the levels of mild ...

Most of the basic, preclinical, and translational scientific neuroimaging investigations are acquired from anesthetized animals1,2. Anesthetics ease experimentation but continuously influence the brain&#39;s and body&#39;s metabolism, blood pressure, and heart rate3. The type of anesthetic and the duration and route of administration add confounding variables to data interpretation that could contribute to reproducibility and translational failures4. A major bottleneck of awake, head-fixed rat neuroimaging studies is the requirement to keep the rat stationary and calm throughout the preparation and data acquisition processes. Small movements produce unwarranted motion artifacts, which can adversely affect data analysis and interpretations.
A new model of neuroimaging from awake, head-fixed rats using customized slings, three-dimensional (3D)-printed head implants, head caps, and a headframe has been devised that offers several advantages for easy experimentation. The 3D head implant is light and covers a small portion of the skull needed for transfixing. The 3D-printed head implants and caps are designed using computer-aided design (CAD) software. The protocols of whisker stimulation, data acquisition, data analysis, and results from anesthetized rats have been described in detail in previous work5,6,7.

<table><tbody><tr><td>Rats</td><td>Charles River</td><td>Sprague Dawley</td><td></td></tr><tr><td>Isoflurane</td><td>Pivetal</td><td>21295098</td><td>General anesthetic</td></tr><tr><td>Lidocaine HCl 2% injection</td><td>Phoenix</td><td>L-2000-04</td><td>Local anesthetic</td></tr><tr><td>Atropine sulfate injection</td><td>Vedco</td><td>5098907512</td><td>Help in respiration</td></tr><tr><td>Lactated Ringer&#039;s injection solution</td><td>Vedco</td><td>50989088317</td><td></td></tr><tr><td>Flunixin injection</td><td>Vedco</td><td>6064408670</td><td>Pain management</td></tr><tr><td>Enrosite injection (Enrofloxacin 2.27%)</td><td>VetOne</td><td>501084</td><td>Avoid infection</td></tr><tr><td>PromAce injection (Acepromazine maleate)</td><td>Beohringer Ingelheim</td><td>136059</td><td></td></tr><tr><td>Animax ointment</td><td>Dechra Veterinary Products</td><td>122-75</td><td>active ingredients of nystatin 1000units per gram, neomycin sulfate 2.5mg per gram, thiostrepton 2500 units per gram, and triamcinolone acetonide 1mg per gram</td></tr><tr><td>Puralube ophthalmic ointment</td><td>Dechra Veterinary Products</td><td>211-38</td><td></td></tr><tr><td>Povidone-iodine PVP prep pads</td><td>Medline</td><td>MDS093917</td><td>Betadine generic</td></tr><tr><td>Isopropyl alcohol swabs</td><td>BD</td><td>326895</td><td></td></tr><tr><td>Vetbond tissue adhesive</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Bur (drill bit), standard operatory carbide</td><td>SS White Burs</td><td>14829</td><td>#3 bur</td></tr><tr><td>Screws, 00-90 x 1/8 flat head stainless steel</td><td>J.I. Morris</td><td>F0090CE125</td><td>Anchor screws</td></tr><tr><td>Stereotaxic system</td><td>Kopf Instruments</td><td>1430</td><td></td></tr><tr><td>Homeothermic heating blanket</td><td>Harvard Apparatus</td><td>50-7220-F</td><td></td></tr><tr><td>Pulse oximeter &amp;amp; heart rate monitor</td><td>Kent Scientific</td><td>MouseStat Jr.</td><td></td></tr><tr><td>Petrolatum</td><td>Fisher Scientific</td><td>P66-1LB</td><td>Vaseline generic</td></tr><tr><td>Wire, bare copper</td><td>Fisher Scientific</td><td>15-545-2C</td><td>20 gauge</td></tr><tr><td>Teets Cold Cure powder</td><td>Pearson Dental</td><td>C73-0054&amp;nbsp;</td><td>active ingredient: Methyl Methacrylate</td></tr><tr><td>Teets Cold Cure liquid</td><td>Pearson Dental</td><td>C73-0078&amp;nbsp;</td><td>active ingredient: Methyl Methacrylate</td></tr><tr><td>Silicone mold rubber</td><td>Smooth-On</td><td>Body Double Fast</td><td>silicon polymer</td></tr><tr><td>Metricide 28 (Germicide)</td><td>Metrex</td><td>Oct-05</td><td></td></tr><tr><td>India ink, black</td><td>Pelikan</td><td>301051</td><td></td></tr><tr><td>Dental drill</td><td>NSK Dental</td><td>Ultimate XL-F</td><td></td></tr><tr><td>3D printer</td><td>Prusa Research</td><td>i3 MK3S+</td><td></td></tr><tr><td>Sew on fasteners</td><td>Velcro</td><td>90030</td><td></td></tr><tr><td>Pet screening utility fabric</td><td>Joann</td><td>10173334</td><td>Netting material</td></tr><tr><td>Bur (drill bit), standard operatory carbide</td><td>SS White Burs</td><td>14829</td><td>#1 bur</td></tr></tbody></table>

head implants for the neuroimaging of awake, head-fixed rats

Anesthetics, commonly used in preclinical and fundamental scientific research, have a depressive influence on the metabolic, neuronal, and vascular functions of the brain and can adversely influence neurophysiological results. The use of awake animals for research studies is advantageous but poses the major challenge of keeping the animals calm and stationary to minimize motion artifacts throughout data acquisition. Awake imaging in smaller-sized rodents (e.g., mice) is very common but remains scant in rats as rats are bigger, stronger, and have a greater tendency to oppose movement restraints and head fixation over the long durations required for imaging. A new model of neuroimaging of awake, head-fixed rats using customized hand-sewn slings, 3D-printed head implants, head caps, and a headframe is described. The results acquired following a single trial of single-whisker stimulation suggest an increase in the intensity of the evoked functional response. The acquisition of the evoked functional response from awake, head-fixed rats is faster than that from anesthetized rats, reliable, reproducible, and can be used for repeated longitudinal studies.

The representative optical imaging signals from a single trial of an anesthetized rat and the summed response (of 40 collected trials) of an awake rat are shown (Figure 4). The signal intensity for single-whisker stimulation of an awake rat can be visualized at a higher threshold than for the anesthetized rat, showing a stronger signal from the awake animal. The C2 whiskers of rats are stimulated at 5 Hz for 1 s, and the functional response is displayed as a fractional change compared to the...

Watch this Scientific Journal Video about Head Implants for the Neuroimaging of Awake, Head-Fixed Rats at JoVE.com

Head Implants for the Neuroimaging of Awake, Head-Fixed Rats

A detailed new procedure for functional imaging of awake, head-fixed rats is described.

Anesthetics, commonly used in preclinical and fundamental scientific research, have a depressive influence on the metabolic, neuronal, and vascular ...

head-implants-for-the-neuroimaging-of-awake-head-fixed-rats

Research

JoVE Journal

Neuroscience

2.4K Views.  University of California Irvine. A detailed new procedure for functional imaging of awake, head-fixed rats is described.

Video: Head Implants for the Neuroimaging of Awake, Head-Fixed Rats

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 ...

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both laboratory and clinical settings. Currently, preclinical ERG and VEP measurements are performed with anesthesia to ensure stable electrode placements. However, the very presence of anesthesia has been shown to contaminate normal physiological responses. To overcome these anesthesia confounds, we develop a novel platform to assay ERG and VEP in conscious rats. Electrodes are surgically implanted sub-conjunctivally on the eye to assay the ERG and epidurally over the visual cortex to measure the VEP. A range of amplitude and sensitivity/timing parameters are assayed for both the ERG and VEP at increasing luminous energies. The ERG and VEP signals are shown to be stable and repeatable for at least 4 weeks post surgical implantation. This ability to record ERG and VEP signals without anesthesia confounds in the preclinical setting should provide superior translation to clinical data.

We show surgical implantation and Recording procedures to measure visual electrophysiological signals from the eye (electroretinogram) and brain (visual evoked potential) in conscious rats, which is more analogous to the human condition where recordings are conducted without anesthesia confounds.

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both ...

Behavior

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

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 ...

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal&#8217;s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

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 ...

Open&#45;Source Implant for Multi&#45;Region Electrophysiological Monitoring in Rats

The TD Drive: A Parametric, Open-Source Implant for Multi&#45;Area Electrophysiological Recordings in Behaving and Sleeping Rats

Intricate interactions between multiple brain areas underlie most functions attributed to the brain. The process of learning, as well as the formation and consolidation of memories, are two examples that rely heavily on functional connectivity across the brain. In addition, investigating hemispheric similarities and/or differences goes hand in hand with these multi-area interactions. Electrophysiological studies trying to further elucidate these complex processes thus depend on recording brain activity at multiple locations simultaneously and often in a bilateral fashion. Presented here is a 3D-printable implant for rats, named TD Drive, capable of symmetric, bilateral wire electrode recordings, currently in up to ten distributed brain areas simultaneously. The open-source design was created employing parametric design principles, allowing prospective users to easily adapt the drive design to their needs by simply adjusting high-level parameters, such as anterior-posterior and mediolateral coordinates of the recording electrode locations. The implant design was validated in n = 20 Lister Hooded rats that performed different tasks. The implant was compatible with tethered sleep recordings and open field recordings (Object Exploration) as well as wireless recording in a large maze using two different commercial recording systems and headstages. Thus, presented here is the adaptable design and assembly of a new electrophysiological implant, facilitating fast preparation and implantation.

Author Spotlight: Deciphering Memory and Learning Through Neural Implants for Multi&#45;Region Brain Studies

Here, we present a unique, 3D-printable implant for rats, named TD Drive, capable of symmetric, bilateral wire electrode recordings, currently in up to ten distributed brain areas simultaneously.

Intricate interactions between multiple brain areas underlie most functions attributed to the brain. The process of learning, as well as the formation and ...

A Cost-Effective, Versatile Probe Implant System for Electrophysiology in Mice

The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. However, current methods for chronically implanting probes present steep trade-offs between cost, ease of use, size, adaptability, and long-term stability.
This protocol introduces a novel chronic probe implant system for mice called the DREAM (Dynamic, Recoverable, Economical, Adaptable, and Modular), designed to overcome the trade-offs associated with currently available options. The system provides a lightweight, modular and cost-effective solution with standardized hardware elements that can be combined and implanted in straightforward steps and explanted safely for recovery and multiple reuse of probes, significantly reducing experimental costs.
The DREAM implant system integrates three hardware modules: (1) a microdrive that can carry all standard silicon probes, allowing experimenters to adjust recording depth across a travel distance of up to 7 mm; (2) a three-dimensional (3D)-printable, open-source design for a wearable Faraday cage covered in copper mesh for electrical shielding, impact protection, and connector placement, and (3) a miniaturized head-fixation system for improved animal welfare and ease of use. The corresponding surgery protocol was optimized for speed (total duration: 2 h), probe safety, and animal welfare.
The implants had minimal impact on animals&#39; behavioral repertoire, were easily applicable in freely moving and head-fixed contexts, and delivered clearly identifiable spike waveforms and healthy neuronal responses for weeks of post-implant data collection. Infections and other surgery complications were extremely rare.
As such, the DREAM implant system is a versatile, cost-effective solution for chronic electrophysiology in mice, enhancing animal well-being, and enabling more ethologically sound experiments. Its design simplifies experimental procedures across various research needs, increasing accessibility of chronic electrophysiology in rodents to a wide range of research labs.

Here, we introduce a lightweight, cost-effective probe implant system for chronic electrophysiology in rodents optimized for ease of use, probe recovery, experimental versatility, and compatibility with behavior.

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. ...

Implanting a Wireless Bidirectional Microelectrode System in a Rat Brain

Source: Melo-Thomas, L., et al., <a href="https://app.jove.com/v/56299">A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats.</a> J. Vis. Exp. (2017)
The video demonstrates a procedure for implanting a wireless bidirectional multichannel electrode unit into a rat brain to record and stimulate multichannel neural networks.

Wszczepienie bezprzewodowego dwukierunkowego systemu mikroelektrod do mózgu szczura

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Head Implants for the Neuroimaging of Awake, Head-Fixed Rats

Podsumowanie

Przeglądaj więcej filmów

Simultaneous fMRI and Electrophysiology in the Rodent Brain

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

Deep Brain Stimulation with Simultaneous fMRI in Rodents

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

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

The TD Drive: A Parametric, Open-Source Implant for Multi-Area Electrophysiological Recordings in Behaving and Sleeping Rats

The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice

Wszczepienie bezprzewodowego dwukierunkowego systemu mikroelektrod do mózgu szczura