Name: A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning (Video) | JoVE Journal
Uploaded: 2015-06-22T14:00:00.000+00:00
Duration: 9 min 22 s
Description: Learn to silence neural activity in specific brain regions during learning tasks using pharmacogenetics in rats. This method combines designer receptors with sensory preconditioning to study memory mechanisms.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

playerTrialDesktop

We thank the authors of Robinson et al.12 for their contributions to the manuscript from which this protocol is partially derived.

The use of animals are approved by the Oberlin College Institutional Animal Care and Use committee and are in accordance with the Guide for the Care and Use of Laboratory Animals21.
1. Preparation for Viral Infusion
Note: This protocol uses BSL-1 precautions. When employing BSL-2 precautions, a disposable lab coat, gloves, shoe covers, eye protections and a particulate respirator (type N95) are required. All individuals handling BSL-2 compounds must be fit tested for a particulate respirator by a local public health agency. Refer to Lowery &#38; Majewska (2010)22 for additional details on handling and storage of viral vectors.
<ol>
	<li>Upon first use, aliquot and store unused virus in 20 &#181;l microcentrifuge tubes to avoid repeated freezing and thawing.</li>
	<li>Prepare the work surface by removing any unnecessary objects and sterilizing the surface with 70% ethanol. Prepare a 10% bleach solution for decontaminating AAV waste. Place a beaker full of the bleach solution and a sterile 10 &#181;l syringe onto the work surface. Place the microcentrifuge tube with the AAV in a container with crushed ice during set up.</li>
	<li>Place the microcentrifuge tube with the virus into a standard bench vice. Load a 10 &#181;l syringe with at least 4 &#181;l of AAV. Take care not to bend the tip of the syringe against the bottom of the microcentrifuge tube during loading. Ensure that there are no air bubbles in the 4 &#181;l of AAV solution in the syringe.</li>
	<li>Dispose of the empty virus tube in the beaker containing 10% bleach. Store unused portions of the virus as specified by the supplier.</li>
	<li>Add additional 10% bleach to the waste container, and allow waste to sit for 30 min before disposing the decontaminated liquid waste.</li>
	<li>Dispose of all protective equipment and plastic waste in a biohazard container as instructed by institutional guidelines. Decontaminate any equipment or surfaces that came in contact with the virus using 10% bleach.</li>
</ol>
2. Surgery
<ol>
	<li>Prepare the surgical area by placing absorbent bench paper under the stereotaxic apparatus and on an adjacent space designated as a dedicated virus handling area.
	<ol>
		<li>Place a 10% bleach waste container in the dedicated virus handling area near the surgical apparatus.</li>
	</ol>
	</li>
	<li>Induce a steady level of anesthesia and prepare the rat for surgery.
	<ol>
		<li>Place the rat into an induction chamber that contains a mixture of isoflurane gas (range of 1 to 3%) and oxygen (100% at approximately 1 L/min) until there is a loss of consciousness and lack of gross purposeful movement. Maintain isoflurane anesthesia throughout the surgical procedure.</li>
		<li>After 6 deep anesthesia is attained, shave the rat from between the eyes to slightly behind the ears.&#160;</li>
		<li>Place the rat back into the induction chamber for an additional 1-3 min.</li>
		<li>Ensure that the isoflurane anesthesia system is connected to the nose cone of the stereotaxic surgery. Open the stopcock on the isoflurane tubing to begin the flow of isoflurane to the stereotax. Maintain the isoflurane and oxygen at the levels specified above.</li>
		<li>Place the rat in a stereotaxic apparatus by securing the mouth onto the bite bar and securing the ear bars.</li>
		<li>Apply eye lubricant to the eyes and betadine to the surgical site prior to incision.</li>
	</ol>
	</li>
	<li>Make a 2.4 cm midline incision of the skin on the dorsal surface of the skull starting approximately 2 mm caudal to the eyes. Retract the skin to expose the skull. Periodically instill sterile 0.9% sterile saline on the margins of the surgical site to prevent tissues from drying.&#160;</li>
	<li>Clear membranous tissue from the skull until bregma and lambda are clearly visible.</li>
	<li>Ensure that the skull is level by measuring the dorsal-ventral coordinates at bregma and at lambda. Adjust the stereotaxic device accordingly until dorsal-ventral coordinates at bregma and lambda differ by no more than 0.04 mm.</li>
	<li>At this time, reduce the isoflurane gas to between 2 and 2.5%.</li>
	<li>Return the drill to bregma, rezero the coordinates and then move the drill to the desired medial-lateral and anterior-posterior coordinate.</li>
	<li>Drill a hole at the coordinate using a 0.9 mm drill bit as it produces an appropriate size hole for a 28 G infusion syringe.</li>
	<li>Set the infusion pump to deliver virus at a rate of 0.2 &#181;l/min. Place the syringe into the infusion arm of the stereotaxic device and lower the syringe into the desired coordinate. Deliver the desired amount of virus (range 0.1 to 0.8 &#181;l). For example, deliver 0.8 &#181;l of AAV over 4 min. 
	Note: conduct pilot studies to determine the ideal infusion volume and the typical spread of virus in each region of interest.
	<ol>
		<li>After the delivery of virus is complete leave the syringe in place for 10 min to prevent backflow into the needle tract. Slowly raise the syringe at a rate of about 5 mm/min. Repeat until all coordinates have been infused with AAV.</li>
	</ol>
	</li>
	<li>Close the wound using surgical staples and coat the wound with topical antibiotic ointment.</li>
	<li>Follow institutional guidelines for post-operative pain management.</li>
	<li>Place the rat in a cage with bedding, a lid and proper signage and return the rat to the animal facility.
	<ol>
		<li>If working under BSL-2 precautions, collect bedding for the amount of time (typically 48-72 hr) specified by institutional guidelines. Place bedding into a biohazard waste container.</li>
		<li>Dispose of all AAV-contaminated materials as specified by guidelines for their safe disposal.</li>
	</ol>
	</li>
</ol>
3. Behavioral Apparatus
Note: The sensory preconditioning apparatus consists of a standard operant conditioning chamber (12&#8221; L x 9.5&#8221; W x 11.5&#8221; H) with a stainless steel grid floor, 2 Plexiglas sidewalls and 2 metal walls.
<ol>
	<li>Mount a 2.8 W light on one of the aluminum walls to serve as the house light and as the visual stimulus when flashed at 2 Hz. Mount a speaker on the chamber to deliver the auditory stimuli (a 10 sec, 1500 Hz, 78 dB pure tone and a 10 sec 78 dB white noise).
	<ol>
		<li>Use a food hopper to deliver 45 mg food pellets into the food cup. Within the food cup, infrared photocells detect the total time spent in the food cup before, during and after presentations of the stimuli and food rewards.</li>
		<li>House the chambers in sound-attenuating cabinets (22&#8221; W x 22&#8221; H x 16&#8221; D) outfitted with exhaust fans (~68 dB).</li>
		<li>Use a PC computer with Med Associates software to control the operant chamber and acquire the data. During the Conditioning sessions, collect data on the total time spent with the head in the food cup during presentation of the light stimulus (5 sec epoch) and during presentation of the food reward (5 sec epoch). During the Test session, collect data on the total time spent in the food cup in response to presentations of the auditory stimuli (10 sec epoch beginning when the auditory stimuli are terminated).</li>
	</ol>
	</li>
</ol>
4. Overview of the Sensory Preconditioning Paradigm
<ol>
	<li>Following recovery from surgery, gradually food restrict the rats to 85% of their free feeding body weight. Consult veterinary staff for an appropriate food restriction paradigm. 
	Note: The 3-phase sensory preconditioning paradigm is illustrated in Figure 2.</li>
	<li>Preconditioning Sessions (Phase 1; Daily 64 min training sessions conducted across 4 consecutive days):
	<ol>
		<li>Present rats with 12 intermixed trials that consist of delivery of auditory and visual stimuli. Include inter-trial intervals that average 4.5 min (range 4.0 to 5.0 min) after each auditory presentation.</li>
		<li>On 6 of the trials, present a tone (preconditioned stimulus) for 10 sec, followed immediately by a 5 sec presentation of the flashing light stimulus.</li>
		<li>On the other 6 trials, present the white noise (unpaired stimulus) alone for 10 sec.</li>
	</ol>
	</li>
	<li>Conditioning sessions (Phase 2; Daily 64 min training sessions conducted across 5 consecutive days):
	<ol>
		<li>Present rats with 8 trials that consist of delivery of the visual stimulus (the flashing light stimulus) for 5 sec followed immediately by delivery of two 45 mg food pellets. Include inter-trial intervals that average 7 min (range 6.0 to 8.0 min) after each trial. Occasionally rats require more than 5 conditioning sessions to learn the light food association.</li>
	</ol>
	</li>
	<li>Test Session (Phase 3; a single 78 min session):
	<ol>
		<li>Present the rats with 12 intermixed trials that consist of delivery of the auditory stimuli alone. Include inter-trial intervals that average 4.5 min (range 4.0 to 5.0 min) after each auditory presentation.</li>
		<li>On 6 of the trials, present the tone stimulus (the preconditioned stimulus) alone for 10 sec.</li>
		<li>On the other 6 trials, present the white noise (the unpaired stimulus) alone for 10 sec.</li>
	</ol>
	</li>
	<li>Counterbalance the stimuli by completing the study with a second set of rats that receive the white noise as the preconditioned stimulus and the tone as the unpaired stimulus.</li>
</ol>
5. Pharmacological and Behavioral Procedure
<ol>
	<li>Turn on the power to the behavioral apparatus and load the Med-PC code for the appropriate behavioral session. Wire and program the behavioral apparatus such that the fans that are mounted on the sound attenuating cabinets turn on when the power is switched on.</li>
	<li>To prepare a 1 mg/ml solution of clozapine N-oxide (CNO), weigh 5.0 mg of CNO into a 15 ml conical tube and add 5 ml of sterile water or 5 ml of sterile 0.9% saline. Vortex until the solution is clear.
	<ol>
		<li>If CNO does not dissolve into solution or if more concentrated solutions of CNO are desired add a solubilizing agent, such as DMSO23. Specifically, to prepare a 1 mg/ml solution of CNO with 0.5% DMSO, weigh 5.0 mg of CNO and add 25 &#181;l of DMSO to a 15 ml conical tube. Flick gently ensuring that the contents remain at the base of the tube. When the solution becomes clear, add 5 ml of sterile water or 5 ml of sterile 0.9% saline. Further instructions for preparing solutions of CNO are available on the DREADD wiki resource page25.</li>
		<li>Prepare a new CNO solution each day that it is administered.</li>
	</ol>
	</li>
	<li>Inject the CNO intraperitoneally at a dose of 1 mg/kg approximately 30 min prior to behavioral testing. For example, inject a 200 g rat with 0.2 ml of the 1 mg/ml CNO solution. The dose and time course of CNO administration were selected based on previously published reports2,12.
	<ol>
		<li>Inject CNO 30 min prior to each preconditioning session (Phase 1; total of 4 days of injections) but administer the vehicle prior to all subsequent behavioral sessions.</li>
	</ol>
	</li>
	<li>After 30 min, place the rats into the behavioral testing chambers and initiate the computer code for the appropriate behavioral session.</li>
	<li>Upon completion of the behavioral task, immediately remove the rats from the chambers and return the rats to the animal colony.</li>
</ol>
6. Analyses of Behavioral Data
Note: The dependent variable for all behavioral sessions is the amount of time that the rat&#8217;s head is inside the food cup as detected by interruption of the infrared photocells in the food cup. The data (in sec) are collected and recorded by the computer software.
<ol>
	<li>Do not conduct analyses on data generated during the Preconditioning phase.</li>
	<li>For the conditioning sessions: analyze each rats&#8217; ability to learn the light food association by comparing food cup behavior during presentation of the visual stimulus across the 5 behavioral sessions. Specifically, calculate the average time spent in the food cup during the 5 sec light epoch for each of 8 trials/session (i.e., an average of 8 trials that are each 5 sec in duration/rat). Compare average control and experimental values for each of 5 daily sessions (see Figure 3A).
	<ol>
		<li>Analyze the rats&#8217; motivation to obtain food reward by comparing food cup behavior during delivery of the food reward (5 sec epoch) across the 5 behavioral sessions using the same calculation as specified for the 5 sec light epoch above. These values will be consistently higher than those acquired during presentation of the visual stimulus (see Figure 3B).</li>
	</ol>
	</li>
	<li>For the test session: calculate a discrimination ratio that describes the rats&#8217; food cup behavior in response to presentations of each auditory stimulus.
	<ol>
		<li>Specifically, for each rat, divide the average time spent in the food cup following each of 6 presentations of the sensory preconditioned stimulus (i.e., the tone) by the sum of the average time spent in the food cup following each of 6 presentations of the sensory preconditioned stimulus (i.e., the tone stimulus) plus the average of the total time spent in the food cup following each of 6 presentations of the unpaired stimulus (i.e., the white noise stimulus). Each epoch is 10 sec in duration. 
		Note: A discrimination score of 0.5 or greater demonstrates that rats learned the associations inherent in the sensory preconditioning paradigm.</li>
	</ol>
	</li>
</ol>
7. Verification of AAV Placement and Expression
<ol>
	<li>Anesthetize and perfuse the rats transcardially using 4% paraformaldehyde. Due to the use of fluorescent labels, do not exceed a post-fixation time of 2 hr to minimize loss of antigenicity and to preserve the fluorescent signal.</li>
	<li>Dehydrate perfused brains by transferring them into a brain jar containing 30 ml of a 20% sucrose solution in phosphate buffered saline (1x) for a minimum of 2 days or until the brain sinks to the bottom of the brain jar.</li>
	<li>Use a freezing sliding microtome to make coronal brain sections (20-40 &#181;m) throughout the entire rostrocaudal extent of the region of interest.</li>
	<li>Conduct immunohistochemistry directed against the tagged receptor or the reporter protein to ascertain the location and expression of the designer receptor and/or reporter12. An immunohistochemistry staining protocol is provided on the DREADD wiki resource page25.</li>
	<li>Mount the sections onto superfrost-plus slides and coverslip using 100-200 &#181;l of an aqueous mounting medium.</li>
	<li>Perform fluorescence microscopy on brain tissue to verify AAV placement and protein expression12,24.</li>
</ol>

<ol><li>Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S., Roth, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evolving+the+lock+to+fit+the+key+to+create+a+family+of+G-protein+coupled+receptors+potently+activated+by+an+inert+ligand%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Evolving the lock to fit the key to create a family of G-protein coupled receptors potently activated by an inert ligand.</a> Proc. Natl. Acad. Sci. USA. 104 (12), 5163-5168 (2007).</li>
<li>Urban, D. J., Roth, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DREADDs+%28Designer+Receptors+Exclusively+Activated+by+Designer+Drugs%29%3A+Chemogenetic+tools+with+therapeutic+utility%5BTitle%5D)+AND+%22Annu.+Rev.+Pharmacol.Toxicol%22%5BJournal%5D)">DREADDs (Designer Receptors Exclusively Activated by Designer Drugs): Chemogenetic tools with therapeutic utility.</a> Annu. Rev. Pharmacol.Toxicol. 55, 399-417 (2015).</li>
<li>Weiner, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+M1+muscarinic+receptor+agonism+of+N-desmethylclozapine+in+the+unique+clinical+effects+of+clozapine%5BTitle%5D)+AND+%22Psychopharm.+%28Berl%22%5BJournal%5D)">The role of M1 muscarinic receptor agonism of N-desmethylclozapine in the unique clinical effects of clozapine.</a> Psychopharm. (Berl. 177 (1-2), 1-2 (2004).</li>
<li>Cohen, N. J., Memory Eichenbaum, H. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCohen%2C+NJ+%22amnesia+and+the+hippocampal+system%22">amnesia and the hippocampal system</a>. , MIT Press. Cambridge, MA. (1993).</li>
<li>Strien, N. M., Cappaert, N. L., Witter, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+anatomy+of+memory%3A+an+interactive+overview+of+the+parahippocampal-hippocampal+network%29%5BTitle%5D)+AND+%22Nat.+Rev.+Neurosci%22%5BJournal%5D)">The anatomy of memory: an interactive overview of the parahippocampal-hippocampal network).</a> Nat. Rev. Neurosci. 10 (4), 272-282 (2009).</li>
<li>Agster, K. L., Burwell, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortical+efferents+of+the+perirhinal%2C+postrhinal+and+entorhinal+cortices+of+the+rat%5BTitle%5D)+AND+%22Hippocampus%22%5BJournal%5D)">Cortical efferents of the perirhinal, postrhinal and entorhinal cortices of the rat.</a> Hippocampus. 19 (12), 1159-1186 (2009).</li>
<li>Aggleton, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+anatomical+systems+embedded+within+the+primate+medial+temporal+lobe%3A+implications+for+hippocampal+function%5BTitle%5D)+AND+%22Neurosci.+Biobehav.+Rev%22%5BJournal%5D)">Multiple anatomical systems embedded within the primate medial temporal lobe: implications for hippocampal function.</a> Neurosci. Biobehav. Rev. 36, 1579-1596 (2012).</li>
<li>Robinson, S., Poorman, C. E., Marder, T. J., Bucci, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+functional+circuitry+between+retrosplenial+and+postrhinal+cortices+during+fear+conditioning%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Identification of functional circuitry between retrosplenial and postrhinal cortices during fear conditioning.</a> J. Neurosci. 32 (35), 12076-12086 (2012).</li>
<li>Bucci, D. J., Saddoris, M. P., Burwell, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Corticohippocampal+contributions+to+spatial+and+contextual+learning%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Corticohippocampal contributions to spatial and contextual learning.</a> J. Neurosci. 24 (15), 3826-3836 (2004).</li>
<li>Kaut, K. P., Bunsey, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+lesions+to+the+rat+hippocampus+or+rhinal+cortex+on+olfactory+and+spatial+memory%3A+retrograde+and+anterograde+findings%5BTitle%5D)+AND+%22Cogn.+Affect.+Behav.+Neurosci%22%5BJournal%5D)">The effects of lesions to the rat hippocampus or rhinal cortex on olfactory and spatial memory: retrograde and anterograde findings.</a> Cogn. Affect. Behav. Neurosci. 1 (3), 270-286 (2001).</li>
<li>Brogden, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensory+preconditioning%5BTitle%5D)+AND+%22J.+Exp.+Psychol%22%5BJournal%5D)">Sensory preconditioning.</a> J. Exp. Psychol. 25, 323-332 (1939).</li>
<li>Robinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemogenetic+silencing+of+neurons+in+retrosplenial+cortex+disrupts+sensory+preconditioning%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Chemogenetic silencing of neurons in retrosplenial cortex disrupts sensory preconditioning.</a> J. Neurosci. 34 (33), 10982-10988 (2014).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NIH+guidelines+for+research+involving+recombinant+or+synthetic+nucleic+acid+molecules%5BTitle%5D)+AND+%22Available+from%3A+http%3A%2F%2Foba.od.nih.gov%2Frdna%2Fnih_guidelines_oba.html%22%5BJournal%5D)">NIH guidelines for research involving recombinant or synthetic nucleic acid molecules.</a> Available from: http://oba.od.nih.gov/rdna/nih_guidelines_oba.html. , (2013).</li>
<li>Samulski, R. J., Muzyczka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AAV-mediated+gene+therapy+for+research+and+therapeutic+purposes%5BTitle%5D)+AND+%22Annu.+Rev.+Virol%22%5BJournal%5D)">AAV-mediated gene therapy for research and therapeutic purposes.</a> Annu. Rev. Virol. 1, 427-451 (2014).</li>
<li>Tenenbaum, L., Lehtonen, E., Monahan, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+risks+related+to+the+use+of+adeno-associated+virus-based+vectors%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">Evaluation of risks related to the use of adeno-associated virus-based vectors.</a> Gene Ther. 3, 545-565 (2003).</li>
<li>Arechavaleta-Velasco, F., Ma, Y., Zhang, J., McGrath, C. M., Parry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adeno-associated+virus-2+%28AAV-2%29+causes+trophoblast+dysfunction%2C+and+placental+AAV-2+infection+is+associated+with+preeclampsia%5BTitle%5D)+AND+%22Am+J+Path%22%5BJournal%5D)">Adeno-associated virus-2 (AAV-2) causes trophoblast dysfunction, and placental AAV-2 infection is associated with preeclampsia.</a> Am J Path. 168 (6), 1951-1959 (2006).</li>
<li>Erles, K., Rohde, V., Thaele, M., Roth, S., Edler, L., Schlehofer, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+of+adeno-associated+virus+%28AAV%29+in+testicular+tissue+and+in+abnormal+semen+samples%5BTitle%5D)+AND+%22Hum.+Reprod%22%5BJournal%5D)">DNA of adeno-associated virus (AAV) in testicular tissue and in abnormal semen samples.</a> Hum. Reprod. 16 (11), 2333-2337 (2001).</li>
<li>Donsante, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AAV+vector+integration+sites+in+mouse+hepatocellular+carcinoma%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">AAV vector integration sites in mouse hepatocellular carcinoma.</a> Science. 317 (5837), 477-47 (2007).</li>
<li>Donsante, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Observed+incidence+of+tumorigenesis+in+long-term+rodent+studies+of+rAAV+vectors%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors.</a> Gene Ther. 8 (17), 1343-1346 (2001).</li>
<li>Wu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+expression+of+Pctk1%2C+Tcf12+and+Ccnd+in+hippocampus+of+rats%3A+impact+on+cognitive+function%2C+synaptic+plasticity+and%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Enhanced expression of Pctk1, Tcf12 and Ccnd in hippocampus of rats: impact on cognitive function, synaptic plasticity and.</a> 97 (1), 69-80 (2011).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((National+Academy+Press.+Guide+for+the+Care+and+Use+of+Laboratory+Animals%5BTitle%5D)+AND+%22National+Academy+Press%22%5BJournal%5D)">National Academy Press. Guide for the Care and Use of Laboratory Animals.</a> National Academy Press. , Washington, DC. (1996).</li>
<li>Lowery, R. L., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracranial+injection+of+adeno-associated+viral+vectors%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Intracranial injection of adeno-associated viral vectors.</a> J. Vis. Exp. (45), (2010).</li>
<li>Cavaletti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+in+the+peripheral+nervous+system+of+systemically+administered+dimethylsulfoxide+in+the+rat%3A+a+neurophysiological+and+pathological%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Effect in the peripheral nervous system of systemically administered dimethylsulfoxide in the rat: a neurophysiological and pathological.</a> 119 (1-2), 1-2 (2000).</li>
<li>Parnaudeau, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mediodorsal+thalamus+hypofunction+impairs+flexible+goal-directed+behavior%5BTitle%5D)+AND+%22Biol.+Psychiatry%22%5BJournal%5D)">Mediodorsal thalamus hypofunction impairs flexible goal-directed behavior.</a> Biol. Psychiatry. 77 (5), 445-453 (2014).</li>
<li>wiki, D. R. E. A. D. D. , Available from: http://www.dreadd.org (2014).</li>
<li>Ferguson, S. M., Phillips, P. E. M., Roth, B. L., Wess, J., Neumaier, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct-pathway+striatal+neurons+regulate+the+retention+of+decision-making+strategies%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Direct-pathway striatal neurons regulate the retention of decision-making strategies.</a> J. Neurosci. 33 (28), 11668-11676 (2013).</li>
<li>Cassatarro, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reverse+pharmacogenetic+modulation+of+the+nucleus+accumbens+reduces+ethanol+consumption+in+a+limited+access+paradigm%5BTitle%5D)+AND+%22Neuropsychopharm%22%5BJournal%5D)">Reverse pharmacogenetic modulation of the nucleus accumbens reduces ethanol consumption in a limited access paradigm.</a> Neuropsychopharm. 39, 283-290 (2014).</li>
<li>Krashes, M. J., Shah, B. P., Koda, S., Lowell, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+versus+delayed+stimulation+of+feeding+by+the+endogenously+released+AgRP+neuron+mediators%5BTitle%5D)+AND+%22GABA%2C+NPY+and+AgRP.+Cell+Metab%22%5BJournal%5D)">Rapid versus delayed stimulation of feeding by the endogenously released AgRP neuron mediators.</a> GABA, NPY and AgRP. Cell Metab. 18 (4), 588-595 (2014).</li>
<li>Gage, F. H., Bjorklund, A., Stenevi, U., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+correlates+of+compensatory+collateral+sprouting+by+aminergic+and+cholinergic+afferents+in+the+hippocampal+formation%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Functional correlates of compensatory collateral sprouting by aminergic and cholinergic afferents in the hippocampal formation.</a> Brain Res. 268 (1), 39-47 (1983).</li>
<li>Nelson, R. J., Young, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavior+in+mice+with+targeted+disruption+of+single+genes%5BTitle%5D)+AND+%22Neurosci.+Biobehav.+Rev%22%5BJournal%5D)">Behavior in mice with targeted disruption of single genes.</a> Neurosci. Biobehav. Rev. 22 (3), 453-462 (1998).</li>
</ol>

The authors declare that they have no competing financial interests.

This protocol describes how to apply a pharmacogenetic approach (DREADD) to investigate how a specific brain region contributes to a multi-phase complex learning task. With the ability to temporarily and remotely silence neural activity in discrete brain regions across phases of learning, this combination of approaches provides a platform to investigate a wide range of behaviors, including more nuanced or masked forms of learning. In the example described in this protocol, control rats and rats that express the designer ...

An exciting challenge within the field of behavioral neuroscience is to determine the neural substrates of complex behaviors. A number of techniques such as permanent lesions, temporary brain inactivation via cannulae implants and optogenetics have been employed to identify the contributions of discrete brain regions to subcomponents of complex behaviors. While these approaches inform our understanding of regional specificity during learning, each technique is not without limitations. Specifically, permanent lesions are typically conducted prior to behavioral testing, thus their effects are present throughout the duration of the paradigm. Cannulation studies that involve the presentation of a short-term neural inactivator (e.g., tetrodotoxin) can produce substantial damage to brain tissue and can induce stress in subjects just prior to behavioral testing. Furthermore, inactivation through cannulation is limited to the region of tissue that surrounds the tip of the cannulae. Lastly, while optogenetics offers a range of flexibility for the temporal control of activity in specific brain regions, it is cost prohibitive and technically demanding.
These limitations can be surmounted using a pharmacogenetic approach (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs, DREADDs)1,2. Importantly, while the concept of pharmacogenetics is sophisticated, the execution of the technique is straightforward. Similar to traditional stereotaxic surgical methods that involve infusion of toxin (e.g., NMDA, ibotenic acid) into discrete brain regions, this technique involves infusing an adeno-associated virus (AAV) that contains a DNA fragment for a modified inhibitory G-protein coupled receptor (hM4Di; the designer receptor) into the region of interest of standard laboratory rodents (see Figure 1). The viral vector also contains a fluorescent reporter (mcitrine). Once incorporated into cells, the designer receptor (and reporter protein) are maximally expressed ~3 weeks post-infusion and can be selectively activated for 2-5 hr by systemic administration of the otherwise biologically inert designer drug, clozapine-N-oxide (CNO)1,3. Because the experimenter is endowed with precise, yet remote temporal control over neural activity in specific brain regions, pharmacogenetics combines particularly well with behavioral paradigms that are conducted in multiple phases. In this example, the contribution of the retrosplenial cortex (RSC) to stimulus-stimulus learning is compared to its role in Pavlovian learning, however this combination of approaches is well suited to any number of questions that seek to identify how specific brain regions contribute to complex behavior.
In addition, while not described in the present protocol, viral and transgenic approaches can be used to achieve cell type-specific DREADD expression2. As is inherent in behavioral paradigms that involve pharmacological and/or other types of experimental manipulations, careful consideration of experimental design and subsequent quantitative analysis is required when employing the DREADD approach. Experimenters new to the DREADD approach are referred to a comprehensive review of current DREADD technology2.
Each day, organisms learn about new stimuli and events and their relationships to one another. Even in a familiar environment, such as home, one is quick to detect alterations in the relationships between stimuli because these changes may be predictive of meaningful events. Such stimulus-stimulus (i.e., relational) learning involves the conjoining of multiple stimuli and has traditionally been associated with the hippocampus, which resides centrally within the medial temporal lobe4. However, the hippocampus does not exist nor act in isolation; cortical regions both within and outside of the medial temporal lobe provide critical sensory information to the hippocampal formation5-7. Traditional permanent lesion studies provide compelling evidence for the involvement of a number of cortical regions (e.g., the retrosplenial, postrhinal and entorhinal cortices) in hippocampal-dependent learning but are limited in their ability to discern the role of a particular region during discrete phases of learning8-10.
The present protocol tests the hypothesis that the RSC is necessary for stimulus-stimulus learning by silencing the RSC during a single phase of a 3-phase sensory preconditioning paradigm11,12. Briefly, rats receive infusions of an AAV that contains the designer receptor and ~3 weeks later are administered the designer drug (CNO) 30 min prior to the start of behavioral testing. In the present protocol, experimental rats receive CNO during the first phase of testing (when stimulus-stimulus learning occurs) and they receive vehicle during the next 2 phases of testing. To control for inadvertent effects of CNO on behavior, infuse rats with the designer receptor (hM4Di) and inject with vehicle instead of CNO. To account for general effects of viral infusion and receptor expression, infuse a control virus that does not contain the designer receptor and administer CNO.
A number of different serotypes of AAV are used to deliver genetic material. The current NIH Guidelines for Research Involving Recombinant or Synthetic Molecules maintains that AAV (all serotypes) and recombinant or synthetic AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and are produced in the absence of a helper virus, require BSL-1 precautions (Appendix B-1. Risk Group 1 (RG1) Agents)13. A number of reviews pertaining to AAV structure, utility and safety are available14,15. Notably, though, due to concerns pertaining to possible reproductive16,17 and potential carcinogenic mechanisms18-20 in rodents, some institutions require the use of BSL-2 precautions when working with AAV. Verify the appropriate BSL prior to use by consulting with oversight committees at individual institutions where the research will be conducted, the Centers for Disease Control and the NIH Guidelines for Research Involving Recombinant DNA Molecules13 when using viral vectors for gene manipulation in the United States. Personal protection, investigator training, vector containment, decontamination, disposal of decontaminated materials, and post-injection animal housing requirements are specified by these guidelines. In addition, consult and follow appropriate Institutional Animal Care and Use committee guidelines or equivalent institutional oversight committee guidelines to ensure the safe handling, administration and disposal of AAV.

<table><tbody><tr><td>Male, Long Evans Rats, 55-60 days old</td><td>Hilltop Lab Animals Inc</td><td></td><td></td></tr><tr><td>rAAV8/hSyn-HA-hM4D(Gi)-IRES-mCitrine</td><td>Virus Vector Core</td><td></td><td>Caution: This is a BSL-1 compound</td></tr><tr><td>rAAV8/hSyn-GFP</td><td>Virus Vector Core</td><td></td><td>Caution: This is a BSL-1 compound</td></tr><tr><td>Clozapine-N-oxide</td><td>R&amp;amp;D Systems</td><td>4936-10</td><td>Designer drug</td></tr><tr><td>Rat Cage lid (polycarbonate)</td><td>Alternative Design&amp;nbsp;</td><td>FT 8XL-PC</td><td>Used to cover animal cages 48-72 hr post infusion</td></tr><tr><td>Filter paper (replacement)</td><td>Alternative Design</td><td>FP-R-1018XAD</td><td>Filter paper that goes with cage lids</td></tr><tr><td>Table top vise</td><td>JETS</td><td>2201-265</td><td>For holding microscentrifuge tubes containing AAV in the hood</td></tr><tr><td>Biohazard trash bags</td><td>Staples</td><td>113444</td><td>Medline biohazard liners</td></tr><tr><td>Biohazard trash can</td><td>Amazon.com</td><td></td><td>United Solutions 34 gallon rectangular wheeled trashcan with hook and lock handle</td></tr><tr><td>Isoflurane, 100 ml</td><td>Patterson Veterinary Supply Inc.</td><td>07-890-8540</td><td>Anesthetic</td></tr><tr><td>Dual small animal stereotaxic with digital display readout console</td><td>David Kopf</td><td>Model 942</td><td>Surgical equipment</td></tr><tr><td>Non-rupture ear bars, set of 2 (rat)</td><td>David Kopf</td><td>Model 955</td><td>Surgical equipment</td></tr><tr><td>Anesthesia mask (rat)</td><td>David Kopf</td><td>Model 906</td><td>Surgical equipment</td></tr><tr><td>High speed stereotaxic drill includes table top motor controller, foot pedal, handpiece, stereotaxic handpiece holder</td><td>David Kopf</td><td>Model 1474</td><td>Surgical equipment</td></tr><tr><td>Microdrill burrs, 0.9 mm</td><td>Fine Science Tools Inc</td><td>19007-09</td><td>Surgical supply</td></tr><tr><td>Automated syringe pump with micro4 controller</td><td>David Kopf</td><td>Model UMP3-1</td><td>Surgical equipment</td></tr><tr><td>Pro-animal detachable ceramic blade clipper kit</td><td>Ahdis</td><td>21420</td><td>Surgical supply</td></tr><tr><td>Betadine skin cleanser</td><td>Perdue Products L.P</td><td>67618-149-04</td><td>Surgical supply</td></tr><tr><td>Triple antiobiotic ointment</td><td>Medline Supply</td><td>53329-087-01</td><td>Surgical supply</td></tr><tr><td>Puralube vet ointment</td><td>Only Veterinary Supply</td><td>17033-211-38</td><td>Surgical supply</td></tr><tr><td>Dino-lite</td><td>Microscope</td><td>AD7013MTL</td><td>An alternative to the traditional disection scope</td></tr><tr><td>Dino-lite rigid table-top boom stand</td><td>Microscope</td><td>MS36B</td><td>Surgical equipment</td></tr><tr><td>28 G 10 &amp;mu;l syringe</td><td>Hamilton</td><td>80308-701SN</td><td>Surgical equipment</td></tr><tr><td>Extra tall MDF sound attenuating cubicle</td><td>Med Associates, Inc</td><td>ENV-018MD</td><td>22&#039; W x 22&quot; H x 16&quot; D</td></tr><tr><td>Extra tall modular test chamber</td><td>Med Associates, Inc</td><td>ENV-007</td><td>Behavioral equipment</td></tr><tr><td>Stainless steel grid floor</td><td>Med Associates, Inc</td><td>ENV-005</td><td>Behavioral equipment</td></tr><tr><td>House light</td><td>Med Associates, Inc</td><td>ENV-215M</td><td>Used as the house light and stimulus light</td></tr><tr><td>Modular pellet dispenser</td><td>Med Associates, Inc</td><td>ENV-203M-45</td><td>Behavioral equipment</td></tr><tr><td>Pellet receptacle, cup type</td><td>Med Associates, Inc</td><td>ENV-200R1M</td><td>Behavioral equipment</td></tr><tr><td>Head entry detector for rat</td><td>Med Associates, Inc</td><td>ENV-254-CB</td><td>Behavioral equipment</td></tr><tr><td>Dustless precision food pellets, 45 mg</td><td>Bio-Serv</td><td>F0165</td><td>Behavioral supply</td></tr><tr><td>Cage speaker for rat chamber</td><td>Med Associates, Inc</td><td>ENV-224AM</td><td>Behavioral equipment</td></tr><tr><td>Programmable audio generator</td><td>Med Associates, Inc</td><td>ANL-926</td><td>Behavioral equipment</td></tr><tr><td>Smart ctrl 8 input/16 output package</td><td>Med Associates, Inc</td><td>DIG-716P2</td><td>Behavioral equipment</td></tr><tr><td>Large table-top cabinet and power supply</td><td>Med Associates, Inc</td><td>SG-6510D</td><td>Behavioral equipment</td></tr><tr><td>PCI interface package</td><td>Med Associates, Inc</td><td>DIG-700P2-R2</td><td>Behavioral equipment</td></tr><tr><td>MED Intel core computer pkg with X Pro 19&quot; monitor</td><td>Med Associates, Inc</td><td>COM-103V</td><td>Behavioral equipment</td></tr><tr><td>Paraformaldehyde (grannular), 1 kg</td><td>Electron Microsopy Sciences</td><td>19210</td><td>Hazard: carcinogen, weigh in hood</td></tr><tr><td>Rabbit monoclonal antibody (HA-Tag)</td><td>Cell Signaling Technologies</td><td>3724S</td><td>Histology reagent</td></tr><tr><td>XP Rabbit monoclonal antibody (GFP)</td><td>Cell Signaling Technologies</td><td>2956S</td><td>Histology reagent</td></tr><tr><td>Anti-rabbit IgG</td><td>Cell Signaling Technologies</td><td>4412S</td><td>Histology supplies</td></tr><tr><td>Superfrost plus slides</td><td>VWR international</td><td>483111-703</td><td>Histology supplies</td></tr></tbody></table>

a method for remotely silencing neural activity in rodents during discrete phases of learning

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different forms of learning. Specifically, viral-mediated delivery is used to express a genetically modified inhibitory G-protein coupled receptor (the Designer Receptor) into a discrete brain region in the rodent. Three weeks later, when designer receptor expression levels are high, a pharmacological agent (the Designer Drug) is administered systemically 30 min prior to a specific behavioral session. The drug has affinity for the designer receptor and thus results in inhibition of neurons that express the designer receptor, but is otherwise biologically inert. The brain region remains silenced for 2-5 hr (depending on the dose and route of administration). Upon completion of the behavioral paradigm, brain tissue is assessed for correct placement and receptor expression. This approach is particularly useful for determining the contribution of individual brain regions to specific components of behavior and can be used across any number of behavioral paradigms.

Behavioral Results
Upon completion of the experiment, the effectiveness of the region-specific temporary inactivation should be quantitatively and qualitatively assessed. The present example involves a 3-phase behavioral paradigm (sensory preconditioning), in which CNO was administered to attenuate neural activity in the RSC during the Preconditioning sessions to test the hypothesis that the RSC is necessary for the formation of associations among neutral stimuli12. Importantly, experi...

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and ...

The goal of this procedure is to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and memory tasks. This is accomplished by first loading the a a V that contains a designer receptor and a reporter into a syringe for infusion. The second step is to infuse a a V into the region of interest in the rat brain.Next, the biologically inert designer drug clozapine and oxide is prepared and injected systemically into the rat. The final step is to conduct behavioral testing using a sensory preconditioning paradigm. Ultimately, immunofluorescence microscopy on the rat brain tissue is used to verify placement of the designer receptor.This method can help answer key questions in the field of behavioral neuroscience, including but not limited to regulation of motivation, attention, and learning and memory Ensure that all institutional precautions for working with adeno-associated viruses are followed throughout the virus used in this video is classified as a biosafety level one compound. Prepare a 10%bleach solution. The decontaminating a a v waste place, a bega containing bleach solution and a sterile 10 microliter syringe.In the

a-method-for-remotely-silencing-neural-activity-rodents-during

Research

JoVE Journal

Behavior

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

Place an anesthetized neonatal pup on a heating pad.Disinfect the scalp with ethanol.Fill a syringe with a viral solution containing adeno-associated viruses carrying the gene for a drug-activated inhibitory receptor and a tracking dye.Align the syringe tip with the pup’s head and slowly inject the virus into the brain for even distribution.Allow the pup to recover.During incubation, viral particles enter neurons and release their genetic material into the nucleus.It leads to inhibitory...

Chemogenetic Silencing of Neuronal Activity in a Neonatal Mouse

Source: Lai, J., et al. Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain. J. Vis. Exp. (2015)This video demonstrates a microinjection-based technique for simultaneous neuronal recording and inhibition in the rat superior colliculus. The procedure involves stereotaxic targeting, skull drilling, and the insertion of an injectrode, which delivers an inhibitory neurotransmitter while recording neuronal activity. Light stimulation evokes...

Inhibition of Neuronal Activity and Its Detection Using Electrophysiology in a Rat

A fully automated protocol for rodent operant conditioning is proposed. The protocol relies on precise temporal control of behavioral events to investigate the extent to which this control influences neural activity underlying sensorimotor integration and cognitive control experiments.

A Fully Automated Rodent Conditioning Protocol for Sensorimotor Integration and Cognitive Control Experiments

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Shuttle-box avoidance learning is well-established in behavioral neuroscience. This protocol describes how shuttle-box learning in rodents can be combined with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical in vivo recordings as a tool to study multiple aspects of learning and perception.

Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

The design and assembly of microdrives for in vivo electrophysiological recordings of brain signals from the mouse is described. By attaching microelectrode bundles to sturdy driveable carriers, these techniques allow for long-term and stable neural recordings. The lightweight design allows for unrestricted behavioral performance by the animal following drive implantation.

Construction of Microdrive Arrays for Chronic Neural Recordings in Awake Behaving Mice

Learn to silence neural activity in specific brain regions during learning tasks using pharmacogenetics in rats. This method combines designer receptors with sensory preconditioning to study memory mechanisms.

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

Explore More Videos