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 ...

<ol>
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Reprod. 16 (11), 2333-2337 (2001).</li><li>Donsante, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV+vector+integration+sites+in+mouse+hepatocellular+carcinoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observed+incidence+of+tumorigenesis+in+long-term+rodent+studies+of+rAAV+vectors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+expression+of+Pctk1,+Tcf12+and+Ccnd+in+hippocampus+of+rats:+impact+on+cognitive+function,+synaptic+plasticity+and.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+Academy+Press.+Guide+for+the+Care+and+Use+of+Laboratory+Animals.">National Academy Press. Guide for the Care and Use of Laboratory Animals.</a> National Academy Press. , (1996).</li><li>Lowery, R. L., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+injection+of+adeno-associated+viral+vectors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+in+the+peripheral+nervous+system+of+systemically+administered+dimethylsulfoxide+in+the+rat:+a+neurophysiological+and+pathological.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mediodorsal+thalamus+hypofunction+impairs+flexible+goal-directed+behavior.">Mediodorsal thalamus hypofunction impairs flexible goal-directed behavior.</a> Biol. Psychiatry. 77 (5), 445-453 (2014).</li><li>wiki, D. R. E. A. D. 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Neurosci. 33 (28), 11668-11676 (2013).</li><li>Cassatarro, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+pharmacogenetic+modulation+of+the+nucleus+accumbens+reduces+ethanol+consumption+in+a+limited+access+paradigm.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+versus+delayed+stimulation+of+feeding+by+the+endogenously+released+AgRP+neuron+mediators.">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. 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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 border="0" cellpadding="0" cellspacing="0" >
	
	
	<tbody>
		<tr >
			<td >Male, Long Evans Rats, 55-60 d</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:&#160; This is a BSL-1 compound</td>
		</tr>
		<tr >
			<td >rAAV8/hSyn-GFP</td>
			<td >Virus Vector Core</td>
			<td ></td>
			<td>Caution:&#160; This is a BSL-1 compound</td>
		</tr>
		<tr >
			<td >Clozapine-N-Oxide</td>
			<td >R&#38;D Systems</td>
			<td >4936-10</td>
			<td>Designer Drug</td>
		</tr>
		<tr >
			<td >Rat Cage lid (Polycarbonate)</td>
			<td >Alternative Design&#160;</td>
			<td >FT 8XL-PC</td>
			<td>Used to cover animal cages 48-72 hours post infusion</td>
		</tr>
		<tr >
			<td >Filer Paper (Replacement)</td>
			<td >Alternative Design&#160;</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 >Medline Biohazard liners&#160;</td>
			<td >Staples</td>
			<td >113444</td>
			<td>Biohazard Trash Bags</td>
		</tr>
		<tr >
			<td >United Solutions 34 gallon rectangular wheeled trashcan with hook and lock handle&#160;</td>
			<td >Amazon.com</td>
			<td ></td>
			<td>Biohazard Trash Can</td>
		</tr>
		<tr >
			<td >Isoflurane, 100 mL</td>
			<td >Patterson Veterinary Supply Inc.&#160;</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&#160;</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&#160; 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&#160;</td>
			<td >Microscope</td>
			<td >AD7013MTL</td>
			<td>An alternative to the traditional disection scope</td>
		</tr>
		<tr >
			<td >Dino-lite Rigid TableTop Boom Stand</td>
			<td >Microscope</td>
			<td >MS36B</td>
			<td>Surgical equipment</td>
		</tr>
		<tr >
			<td >28 Gauge 10uL Syringe</td>
			<td >Hamilton&#160;</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&#39;Wx22&#34;Hx16&#34;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&#160;</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&#160;</td>
			<td >Med Associates, Inc</td>
			<td >ENV-203M-45</td>
			<td>Behavioral equipment</td>
		</tr>
		<tr >
			<td >Pellet Recetacle, 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>
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			<td >Large Table Top Cabinet and Power Supply</td>
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			<td >PCI Interface Package</td>
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			<td >MED Intel core Computer Pkg with X Pro 19&#34; Monitor</td>
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			<td >Paraformaldehyde (grannular), 1 kg</td>
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			<td>Hazard:&#160; carcinogen, weigh in hood</td>
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			<td >Rabbit Monoclonal antibody (HA-Tag)</td>
			<td >Cell Signaling Technologies&#160;</td>
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			<td >XP Rabbit monoclonal antibody (GFP)</td>
			<td >Cell Signaling Technologies&#160;</td>
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			<td >Anti-Rabbit IgG</td>
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			<td >Superfrost Plus slides&#160;</td>
			<td >VWR international</td>
			<td >483111-703</td>
			<td>Histology supplies</td>
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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...

Watch this Scientific Journal Video about A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning at JoVE.com

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 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 ...

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

Research

JoVE Journal

Behavior

14.5K ビュー.  Oberlin College. 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.