The authors wish to acknowledge support from USPHS grants K01DA031745 (MMT), R01DA042029 (MMT), DA035805 (YHH), F31DA039646 (MTR), T32031111 (MTR), and the Pennsylvania Department of Health.

The experiments described in this protocol were consistent with the guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Pittsburgh&#39;s Institutional Animal Care and Use Committee. All procedures were performed using adult, na&#239;ve Sprague-Dawley rats that weighed 275-325 g upon arrival.
1. Construction of Optic Fiber Implants and Patch Cables
<ol>
	<li>Prepare optic fi...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+treatments+for+cocaine+toxicity:+A+difficult+transition+to+the+bedside.">Experimental treatments for cocaine toxicity: A difficult transition to the bedside.</a> Journal of Pharmacology and Experimental Therapeutics. 347 (2), 251-257 (2013).</li><li>Makani, R., Pradhan, B., Shah, U., Parikh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+repetitive+transcranial+magnetic+stimulation+(rTMS)+in+treatment+of+addiction+and+related+disorders:+A+systematic+review.">Role of repetitive transcranial magnetic stimulation (rTMS) in treatment of addiction and related disorders: A systematic review.</a> Current Drug Abuse Reviews. 10 (1), 31-43 (2017).</li><li>Shaham, Y., Shalev, U., Lu, L., De Wit, H., Stewart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reinstatement+model+of+drug+relapse:+History,+methodology+and+major+findings.">The reinstatement model of drug relapse: History, methodology and major findings.</a> Psychopharmacology. 168 (1-2), 3-20 (2003).</li><li>Rich, M. T., Huang, Y. H., Torregrossa, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+at+Thalamo-amygdala+Synapses+Regulates+Cocaine-Cue+Memory+Formation+and+Extinction.">Plasticity at Thalamo-amygdala Synapses Regulates Cocaine-Cue Memory Formation and Extinction.</a> Cell Reports. 26 (4), 1010-1020 (2019).</li><li>Rich, M. T., Huang, Y. H., Torregrossa, M. 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As described above, there are several critical steps that are important for achieving the proper experimental results. The protocol will likely only be effective in animals that properly acquire cocaine self-administration, and to date, it has only been tested using the parameters outlined above. It is possible that cocaine dose, schedule of reinforcement, and cue parameters can be modified with likely little effect on behavioral outcomes, with the exception that a second-order schedule of reinforcement may lead to amygd...

Substance abuse is a very serious public health issue in the U.S. and worldwide. Despite decades of intense research, there are very few effective therapeutic options1,2. A major setback to treatment is the fact that chronic drug use generates long-term associative memories between environmental cues and the drug itself. Re-exposure to drug-related cues drives physiological and behavioral responses that motivate continued drug use and relapse3. A novel therapeutic strategy is to enact memory-based treatments that aim to manipulate the circuits involved in regulating drug-cue associations. Recently, it was observed that synapses in the lateral amygdala (LA), specifically those arising from the medial geniculate nucleus (MGN) of the thalamus, are strengthened by repeated cue-associated cocaine self-administration, and that this potentiation can support cocaine seeking behavior4,5. Therefore, it was proposed that cue-induced reinstatement could be attenuated by reversing plasticity at MGN-LA synapses.
The ability to precisely target the synaptic plasticity of a specific brain circuit has been a major challenge to the field. Traditional pharmacological tools have had some success in decreasing relapse behaviors, but are limited by the inability to manipulate individual synapses. However, the recent development of in vivo optogenetics has provided the tools needed to overcome these limitations and control neural pathways with temporal and spatial precision6,7,8. By expressing light-sensitive opsins in a specific brain circuit, laser light can then be used to activate or inhibit the circuit. Frequency-dependent optical stimulation can be utilized to specifically manipulate the synaptic plasticity of the circuit in a behaving animal.
This manuscript outlines the procedure taken to manipulate the behaviorally-relevant MGN-LA circuit using in vivo optogenetics. First, the excitatory opsin oChIEF was expressed in the MGN and optical fibers were bilaterally implanted in the LA. Animals were then trained to self-administer cocaine in a cue-dependent fashion, which potentiates the MGN-LA pathway. Next, sustained, low frequency stimulation with 473 nm laser light was used to produce circuit-specific LTD. Reversing the plasticity induced by cocaine use generated a long-lasting reduction in the capacity of cues to trigger actions that are associated with drug seeking behavior.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% Saline</td>
			<td>Fisher Scientific</td>
			<td>NC0291799</td>
			<td></td>
		</tr>
		<tr>
			<td>A.M.P.I. Stimulus Isolator</td>
			<td>Iso-Flex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AAV5.hSyn.oChIEF.tdTomato</td>
			<td>Duke Viral Vector Core (via Roger Tsien)</td>
			<td>#268</td>
			<td>See Lin et al., 2009; Nabavi et al., 2014</td>
		</tr>
		<tr>
			<td>AAV5.hSyn.tdTomato (Control)</td>
			<td>Duke Viral Vector Core Control</td>
			<td></td>
			<td>See Lin et al., 2009; Nabavi et al., 2014</td>
		</tr>
		<tr>
			<td>Artificial Tears (Opthalmic Ointment)</td>
			<td>Covetrus</td>
			<td>70349</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP Magnesium Salt</td>
			<td>Fisher Scientific</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Butler Schein</td>
			<td>38250</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Cesium chloride</td>
			<td>Fisher Scientific</td>
			<td>289329</td>
			<td></td>
		</tr>
		<tr>
			<td>Cesium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>516988</td>
			<td></td>
		</tr>
		<tr>
			<td>Cesium methanesulfonate</td>
			<td>Fisher Scientific</td>
			<td>C1426</td>
			<td></td>
		</tr>
		<tr>
			<td>Cocaine HCl</td>
			<td>NIDA Drug Supply Center</td>
			<td>9041-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
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			<td>D-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher Scientific</td>
			<td>BP231-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-Channel Temperature Controller</td>
			<td>Warner Instruments</td>
			<td>TC-344C</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Fisher Scientific</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>University of Pittsburgh Chemistry Stockroom</td>
			<td>200C5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferrule Dust Caps</td>
			<td>Thor Labs</td>
			<td>CAPL</td>
			<td>White plastic dust caps for 1.25 mm Ferrules</td>
		</tr>
		<tr>
			<td>Ferrule Mating Sleeves</td>
			<td>Doric Lenses</td>
			<td>F210-3011</td>
			<td>Sleeve_BR_1.25, Bronze, 1.25 mm ID</td>
		</tr>
		<tr>
			<td>Ferrules</td>
			<td>Precision Fiber Products</td>
			<td>MM-FER2007C-2300</td>
			<td>&#216;1.25 mm Multimode LC/PC Ceramic ferrule, &#216;230 &#956;m hole size</td>
		</tr>
		<tr>
			<td>Fiber Optic</td>
			<td>Thor Labs</td>
			<td>FP200URT</td>
			<td>200 &#956;m core multimode fiber (0.5 NA)</td>
		</tr>
		<tr>
			<td>Fiber Optic Rotary Joint</td>
			<td>Prizmatix</td>
			<td>(Ordered from Amazon)</td>
			<td>18 mm diameter, FC-FC connector for fiber</td>
		</tr>
		<tr>
			<td>Fiber Stripping Tool</td>
			<td>Thor Labs</td>
			<td>T12S21</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>F6057</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Henry Schein</td>
			<td>6913</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP Sodium Salt</td>
			<td>Fisher Scientific</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>80085</td>
			<td>10 &#956;L volume, 26 gauge, 2 inch, point style 3</td>
		</tr>
		<tr>
			<td>Heat Gun</td>
			<td>Allied Electronics</td>
			<td>972-6966</td>
			<td>250 V, 750-800 &#176;F</td>
		</tr>
		<tr>
			<td>Heat-Curable Epoxy</td>
			<td>Precision Fiber Products</td>
			<td>PFP-353ND-8OZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Henry Schein</td>
			<td>55737</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher Scientific</td>
			<td>219405490</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein</td>
			<td>29405</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine HCl</td>
			<td>Henry Schein</td>
			<td>55853</td>
			<td>Ketamine is a controlled substance and should be handled according to institutional guidelines</td>
		</tr>
		<tr>
			<td>Lactated Ringer&#8217;s</td>
			<td>Henry Schein</td>
			<td>9846</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser, driver, and laser-to-fiber coupler</td>
			<td>OEM Laser Systems</td>
			<td>BL-473-00100-CWM-SD-xx-LED-0</td>
			<td>100 mW, 473-nm, diode-pumped solid-state laser (One option)</td>
		</tr>
		<tr>
			<td>L-glutathione</td>
			<td>Fisher Scientific</td>
			<td>G4251</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>Butler Schein</td>
			<td>14583</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Sensor</td>
			<td>Thor Labs</td>
			<td>PM100D</td>
			<td>Compact energy meter console with digital display</td>
		</tr>
		<tr>
			<td>Loctite instant adhesive</td>
			<td>Grainger</td>
			<td>5E207</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>203726</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Amplifier/Data Acquisition</td>
			<td>Molecular Devices</td>
			<td>MULTICLAMP700B / Digidata 1440A</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4501</td>
			<td>Dual syringe</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MPC-200/ROE-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>Upright microscope for electrophysiology</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX61VS</td>
			<td>Epifluorescent slide-scanning microscope</td>
		</tr>
		<tr>
			<td>N-methyl-D-glucamine</td>
			<td>Sigma-Aldrich</td>
			<td>M2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Orthojet dental cement, liquid</td>
			<td>Lang Dental</td>
			<td>1504BLK</td>
			<td>black</td>
		</tr>
		<tr>
			<td>Orthojet dental cement, powder</td>
			<td>Lang Dental</td>
			<td>1530BLK</td>
			<td>Contemporary powder, black</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch Cables</td>
			<td>Thor Labs</td>
			<td>FP200ERT</td>
			<td>Multimode, FT030 Tubing</td>
		</tr>
		<tr>
			<td>Picrotoxin</td>
			<td>Fisher Scientific</td>
			<td>AC131210010</td>
			<td></td>
		</tr>
		<tr>
			<td>Polishing Disc</td>
			<td>Thor Labs</td>
			<td>D50FC</td>
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			<td>Fisher Scientific</td>
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			<td>Sigma-Aldrich</td>
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			<td>Henry Schein</td>
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			<td>Fisher Scientific</td>
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			<td>Sigma-Aldrich</td>
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using optogenetics to reverse neuroplasticity and inhibit cocaine seeking in rats

This protocol demonstrates the steps needed to use optogenetic tools to reverse cocaine-induced plasticity at thalamo-amygdala circuits to reduce subsequent cocaine seeking behaviors in the rat. In our research, we had found that when rats self-administer intravenous cocaine paired with an audiovisual cue, synapses formed at inputs from the medial geniculate nucleus of the thalamus (MGN) onto principal neurons of the lateral amygdala (LA) become stronger as the cue-cocaine association is learned. We hypothesized that reversal of the cocaine-induced plasticity at these synapses would reduce cue-motivated cocaine seeking behavior. In order to accomplish this type of neuromodulation in vivo, we wanted to induce synaptic long-term depression (LTD), which decreases the strength of MGN-LA synapses. To this end, we used optogenetics, which allows neuromodulation of brain circuits using light. The excitatory opsin oChiEF was expressed on presynaptic MGN terminals in the LA by infusing an AAV containing oChiEF into the MGN. Optical fibers were then implanted in the LA and 473 nm laser light was pulsed at a frequency of 1 Hz for 15 minutes to induce LTD and reverse cocaine induced plasticity. This manipulation produces a long-lasting reduction in the ability of cues associated with cocaine to induce drug seeking actions.

A timeline outlining the order of experiments is shown in Figure 1. Throughout behavioral experiments, the number of cocaine infusions as well as the number of responses made on the active lever serves as a measure of the intensity of cocaine-seeking behavior. During the initial days of cocaine self-administration, the number of active responses should gradually increase across each acquisition day, before stabilizing during the second week. Conversely, inactive lever responses should remain...

Watch this Scientific Journal Video about Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats at JoVE.com

Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats

The methods described here outline a procedure used to optogenetically reverse cocaine-induced plasticity in a behaviorally-relevant circuit in rats. Sustained low-frequency optical stimulation of thalamo-amygdala synapses induces long-term depression (LTD). In vivo optogenetically-induced LTD in cocaine-experienced rats resulted in the subsequent attenuation of cue-motivated drug seeking.

This protocol demonstrates the steps needed to use optogenetic tools to reverse cocaine-induced plasticity at thalamo-amygdala circuits to reduce ...

This protocol is significant because it uses novel advances in optogenetic techniques and a preclinical model of substance abuse to reverse plasticity in neural circuits that are responsible for promoting relapse. The main advantage of this technique is that it permits circuit specific manipulation of synaptic activity in an awake animal, which has long lasting inhibitory effects on future cue motivated cocaine seeking behavior. This method could be utilized across many other systems and circuits to specifically manipulate behaviorally relevant neuroplasticity.Start preparing for the surgery by connecting a 26 gauge stainless steel injection cannula to a Hamilton syringe filled with one microliter of concentrated AAV solution. After anesthetizing the animal, shave the surgical area and lubricate the eyes to prevent drying as described in the text manuscript. Next, inject into the rat a body weight volume of the analgesic carprofen subcutaneously through the skin of the upper back, and inject five milliliters of lactated Ringer's solution subcutaneously through the skin of the lower back.Then perform the intravenous catheter implantation. Immediately following catheter implantation, secure rat in a stereotaxic frame to perform adeno-associated viral injections. Inject a bolus of lidocaine subcutaneously through the skin above the skull in order to locally anesthetize the area, and then make a 0.5 millimeter incision from the front to the rear of the skull.Remove overlying tissue to expose the surface of the skull. Level the rat's head along the anterior posterior axis and zero stereotaxic coordinates to bregma, then use a dremel tool equipped with a small drill bit to drill three small holes through the skull, and mount stainless steel screws in place with a screwdriver. For the injection of AAV, drill bilateral holes based on the coordinates from the Rat Brain Atlas"for the medial geniculate nucleus or MGN.The coordinates used relative to bregma are minus 5.4 millimeters on the anterior posterior axis, plus three millimeters on the medial lateral axis, and minus 6.6 millimeters on the dorsal ventral axis. Slowly lower the injection cannula at approximately four millimeters per minute, until cannula are properly positioned in the MGN, and inject concentrated AAV solution at a rate of 0.1 microliters per minute. After infusions are complete, leave injection cannula in place for five minutes to allow for diffusion of the virus away from the cannula and then slowly withdraw cannula from the brain.Next, proceed to the implantation of optic fibers targeting medial geniculate nucleus lateral amygdala terminals. Use a dremel tool to drill bilateral holes above the lateral amygdala, at coordinates as explained in the manuscript. Use forceps to grasp the ferrule of the optic fiber implant and secure the ferrule to the stereotaxic adapters in place.Slowly lower fibers at a rate of two millimeters per minute until the tip of the fiber sits in the dorsal portion of the LA.Secure ferrules to the skin, first using a thin layer of Loctite instant adhesive, followed by dental cement. Once dental cement has sufficiently dried, cover ferrules with ferrule sleeves and dust covers. Following surgical procedures, house the rat individually and flush catheters daily with saline containing five milligrams per milliliter of gentamicin and 30 USP units per milliliter of heparin.24 hours prior to the start of behavioral experiments, food restrict rats to 90%of their free feeding weight. Place the rat in an operant chamber to undergo daily one hour training sessions for self-administration of two milligrams per milliliter of cocaine, under a fixed ratio one schedule of reinforcement. In the chamber, allow the rat to lever press.A press on the designated active lever results in a cocaine infusion at one milligram per kilogram dose, and a ten second presentation of a compound light and tone cue. A press on the designated inactive lever has no programmed effects. Continue the self-administration experiments for at least 10 days, until the rat successfully earns at least eight infusions per day across three consecutive days.Failure to reach acquisition criteria by day 20, results in exclusion from the study. After acquisition criteria are successfully met, subject the rat to one hour instrumental extinction sessions for six to 10 days by allowing rats to freely lever press in operant chambers. Continue instrumental extinction daily until an average of maximum 25 lever presses over two consecutive days occurs.For optogenetic induction of long-term depression or LTD, connect patch cables to a 473 nanometer blue laser diode via a rotary joint suspended above a clean, standard rodent housing cage. Turn on the laser according to operating instructions and connect the laser to a pulse generator. Adjust settings to give the rat the 900 pulses of light at two microsecond, at one hertz.Measure the light output through the patch cord using a light sensor, and adjust the laser intensity so that the light output through the patch cable is approximately five to seven milliwatts. Place the rat in the clean housing cage and remove dust covers and ferrule sleeves exposing the ferrules. Then connect the patch cords bilaterally to each ferrule.If set up properly, rodents will be able to move freely around the cage during optogenetic stimulation. After allowing the rat to explore the environment for three minutes, turn on the pulse generator to initiate optogenetic stimulation. Following long-term depression induction, allow rat to remain in the cage for three minutes, before placing it back in the home cage.24 hours after in vivo optogenetic stimulation, place the rat back into the operant conditioning chambers to undergo a one hour standard cue induced reinstatement session to assess cocaine seeking behavior. A press on the active lever results in a ten second presentation of the light and tone cue. At least one week after the first test, give a second reinstatement test to determine if optogenetic LTD induction results in a long-term suppression of cocaine seeking.In the representative analysis, acquisition and extinction of cocaine self-administration are shown. Cocaine infusions and active lever responses increased across acquisition, with few inactive lever responses. During cocaine self-administration, the number of active responses gradually increased across each acquisition day before stabilizing during the second week.On day one of the extinction, and initial boost in lever pressing was observed, followed by a decrease in responding on both levers. Following instrumental extinction, reintroduction of cues reinstated cocaine seeking represented by an increase in the number of active lever responses. Optical LTD caused a significant reduction in active lever presses relative to controls.Seven days later, rats were tested again. Animals that previously underwent MGN-LA LTD had significantly reduced active lever pressing compared to controls, revealing that optical LTD produced a long-term decrease in cocaine seeking. Ex vivo electrophysiological recordings showed a decrease in the amplitude of optically evoked excitatory postsynaptic current in LA neurons, following optical LTD.Additionally, the rise slope of excitatory postsynaptic potentials was reduced by ex vivo optical stimulation in the neurons, from animals that had sham stimulation, but not in neurons from animals that had in vivo optical LTD. The placement of optic fiber implants and viral expression were verified histologically in the lateral amygdala and MGN. To successfully reduce cocaine seeking behavior, it is critical to achieve proper light output through the optic fiber implants, and to use sustained, low-frequency stimulation to promote long-term depression.Following this procedure, neural recording techniques could be used to determine changes in neural activity, and other behavioral tests of cocaine seeking or other learned behaviors could be conducted.

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Neuroscience

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has great importance for distal muscle control in humans and, to a lesser extent, in rodents. A unilateral cut of one pyramid results in loss of CST innervation of the spinal cord mainly on the contralateral side of the spinal cord leading to transient motor disability in the forelimbs and sustained loss of dexterity. Ipsilateral projections of the corticospinal tract are minor. We have refined our surgical method to increase the chances of lesion completeness. We describe postsurgical care. Deficits on the Montoya staircase pellet reaching test and the horizontal ladder test shown here are detected up to 8 weeks postinjury. Deficits on the cylinder rearing test are only detected transiently. Therefore, the cylinder test may only be suitable for detection of short term recovery. We show how, electrophysiologically and anatomically, one may assess lesions and plastic changes. We also describe how to analyse fibers from the uninjured CST sprouting across the midline into the deprived areas. It is challenging to obtain &#62;90% complete lesions consistently due to the proximity to the basilar artery in the medulla oblongata and survival rates can be low. Alternative surgical approaches and behavioural testing are described in this protocol. The pyramidotomy model is a good tool for assessing neuroplasticity-inducing treatments, which increase sprouting of intact fibers after injury.

The corticospinal tract, one of the major sensorimotor tracts, can be lesioned unilaterally in the rodent brainstem in order to test neuroplasticity-inducing therapies for the central nervous system. This surgical procedure (&#8220;pyramidotomy&#8221;) and postoperative assessments are described in this protocol.

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

In this report, the advantages of organotypic cultures and dissociated primary cultures of mouse-derived dorsal root ganglia are highlighted to investigate a wide range of mechanisms associated with neuron-glial interaction, neuroplasticity, neuroinflammation, and response to viral infection.

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...