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>
			<td>P5958</td>
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			<td>Fisher Scientific</td>
			<td>83000</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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			<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.

using-optogenetics-to-reverse-neuroplasticity-inhibit-cocaine-seeking

Research

JoVE Journal

Neuroscience

2.3K visualizações.  University of Pittsburgh. Este protocolo é significativo porque utiliza novos avanços em técnicas optogenéticas e um modelo pré-clínico de abuso de substâncias para reverter a plasticidade em circuitos neurais que são responsáveis por promover a recaída. A principal vantagem desta técnica é que ela permite a manipulação específica do circuito da atividade sináptica em um animal acordado, o que tem efeitos inibitórios duradouros sobre o comportamento de busca de cocaína motivado por pistas futuras. Es...