All behavioral testing was performed in the University of Texas Medical Branch (UTMB) Rodent In Vivo Assessment (RIVA) Core, directed by Dr. Kelly Dineley and housed within the Center for Addiction Research, directed by Dr. Kathryn Cunningham. Support for this work came from the Peter F. McManus Charitable Trust, National Institute of Environmental Health Sciences Center for Environmental Toxicology at UTMB (T32ES007254), Institute for Translational Sciences at UTMB (UL1TR001439), Mitchell Center for Neurodegenerative Diseases, and Center for Addiction Research at UTMB (DA007287, DA070087, and pilot study funds).

All animal manipulations are carried out in accordance with the Guide for the Care and Use of Laboratory Animals (2011) and with approval from the Institutional Animal Care and Use Committee.
1. Animals
<ol>
	<li>Acclimate male Sprague Dawley rats approximately 8-9 weeks of age (250-260 g) for a minimum of seven days in a colony room maintained at 21-23 &#176;C and 45-50% humidity on a 12 h light-dark cycle (lights on 6:00-18:00 h).</li>
	<li>House rats two/cage and handle daily throughout the study. Provide food and water ad libitum in the home cage throughout all phases of the study. 
	NOTE: Food restriction increases the saliency of the cocaine reward48, potentiates cocaine-induced locomotor activity49, and acts as a stressor for reinstatement50 and thus may also impact cue reactivity.</li>
	<li>Randomly assign rats to saline or cocaine treatment groups.</li>
</ol>
2. Surgery
<ol>
	<li>Place rat under anesthesia using a cocktail containing 8.6 mg/kg of xylazine, 1.5 mg/kg of acepromazine, and 43 mg/kg of ketamine suspended in bacteriostatic saline. Confirm that the rat is sufficiently anesthetized by pinching its toe and monitoring motor reflexes and respiration. Apply ophthalmic ointment directly to eyes to prevent eye dryness. 
	NOTE: If the rat is not sufficiently anesthetized it will exhibit a motor response to the toe pinch, and the rat&#39;s breathing rate may rapidly increase.
	<ol>
		<li>Using aseptic technique while the rat is under anesthesia, implant an intravenous catheter into the jugular vein connected to a back mount with a cannula. After surgical implantation, allow rats to recover for 5-7 days 28,36,37,51. 
		NOTE: The intravenous route of cocaine self-administration in humans results in rapid increases in brain cocaine levels52,53. Thus, in one of its most common preclinical variants, rats are trained to self-administer cocaine via an intra-jugular catheter.</li>
	</ol>
	</li>
	<li>Maintain catheter patency with daily flushes using a solution of 0.1 mL of bacteriostatic saline containing heparin sodium (10 U/mL), streptokinase (0.67 mg/mL), and ticarcillin disodium (66.67 mg/mL). Begin catheter flushing the day after surgery and continue each day following self-administration sessions.
	<ol>
		<li>Fill a syringe with the solution and connect the back mount to the syringe via polyethylene tubing to flush.</li>
		<li>Verify proper catheter function periodically throughout the experiment by intravenous administration of 10 mg/kg of methohexital sodium. 
		NOTE: This is a dose sufficient to briefly anesthetize the animal only when administered intravenously.</li>
	</ol>
	</li>
</ol>
3. Self-Administration
<ol>
	<li>Use software programming to generate 4 separate programs as described below.
	<ol>
		<li>Code two programs for self-administration on a fixed ratio (FR) 1 schedule, i.e., where one active lever press results in one infusion. For program #1, code the left lever as the active lever; for program #2, code the right lever as the active lever. Construct both programs to be 180-min in length, and deliver the cocaine stimulus and discrete cue complex in the following manner:
		<ol>
			<li>Have one active lever press cause simultaneous illumination of both the house light and the stimulus light above the active lever.</li>
			<li>After a 1-s delay, have the infusion pump deliver 0.1 mL of solution over 6-s.</li>
			<li>Have the stimulus light shut off after 7-s (inactivating at the same time as the infusion pump).</li>
			<li>Have the house light shut off after 27-s, with the last 20-s indicating a time-out period during which active lever presses have no scheduled consequences, yet these are still recorded.</li>
			<li>For data output, collect infusions, active lever presses, inactive lever presses, and latency to first active lever press.</li>
		</ol>
		</li>
		<li>Code two more programs for self-administration on a FR5 schedule, i.e., where five lever presses result in one infusion. Again, make one program with the active lever as left, and one program with the active lever as right. Make both programs 180-min in length, and deliver the reinforcer and discrete cue complex in the following manner:
		<ol>
			<li>Have five active lever presses cause simultaneous illumination of both the house light and the stimulus light above the active lever.</li>
			<li>Repeat Steps 3.1.1.2 through 3.1.1.5.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Self-Administration Training
	<ol>
		<li>Prepare the cocaine solution daily in sterile 0.9% NaCl to a dose of 0.75 mg/kg/0.1 mL. 
		NOTE: Over the duration of each 180 min self-administration session, rats administer approximately 4-5 mL of cocaine solution, thus to prepare in excess, anticipate approximately 8 mL/rat daily.</li>
		<li>Fill the catheter syringe (10 mL) with at least 8 mL of cocaine or saline solution. After ensuring that the cocaine/saline solution is flowing, insert the syringes into the infusion pump and connect to the polyethylene tubing encased by a metal spring leash at the start of each self-administration session. Carefully adjust the syringe in each infusion pump such that the solution is evenly and completed distributed in the polyethylene tubing prior to attaching to each catheter.</li>
		<li>Place rats into respective standard operant conditioning chambers housed in ventilated, sound-attenuating cubicles with fans. Consistently place each rat in the same operant chamber, and keep the active lever on the same side throughout all experiments. Counterbalance assignment of rats between left and right active levers. 
		NOTE: Each chamber is equipped with a pellet receptacle flanked by two retractable response levers, a stimulus light above each response lever, and a house light opposite the levers. Cocaine/saline syringes are connected to a 23g needle (filed to avoid poking tubing) attached to polyethylene tubing encased inside a metal spring leash which connects to each rat&#39;s catheter and is operated on a liquid swivel. Syringes are placed into infusion pumps located adjacent to the cubicle.</li>
		<li>Train rats to lever press for cocaine infusions (0.75 mg/kg/0.1 mL infusion) or saline infusions (0.1 mL) during daily 180-min sessions using established methods28,36,37,51. 
		NOTE: This training is performed simply by placing the rat into the operant chamber and allowing it to lever press; pressing the active lever will result in receipt of the discrete cue complex and cocaine infusion, while pressing the inactive lever bears no scheduled consequences.
		<ol>
			<li>Schedule completions on the active lever result in delivery of a cocaine or saline infusion over a 6-s period paired simultaneously with illumination of the house light and stimulus light above the active lever and activation of the infusion pump (this is the discrete cue complex paired with cocaine delivery); inactive lever presses produce no scheduled consequences. Following reinforcer (cocaine) delivery, the stimulus light as well as the infusion pump are inactivated; the house light remains on for an additional 20-s to indicate a timeout period during which lever presses have no scheduled consequences. 
			NOTE: A description of how to set this up is found in Steps 3.1.1.1-3.1.1.4</li>
		</ol>
		</li>
		<li>Upon completion of the 180-min session, remove each rat from its operant conditioning chamber and flush its catheter (Step 2.2). Carefully clean the chambers between sessions, wiping each surface with a 70% ethanol solution. 
		NOTE: Rats that receive cocaine during the self-administration session may be difficult to remove from the chamber and are more likely to be aggressive. Use slow, careful&#160;motions to disconnect each rat from its tether.</li>
		<li>Train rats on a FR1 schedule of reinforcement and progress to a FR5 schedule after achieving a criterion of seven infusions/h with less than 10% variability in the total number of infusions per session for three consecutive days. Continue self-administration sessions until rats have reached a total of 14 days of self-administration training. 
		NOTE: This is one variant of operant learning; some protocols prefer to have rats remain at FR1 throughout the entirety of the experiment. Changes in fixed ratio schedule from FR1 to FR5 do not impact the number of infusions an animal self-administers54,55. However, increasing the fixed ratio size helps to ensure stability of responding in the rat, and may increase the saliency of the reward56. Additionally, if a treatment during forced abstinence is desired, rats should be pseudo-randomly assigned to treatment groups upon achieving stability (less than 10% variability in the total number of infusions per session for a minimum of 3 consecutive days) at the FR5 schedule. Additionally, saline rats will not progress from FR1 to FR5, as the saliency of saline is not sufficient to achieve the stability required to progress from FR1 to FR5.</li>
	</ol>
	</li>
</ol>
4. Forced Abstinence
<ol>
	<li>Place rats into forced abstinence for 30 days; that is, do not provide rats with the opportunity to self-administer cocaine or saline for a duration of 30 days. Importantly, during this period do not return rats to the operant chambers (the previous context of self-administration). However, remove rats from home cages for daily handling and weighing, as a measure of general health. 
	NOTE: The duration of forced abstinence can be altered, but it is important to choose a duration that is sufficient to avoid ceiling and floor effects. Factors to be cognizant of when choosing the duration of the forced abstinence period include rodent strain, age, self-administration session length and duration, drug, drug dose and infusion duration, and the sex of the animal.</li>
</ol>
5. Cocaine Cue Reactivity
<ol>
	<li>Code two software programs for the cue reactivity task at a FR1 schedule; one program with the left lever as the active lever, and one with the right lever as the active lever. Construct each program to collect lever pressing data for 60-min and deliver the discrete cue complex as described below.
	<ol>
		<li>Repeat Steps 3.1.1.1-3.1.1.4</li>
		<li>For data output, collect cue presentations (previously known as infusions), previously-active lever presses, inactive lever presses, and latency to first&#160;lever press.</li>
	</ol>
	</li>
	<li>Cocaine Cue Reactivity Test Session
	<ol>
		<li>Assess cue reactivity on day 30 of forced abstinence in a 60-min operant test session. Stagger start times of rats to facilitate rapid decapitation and brain harvest. Importantly, for the cue reactivity test, do not place the syringe in the infusion pump so that the sound of the infusion pump serves as an auditory cue but no cocaine reinforcement is delivered. 
		NOTE: Cue reactivity can be assessed on days other than day 30 of forced abstinence, however the fact that responsiveness to cocaine-paired cues varies over time34 should be kept in mind when choosing the date for the cue reactivity test.</li>
		<li>Place each rat in the self-administration chamber in which it was trained and tether to its cannula as during the daily self-administration sessions. Presses on the lever that previously delivered cocaine or saline are now only reinforced by delivery of the discrete cue complex (i.e., the stimulus light and house are illuminated, and the infusion pump is activated) on a FR1 schedule (i.e., one discrete cue complex is presented per active lever press); count the cue presentations and previously-active lever presses during one 60-min session, and also record the number of inactive lever presses. However, as with self-administration, inactive lever presses produce no scheduled consequences. 
		NOTE: An additional control option is to record previously-active and inactive lever presses in the absence of scheduled consequences (i.e., no discrete cue complex reinforcer).</li>
		<li>Upon completion of the cue reactivity test session, immediately euthanize rats to capture the biochemical effects of re-exposure to the operant conditioning chamber and discrete cue complex.</li>
	</ol>
	</li>
</ol>
6. Data Collection, Organization, and Analysis
<ol>
	<li>Break down data into three conventional categories: general health, self-administration, and cue reactivity.
	<ol>
		<li>Monitor general health (e.g., animal weight, fur condition) throughout the experiment, and provide animals with veterinary care, if necessary, or remove from the study if lever pressing appears affected by health.</li>
		<li>Collect self-administration data during the 14 days of self-administration. Use an unpaired, two-tailed t-test to determine statistical differences between cocaine and saline self-administration groups for total number of infusions, active lever presses, inactive lever presses, and latency to first active lever press. 
		NOTE: Self-administration data needs to be analyzed prior to forced abstinence if one desires to study&#160;the effect of a treatment during forced abstinence on cue reactivity (so that groups can have an equal average number of infusions, as well as active and inactive lever presses). If a two-by-two design is chosen (cocaine &#177; treatment, saline &#177; treatment), a two-way ANOVA with appropriate post hoc analysis should be used to analyze this data.</li>
		<li>For cue reactivity data to collect and analyze for the total number of cue presentations, previously-active lever presses, inactive lever presses, and latency to first&#160;lever press. Also analyze this data with an unpaired, two-tailed t-test to determine differences between animals self-administering cocaine and those self-administering saline. 
		NOTE: Again, if a two-by-two design is chosen (cocaine &#177; treatment, saline &#177; treatment), a two-way ANOVA with appropriate post hoc analysis should be used to analyze this data.</li>
	</ol>
	</li>
</ol>

The authors have no conflicts of interest to disclose.

Exposure to drug-paired cues and physiological changes in response to these cues16 are associated with relapse,11,16 and the cocaine cue reactivity test employed above contingently presents cocaine-paired cues in the absence of drug; thus, drug-seeking behavior in the form of previously-active lever presses&#160;serves as a measure of relapse vulnerability. The cue reactivity protocol described herein is a preclinical means by which relaps...

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value1. Cue reactivity is the sensitivity to cues previously linked with the drug-taking experience, and it plays a prominent role in human craving2,3,4,5. The risk of progression to CUD, as well as relapse during abstinence, is thought to be higher for individuals who express high sensitivity to drug-associated cues6,7. Both environmental contexts (e.g., people, buildings, music genres) and discrete drug-associated stimuli (e.g., paraphernalia) become associated with the cocaine reward; exposure to these cues can trigger changes in peripheral physiology (e.g., heart rate, skin temperature, and skin resistance), brain plasticity, and brain functional connectivity2,8,9,10. In other words, re-exposure to cocaine-associated cues activates limbic corticostriatal circuits to evoke conditioned physiological and subjective responses that drive appetitive approach (drug-seeking) behavior11,12,13,14,15.
Cue reactivity measured with functional brain imaging analyses is predictive of relapse vulnerability in subjects with CUD16. Cue reactivity measurements in rodent models serve as a surrogate measure for relapse risk and can be exploited for translational studies. Thus, a pharmacotherapy that decreases cue reactivity in rodents may be carried forward as a relapse-prevention treatment in human clinical trials. Preclinical models with the necessary translational merit and predictive validity are especially important since there are currently no FDA-approved pharmacotherapies for CUD17.
The rodent self-administration procedure is the gold standard, translational model with predictive validity for human drug-taking18 and critically important to understanding the molecular and physiological processes underlying CUD. Response-independent delivery of cocaine results in distinct behavioral, molecular, and neurochemical effects relative to response-dependent cocaine exposure; e.g., response-independent cocaine delivery evokes significantly higher mortality19. Furthermore, the neurochemical consequences of abstinence from response-dependent cocaine self-administration are distinct from those triggered by abstinence from response-independent cocaine delivery20,21. Thus, CUD models based upon response-dependent delivery of cocaine are superior translational models when assessing cue reactivity and associated mechanisms of action.
In the protocol outlined below, cocaine is delivered intravenously through an indwelling intra-jugular catheter. However, alternative methods to self-administer drug via oral and inhalation routes have been developed. Importantly, rodents control delivery of the drug, analogous humans, through operant responses. Therefore, there is high concordance between drugs self-administered by rodents and humans22. The preclinical drug self-administration procedure below employs lever pressing, reinforced by drug delivery, to motivate response rates higher than vehicle control. Drug-seeking behavior is trained by pairing originally &#34;neutral&#34; cues (e.g., a stimulus light or tone and the contextual environment in which cocaine self-administration occurs) with cocaine infusion; these cues become conditioned reinforcers (for review:&#160;Cunningham &#38; Anastasio, 201423). Subsequent re-exposure to cocaine-associated cues triggers drug-seeking behavior in rodents (i.e., attempts to deliver cocaine through pressing on the previously-active lever) as well as craving and relapse in CUD subjects24,25,26,27.
Typically, preclinical rodent studies of drug-seeking behavior following cocaine self-administration utilize extinction training and/or drug reinstatement conducted within the drug-associated environment28,29,30,31,32. Presses on the previously-active lever, in the absence of drug and/or cue delivery, typically constitute the measure of reinstatement following extinction33,34,35. On the contrary, cue reactivity drug-seeking behavior is assessed following forced abstinence without prior extinction training28,36,37,38,39.
Outcome measures and experimental variables have been carefully chosen and validated to dissect different aspects of the neurobiology of drug-seeking and relapse-like behavior, and it is well-established that neuroadaptations differ between models with and without extinction training 40,41,42,43. Furthermore, from a translational perspective, rodent extinction training is not mirrored in clinical settings for CUD since drug-related cues include mood states, places, and people44; the unique combination of these cues are likely not available in a clinical environment45,46,47. Thus, the rodent model described herein acts as a better parallel to the human condition than many of the models currently available.
The following describes a validated cocaine self-administration training, forced abstinence and cue reactivity test protocol for rats. Briefly, rats are implanted with intra-jugular catheters, trained to self-administer cocaine or saline via &#39;active&#39; lever press, and receipt of the cocaine or saline stimulus is paired with discrete light and sound cues which serve as conditioned reinforcers. Following 14 days of cocaine self-administration, rats are subjected to 30 days of forced abstinence and a subsequent 60-min cue reactivity test in which lever pressing is measured. The cue reactivity test is a surrogate measure for cocaine relapse vulnerability in humans.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Catheter Tubing: 0.50mm ID x 0.94mm OD x 0.2mm width</td><td>Fisher Scientific, Hampton, NH, USA</td><td>11-189-15A</td><td>1/experiment</td></tr><tr><td>Cue Light</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>ENV-229M</td><td>2/operant chamber</td></tr><tr><td>Guide Cannulae (22 gauge, pedestal size-8mm, cut length 11 mm, 5 mm above the pedestal)</td><td>Plastics One, Roanoke, VA, USA</td><td>8IC313G5UPXC</td><td>1/rat</td></tr><tr><td>House Light</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>ENV-227M</td><td>1/operant chamber</td></tr><tr><td>Infusion Pump</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>PHM-100</td><td>1/operant chamber</td></tr><tr><td>Levers</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>ENV-110M</td><td>2/operant chamber</td></tr><tr><td>Liquid Swivels</td><td>Instech, Plymouth Meeting, PA, USA</td><td>375/22</td><td>1/operant chamber</td></tr><tr><td>MED-PC Package with Infusion Pump Software</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>SOF-735 (infusions software SOF-700RA-10 version 1.04)</td><td>1</td></tr><tr><td>Metal Spring Leash</td><td>Plastics One, Roanoke, VA, USA</td><td>C313CS/SPC</td><td>1/operant chamber</td></tr><tr><td>Needle (23g, 1 in)</td><td>Becton Dickinson, Franklin Lakes, NJ, USA</td><td>305193</td><td>1/operant chamber</td></tr><tr><td>Nitex Mesh (6/6 woven mesh sheet, 12&quot;x12&quot;, 500 microns thick, 38% Open Area)</td><td>Amazon, Seattle, WA, USA</td><td>CMN-0500-C, B000FMUNE6</td><td>~1 sheet/100 rats</td></tr><tr><td>PCI Interface Package</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>DIG-700P2-R2, MED-SYST-16</td><td>1/16 operant chambers</td></tr><tr><td>Power Supply for Interface Modules</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>SG-6510D</td><td>1/16 operant chambers</td></tr><tr><td>Sound-attenuating Cubicle</td><td>Med Associates Inc., St. Albans, VT, USA</td><td>ENV-018V</td><td>1/operant chamber</td></tr><tr><td>Syringes, 10 mL Luer-Lok&amp;trade; tip</td><td>Fisher Scientific, Hampton, NH, USA</td><td>14-827-52</td><td>1 case/experiment (1/operant chamber)</td></tr><tr><td>Tygon Tubing for flushes: 0.51mmID x 1.52mm OD 0.51mm width x 152.4m length</td><td>Fisher Scientific, Hampton, NH, USA</td><td>14-170-15B</td><td>1/experiment</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acepromazine (10mg/mL)</td><td>Henry Schein (Animal Health), Melville, NY, USA</td><td>003845</td><td>~0.5mg/rat*</td></tr><tr><td>Acraweld Repair Resin</td><td>Henry Schein (Dental), Melville, NY, USA</td><td>1013959</td><td>1/experiment</td></tr><tr><td>Altalube (ophthalmic ointment)</td><td>Henry Schein (Dental), Melville, NY, USA</td><td>6050059</td><td>1/experiment</td></tr><tr><td>Cocaine</td><td>NIDA North Bethesda, MD, USA</td><td>N/A</td><td>~350mgs/rat for whole experiment*; requires DEA License</td></tr><tr><td>Heparin (10,000 USP units/10 mL)</td><td>Sagent Pharmaceuticals, Schaumburg, IL, USA</td><td>NDC 25021-400-10</td><td>1/experiment (~21 units/rat*)</td></tr><tr><td>Jet Liquid</td><td>Henry Schein (Dental), Melville, NY, USA</td><td>1256401</td><td>1/experiment</td></tr><tr><td>Ketamine (100mg/mL, 10mL)</td><td>Henry Schein (Dental), Melville, NY, USA</td><td>1049007</td><td>~15mg/rat*; requieres DEA license</td></tr><tr><td>Methohexital Sodium (Brevital&amp;reg;, 500 mg/50 mL)</td><td>Patterson Dental, Saint Paul, MN, USA</td><td>043-5461</td><td>1/experiment; requires DEA License</td></tr><tr><td>Saline (0.9%, USP)</td><td>Baxter, Deerfield, IL, USA</td><td>2B1307</td><td>1 case/experiment</td></tr><tr><td>Streptokinase from &amp;beta;-hemolytic Streptococcus (Lancefield Group C) &amp;ge;3,000 units/mg</td><td>Sigma Aldrich, St. Louis, MO, USA</td><td>S3134-250KU</td><td>1 vial/experiment (~1.5mg/rat/experiment*)</td></tr><tr><td>Ticarcillin Disodium Salt</td><td>Fisher Scientific, Hampton, NH, USA</td><td>50-213-695</td><td>~4 vials/exeriment or purchase the 25g vial cat.# 50-489-093 (~150mg/rat/experiment*)</td></tr><tr><td>Xylazine (100mg/mL)</td><td>Henry Schein (Animal Health), Melville, NY, USA</td><td>033198</td><td>~3mg/rat*</td></tr><tr><td colspan="4">*Assumes rat age is that described in the protocol, rats self-administer for 14 days, and flushes occur for 21 days.</td></tr></tbody></table>

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.

Results of a cocaine self-administration and abstinence experiment followed by a cue reactivity test from a previously published study57 are shown in Figure 1. The study timeline is depicted in Figure 1A.
Rats individually transition from FR1 to FR5 as they meet criteria. As operant conditioning proceeds in the cocaine-administering group, rats g...

Watch this Scientific Journal Video about A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder at JoVE.com

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

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 ...

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Research

JoVE Journal

Neuroscience

10.5K Views.  University of Texas Medical Branch. Cue reactiviteit is geconceptualiseerd als gevoeligheid voor signalen die verband houden met drugsgebruik ervaringen die bijdragen aan verlangen en terugvallen in abstinent mens. Cue reactiviteit is gemodelleerd in ratten door meten attentional oriëntatie richting van drug-geassocieerde signalen, waardoor de werking van de aanpak van de appetitive in een cue reactiviteit test volgend op zelfbestuur en gedwongen onthouding.

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

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

Behavior

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

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.

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

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 ...

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

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 ...

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.

This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...

A Conflict Model of Reward-seeking Behavior in Male Rats

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual stimulus) that represents a high-value reward, and the aversive stimuli (pins), are concurrently presented. The male rats have to climb or jump over the obstacle full of pins to approach and investigate the sexual partner. If the animal persists in their approaching behavior regardless of the aversive stimuli, it is considered as a maladaptive or risky reward-seeking behavior. The conflict task permits the evaluation of deficit in inhibitory control resulting from exposure to abused drug, such as morphine, or a stressful event. 
The main advantage of this model is that it provides a simple and quick way to discover the deficit in inhibitory control after exposure to opiate drugs or other stressful events. In addition to opiates, this behavioral model would also be useful for quickly discovering the inhibitory control deficits induced by other addictive drugs. However, the limitation is that the male rats' performance may be subject to exercising effects with repeated testing under this conflict task. In the future, one can hope that the individuals with the compulsive phenotype of reward-seeking behavior after exposure to opiates will be identified based on modifying this conflict model.

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.

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

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