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 thr...

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>Equipment</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&#34;x12&#34;, 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&#8482; 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 1.52mmOD 0.51mm thick x 152.4m</td>
			<td>Fisher Scientific, Hampton, NH, USA</td>
			<td>14-170-15B</td>
			<td>1/experiment</td>
		</tr>
		<tr>
			<td>Chemicals</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&#174;, 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 &#946;-hemolytic Streptococcus (Lancefield Group C) &#8805;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></td>
			<td></td>
			<td></td>
			<td>*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 ...

In this protocol, cue reactivity measures an animal's motivation to seek cocaine following absence from cocaine self-administration. Cue reactivity occurs when the rat is re-exposed to the context and cues previously associated with intravenous cocaine delivery. Cue reactivity is a measure for relapse risk.The cue reactivity protocol described here is a pre-clinical means to test relapse modifying pharmacotherapies as well as investigate genetic and environmental factors that contribute to relapse risk. This is an operant task for drug-seeking behavior that aligns with many properties of human drug taking and relapse. Optimization of key parameters such as cocaine dose, delivery volume, and infusion duration have been made to establish phase validity and translational productive power.For this protocol, use male Sprague Dawley rats that are roughly eight to nine weeks old which have been acclimated to the colony room for at least seven days. During the acclimation period, house the rats in pairs and handle them daily throughout the study. Provide unlimited access to food in their home cages throughout all phases of the study.Implant jugular vein catheters using the method outlined by Kmiotek and collaborators in their JoVE video, then randomly assign the rats to saline or cocaine treatment groups. For training, program schedules for cocaine self-administration as described in the text protocol. Equip the training chambers with two retractable response levers, a stimulus light above each response lever, and a house light opposite the levers.Deliver cocaine or saline from syringe infusion pumps outside of the cubicle. Use 23 gauge needles attached to the syringes and polyethylene tubing encased in a metal spring leash attached to the rat's catheters. Cocaine or saline is delivered after an active lever press, but not after inactive lever press.Change the solutions daily. Use freshly made cocaine dilutions in sterile 0.9%sodium chloride tailored to each rat's weight. Load at least eight milliliters of solution into each rat's syringe.Begin with training the rats on an FR1 schedule of reinforcement and progress to an FR5 schedule after three straight days of seven infusions per hour with less than 10%variability in the total number of infusions per session. It is imperative to analyze self-administration behavior data on a daily basis to determine when to advance an animal to FR5 and to check for loss of catheter patency. One of the most critical steps of the protocol is to monitor self-administration during the first 14 days of training to ensure the animal self-administer normally.If self-administration dramatically decreases, the rat's catheter has likely lost patency. At the start of a session, check that there are no bubbles in the syringe. Attach the syringe to the polyethylene tubing and insert it into the infusion pump.Carefully adjust the syringe so that the solution is evenly and completely distributed in the polyethylene tubing. Next, connect the rats to the delivery system and place them into their respective standard operant conditioning chambers housed in ventilated sound-attenuating cubicles with fans. Consistency throughout the experiment is vital.Always place a rat into the same chamber and keep the active lever on the same side within that chamber. Over 14 daily 180-minute sessions, the rats have free access to the levers and will readily learn which lever provides stimulation provided the system is working. Each trigger of the active lever on a fixed ratio schedule of one releases 0.1 milliliters of solution over six seconds and simultaneously illuminates the cage light and the stimulus light above the active lever.There is a one-second delay before solution is released. Following each cocaine or saline delivery, the stimulus light and infusion pump are deactivated for 20 seconds during which the house light remains on and pressing the lever does nothing but is recorded. When the light goes off, the lever is reactivated.Any triggering of the inactive lever produces no scheduled consequences. At the end of that 180-minute session, use slow careful movements to remove the potentially aggressive rat from the chamber. Then flush its catheter.Between sessions, carefully clean the chambers. Wipe down every surface with a 70%ethanol solution. At the end of the training sessions, put the rats into forced abstinence for 30 days with no access to the operant chamber.Continue to handle the rats on a daily basis and monitor the general health. After the long abstinence period, allow the rats 60 minutes in the operant chamber immediately followed by brain harvesting. For this session, run the cue reactivity scheduled program.Stagger the session start times to allow time to harvest brains between sessions. Importantly, operate the pump without the syringe attached so the auditory cue of the pump is still a part of the stimulus without the substance delivery. Begin this session as usual with the rat in the self-administration chamber in which it was trained.In this test, when the active lever that previously delivered cocaine or saline is pressed, only the cues are delivered on an FR1 schedule. The inactive lever still does nothing. Record all the cue presentations, previously active lever presses and inactive lever presses as well as the latency to first lever press.Using the described protocol, a study was conducted and rats were individually transitioned from FR1 to FR5 as they met criteria. In the cocaine-administering group, rats gradually increased number of infusions until they reached criterion, then they were transitioned from FR1 to FR5. Multiple output measures were gathered from the cocaine cue reactivity test.For example, rats which previously self-administered cocaine pressed the previously active lever much more than rats which self-administered saline. Another important output measure is latency to first level press. When a change in this is observed, the animal may be physically stressed or have decreased motivation for cocaine self-administration.Locomotor behavior tasks can be used to assess changes in physical health and differentiate motor impairment from motivational changes. After watching this video, you should have a good understanding of how to train rats to self-administer a drug, maintain catheter patency during self-administration, and assess cue reactivity to drug-associated cues following abstinence as a measure of relapse risk. While attempting this procedure, it's important to remember to carefully program the software, properly handle the animals, and regularly flush intravenous catheters.Following this procedure, one can assess the cellular and molecular mechanisms that drive cocaine cue reactivity as well as test potential pharmacotherapuetics that modify cocaine cue reactivity.

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JoVE Journal

Neuroscience

10.5K Views.  University of Texas Medical Branch. In this protocol, cue reactivity measures an animal's motivation to seek cocaine following absence from cocaine self-administration. Cue reactivity occurs when the rat is re-exposed to the context and cues previously associated with intravenous cocaine delivery. Cue reactivity is a measure for relapse risk.The cue reactivity protocol described here is a pre-clinical means to test relapse modifying pharmacotherapies as well as investigate genetic and environmental factors that contribut...