Name: a protocol for measuring cue reactivity in a rat model of cocaine use disorder
Uploaded: 2018-06-18T12:30:00
Description: Watch this Scientific Journal Video about A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder at JoVE.com

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...

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 ...

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