This work was supported by the Natural Sciences &#38; Engineering Council Discovery Program (US: 298915), the Fonds de Recherche du Quebec - Sant&#233; (CSBN), and the Canada Research Chairs Program (US).

1. Animals
All rats are treated in accordance with the guidelines of the Canadian Council on Animal Care and approval for all the experimental procedures was granted by the Concordia University Animal Research Ethics Committee.
<ol>
	<li>Obtain rats at 275-300 g (males) or 225-250 g (females). House rats in pairs until surgery in standard plastic cages containing corncob bedding and shredded paper.</li>
	<li>Maintain rats on a reversed light-dark cycle (light off at 9:30 AM).</li>
	<li>Make sure food and water are available ad libitum, except during the abstinence period. We use a regular rat chow (20.9% protein, 67.2% carbohydrates and 11.8% fat).</li>
</ol>
2. Catheters and Connectors
<ol>
	<li>Make catheters in bulk and use as needed over a long period of time following autoclave sterilization.</li>
	<li>Cut the Silastic tubing to 12 cm long pieces.
	<ol>
		<li>Use a toothpick, to place a small drop of silicon around the catheter, 3 cm from the tip.</li>
		<li>Let the silicon dry for 24 hr and then autoclave and store the catheters.</li>
	</ol>
	</li>
	<li>Bend the long tube of the &#34;5-up&#34; cannula, that will serve as a connector, in a right angle to the plastic pedestal, then autoclave and store.</li>
	<li>Assemble, aseptically, the catheters on the day of the surgery, by pushing the &#34;long&#34; side of the Silastic tube over the bent metal tube of the &#34;5-up&#34; cannula, about half way to the plastic pedestal.
	<ol>
		<li>Connect the &#34;5-up&#34; tube via Tygon tubing and a blunted 22 G needle to a 1 ml syringe filled with sterile saline.</li>
		<li>Flush saline through the catheter to ensure unobstructed flow.</li>
	</ol>
	</li>
</ol>
3. Intravenous Catheterization Surgery
<ol>
	<li>Allow the rats one week of acclimatization in the animal care facility (ACF) before surgery. Do not perform the surgery on rats with body weights lower than 300-325 g (males) or 250-275 g (females).</li>
	<li>Before surgery weigh and anesthetize the rats with a ketamine/xylazine mixture (90/13 mg/kg; i.p.).
	<ol>
		<li>Shave, and then clean the head and the area on the right shoulder (close to base of the neck) with alcohol and surgical scrub.</li>
		<li>Inject penicillin (450,000 IU/rat, s.c.) to protect against infection, and 2 ml of saline (s.c.) for hydration. Apply tear gel to the rat&#39;s eyes to prevent dryness induced by anesthesia.</li>
	</ol>
	</li>
	<li>Place the rat on its abdomen and make an incision, using a scalpel, at the top of the head, between the eyes, and caudally towards the ears of the rat.</li>
	<li>Place the rat on its back with the forearms taped to the surface. Lift the skin in the incision area, just rostral to the clavicle, with forceps and make a 1 cm longitudinal cut with small scissors.</li>
	<li>Using two straight forceps, separate&#160;the fat and other&#160;tissue&#160;until the external jugular vein is exposed.</li>
	<li>Push the tips of straight forceps under the vein to lift and separate the vein from surrounding tissue. Carefully clean the vein from any adhering tissue and then insert curved forceps under the vein to keep it lifted and slightly stretched.</li>
	<li>Make a small, v-shaped, cut on the upper surface of the vein using small spring scissors.
	<ol>
		<li>Hold the cut open using a vessel dilator, and advance the &#34;short&#34;, 3 cm tip of the catheter into the vein towards the heart until the silicone drop is at the incision point.</li>
		<li>Tie the catheter to the vein with 3 sutures: one as far caudal as possible, one just rostral to the silicone drop and the third one as far rostral as possible.</li>
		<li>Pass saline through the catheter following each suture to ensure that it remains unobstructed and that the sutures are not too tight.</li>
	</ol>
	</li>
	<li>Remove the tape around the arms and place the rat ventral side down.
	<ol>
		<li>Insert a hemostat through the caudal side of the head incision and push to separate the skin from the underlying tissue to create a subcutaneous &#34;pocket&#34;.</li>
		<li>Guide the hemostat under the skin towards the opening on the shoulders where the catheter was inserted.</li>
		<li>Break through the connective tissue using the hemostat, and tightly clamp the catheter at the most rostral point, below the base of the &#34;5-up.&#34;</li>
	</ol>
	</li>
	<li>Detach the &#34;5-up&#34; and thread the long end of the catheter under the skin and out the incision located at the top of the rat&#39;s head.
	<ol>
		<li>Use scissors to cut the catheter below where the hemostat was clamped and reattach the long end of the &#34;5-up.&#34;</li>
		<li>Pull the catheter along the metal shaft of the &#34;5-up&#34; all the way to the plastic pedestal. Pass saline through to ensure free flow.</li>
	</ol>
	</li>
	<li>Place the rat on its back, and suture only the incision near the clavicle. The head incision should remain&#160;open.&#160;</li>
	<li>Place the rat in a stereotaxic apparatus. Pull the skin away at the incision at the top of the head using bulldog clamps and clean and dry the surface of the skull.</li>
	<li>Use a manual drill bit, to drill four to five holes into the skull and insert machine screws into the holes to act as scaffolding for the head-cap.
	<ol>
		<li>Place the &#34;5-up&#34; connector between the screws and tuck the excess length of the catheter into the subcutaneous &#34;pocket&#34; previously made using the hemostat.</li>
	</ol>
	</li>
	<li>Use dental cement to affix the &#34;5-up&#34; to the top of the skull. Remove the rat from the stereotaxic apparatus after the cement is fully dried.</li>
	<li>Give the rat post-operative care, which consists of ketoprofen (analgesic; 5 mg/kg, s.c.) and 2 ml of saline (s.c.). Administer ketoprofen for an additional 2 days following surgery to aid with pain management.</li>
	<li>Beginning 48 hr&#160;following surgery, flush rats daily with a mixture of heparin and gentamicin in sterile saline (7.5 IU + 12 &#181;g; 0.2 - 0.3 ml) throughout the recovery period of ~2 days and through the heroin self-administration phase.</li>
</ol>
4. Behavioral Procedure
The timeline for the procedure of food restriction-induced augmentation of heroin seeking is presented in Figure 1.
<ol>
	<li>Operant conditioning chambers.
	<ol>
		<li>House rats individually in operant conditioning chambers enclosed in sound attenuating wooden compartments equipped with a fan.</li>
		<li>Each chamber consists of a stainless steel metal grid floor, a front and back Plexiglas wall and two metal panel sidewalls. Mount two retractable levers 9 cm above the floor of the right sidewall, and locate a cue-light above each lever. Responding on the drug-paired (active) lever activates an infusion pump while responding on the nondrug-paired (inactive) lever has no programmable consequences.</li>
		<li>Locate a tone module (2.9 kHz) above the active lever, and position a red house-light at the top-center of the left sidewall.</li>
		<li>Connect the drug pump to the catheter via a liquid swivel and Tygon tubing protected with a metal spring. In addition, equip each chamber with a food hopper and a water bottle allowing unrestricted access to food and water.</li>
	</ol>
	</li>
	<li>Habituation day:
	<ol>
		<li>Following recovery from surgery move the rats to the operant training chambers and allow for a 24 hr habituation period.</li>
		<li>Do not connect the rats to the metal spring and do not activate the cue-light/tone complex.</li>
		<li>Make sure that the active lever remains retracted, but ensure the inactive lever is extended to help with the discrimination between the two levers during training.</li>
	</ol>
	</li>
	<li>Heroin self-administration:
	<ol>
		<li>Connect the 5-up connector to the Tygon tube and attach the metal spring.</li>
		<li>Allow the rats to self-administer heroin (0.1 mg/kg/infusion) for 10 days during three 3 hr sessions separated by 3 hr intervals.</li>
		<li>Begin each session following the onset of the dark phase (around 9:30 AM in our laboratory) with the extension of the active and inactive levers, as well as illumination of the house-light and activation of the cue-light/tone complex for 30 sec.</li>
		<li>Responses on the active lever result in activation of the drug pump (5 sec, 0.13 ml / infusion) and initiation of a 20 sec timeout period during which the house-light is turned off and the cue light/tone complex above the active lever is activated (FR-1, 20 sec timeout). During the timeout period, active lever responses are recorded but not reinforced. Inactive lever responses are recorded but have no programmable consequences.</li>
		<li>Following each 3 hr session, the active lever is retracted whereas the inactive lever remains extended until 1 hr before the first session of the following day.</li>
	</ol>
	</li>
	<li>Abstinence and food restriction:
	<ol>
		<li>Following self-administration training, remove the rats from the operant conditioning chambers and individually house them in the ACF, and give unrestricted access to food and water for one drug-washout day.</li>
		<li>Divide the rats into two groups: food restricted (FDR) or sated, matched according to body weight, number of infusions and active lever responses across the last 5 days of training.</li>
		<li>Following the drug-washout day, remove the FDR rats&#39; food, and feed them approximately 15 g of rat chow at 1:30 PM. Adjust the amount of food across 14 days of abstinence to maintain the FDR rats&#39; body weight (BW) to approximately 90% of their own baseline BW.</li>
	</ol>
	</li>
	<li>Drug-seeking test: On the morning of abstinence day 14, return the rats to the operant conditioning chambers and attach them to the metal spring. Use a 1 hr or 3 hr session during which active and inactive lever responses have the same consequences as in self-administration training, excluding the availability of the drug (i.e.&#160;under extinction conditions).</li>
</ol>

<ol>
	<li>Jaffe, J. H., Gilman, A. G., Rall, T. W., Nies, A. S., Taylor, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+addiction+and+drug+abuse.">Drug addiction and drug abuse.</a> Goodman &amp; Gilman's the pharmacological basis of therapeutics. , 522-573 (1990).</li><li>O'Brien, C. P., McLellan, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myths+about+the+treatment+of+addiction.">Myths about the treatment of addiction.</a> Lancet. 347, 237-240 (1996).</li><li>Childress, A. R., Ehrman, R., Rohsenow, D. J., Robbins, S. J., O'Brien, C. P., Lowinson, J. W., Luiz, P., Millman, R. B., Langard, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classically+conditioned+factors+in+drug+dependence.">Classically conditioned factors in drug dependence.</a> Substance abuse: A comprehensive textbook. , 56-69 (1992).</li><li>Sinha, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+does+stress+increase+risk+of+drug+abuse+and+relapse?.">How does stress increase risk of drug abuse and relapse?.</a> Psychopharmacology. 158, 343-359 (2001).</li><li>Cheskin, L. J., Hess, J. M., Henningfield, J., Gorelick, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calorie+restriction+increases+cigarette+use+in+adult+smokers.">Calorie restriction increases cigarette use in adult smokers.</a> Psychopharmacology. 179, 430-436 (2005).</li><li>Krahn, D., Kurth, C., Demitrack, M., Drewnowski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+of+dieting+severity+and+bulimic+behaviors+to+alcohol+and+other+drug+use+in+young+women.">The relationship of dieting severity and bulimic behaviors to alcohol and other drug use in young women.</a> J. Subst. Abuse. 4, 341-353 (1992).</li><li>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=Reinstatement+of+cocaine-reinforced+responding+in+the+rat.">Reinstatement of cocaine-reinforced responding in the rat.</a> Psychopharmacology. 75, 134-143 (1981).</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, 3-20 (2003).</li><li>Shalev, U., Highfield, D., Yap, J., Shaham, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+and+relapse+to+drug+seeking+in+rats:+studies+on+the+generality+of+the+effect.">Stress and relapse to drug seeking in rats: studies on the generality of the effect.</a> Psychopharmacology. 150, 337-346 (2000).</li><li>Tobin, S., Newman, A. H., Quinn, T., Shalev, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+dopamine+D1-like+receptors+in+acute+food+deprivation-induced+reinstatement+of+heroin+seeking+in+rats.">A role for dopamine D1-like receptors in acute food deprivation-induced reinstatement of heroin seeking in rats.</a> Int. J. Neuropsychopharmacol. 12, 217-226 (2009).</li><li>Katz, J. L., Higgins, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+validity+of+the+reinstatement+model+of+craving+and+relapse+to+drug+use.">The validity of the reinstatement model of craving and relapse to drug use.</a> Psychopharmacology. 168, 21-30 (2003).</li><li>Epstein, D. H., Preston, K. L., Stewart, J., Shaham, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+model+of+drug+relapse:+an+assessment+of+the+validity+of+the+reinstatement+procedure.">Toward a model of drug relapse: an assessment of the validity of the reinstatement procedure.</a> Psychopharmacology. 189, 1-16 (2006).</li><li>Zacny, J. P., de Wit, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+a+restricted+feeding+regimen+on+cigarette+smoking+in+humans.">The effects of a restricted feeding regimen on cigarette smoking in humans.</a> Addict. Behav. 17, 149-157 (1992).</li><li>Fuchs, R. A., Lasseter, H. C., Ramirez, D. R., Xie, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relapse+to+drug+seeking+following+prolonged+abstinence:+the+role+of+environmental+stimuli.+Drug+Discov.+Today+Dis.">Relapse to drug seeking following prolonged abstinence: the role of environmental stimuli. Drug Discov. Today Dis.</a> Models. 5, 251-258 (2008).</li><li>D'Cunha, T. M., Sedki, F., Macri, J., Casola, C., Shalev, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+chronic+food+restriction+on+cue-induced+heroin+seeking+in+abstinent+male+rats.">The effects of chronic food restriction on cue-induced heroin seeking in abstinent male rats.</a> Psychopharmacology. 225, 241-250 (2013).</li><li>Caprioli, D., Celentano, M., Dubla, A., Lucantonio, F., Nencini, P., Badiani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambience+and+drug+choice:+cocaine-+and+heroin-taking+as+a+function+of+environmental+context+in+humans+and+rats.">Ambience and drug choice: cocaine- and heroin-taking as a function of environmental context in humans and rats.</a> Biol. Psychiatry. 65, 893-899 (2009).</li></ol>

Approval for all the experimental procedures was granted by the Concordia University Animal Research Ethics Committee, in accordance with the guidelines of the Canadian Council on Animal Care. The authors declare that they have no competing financial interests. FS and TD equally contributed to the development of the procedure presented here and to the writing of the manuscript.

The procedure described here has strong face validity because 1) it allows for voluntary drug intake and control over drug administration, 2) it uses a route of administration that is common in human addicts, 3) it reflects (relatively) prolonged periods of drug exposure and abstinence, and 4) it avoids the extinction training before the drug-seeking test that is integral to the reinstatement procedure but is rarely observed in human addicts14. In addition, the procedure models a prolonged food restriction con...

One of the major difficulties in the treatment of drug addiction is the high rate of relapse, even after prolonged periods of abstinence1,2. Studies in human addicts have identified a critical contribution of environmental challenges, such as the exposure to drug-associated discrete cues or environments3 and stressful life events4, to relapse. A common environmental challenge that was found to have an effect on drug taking and craving is chronic food restriction. For example, it has been reported that chronic caloric restriction is associated with increased cigarette smoking5.&#160;Furthermore,&#160;dieting severity (caloric restriction) is positively correlated with an increased prevalence of drug use in young women6. However, the underlying brain mechanisms are not fully understood.
Due to practical and ethical considerations related to human research, animal models have been developed to identify the relevant neural mechanisms. We have previously used the reinstatement procedure7,8 to demonstrate resumption of extinguished drug seeking in rats that are exposed to acute (24-48 hr) food deprivation9,10. However, an integral part of the reinstatement procedure is an extinction period that is not a common feature of drug addiction treatments11 (but see Epstein et al.12 for a comprehensive discussion on the validity of the reinstatement procedure). Moreover, in the particular case of dietary manipulations in humans, only chronic food restriction, but not acute food deprivation, was shown to effectively increase drug-taking5,13.
Consequently, we have suggested an &#34;abstinence procedure&#34;14 that allows the study of the effects of chronic food restriction on drug seeking. Using this procedure we have recently demonstrated a robust augmentation of heroin seeking in abstinent rats following exposure to 14 days of mild food restriction15.

<table><tbody><tr><td>Name</td><td>Company</td><td>Catalogue Number</td><td></td></tr><tr><td>Ketamine (Vetalar, 100 mg/ml)</td><td>Bioniche Animal Health</td><td>Obtained through local distributer</td><td></td></tr><tr><td>Xylazine (Rompun, 100 mg/ml)</td><td>Bayer</td><td>Obtained through local distributer</td><td></td></tr><tr><td>Heroin HCl</td><td>National Institute for Drug Abuse, Research Triangle Park, NC, USA</td><td></td><td></td></tr><tr><td>Anafen injection 1 mg/ml Vial/20 ml</td><td>CDMV, Canada</td><td>12868</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Name</td><td>Company</td><td>Catalogue Number</td><td></td></tr><tr><td>Silastic tubing (ID 0.02, OD 0.037)</td><td>Fisher Scientific</td><td>1118915A</td><td></td></tr><tr><td>GE marine silicon</td><td>GE</td><td>SE-1134</td><td></td></tr><tr><td>Tygon tubing (ID 0.02, OD 0.060)</td><td>VWR</td><td>63018-044</td><td></td></tr><tr><td>Cannulae (22 G, 5-up)</td><td>Plastics One</td><td>C313G-5up</td><td></td></tr><tr><td>Liquid swivels, plastic, 22 G</td><td>Lomir Biomedical, Inc.</td><td>RSP1</td><td></td></tr><tr><td>Fixed speed infusion pump (3.3 rpm)</td><td>Coulbourn Instruments</td><td>A73-01-3.3</td><td></td></tr><tr><td>Rat test cage</td><td>Coulbourn Instruments</td><td>H10-11R-TC</td><td></td></tr><tr><td>Stainless steal grid floor</td><td>Coulbourn Instruments</td><td>H10-11R-TC-SF</td><td></td></tr><tr><td>House light-rat</td><td>Coulbourn Instruments</td><td>H11-01R</td><td></td></tr><tr><td>Single high-bright cue-rat</td><td>Coulbourn Instruments</td><td>H11-03R</td><td></td></tr><tr><td>Tone module 2.9 kHz</td><td>Coulbourn Instruments</td><td>H12-02R-2.9</td><td></td></tr><tr><td>Retractable lever-rat</td><td>Coulbourn Instruments</td><td>H23-17RA</td><td></td></tr><tr><td>Balance arm</td><td>Coulbourn Instruments</td><td>H29-01</td><td></td></tr><tr><td>Sound attenuation boxes</td><td>Concordia University</td><td>Home made</td><td></td></tr><tr><td>System controller 2</td><td>Coulbourn Instruments</td><td>SYS CTRL 2</td><td></td></tr><tr><td>System power base</td><td>Coulbourn Instruments</td><td>H01-01</td><td></td></tr><tr><td>Habitest Universal Linc</td><td>Coulbourn Instruments</td><td>H02-08</td><td></td></tr><tr><td>Environment connection board &amp;amp; Linc cable</td><td>Coulbourn Instruments</td><td>H03-04</td><td></td></tr><tr><td>Graphic State Notation 3</td><td>Coulbourn Instruments</td><td>GS3</td><td></td></tr></tbody></table>

a procedure to study the effect of prolonged food restriction on heroin seeking in abstinent rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

Body weights over time: Typical results demonstrate that body weights remain stable throughout heroin self-administration training. During abstinence, rats with unrestricted access to chow increase their body weight, while those that are food restricted decrease their body weight to approximately 90% of their own original body weight or 75-80% of the sated rats&#39; body weight (Figure 2).
Infusions, active and inactive lever responses over training: Infusions an...

Watch this Scientific Journal Video about A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats at JoVE.com

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

a-procedure-to-study-effect-prolonged-food-restriction-on-heroin

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11.0K Views.  Concordia University. A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

Video: A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

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 ...

Neuroscience

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods ...

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.

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual ...

A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with drug-taking. This model has been implemented in experiments using different types of substances of abuse such as methamphetamine, cocaine, and alcohol. However, punishment-induced abstinence in heroin-trained animals has not been demonstrated. Furthermore, acute stress is a key trigger for relapse in humans and animal models. It was previously demonstrated that acute food deprivation robustly induced reinstatement of extinguished cocaine and heroin seeking. The procedure described here can be used to assess the effects of acute stress exposure on heroin seeking after punishment-imposed abstinence. A total of 8 rats were implanted with chronic intravenous (i.v.) catheters and trained to self-administer heroin (0.1 mg/kg/infusion) for 18 days under a seek-take chained schedule. Completing the seek link gave access to the take lever, which was paired with a heroin infusion. The seek lever was programmed with a variable interval 60 schedule of reinforcement (VI60), and the take lever was programmed with a fixed-ratio 1 reinforcement schedule (FR1). Following self-administration training, a mild foot shock was delivered on 30% of the completed seek links instead of the extension of the take lever. Footshock intensity was increased by 0.1 mA per daily session from 0.2 mA to 1.0 mA. Heroin-seeking tests were performed after 24 h of food deprivation (FD) or sated conditions. Rats under acute food deprivation condition robustly increased heroin seeking after punishment-imposed abstinence.

A procedure that demonstrates a robust acute food deprivation-induced relapse to heroin seeking after punishment-imposed abstinence is described. A punishment-imposed abstinence model was successfully implemented using the seek and take chain schedule for heroin self-administration. Heroin-seeking tests are then performed following 24 h of food-deprivation stress.

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with ...

Investigating Binge Eating Disorder with a Palatable Food Access Mouse Model

Intermittent Binge-Intake Model in Mice

Obesity affects one in eight individuals globally. Overeating, especially high-calorie foods, plays a significant role in obesity. Binge eating disorder (BED) is a complex condition caused by a combination of biological, psychological, and social factors. It involves excessive consumption of high-calorie foods in a short period and is often linked to anxiety and cravings.
Here, we present a protocol employing continuous and intermittent access to a new highly palatable food (HPF) -- M&amp;M&#39;s -- to develop a mouse model for investigating binge eating disorder (BED). The HPF, chosen for its high fat and sugar content, represents an optimal food source to study due to its strong palatability, which makes it particularly suited for examining compulsive eating behaviors. Using C57BL/6 mice, we provided continuous or intermittent access to M&amp;M&#39;s while allowing unrestricted access to standard chow and water. By the 8th day, the mice in the intermittent access group exhibited pronounced binge-eating behaviors, which persisted through the 26th day. These mice also consumed significantly more calories -- predominantly from the confectionary -- than those in the continuous access and control groups. The contrast between continuous and intermittent access underscores the critical role of feeding schedules in promoting the overconsumption of palatable foods.
Furthermore, behavioral assessments revealed intermittent access to the HPF-induced anxiety-like behaviors, highlighting the psychological impact of access patterns on both eating behavior and emotional states. By incorporating these two distinct feeding paradigms, this study offers valuable insights into how the availability of highly palatable foods can exacerbate binge-eating tendencies. This model provides a more realistic approach to studying binge-eating behaviors and their metabolic consequences. It highlights the importance of standardized models in biomedical research. Our findings offer insights into the physiological and neural mechanisms underlying BED, which could pave the way for developing effective therapeutic interventions for BED and obesity.

Author Spotlight: Accessible M&amp;M-Based Mouse Model for Investigating Binge Eating Disorder - Insights into Eating Behaviors, Anxiety, and Neural Mechanisms

A binge-eating model was established in mice using intermittent access to M&amp;M's (a highly palatable food made of fat and sugar). Mice with intermittent access to this high-fat, high-sugar food showed stress-like behaviors, increased caloric intake, and a preference for that food over standard chow compared to mice with continuous or no access to the food.

Obesity affects one in eight individuals globally. Overeating, especially high-calorie foods, plays a significant role in obesity. Binge eating disorder ...

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

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

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

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

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

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Fat Preference: A Novel Model of Eating Behavior in Rats

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

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

A General Method for Evaluating Incubation of Sucrose Craving in Rats

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

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

A Conflict Model of Reward-seeking Behavior in Male Rats

A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence

Intermittent Binge-Intake Model in Mice

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