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 condition that is thought to be critical when considering the effects of caloric restriction on drug seeking5.
In the protocol we describe here, the rats are housed in the operant conditioning chambers through the drug self-administration period (24 hr/day). It might be possible to increase the efficiency of data collection by using a &#34;squads&#34; protocol in which the rats are transferred back to the ACF after a single daily training session, allowing for multiple groups to be trained every day. However, this approach might be more suitable for cocaine training, since heroin is preferably administered in the &#34;home&#34; environment16. We have not assessed the efficiency of the procedure using the squads option. Another way to increase the throughput of the procedure is by staggering the drug self-administration training for two groups of rats by 10-12 days. Using this design, rats in the second group are trained for self-administration while the first group goes through the abstinence period in the ACF. Drug self-administration training for the second group must be concluded at least a day before the planned test for the first group to allow for thorough cleaning of the chambers.
We have reported before15 that the length of the restriction period is critical, with 3-5 days of food restriction failing to augment heroin seeking at the end of the abstinence period. We have not observed an increase in the efficacy of the food restriction manipulation with increased exposure periods (~28 days; unpublished data).
It is also worth noting that the change in context between the self-administration phase and the abstinence period seems to be important for the augmentation of drug seeking. We have collected data that suggest a much-reduced effect for food restriction when the rats are maintained in the drug-training environment during the abstinence period (unpublished observation).
In conclusion, the food restriction-induced augmentation of heroin seeking is a robust, easily replicable effect. It therefore allows for efficient designs of studies on the neural mechanisms that underlie relapse following abstinence, using pharmacological pretreatments, online monitoring of neurochemical or neuronal activity using microdialysis or electrophysiological techniques, and post-test histological analyses.

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 border="0" cellpadding="0" cellspacing="0" width="840">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="69">
			<td height="69" style="height:69px;width:177px;">Name</td>
			<td style="width:411px;">Company</td>
			<td style="width:252px;">Catalogue Number</td>
		</tr>
		<tr height="103">
			<td height="103" style="height:103px;width:177px;">Ketamine (Vetalar, 100 mg/ml)</td>
			<td style="width:411px;">Bioniche Animal Health</td>
			<td style="width:252px;">Obtained through local distributer</td>
		</tr>
		<tr height="103">
			<td height="103" style="height:103px;width:177px;">Xylazine (Rompun, 100 mg/ml)</td>
			<td style="width:411px;">Bayer</td>
			<td style="width:252px;">Obtained through local distributer</td>
		</tr>
		<tr height="111">
			<td height="111" style="height:111px;width:177px;">Heroin HCl</td>
			<td style="width:411px;">National Institute for Drug Abuse, Research Triangle Park, NC, USA</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="137">
			<td height="137" style="height:137px;width:177px;">Anafen injection 1 mg/ml Vial/20 ml</td>
			<td style="width:411px;">CDMV, Canada</td>
			<td align="right" style="width:252px;">12868</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:177px;">Name</td>
			<td style="width:411px;">Company</td>
			<td style="width:252px;">Catalogue Number</td>
		</tr>
		<tr height="137">
			<td height="137" style="height:137px;width:177px;">Silastic tubing (ID 0.02, OD 0.037)</td>
			<td style="width:411px;">Fisher Scientific</td>
			<td style="width:252px;">1118915A</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:177px;">GE marine silicon</td>
			<td style="width:411px;">GE</td>
			<td style="width:252px;">SE-1134</td>
		</tr>
		<tr height="137">
			<td height="137" style="height:137px;width:177px;">Tygon tubing (ID 0.02, OD 0.060)</td>
			<td style="width:411px;">VWR</td>
			<td style="width:252px;">63018-044</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:177px;">Cannulae (22 G, 5-up)</td>
			<td style="width:411px;">Plastics One</td>
			<td style="width:252px;">C313G-5up</td>
		</tr>
		<tr height="103">
			<td height="103" style="height:103px;width:177px;">Liquid swivels, plastic, 22 G</td>
			<td style="width:411px;">Lomir Biomedical, Inc.</td>
			<td style="width:252px;">RSP1</td>
		</tr>
		<tr height="137">
			<td height="137" style="height:137px;width:177px;">Fixed speed infusion pump (3.3 rpm)</td>
			<td style="width:411px;">Coulbourn Instruments</td>
			<td style="width:252px;">A73-01-3.3</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:177px;">Rat test cage</td>
			<td style="width:411px;">Coulbourn Instruments</td>
			<td style="width:252px;">H10-11R-TC</td>
		</tr>
		<tr height="103">
			<td height="103" style="height:103px;width:177px;">Stainless steal grid floor</td>
			<td style="width:411px;">Coulbourn Instruments</td>
			<td style="width:252px;">H10-11R-TC-SF</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:177px;">House light-rat</td>
			<td style="width:411px;">Coulbourn Instruments</td>
			<td style="width:252px;">H11-01R</td>
		</tr>
		<tr height="103">
			<td height="103" style="height:103px;width:177px;">Single high-bright cue-rat</td>
			<td style="width:411px;">Coulbourn Instruments</td>
			<td style="width:252px;">H11-03R</td>
		</tr>
		<tr height="103">
			<td height="103" style="height:103px;width:177px;">Tone module 2.9 kHz</td>
			<td style="width:411px;">Coulbourn Instruments</td>
			<td style="width:252px;">H12-02R-2.9</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:177px;">Retractable lever-rat</td>
			<td style="width:411px;">Coulbourn Instruments</td>
			<td style="width:252px;">H23-17RA</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:177px;">Balance arm</td>
			<td style="width:411px;">Coulbourn Instruments</td>
			<td style="width:252px;">H29-01</td>
		</tr>
		<tr height="103">
			<td height="103" style="height:103px;width:177px;">Sound attenuation boxes</td>
			<td style="width:411px;">Concordia University</td>
			<td style="width:252px;">Home made</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:177px;">System controller 2</td>
			<td style="width:411px;">Coulbourn Instruments</td>
			<td style="width:252px;">SYS CTRL 2</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:177px;">System power base</td>
			<td style="width:411px;">Coulbourn Instruments</td>
			<td style="width:252px;">H01-01</td>
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		<tr height="103">
			<td height="103" style="height:103px;width:177px;">Habitest Universal Linc</td>
			<td style="width:411px;">Coulbourn Instruments</td>
			<td style="width:252px;">H02-08</td>
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		<tr height="137">
			<td height="137" style="height:137px;width:177px;">Environment connection board &#38; Linc cable</td>
			<td style="width:411px;">Coulbourn Instruments</td>
			<td style="width:252px;">H03-04</td>
		</tr>
		<tr height="103">
			<td height="103" style="height:103px;width:177px;">Graphic State Notation 3</td>
			<td style="width:411px;">Coulbourn Instruments</td>
			<td style="width:252px;">GS3</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 and active lever responding increase over training days, whereas inactive lever responding does not (Figure 3). Typically, active lever responding increases throughout training and stabilizes at a higher rate of responding. Inactive lever responding remains minimal throughout training, signifying that the rats have properly discriminated between both levers. Finally, the number infusions taken will increase throughout training and remain stable at a rate lower than those of the active lever responses. This dissociation is possible due to the 20 sec timeout following drug infusions.
Active and Inactive lever responses on test day: Active lever responding for the food restricted rats significantly increases compared to the sated rats (we often observed a 250% difference between the treatment groups, see15). Inactive lever responding is minimal and comparable between the food restricted and sated rats (Figure 4).
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/50751/50751fig1.jpg" width="500px" /> 
Figure 1. A timeline of the experimental procedure. The procedure consists of three phases. Animals are first trained to self-administer a drug in the presence of a cue/tone complex (heroin self-administration phase), then moved to a different context and undergo a one-day drug washout period. Washout day is followed by a prolonged period of abstinence, where rats are given a regimen of food restriction or unlimited access to food. On the 14th day of abstinence, rats are returned to the self-administration environment for a drug-seeking test in the presence of drug-paired cues under extinction conditions (drug-seeking test phase). <a href="https://www.jove.com/files/ftp_upload/50751/50751fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/50751/50751fig2.jpg" width="500px" /> 
Figure 2.&#160;Body weights in the food restricted (FDR) and Sated groups, across experimental days. All rats undergo 10 days of heroin self-administration training in operant training chambers and a 1 day drug washout in the animal care facility. Rats are matched according to body weight, active lever responding and drug infusions before separation into the FDR or Sated group. <a href="https://www.jove.com/files/ftp_upload/50751/50751fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/50751/50751fig3.jpg" width="500px" /> 
Figure 3.&#160;The number of infusions, active, and inactive lever responses made during heroin self-administration training. Heroin (0.13 mg/kg/infusion) is self-administered in three 3 hr sessions, over 10 days, under a FR-1, 20 sec time out schedule of reinforcement. <a href="https://www.jove.com/files/ftp_upload/50751/50751fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/50751/50751fig4.jpg" width="500px" /> 
Figure 4.&#160;The effect of exposure to prolonged food restriction (FDR) on heroin seeking in abstinent rats. The data represents typical active and inactive lever responding on test day. Test day consists of one 1 hr (or 3 hr) test session under extinction conditions, following heroin self- administration training and 14 days of abstinence under FDR or sated conditions. <a href="https://www.jove.com/files/ftp_upload/50751/50751fig4highres.jpg" target="_blank">Click here to view larger image</a>.

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

Nanyang Technological University

Research

JoVE Journal

Behavior

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

A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste reactivity with the behavioral aspects of operant self-administration of drugs. Under very brief general anaesthesia, rats are implanted with an intraoral (IO) cannula that allows delivery of test solutions directly in the oral cavity. Animals are then tested in operant self-administration chambers whereby they can press a lever to receive IO infusions of test solutions. IO self-administration has several advantages over experimental procedures that involve drinking a solution from a spout&#160;or operant responding for solid pellets or solutions delivered in a receptacle. Here, we show that IO self-administration can be employed to study self-administration of high fructose corn syrup (HFCS). Rats were first tested for self-administration on a progressive ratio (PR) schedule, which assesses the maximum amount of operant behavior that will be emitted for different concentrations of HFCS (i.e.&#160;8%, 25%, and 50%). Following this test, rats self-administered these concentrations on a continuous schedule of reinforcement (i.e.&#160;one infusion for each lever press) for 10 consecutive days (1 session/day; each lasting 3 hr), and then they were retested on the PR schedule. On the continuous reinforcement schedule, rats took fewer infusions of higher concentrations, although the lowest concentration of HFCS (8%) maintained more variable self-administration. Furthermore, the PR tests revealed that 8% had lower reinforcing value than 25% and 50%. These results indicate that IO self-administration can be employed to study acquisition and maintenance of responding for sweet solutions. The sensitivity of the operant response to differences in concentration and schedule of reinforcement makes IO self-administration an ideal procedure to investigate the neurobiology of voluntary intake of sweets.

The current study evaluates a novel procedure for assessing the reinforcing effects of palatable solutions in laboratory rats: intraoral self-administration. To this end, operant responding (i.e.&#160;lever pressing) for intraoral infusions of sweet solutions at different concentrations was measured on continuous and progressive ratios schedules of reinforcement.

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

A Treatment Package without Escape Extinction to Address Food Selectivity

Feeding difficulties and feeding disorders are a commonly occurring problem for young children, particularly children with developmental delays including autism. Behavior analytic interventions for the treatment of feeding difficulties oftentimes include escape extinction as a primary component of treatment. The use of escape extinction, while effective, may be problematic as it is also associated with the emergence of challenging behavior (e.g., extinction burst). Such challenging behavior may be an acceptable side effect in treatment cases where feeding problems are severe and chronic (e.g., failure to thrive). However, in more acute cases (e.g., selective eating), the negative side effect may be unwarranted and undesired. More recent research on the behavioral treatment of food selectivity has begun to evaluate treatments for feeding difficulties that do not include escape extinction (e.g., demand fading, behavioral momentum), with some success. However, research to date reveals individual differences in responsiveness to such treatments and no clear preferable treatment has emerged. This manuscript describes a multi-component treatment package that includes shaping, sequential presentation and simultaneous presentation, for the treatment of food selectivity in four young children with developmental delays. This treatment package extends the literature on the behavioral treatment for food selectivity and offers a multi-component treatment protocol that may be clinically applicable across a range of treatment scenarios and settings.

Feeding difficulties are a common problem for children with developmental disorders, including autism, and behavioral interventions often include escape extinction. Recent research has begun to evaluate treatments that do not include escape extinction. This manuscript describes a multicomponent treatment package that does not use escape extinction to treat feeding difficulties.

Feeding difficulties and feeding disorders are a commonly occurring problem for young children, particularly children with developmental delays including ...

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 ...

Feeding Experimentation Device (FED): Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in mice: the Feeding Experimentation Device (FED). FED is an open-source system that was designed to facilitate flexibility in food intake studies. Due to its compact and battery powered design, FED can be placed within standard home cages or other experimental equipment. Food intake measurements can also be synchronized with other equipment in real-time via FED's transistor-transistor logic (TTL) digital output, or in post-acquisition processing as FED timestamps every event with a real-time clock. When in use, a food pellet sits within FED's food well where it is monitored via an infrared beam. When the pellet is removed by the mouse, FED logs the timestamp onto its internal secure digital (SD) card and dispenses another pellet. FED can run for up to 5 days before it is necessary to charge the battery and refill the pellet hopper, minimizing human interference in data collection. Assembly of FED requires minimal engineering background, and off-the-shelf materials and electronics were prioritized in its construction. We also provide scripts for analysis of food intake and meal patterns. Finally, FED is open-source and all design and construction files are online, to facilitate modifications and improvements by other researchers.

Feeding Experimentation Device FED: Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Feeding Experimentation Device (FED) is an open-source device for measuring food intake in mice. FED can also synchronize food intake measurements with other techniques via a real-time digital output. Here, we provide a step-by-step tutorial for the construction, validation, and usage of FED.

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in ...

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

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 ...

Tickling, a Technique for Inducing Positive Affect When Handling Rats

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk for both the animals and the hands of their handlers, decreased animal welfare, and less valid research data. To minimize negative effects on experimental results and human-animal relationships, research animals are often habituated to being handled. However, the methods of habituation are highly variable and often of limited effectiveness. More potently, it is possible for humans to mimic aspects of the animals&#39; playful rough-and-tumble behavior during handling. When applied to laboratory rats in a systematic manner, this playful handling, referred to as tickling, consistently gives rise to positive behavioral responses. This article provides a detailed description of a standardized rat tickling technique. This method can contribute to future investigations into positive affective states in animals, make it easier to handle rats for common husbandry activities such as cage changing or medical/research procedures such as injection, and be implemented as a source of social enrichment. It is concluded that this method can be used to efficiently and practicably reduce rats&#39; fearfulness of humans and improve their welfare, as well as reliably model positive affective states.

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk ...

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 ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...