This work was supported by the Natural Sciences &#38; Engineering Council Discovery Program (US: RGPIN-2016-06694).

All rats are treated according to the guidelines of the Canadian Council on Animal Care. Approval for all the experimental procedures was granted by the Concordia University Animal Research Ethics Committee.
1. Animals
<ol>
	<li>Acquire Long Evans rats weighing 275-300 g (males) or 225-250 g (females). Double house rats in standard plastic cages with corncob bedding and shredded paper until surgery.</li>
	<li>Keep the rats on a reversed light-dark cycle (lights off at 9:30 AM, light on at 9:30 PM).</li>
	<li>Allow unlimited access to food and water, except during the food-deprivation test. Use regular rat chow (20.9% protein, 67.2% carbohydrates, and 11.8% fat).</li>
</ol>
2. Intravenous surgeries
<ol>
	<li>Build catheters and perform intravenous surgery as described in Sedki et al. 201317 with the following differences: 1) Use Isofluorane 2-2.5% for anesthesia, 2) Change the dose of penicillin administered during surgery to 60 000 IU/rat, via subcutaneous injection, and 3) Increase the dose of gentamicin in the mixture of heparin and gentamicin used to flush the catheters to 7.5 IU + 800 &#181;g; 0.2- 0.3 mL.</li>
	<li>Inject ketoprofen or carprofen, an anti-inflammatory analgesic, intraperitoneally or subcutaneously (5 mg/kg) immediately after surgery (before recovery from the anesthesia) and on the following 3 days. During the 2 days recovery period, give mashed food and heat (if necessary) to help with the recovery. Weigh and supervise the animals daily to check for any abrupt loss of weight or any other signs of health issues.</li>
</ol>
3. Behavioral procedure
<ol>
	<li>Operant conditioning chambers:
	<ol>
		<li>Set the operant conditioning chambers as described in Sedki et al. 201317.</li>
	</ol>
	</li>
	<li>Habituation day:
	<ol>
		<li>House the rats in the operant training chambers after recovery from surgery and allow animals to habituate to the chamber for 24 h before initiating any protocol. Keep animals housed in the operant chambers throughout the whole experiment.</li>
		<li>Do not attach the rats to the metal spring and do not initiate any experimental protocol (the cue-light, the tone, and the levers are not available during habituation day).</li>
	</ol>
	</li>
	<li>Heroin self-administration:
	<ol>
		<li>Attach the 5-up connector to the Tygon tube and the metal spring.</li>
		<li>Set up daily training sessions of 6 h of heroin self-administration (0.1 mg/kg/infusion) according to the procedure described below.</li>
		<li>Start daily training sessions on the onset of the dark phase of the reversed light cycle (around 9:30 AM in the representative results) with the extension of the seek lever (or take lever on the first 2 days; see below), as well as turning off the houselight on.</li>
		<li>Self-administration with only the take lever available under fixed-ratio 1 (FR1; 2 days)
		<ol>
			<li>Program each trial to begin with the insertion of the take lever.</li>
			<li>Once the rat presses the take lever one time (FR1), retract the take lever, turn on the cue light above the lever and tone for a 20 s timeout period, turn off the houselight, and allow the delivery of a heroin infusion (0.1 mg/kg in 0.13 mL)</li>
			<li>Program a 30 s inter-trial interval in which no cues are presented, the houselight is turned off, and the lever is retracted. For the beginning of the next trial, insert the take lever, and turn on the houselight.</li>
		</ol>
		</li>
		<li>Self-administration with seek-take chain under FR1 (5 days)
		<ol>
			<li>Program the operant training system to insert only the seek lever and turn on the houselight to signal the beginning of the session. The take lever is in a retracted state.</li>
			<li>Once the rat presses one time (FR1) on the seek lever, the program retracts the seek lever and inserts the take lever.</li>
			<li>One press on the take lever (FR1) retracts the take lever and initiates the same procedure as in step 3.3.4.2 and a 30 s inter-trial interval.</li>
			<li>Program the operant training system to insert the seek lever at the conclusion of the inter-trial interval and turn on the houselight to signal the beginning of the next trial.</li>
			<li>Program the operant training system to initiate the inter-trial interval if a rat fails to accomplish the seek-take chain during 10 min (not pressing the seek or the take lever) to represent the end of a trial and the loss of the opportunity to administer the drug.</li>
		</ol>
		</li>
		<li>Self-administration with seek-take chain under Variable Intervals (VI5, VI30, and VI60)
		<ol>
			<li>Program all steps in section 3.3.5 but introduce a variable interval of 5 (VI5) as the new schedule of reinforcement on the seek lever, instead of FR1. Allow self-administration training under the VI5 schedule for 3 days.</li>
			<li>Set up the software to allow the first press on the seek lever to activate the VI5 schedule. For the VI5 schedule, let the software to randomly select an interval time from a list of 0.1 s, 5 s, and 10 s, which results in an average of 5 s interval. The first seek lever press after the selected interval has elapsed, retracts the seek lever and extends the take lever.</li>
			<li>Follow steps 3.3.4.2, 3.3.5.4., 3.3.5.5.&#160;for take lever and end of trial instructions.</li>
			<li>Next, train rats for 5 days under VI30 schedule for the seek link, followed by 3 days under VI60 schedule.</li>
			<li>For the VI30 schedule, allow the software to randomly select a time from a list of 15 s, 30 s, and 45 s, for an average of 30 s, whereas for the VI60 schedule, the software randomly selects a time from a list of 45 s, 60 s, and 75 s.</li>
			<li>Vary the inter-trial intervals along the different schedules of reinforcement. Start the inter-trial interval at 30 s during FR1 and VI5 but gradually increase to 7 min during VI30 and VI60.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Punishment-imposed abstinence
	<ol>
		<li>Start punishment sessions with insertion of seek lever and turning the houselight on. Allow punishment sessions for 8 days, with regular supervision to guarantee that animals do not show signs of abnormal pain or any health issues.</li>
		<li>Program the operant training system so that after completing the seek link under VI60, the seek lever retracts, the houselight is turned off, and a mild footshock is administered on 30% of the completed seek links instead of insertion of the take lever. The take lever extends on the other 70% of completed seek links, as in a normal self-administration trial.</li>
		<li>Program an inter-trial interval of 7 min with all cues and houselight off before inserting the seek lever and turning the houselight on to signal the beginning of the next trial.</li>
		<li>Set the footshock intensity at 0.2 mA and increase by 0.1 mA per punishment day until 1.0 mA.</li>
		<li>After rats press the take lever (on trials when it is available), repeat step 3.3.4.2 for retraction of the lever, delivery of the heroin infusion, and presentation of cues, followed by a 7 min inter-trial interval.</li>
		<li>Start the next trial with the insertion of the seek lever and turning the houselight on.</li>
		<li>If the rat does not press any lever for 10 min, follow step 3.3.5.5.</li>
	</ol>
	</li>
	<li>Acute food deprivation (24 h)
	<ol>
		<li>On the day following the last day of punishment-imposed abstinence, divide rats into two matched groups (according to body weight and the average number of seek lever and infusions over the last 3 days of self-administration training): Food Deprived (FD) and Sated.</li>
		<li>Remove food hoppers from the chambers (around 9:30 AM) for rats that undergo food deprivation-induced relapse test and keep food hoppers for the sated group that has unlimited access to food. Provide unlimited access to water in both groups and supervise animals to guarantee that they do not show any health problems during the food deprivation period.</li>
	</ol>
	</li>
	<li>Food deprivation-induced heroin seeking tests
	<ol>
		<li>The morning after the 24 h of food deprivation, perform a 3 h heroin-seeking test, under a VI60 schedule with an inter-trial interval of 7 min with no shock or heroin infusions being delivered. Return food hoppers to the food-deprived rats after the 3 h session.</li>
		<li>On the morning after the first heroin seeking test, remove food hoppers from the chambers of the rats that were sated on the first relapse test. Allow the rats that were previously food-deprived unlimited access to food (sated condition) before and during the test.</li>
		<li>Perform another 3 h heroin seeking test on the next day (after 24 h food deprivation), as in step 3.6.1.</li>
		<li>The day after the test euthanize the rats using a carbon dioxide chamber (100% CO2 at fill rate of 30-70% of the chamber volume per minute).</li>
	</ol>
	</li>
</ol>

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A., 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=Challenges+for+translational+psychopharmacology+research--some+basic+principles.">Challenges for translational psychopharmacology research--some basic principles.</a> Psychopharmacology (Berl). 199 (3), 291-301 (2008).</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. 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V., Minier-Toribio, A., Shekara, A., Bossert, J. M., 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=Effect+of+the+dopamine+stabilizer+(-)-OSU6162+on+potentiated+incubation+of+opioid+craving+after+electric+barrier-induced+voluntary+abstinence.">Effect of the dopamine stabilizer (-)-OSU6162 on potentiated incubation of opioid craving after electric barrier-induced voluntary abstinence.</a> Neuropsychopharmacology. 45 (5), 770-779 (2020).</li><li>Pelloux, Y., Everitt, B. J., Dickinson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compulsive+drug+seeking+by+rats+under+punishment:+effects+of+drug+taking+history.">Compulsive drug seeking by rats under punishment: effects of drug taking history.</a> Psychopharmacology (Berl). 194 (1), 127-137 (2007).</li><li>Marchant, N. J., Campbell, E. J., Kaganovsky, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Punishment+of+alcohol-reinforced+responding+in+alcohol+preferring+P+rats+reveals+a+bimodal+population:+Implications+for+models+of+compulsive+drug+seeking.">Punishment of alcohol-reinforced responding in alcohol preferring P rats reveals a bimodal population: Implications for models of compulsive drug seeking.</a> Progress in Neuro-Psychopharmacology &amp; Biological Psychiatry. 87, 68-77 (2018).</li><li>Krasnova, I. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incubation+of+methamphetamine+and+palatable+food+craving+after+punishment-induced+abstinence.">Incubation of methamphetamine and palatable food craving after punishment-induced abstinence.</a> Neuropsychopharmacology. 39 (8), 2008-2016 (2014).</li><li>Panlilio, L. 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The authors declare that they have no competing financial interests.

There are two important demonstrations in this paper. First, the validation of the punishment-imposed abstinence using the seek and take chain with heroin. Second, it was demonstrated that stress-induced relapse could be observed in a punishment-imposed abstinence procedure. These are important demonstrations because (i) The punishment-induced abstinence procedure more closely mimics the human condition as it results in voluntary abstinence, i.e., not due to extinction of drug-seeking or forced removal from the drug-taking environment18, (ii) A reliable relapse was demonstrated using an ecologically relevant stressor: acute food deprivation stress.
When implementing the protocol, care must be given to the proper training of the rats under the seek-take procedure that is more complex compared to the common continuous self-administration using one "active" lever. In addition, the punishment phase should be carefully adjusted to the rat strain used to prevent unnecessary pain and "over-punishment" that could result in a freezing response and would interfere with relapse tests. Note that the analgesic effects of heroin could decrease the response to footshock punishment; however, under the conditions described in this protocol, robust punishment-imposed abstinence from heroin self-administration was found. The conditions of this protocol aimed to separate as much as possible the time between a heroin infusion and the footshock (on punishment trials). To achieve this, the protocol included long inter-trial intervals (7 min) between infusion trials and foot shock trials. 
The bimodal-like distribution found for drug-seeking under food deprivation is not unique when studying relapse. It reflects individual differences that are observed within humans with substance use experience19. These two separate subpopulations could be an advantage for future studies that aim to identify individual differences that predict stress-induced relapse.
In contrast to the training under the more common active/inactive levers procedures, where both drug-seeking and taking are associated with the same active lever, the seek-take chained procedure allows the examination of specific factors related to drug-seeking and drug-taking as two separate sets of actions. Only drug-seeking is punished, thus avoiding possible devaluation of the drug rewarding properties20.
One limitation of this study is the reduced drug intake under the VI60 schedule compared to studies using FR1. In addition, long inter-trial intervals of 7 min also reduce the available time during the session to access the drug. One option to overcome these limitations in the future would be to reduce the inter-trial interval or increase the session length. Another limitation of the model is the pairing physical stress (footshock) to drug-seeking, while human drug users mostly experience psychological stress. Yet, under the probabilistic introduction of the punishment, as set in the procedure, drug-seeking is attenuated by fear of the possible consequences, which can be perceived as a psychological stressor. The conditions in the protocol described here set up a motivational conflict that is experienced at every trial during the punishment phase and are central to the decision process in addiction21.
Relapse to heroin can occur even after a prolonged abstinence period22. Future studies should investigate the effect of exposure to food deprivation stress after longer punishment-imposed abstinence periods. Moreover, this paper was focused on the demonstration of relapse and not the study of individual differences in response to punishment ("punishment-resistant" and "punishment sensitive"23). Footshock intensity was, therefore, increased until 1.0 mA to guarantee that all rats achieved voluntary abstinence. It would be interesting to explore the development of individual differences in heroin-trained rats by keeping the footshock at lower intensities (e.g., 0.4 mA).
Here a relapse procedure is presented in which voluntary abstinence is reached due to negative consequences, and relapse is induced by an ecologically relevant environmental stressor. The food deprivation-induced relapse is robust and easily replicable. The procedure will allow a better understanding of the mechanism underlying relapse to heroin after voluntary abstinence.

Relapse is the most challenging problem in the treatment of drug use1,2. However, only a handful of pharmacological treatments are approved to help avoid relapse in humans3. The opioid epidemic that North America is currently facing is a striking example of it, and it demands considering different approaches on animal models of relapse to opioids.
Acute stress has been shown to be a key trigger to relapse in humans4. One environmental stressor that is often associated with drug addiction is food deprivation. Drug users often choose to allocate resources towards obtaining drugs instead of food. Caloric deficit has been shown to be correlated with higher relapse to cigarettes5 and alcohol6 use. Due to ethical and practical issues, animal models have been developed over the last decades to facilitate research in the field. In animal models, acute food deprivation has been demonstrated to robustly reinstate extinguished heroin seeking7. Currently, most animal models of relapse are based on abstinence procedures that are either not representative of human abstinence (e.g., extinction-based models) or encompass only the small percentage of drug users that are forced to abstain due to incarceration or inpatient treatment (e.g., forced abstinence models). The main reason drug users choose to abstain is the negative consequences associated with drug-seeking and taking8. Punishment-imposed abstinence is an animal model that mimics the negative consequences associated with drug-seeking on self-imposed abstinence in humans. This model introduces an aversive stimulus, e.g., a mild footshock, with drug-seeking or taking, which leads the animal to stop taking the drug voluntarily. Another procedure that incorporates negative consequences for drug-seeking is the electrical barrier conflict model for drug abstinence and relapse9. The rat must cross an electrical barrier to perform the operant behavior linked with drug self-administration. The model was used successfully to demonstrate voluntary abstinence and relapse to psychostimulant and opioid drugs10,11. However, under the electrical barrier procedure, drug-seeking efforts are always associated with an aversive event, unlike the human condition. Moreover, drug-taking itself might overlap with the electrical footshock as the animal returns to the safe area following the infusion by crossing the barrier again.
Punishment-imposed abstinence has been used with other drugs of abuse such as cocaine12, alcohol13, methamphetamine14, remifentanil15, but it was never applied to heroin-trained animals. The model has been used to study relapse induced by priming14 and drug-associated cues16, but it was not integrated into a stress-induced relapse procedure. The procedure described here is used to demonstrate acute food deprivation-induced relapse to heroin seeking after punishment-imposed abstinence in male rats.

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	<tbody>
		<tr>
			<td>Anafen Injection 100 mg/mL Vial/50 mL</td>
			<td>MERIAL Canada, Inc.</td>
			<td>1938126</td>
			<td>anti-inflammatory drug</td>
		</tr>
		<tr>
			<td>Balance arm</td>
			<td>Coulbourn Instruments</td>
			<td>H29-01</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>Environment connection board &#38; Linc cable</td>
			<td>Coulbourn Instruments</td>
			<td>H03-04</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>GE Marine Silicon</td>
			<td>GE</td>
			<td>SE-1134</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphic State Notation 3</td>
			<td>Coulbourn Instruments</td>
			<td>GS3</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Habitest universal Linc</td>
			<td>Coulbourn Instruments</td>
			<td>H02-08</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>House light-Rat</td>
			<td>Coulbourn Instruments</td>
			<td>H11-01R</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane USP 99.9% Vial/250 mL</td>
			<td>Fresenius Kabi Canada Ltd</td>
			<td>2237518</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>Rat test cage</td>
			<td>Coulbourn Instruments</td>
			<td>H10-11R-TC</td>
			<td>Operant conditioning chambers</td>
		</tr>
		<tr>
			<td>Retractable lever-Rat</td>
			<td>Coulbourn Instruments</td>
			<td>H23-17RA</td>
			<td></td>
		</tr>
		<tr>
			<td>Silastic tubing (ID 0.02, OD 0.037)</td>
			<td>Fisher Scientific (Canada)</td>
			<td>1118915A</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>Sound attenuation boxes</td>
			<td>Concordia University</td>
			<td>Home made</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>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>Tone module 2.9 KHz</td>
			<td>Coulbourn Instruments</td>
			<td>H12-02R-2.9</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>
	</tbody>
</table>

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.

Male rats demonstrated an increase in seek lever presses as the schedule of reinforcement increased over the training days, and a reliable, consistent number of heroin infusions over training days (Figure 1). During punishment, rats decreased the number of seek lever presses and infusions with the increase of footshock intensity over 8 punishment days (Figure 2). Food-deprivation condition significantly increased heroin seeking after punishment-imposed abstinence (Feeding condition: F(1, 7) = 7.12, p = .0.03, d = 1.3; Figure 3A). There was no statistically significant effect for time nor for drug-seeking over time across feeding conditions (Time: F(2, 14) = 1.029, p = .38; Time x Feeding condition: F(2, 14) = 1.347, p = .29; Figure 3B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63657/63657fig01.jpg" /> 
Figure 1: Heroin self-administration (N = 8). Mean &#177; SEM of seek lever presses (in red) and the number of infusions (blue), over a gradual increase of reinforcement schedules (FR1, VI5, VI30, VI60) for 18 training days. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63657/63657fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63657/63657fig02.jpg" /> 
Figure 2: Punishment-imposed abstinence (N = 8). Mean &#177; SEM seek lever responses over punishment days. Footshock intensity increased 0.1 mA per day from 0.2 mA to 1.0 mA, excluding 0.9 mA. Inserted graph is the mean (&#177;SEM) infusions over the punishment days. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63657/63657fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63657/63657fig03.jpg" /> 
Figure 3: Food deprivation-induced heroin seeking tests (N = 8). (A) Mean &#177; SEM seek lever presses under food-deprived and sated conditions during a 3 h heroin seeking test, under extinction conditions and no footshock punishment. Counterbalanced within-subject design. * Represents statistically significant difference, p = 0.032. (B) Mean &#177; SEM seek lever presses under food-deprived (FD) and sated conditions during the heroin relapse test (1 h segments). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63657/63657fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence at JoVE.com

A Procedure to Study Stress-Induced Relapse of 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 ...

a-procedure-to-study-stress-induced-relapse-heroin-seeking-after

Nanyang Technological University

Research

JoVE Journal

Behavior

2.5K Views.  Concordia University. 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.

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

Assessment and Evaluation of the High Risk Neonate: The NICU Network Neurobehavioral Scale

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant. The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. These are infants who are at increased risk for poor developmental outcome because of insults during prenatal development, such as substance exposure or prematurity or factors such as poverty, poor nutrition or lack of prenatal care that can have adverse effects on the intrauterine environment and affect the developing fetus. The NNNS assesses the full range of infant neurobehavioral performance including neurological integrity, behavioral functioning, and signs of stress/abstinence. The NNNS is a noninvasive neonatal assessment tool with demonstrated validity as a predictor, not only of medical outcomes such as cerebral palsy diagnosis, neurological abnormalities, and diseases with risks to the brain, but also of developmental outcomes such as mental and motor functioning, behavior problems, school readiness, and IQ. The NNNS can identify infants at high risk for abnormal developmental outcome and is an important clinical tool that enables medical researchers and health practitioners to identify these infants and develop intervention programs to optimize the development of these infants as early as possible. The video shows the NNNS procedures, shows examples of normal and abnormal performance and the various clinical populations in which the exam can be used.

The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. The purpose of this article is to describe the NNNS, provide video examples of the NNNS procedures and discuss the ways in which the exam has been used.

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant.  The NICU Network Neurobehavioral Scale ...

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.

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 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 Mouse Model of Subchronic and Mild Social Defeat Stress for Understanding Stress-induced Behavioral and Physiological Deficits

Stressful life events often increase the incidence of depression in humans. To study the mechanisms of depression, the development of animal models of depression is essential. Because there are several types of depression, various animal models are needed for a deeper understanding of the disorder. Previously, a mouse model of subchronic and mild social defeat stress (sCSDS) using a modified chronic social defeat stress (CSDS) paradigm was established. In the paradigm, to reduce physical injuries from aggressors, the duration of physical contact between the aggressor and a subordinate was reduced compared to in the original CSDS paradigm. sCSDS mice showed increased body weight gain, food intake, and water intake during the stress period, and their social behaviors were suppressed after the stress period. In terms of the face validity of the stress-induced overeating and overdrinking following the increased body weight gain, the sCSDS mice may show some features related to atypical depression in humans. Thus, a mouse model of sCSDS may be useful for studying the pathogenic mechanisms underlying depression. This protocol will help establish the sCSDS mouse model, especially for studying the mechanisms underlying stress-induced weight gain and polydipsia- and hyperphagia-like symptoms.

Here, methods for developing a mouse model of subchronic and mild social defeat stress are described and used to investigate the pathogenic features of depression including hyperphagia- and polydipsia-like symptoms following increased body weight.

Stressful life events often increase the incidence of depression in humans. To study the mechanisms of depression, the development of animal models of ...

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, ...

Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor locomotion after surgery. For example, rats who undergo surgical procedures and are subjected to the Morris water maze may not be able to swim, thus voiding the experiment.
New Method: We established a modified behavioral spontaneous alternation T-maze test. The major advantage of the modified T-maze protocol is its relatively simple design that is powerful enough to assess functional learning/memory after ischemia. Additionally, the data analysis is simple and straightforward. We used the T-maze to determine the rats&#39; learning/memory deficits both in the presence or absence of mild to moderate (6 min) asphyxial cardiac arrest (ACA). Rats have a natural tendency for exploration and will explore the alternate arms in the T-maze, whereas hippocampal-lesioned rats tend to adopt a side-preference resulting in decreased spontaneous alternation ratios, revealing the hippocampal-related functional learning/memory in the presence or absence of ACA.
Results: ACA groups have higher side-preference ratios and lower alternations as compared to control.
Comparison with Existing Method(s): The Morris water and Barnes maze are more prominent for assessing learning/memory function. However, the Morris water maze is more stressful than other mazes. The Barnes maze is widely used to measure reference (long-term) memory, while ACA-induced neurocognitive deficits are more closely related to working (short-term) memory.
Conclusions: We have developed a simple, yet effective strategy to delineate working (short-term) memory via the T-maze in our global cerebral ischemia model (ACA).

This protocol describes the use of a modified T-maze to evaluate functional learning/memory in asphyxia cardiac arrest-induced cerebral ischemia.

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor ...

A Method to Study Adaptation to Left-Right Reversed Audition

An unusual sensory space is one of the effective tools to uncover the mechanism of adaptability of humans to a novel environment. Although most of the previous studies have used special spectacles with prisms to achieve unusual spaces in the visual domain, a methodology for studying the adaptation to unusual auditory spaces has yet to be fully established. This study proposes a new protocol to set-up, validate, and use a left-right reversed stereophonic system using only wearable devices, and to study the adaptation to left-right reversed audition with the help of neuroimaging. Although individual acoustic characteristics are not yet implemented, and slight spillover of unreversed sounds is relatively uncontrollable, the constructed apparatus shows high performance in a 360&#176; sound source localization coupled with hearing characteristics with little delay. Moreover, it looks like a mobile music player and enables a participant to focus on daily life without arousing curiosity or drawing attention of other individuals. Since the effects of adaptation were successfully detected at the perceptual, behavioral, and neural levels, it is concluded that this protocol provides a promising methodology for studying adaptation to left-right reversed audition, and is an effective tool for uncovering the adaptability of humans to a novel environments in the auditory domain.

The present study proposes a protocol to investigate the adaptation to left-right reversed audition achieved only by wearable devices, using neuroimaging, which can be an effective tool for uncovering the adaptability of humans to a novel environment in the auditory domain.

An unusual sensory space is one of the effective tools to uncover the mechanism of adaptability of humans to a novel environment. Although most of the ...

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 ...

Paradigms of Lower Extremity Electrical Stimulation Training After Spinal Cord Injury

Skeletal muscle atrophy, increased adiposity and reduced physical activity are key changes observed after spinal cord injury (SCI) and are associated with numerous cardiometabolic health consequences. These changes are likely to increase the risk of developing chronic secondary conditions and impact the quality of life in persons with SCI. Surface neuromuscular electrical stimulation evoked resistance training (NMES-RT) was developed as a strategy to attenuate the process of skeletal muscle atrophy, decrease ectopic adiposity, improve insulin sensitivity and enhance mitochondrial capacity. However, NMES-RT is limited to only a single muscle group. Involving multiple muscle groups of the lower extremities may maximize the health benefits of training. Functional electrical stimulation-lower extremity cycling (FES-LEC) allows for the activation of 6 muscle groups, which is likely to evoke greater metabolic and cardiovascular adaptation. Appropriate knowledge of the stimulation parameters is key to maximizing the outcomes of electrical stimulation training in persons with SCI. Adopting strategies for long-term use of NMES-RT and FES-LEC during rehabilitation may maintain the integrity of the musculoskeletal system, a pre-requisite for clinical trials aiming to restore walking after injury. The current manuscript presents a combined protocol using NMES-RT prior to FES-LEC. We hypothesize that muscles conditioned for 12 weeks prior to cycling will be capable of generating greater power, cycle against higher resistance and result in greater adaptation in persons with SCI.

Spinal cord injury is a traumatic medical condition that may result in elevated risks of chronic secondary metabolic disorders. Here, we presented a protocol using surface neuromuscular electrical stimulation-resistance training in conjunction with functional electrical stimulation-lower extremities cycling as a strategy to ameliorate several of these medical problems.

Skeletal muscle atrophy, increased adiposity and reduced physical activity are key changes observed after spinal cord injury (SCI) and are associated with ...