1. Animal Housing and Acclimatization Procedures
<ol>
<li>Male Long Evans rats, weighing 300-325 g at the time of arrival in the vivarium, are housed individually in standard plastic cages containing recycled paper pellet bedding, with wire grid lids to accommodate water bottles and food.</li>
<li>Rats are maintained on a reverse light-dark schedule (lights on 19:00 to 07:00), in a temperature- and humidity-controlled vivarium.</li>
<li>Food and water are available ad libitum in the home cages throughout the experiment.</li>
<li>Rats are allowed to acclimatize to the vivarium and housing conditions for one week prior to surgery. During this time rats are left undisturbed with the exception of routine cleaning, feeding, and monitoring of weight.</li>
</ol>
2. Intravenous Catheterization Surgery
<ol>
<li>Under isoflurane gas anesthesia, and using standard aseptic surgical techniques, rats are prepared with silastic catheters in the right jugular vein. The catheter exits into a modified cannula (Plastics One) at the surface of the skull, and the cannula is mounted to the skull with jewelers' screws and dental cement. For details of the surgical procedures, see 7, 8.</li>
<li>At the end of the surgery, catheters are flushed with approximately 0.2 mL of a solution containing 50% heparin (1000 IU) and 50% dextrose (25 g/50 mL). A plastic blocker is placed over the opening of the cannula to protect the catheter from external debris, and to maintain catheter patency.</li>
<li>At the time of surgery, and again on the day after surgery, rats are administered 5 mg/kg, SC, of the analgesic Ketoprofen.</li>
<li>Rats are allowed at least seven days to recover from surgery before experimentation. During this time, rats are left undisturbed in the home cages, with the exception of routine cleaning and feeding, and daily monitoring of weight and general health. On the last day of the recovery period, catheters are flushed with the heparin/dextrose solution (Section 2.2) to verify catheter patency.</li>
</ol>
3. Behavioral Procedures
All behavioral procedures are carried out using equipment supplied by Med Associates Inc. (St Albans, VT). The equipment, housed in a dedicated room, includes a set of standard operant chambers, each contained within a sound-attenuating enclosure. Each operant chamber is equipped with two retractable levers, both elevated 6.5 cm above a stainless steel rod floor. A white stimulus light is located just above one lever, and responses on that lever (the so-called "active" lever) result in the simultaneous illumination of the stimulus light and activation of an infusion pump (Razel Scientific Instruments, St. Albans, VT). The pump is situated on a shelf just outside the operant chamber, but within the sound-attenuating enclosure. The other lever (the so-called "inactive" lever) is left extended in the chamber throughout all experimental procedures; responding on this lever is without consequence. Finally, each chamber is equipped to deliver constant-current, intermittent, inescapable, electric footshock through a scrambler to the steel rod floor. All experimental conditions (e.g., stimulus presentations, drug infusions, shock delivery), as well as data recording and consolidation, are accomplished via interfaces between the test chambers and a central computer operating Med Associates software (MED-IV).
The experiment is carried out in 5 phases: 1) Habituation; 2) Cocaine SA; 3) Drug-free period; 4) Extinction, and 5) Testing for reinstatement. In all phases (except for the drug-free period), rats are transferred for daily sessions from the housing room to the operant test chambers. Animals are transferred individually, in small plastic containers. Food and water are not available in the operant chambers.
<ol>
<li>Habituation
<ol>
<li>Twenty-four hours preceding the first cocaine SA session (see Section 3.2.), rats are allowed to habituate to the operant chambers during a single 2-h session; during this session the active lever is retracted.</li>
</ol>
</li>
<li>Cocaine SA training
<ol>
<li>Before each daily training session, rats are weighed in the housing room. Subsequently they are transferred to the operant chambers.</li>
<li>Upon being placed in the operant chambers, a spring and tubing assembly (extending from the drug infusion pump) is connected to the skull-mounted cannula.</li>
<li>Rats are allowed to self-administer cocaine HCl during daily 3-h sessions, for a total of 8-10 days (either consecutively or with two intervening "days off" between sessions 5 and 6).</li>
<li>SA sessions alternate between the morning (9:00-12:00) and afternoon (13:00-16:00). The timing of SA sessions is alternated because the procedures in subsequent phases of the experiment (i.e. extinction and testing) occur at both times of day for all animals (see Sections 3.4 and 3.5); thus, this arrangement prevents any confounding effects of the time of day on subsequent drug-seeking behavior.</li>
<li>Each training session is preceded by a 5-min acclimatization period, during which animals are placed in the operant chambers with the active lever retracted.</li>
<li>At the beginning of the training session, the active lever is extended into the chamber and the stimulus light (located above the active lever) is illuminated for 30 s. In addition, the house light is activated at this time, and remains illuminated throughout the session. Together, these events signal the availability of cocaine.</li>
<li>During the session, responses on the active lever are reinforced by a 3-s infusion of cocaine (0.23 mg/65 &mu;L, i.v.) on a fixed-ratio-1 schedule of reinforcement. Concurrent with activation of the infusion pump and drug delivery is a 20-s activation of the stimulus light above the active lever. This 20-s light presentation corresponds to a "time-out" period during which additional active lever responses are recorded but not reinforced.</li>
<li>Animals are allowed a maximum of 50 infusions per 3-h session. We routinely impose this criterion in our work and, in our experience, have found that it greatly reduces, if not eliminates, incidents of deaths due to overdose.</li>
<li>At the end of each daily session, rats' catheters are flushed with 0.2-0.3 mL of heparin-dextrose solution (see Section 2.2) and the rats are returned to their home cage.</li>
</ol>
</li>
<li>Drug-free period
<ol>
<li>After the final cocaine SA session, rats are left undisturbed (with the exception of routine cleaning, feeding, and monitoring of weight) in their home cage for a minimum of seven days.</li>
</ol>
</li>
<li>Extinction of cocaine-trained behavior
<ol>
<li>Rats are transferred to the operant chambers between 09:00-10:00 for extinction training.</li>
<li>The spring and tubing assembly is connected to the skull-mounted cannula.</li>
<li>Consistent with the conditions for SA training, each day of extinction training is preceded by a 5-min acclimatization period, during which animals are placed in the operant chambers with the active lever retracted.</li>
<li>Rats are then given four 60-min extinction sessions, separated by 30-min intervals. Each 60-min session is initiated by the same events that occur at the start of training sessions (i.e., illumination of the house light, extension of the active lever, and 30-sec illumination of the stimulus light above the active lever). Throughout the sessions, all conditions that were present during SA training are maintained (i.e., response-contingent illumination of stimulus light for 20 sec and 3-s activation of the infusion pump), except that active lever responses are no longer reinforced with cocaine infusions. During the 30-min intervals that separate each 60-min extinction session, the active lever is retracted and the house light is extinguished.</li>
<li>At the end of the last 60-min session, rats are returned to their home cage.</li>
<li>Steps 3.4.1. to 3.4.5. are repeated for 3 consecutive days.</li>
</ol>
</li>
<li>Reinstatement of cocaine-trained behavior by footshock stress
<ol>
<li>Rats are transferred to the operant chambers between 09:00-10:00</li>
<li>Upon being placed in the operant chambers, the spring and tubing assembly is connected to the skull-mounted cannula.</li>
<li>Consistent with the conditions for training and extinction, a 5-min acclimatization period is provided, during which rats are placed in the operant chambers with the active lever retracted.</li>
<li>Rats are then given two 60-min extinction sessions (see 3.4.4.) separated by a 30-min interval, identical to those given during the extinction phase.</li>
<li>If after the second 60-min session the total number of responses on the previously active lever is less than 15, animals are given a test for reinstatement after an additional 30-min interval. In this 30-min interval, footshock or sham footshock (i.e., no footshock) is administered (see 3.5.6.). If a criterion of 15 or fewer responses on the active lever is not achieved after the second session, an additional extinction session(s) is given prior to testing for reinstatement.</li>
<li>Ten minutes after the second 60-min extinction session (or after any additional 60-min extinction session(s) required to establish the extinction criterion of 15 or fewer responses), animals are exposed to 20 min of intermittent electric footshock stress (or sham footshock) in the operant chamber. Footshock is given during a time when the active lever is retracted and house and stimulus lights are extinguished. Footshock is delivered according to a variable time schedule at a mean interval of 40 s (10-70 s range). Each shock is delivered at an intensity of 0.9 mA, and is 0.5 s in duration. These parameters of footshock are based on extensive pilot work carried out in our lab to establish reliable reinstatement of cocaine seeking, with the minimum intensity and duration of footshock exposure. Under these parameters, animals exhibit a reflexive jumping response to the 0.5 s shock, and occasionally emit a brief vocalization. Although animals are observed throughout the shock period, the intensity of shock is not of sufficient magnitude to induce more pronounced signs of distress.</li>
<li>Immediately following termination of the footshock session, the active lever is extended and rats are allowed to lever press under extinction conditions (i.e., responses are not reinforced with cocaine, but do continue to result in illumination of the stimulus light and activation of the infusion pump). This period of time is defined as the test for reinstatement and is typically 1-3 hours in duration. Most responding occurs in the early part of the test session (first 30 min- 1 h), and subsequently extinguishes over more extended test periods. Thus, a 1 h test is sufficient to capture the reinstatement effect, with longer test periods providing a more complete extinction profile of the effect.</li>
<li>When a repeated measures design is employed, each rat is tested under footshock and sham footshock conditions. Steps 3.5.1. to 3.5.8 are repeated on a subsequent test day, and the alternate test for reinstatement (footshock or sham footshock) is administered.</li>
</ol>
</li>
</ol>
4. Representative Results:
<img src="/files/ftp_upload/2265/2265fig1.jpg" alt="Figure 1" /> 
Figure 1. Stylized response profiles for activity on the active and inactive levers during self-administration training, extinction, and reinstatement testing.
Figure 1 presents stylized operant response profiles, typical of those obtained during the various phases of the experimental protocol described herein.
1. Active lever responses
<ol>
<li>Cocaine SA training. Figure 1 (left panel) shows a typical cocaine SA training curve that includes both number of infusions and total number of active lever presses. Rats generally acquire cocaine SA within 1-3 sessions and, once the behavior is established, maintain a stable rate of drug intake (typically less than 20% between-session variance) over subsequent sessions.</li>
<li>Extinction of cocaine-trained behavior. It can be seen in Figure 1 (middle panel) that on Day 1 of Extinction, when responses are non-reinforced for the first time, total number of responses on the active lever is initially higher than at the end of the training phase (corresponding to a so-called "extinction burst"); gradually, over the four extinction sessions, responding decreases. On Days 2 and 3 of extinction, some recovery of responding (relative to that observed during the last session of the previous day) may be observed; however, responses are generally fewer than at the start of the previous day of extinction, and extinction generally proceeds more rapidly over the four sessions for that day.</li>
<li>Reinstatement of cocaine seeking. It can be seen in Figure 1 (right panel) that 20 min of exposure to intermittent footshock induces an increase in non-reinforced responding on the active lever, relative to that observed at the end of the preceding extinction session. Footshock also induces an increase in responding relative to that observed under conditions in which sham footshock is administered. Thus, the reinstatement of cocaine seeking by footshock stress can be expressed either as: 1) the increased number of non-reinforced responses after exposure to footshock, relative to responses in the extinction session before footshock or 2) the increased number of non-reinforced responses after footshock relative to sham footshock.</li>
</ol>
2. Inactive lever responses
It can be seen in Figure 1 (red line) that, throughout all experimental phases, there are very low response levels associated with the inactive lever. However, a modest increase in responding on this lever is sometimes observed following exposure to footshock stress. Even in cases in which such an increase in responding occurs, the increase in active lever responding tends to be proportionately much greater.

<ol>
	<li>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=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 (1-2), 3-20 (2003).</li><li>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=Priming+effects+with+drugs+and+other+reinforcers.">Priming effects with drugs and other reinforcers.</a> Experimental and Clinical Psychopharmacology. 4, 5-10 (1996).</li><li>Bossert, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+relapse+to+heroin+and+cocaine+seeking:+An+update+and+clinical+implications.">Neurobiology of relapse to heroin and cocaine seeking: An update and clinical implications.</a> Eur J Pharmacol. 526 (1-3), 36-50 (2005).</li><li>Shalev, U., Grimm, J. W., 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=Neurobiology+of+relapse+to+heroin+and+cocaine+seeking:+a+review.">Neurobiology of relapse to heroin and cocaine seeking: a review.</a> Pharmacol Rev. 54 (1), 1-42 (2002).</li><li>Shaham, Y., 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=Stress+reinstates+heroin-seeking+in+drug-free+animals:+an+effect+mimicking+heroin,+not+withdrawal.">Stress reinstates heroin-seeking in drug-free animals: an effect mimicking heroin, not withdrawal.</a> Psychopharmacology. 119, 334-341 (1995).</li><li>Shaham, Y., Erb, S., 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=Stress-induced+relapse+to+heroin+and+cocaine+seeking+in+rats:+a+review.">Stress-induced relapse to heroin and cocaine seeking in rats: a review.</a> Brain Res Brain Res Rev. 33 (1), 13-33 (2000).</li><li>Erb, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+injections+of+CRF+reinstate+cocaine+seeking+in+rats+after+postinjection+delays+of+up+to+3+h:+an+influence+of+time+and+environmental+context.">Central injections of CRF reinstate cocaine seeking in rats after postinjection delays of up to 3 h: an influence of time and environmental context.</a> Psychopharmacology (Berl). 187 (1), 112-120 (2006).</li><li>Erb, S., Shaham, Y., 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=Stress+reinstates+cocaine-seeking+behavior+after+prolonged+extinction+and+a+drug-free+period.">Stress reinstates cocaine-seeking behavior after prolonged extinction and a drug-free period.</a> Psychopharmacology. 128, 408-412 (1996).</li><li>Tran-Nguyen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-dependent+changes+in+cocaine-seeking+behavior+and+extracellular+dopamine+levels+in+the+amygdala+during+cocaine+withdrawal.">Time-dependent changes in cocaine-seeking behavior and extracellular dopamine levels in the amygdala during cocaine withdrawal.</a> Neuropsychopharmacology. 19, 48-59 (1998).</li><li>Stretch, R., Gerber, G. J., Wood, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+behavior+maintained+by+response-contingent+intravenous+infusions+of+amphetamine+in+squirrel+monkeys.">Factors affecting behavior maintained by response-contingent intravenous infusions of amphetamine in squirrel monkeys.</a> Canadian Journal of Physiology and Pharmacology. 49, 581-589 (1971).</li><li>Shaham, Y., Rodaros, D., 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+heroin-reinforced+behavior+following+long-term+extinction+Implications+for+the+treatment+of+relapse+to+drug-taking.">Reinstatement of heroin-reinforced behavior following long-term extinction Implications for the treatment of relapse to drug-taking.</a> Behavioural Pharmacology. 5, 360-364 (1994).</li><li>Wit, H. d. e., 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>Erb, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-2+adrenergic+receptor+agonists+block+stress-induced+reinstatement+of+cocaine+seeking.">Alpha-2 adrenergic receptor agonists block stress-induced reinstatement of cocaine seeking.</a> Neuropsychopharmacology. 23 (2), 138-150 (2000).</li><li>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=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 (Berl). 150, 337-346 (2000).</li></ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Canadian Council of Animal Care and University of Toronto animal care committee.

The production of this video was sponsored by Med Associates, Inc. which produces the instrument used in this article.

Over the past few decades, three variations of the SA reinstatement procedure have been developed to study reinstatement of drug seeking in laboratory rats and monkeys. The differences that define the procedural variations relate primarily to whether drug SA, extinction, and testing sessions occur within the same day or on different days 1. The procedure we have described in this paper is commonly referred to as the "within-between" session variation, because drug SA occurs during daily sessions (i.e., in a between-session manner) that are separate from extinction and test sessions, but extinction and testing occur on the same day (i.e., in a within-session manner) [e.g., 7, 9]. Alternatively, in so-called "between sessions" procedures, all phases of the experiment occur sequentially, such that SA, extinction, and testing sessions are given on different days (i.e., all phases are between sessions) [e.g., 8, 10, 11]. In contrast, in complete "within sessions" procedures, SA training, extinction, and test sessions all occur within the same day [e.g., 12].
Although there are published reports of footshock-induced reinstatement of drug seeking using different variations of the reinstatement procedure, we have found this stressor (at least in animals with a history of cocaine SA) to be most effective when extinction and testing occur on the same day (i.e., in a "within-between" sessions manner). Alternatively, we have had success with obtaining reliable footshock-induced reinstatement of cocaine seeking using a completely "between sessions" procedure, but only under conditions in which animals are housed in the SA chambers throughout the experiment 8, or at least during extinction and testing 13. The disadvantage of this between-session approach, over that described in the present protocol, is that it limits the number of subjects that can be trained and tested at one time and, thereby, decreases the efficiency of data collection. Although under the conditions of the present protocol only one group of animals can be extinguished and tested at a given time, a greater throughput can be achieved by staggering by one week the start time of the SA training phase for two groups of rats. In this scenario, a first group of rats can enter the drug-free phase as the second group enters Week 2 of SA training, and, subsequently, extinction and testing can be carried out in the first group while the second group is in its drug-free phase. This approach permits a more efficient use of resources and accelerates the rate of data acquisition, while leading to very effective and reliable footshock-induced reinstatement.
Importantly, in our procedure, extinction of the previously drug-reinforced behavior and testing for reinstatement occurs in the presence of drug-associated cues. Although we have never carried out extinction training or testing in the absence of drug-associated cues, it is our understanding, based on personal communications, that the stressor may in fact be ineffective in inducing reinstatement when testing occurs in the absence of extinguished cues (at least in the case of animals with a history of cocaine SA). A systematic examination of this variable, and the theoretical implications it poses, is an important question for future research.
A final procedural factor that warrants comment is that the effect of footshock stress on reinstatement appears to be highly sensitive to manipulations of context. For example, footshock stress is only effective in inducing the reinstatement of drug seeking if it is administered in the environment in which drug SA and extinction occur; that is, footshock does not reinstate drug seeking if exposure to the stressor occurs in a novel context, suggesting an important interaction between the stressor and drug environment in its effects on reinstatement 14.
In conclusion, footshock reinstatement procedures, like those described in this paper, have been used with considerable success to elucidate the basic behavioral and neurobiological mechanisms governing the relationship between stress and drug relapse. Indeed, these procedures, when modified to accommodate pharmacological pretreatments or neurochemical manipulations targeting specific neuronal substrates, can serve as powerful tools for characterizing the neurobiology of reinstatement to drug seeking by stress.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Basic Drug Self-Administration Test Package for Rat Package</td>
<td>&nbsp;</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>MED-008-CT-B1</td>
<td>&nbsp;</td>
<tr>
<td>Deep extra tall MDF sound attenuating cubicle</td>
<td>Basic Drug Self-Administration Test Package for Rat Package Component</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>ENV-018MD</td>
<td>&nbsp;</td>
<tr>
<td>Standard modular test chamber with drug infusion top and waste pan</td>
<td>Basic Drug Self-Administration Test Package for Rat Package Component</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>ENV-008CT</td>
<td>&nbsp;</td>
<tr>
<td>Stainless steel grid floor</td>
<td>Basic Drug Self-Administration Test Package for Rat Package Component</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>ENV-005</td>
<td>&nbsp;</td>
<tr>
<td>House light, hooded, 28V</td>
<td>Basic Drug Self-Administration Test Package for Rat Package Component</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>ENV-215M</td>
<td>&nbsp;</td>
<tr>
<td>Stimulus light 1&#x201D; white lens</td>
<td>Basic Drug Self-Administration Test Package for Rat Package Component</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>ENV-221M</td>
<td>&nbsp;</td>
<tr>
<td>Low profile retractable response lever</td>
<td>Basic Drug Self-Administration Test Package for Rat Package Component</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>ENV-112CM</td>
<td>&nbsp;</td>
<tr>
<td>SmartCtrl 8 Input/16 output package</td>
<td>Basic Drug Self-Administration Test Package for Rat Package Component</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>DIG-716P2</td>
<td>&nbsp;</td>
<tr>
<td>Single speed syringe pump, 3.33 RPM</td>
<td>Basic Drug Self-Administration Test Package for Rat Package Component</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>PHM-100</td>
<td>&nbsp;</td>
<tr>
<td>Rat self infusion package</td>
<td>Basic Drug Self-Administration Test Package for Rat Package Component</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>PHM-119B-23-D</td>
<td>&nbsp;</td>
<tr>
<td>Drug Delivery, Arm assembly </td>
<td>Basic Drug Self-Administration Test Package for Rat Package Component</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>PHM-110-SAI</td>
<td>&nbsp;</td>
<tr>
<td>Square wave aversive stimulator</td>
<td>Footshock Stress Component</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>ENV-414S</td>
<td>&nbsp;</td>
<tr>
<td>Patented quick disconnect harness for stainless steel grid floor</td>
<td>Footshock Stress Component</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>ENV-005-QD</td>
<td>&nbsp;</td>
<tr>
<td>Shock output cable</td>
<td>Footshock Stress Component</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>SG-219G-10</td>
<td>&nbsp;</td>
<tr>
<td>MED-PC IV Software</td>
<td>Software</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>SOF-735</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a procedure for studying the footshock-induced reinstatement of cocaine seeking in laboratory rats

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

Watch this Scientific Journal Video about A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats at JoVE.com

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

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

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JoVE Journal

Neuroscience

20.7K Views.  University of Toronto Scarborough. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

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

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

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

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

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

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

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...