This work was supported by National Natural Science Foundation of China (NSFC)31741058 (Y.Z.), Shenzhen Governmental grants (JCYJ20170818162613877 and JCYJ20170818163217196) and China Postdoctoral Science Foundation Grant 2017M622826 (K.Z.).

The experimental procedures were performed in accordance with the guidelines for the National Care and Use of Animals and the experiments were approved by the Institutional Animal Care and Use Committee at Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.
1. Animal Habituation
<ol>
	<li>Obtain male adult rats from animal laboratory and maintain them under a 12 h light/dark cycle (light on from 7:00 to 19:00) with free access to food and water.</li>
	<li>Handle the animals by playing with them or patting them twice per day for two weeks before the experiments.</li>
	<li>Randomly assign animals into saline or morphine groups.</li>
</ol>
2. Drug Preparation
<ol>
	<li>Prepare morphine hydrochloride as 10 mg/mL and inject rats intraperitoneally (i.p.) at a volume of 1 mL/kg.</li>
	<li>As a control, inject rats with saline at a volume of 1 mL/kg.</li>
</ol>
3. CPP Apparatus
<ol>
	<li>Construct a three-chamber CPP apparatus with different tactile (floor) cues and test rats&#39; initial preference to two side chambers. The apparatus consists of two large black side chambers (30 cm &#215; 25 cm &#215; 30 cm, L&#215;W&#215;H) and one middle white chamber (11 cm &#215; 25 cm &#215; 30 cm, L&#215;W&#215;H) with white smooth PVC floor. The two side chambers have different floor textures as somatosensory cues: one with a grid plexiglass floor, the other with a rough PVC floor.</li>
	<li>Vary the combination of tactile cues to make sure that the apparatus is unbiased, since a biased apparatus is susceptible to yielding false positive results4. 
	NOTE: An important methodological issue in the design of the CPP apparatus is whether the apparatus is &#34;biased&#34; or &#34;unbiased&#34;4. If one group of untrained rats shows no initial preference for one side chamber over another, the apparatus is considered &#34;unbiased&#34;. Otherwise, consider it as &#34;biased&#34;13.</li>
</ol>
4. CPP Procedure
<ol>
	<li>Establish the morphine-induced CPP paradigm mainly according to previous studies21,22 with modification of conditioning time points, number of conditioning sessions, conditioning trial period and testing time points20. A short description is given below, of which the experimental schedule is depicted in Figure 1.</li>
	<li>Pre-conditioning phase
	<ol>
		<li>During this phase (day 1 to 3), remove guillotine doors to allow free access to the entire apparatus and place the animal in the middle chamber.</li>
		<li>Record the activity of the animals for 15 min.</li>
		<li>Calculate the time spent in the two side chambers on day 3 as the baseline and refer this as T0.</li>
		<li>Remove rats that enter less than -four times to either of the side chambers from the experiment23 as these rats are likely to have problems in general well-being (e.g., dirty fur and having tumors). Check the health state of the animals during the whole experimental period.</li>
	</ol>
	</li>
	<li>Conditioning phase 
	NOTE: Conditioning phase includes 6 days (day 4 to 9).
	<ol>
		<li>For morphine-conditioned subjects, inject rats i.p. with morphine (10 mg/kg) or saline (1 mL/kg) alternately in the morning (9:00) and evening (19:00) sessions (10 h apart).</li>
		<li>Confine rats immediately after each injection to its drug-paired (non-preferred) or saline-paired (preferred) chamber for 45 min (Figure 1B).</li>
		<li>Keep the video recording system on. 
		NOTE: According to our experience, around 5% of rats have the respiratory depression after the first injection of morphine (10 mg/kg). In such a case, to increase the survival rate of respiratory depressed rats, get the rat out of the conditioning chamber, pull its tongue out and put it on a white tissue paper in its home cage.</li>
		<li>For saline-conditioned controls, give rats saline injections (1 mL/kg) prior to exposure to one side chamber of the CPP apparatus during the morning session and to the other side during the evening session for 45 min/session in line with morphine group (Figure 1C).</li>
	</ol>
	</li>
	<li>Testing phase
	<ol>
		<li>During this phase, remove guillotine doors to allow free access to the entire apparatus and place the animal in the middle chamber to let them freely explore the whole apparatus for 15 min.</li>
		<li>Record their activity during this 15 min.</li>
		<li>Carry out three post-conditioning tests, i.e., test 1 (T1), test 2 (T2) and test 3 (T3), 2 days, 10 days and 18 days after the last conditioning session in a morphine-free state, respectively (Figure 1A).</li>
		<li>Vary testing time points for different experimental goals. 
		NOTE: To minimize the number of rats required, a within-subject design is employed. However, to exclude the extinction effect, a between subject design is required.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58384/58384fig1.jpg" /> 
Figure 1: Experimental procedures. (A) Experimental procedures of the whole CPP process. (B) Experimental schedule of the conditioning phase of morphine group. (C) Experimental schedule of the conditioning phase of saline group. <a href="https://www.jove.com/files/ftp_upload/58384/58384fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Data Analysis
<ol>
	<li>Calculate the CPP score in morphine(saline) group as: CPP score = the time spent in the drug-paired (non-preferred) chamber/ the total time spent in both conditioning chambers.</li>
	<li>Perform data analysis of CPP scores and entrances to side chambers with appropriate statistical methods using SPSS.</li>
	<li>Present all behavioral data as mean &#177; SEM, where SEM is the standard error of mean. Consider values of p&#60;0.05 as statistically significant and indicate by asterisks (*) and octothorpesin (#) in the figures.</li>
</ol>

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</ol>

The authors have nothing to disclose.

Here, a method using a morphine CPP paradigm is described to establish long-term reward memory in rat with somatosensory cues as conditioned context. To build such a long-term reward memory, a previous protocol21 with a balanced CPP apparatus is modified and the saline and morphine sessions are set at least 10 h apart. In addition, more conditioning sessions are added during the conditioning phase to enhance the associative learning. Apart from the CPP scores, the entrances to two side chambers are measured and the pattern of increased entrances to side chambers is similar with that of the increased active responses in the self-administration paradigm.
Several factors influence the establishment of the CPP paradigm mentioned above. One key factor is the design of the apparatus: rats in general should have similar preference to both side chambers to ensure the apparatus is balanced. An unbalanced apparatus can mask the acquisition of CPP due to the initial high preference to one side chamber13. Therefore, extreme preference to one side chamber (more than 90% of the total time in any of the two side chambers) should be avoided when choosing the texture of the floor or other contextual cues. For different experimental purposes, experimenters can balance the CPP apparatus by modifying the contextual stimuli. For balanced apparatus, two ways of experimental design are commonly used4. One biased way is to pair drugs with the non-preferred chamber with all subjects; an unbiased way is to pair half of the animals with drugs in the non-preferred chamber, the other half with the preferred chamber. Both ways can establish CPP13 but studies17,25 with the latter design always exclude many subjects due to their strong preference to one side chamber. As individual vulnerability to drug addiction26,27,28 exists, a biased design was chosen in this study to avoid exclusion of healthy subjects. One limitation of biased design is the effect of inconsistent preference to side chambers. As shown in Figure 2, rats in the saline group have consistent low CPP scores for the initial non-preferred chamber. Thus, the possibility that inconsistent preference to side chambers could affect the CPP scores in the morphine group is excluded by setting the saline controls. To avoid the ceiling effect of the preferred chamber, we use the initial non-preferred chamber as the morphine paired chamber. To avoid the effect of novelty and stress on the baseline preference to side chambers, three pre-conditioning sessions are performed, and the third session is used as the baseline. The fourth factor is the interval between morphine and saline sessions. In our paradigm, we choose intervals of at least 10 hours to avoid the withdrawal effect in the saline conditioning period. The dosage of morphine is also an important factor. Extremely low doses of morphine, such as 0.01 mg/kg6, did not induce place preference. A dose of 10 mg/kg induced the highest CPP score17 in rats and is commonly used in rat morphine CPP studies17,21,23. However, 10 mg/kg morphine (i.p.) could induce respiratory depression in around 5% of rats. Lower doses of morphine, such as 5 mg/kg, could be used in future studies. For other species, such as tree shrew, tolerance to drugs should be considered when building the CPP paradigm. Since tree shrew develops fast tolerance to morphine, increased doses of morphine should be used during the conditioning phase29,30.
With the modified protocol, a long-term reward memory is established for at least 18 days after 6 days of conditioning11 (Figure 2). Future studies should measure whether CPP could be maintained for a longer time. Repeated relapse is the major barrier in treating drug addiction. Stress, environmental cues and drugs could induce relapse. After extinction of the CPP memory, this model could be used to model relapse induced by stress or drugs.
The entrances to the drug-paired chambers in the morphine group increased in the abstinence period11 (Figure 3), which is similar with the increased active responses in the self-administration paradigm. However, entrances to the saline-paired chamber also increased with the CPP paradigm in both rats and tree shrews11, which is different from the inactive responses of the self-administration paradigm. This difference might due to the different underlying principles of the two paradigms: one is classical conditioning, the other is operant conditioning. Besides, tree shrews with food CPP experiences did not show such a pattern of increased entrances30. Therefore, the increased entrances to both side chambers in morphine-experienced animals are thought to represent incubated craving of drugs. Although the limitation of using repeated measures in one subject cannot be excluded, the pattern of increased entries was conserved in rats11, tree shrews11,30 and monkeys31. Nonetheless, a between subject design during the abstinence period is highly recommended for future studies to exclude the extinction effect of repeated measurements.
Apart from the increased entrances, CPP scores with low doses of morphine (1 mg/kg and 3 mg/kg) increased progressively during the abstinence period17. These results suggest that the CPP paradigm could also model the incubation of craving phenomenon. However, an increased CPP score might also represent an increased memory for the conditioned stimulus. In this aspect, entrances to the side chambers in a three-chamber apparatus, which are similar to active responses in the self-administration paradigm, more closely resemble the search for drugs or the rewarding effect of drugs. It would be interesting to check the trends of entrances to side chambers during the abstinence period in rat CPP paradigms of lower doses of morphine. Future studies with other drugs and mice should be done to verify whether this phenomenon is conserved across drugs of abuse/species.

Drug addiction is a chronic disease1 with several different stages2: binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation. In the initial binge/intoxication stage of the drug addiction cycle, subjects take drugs due to the reinforcing effect2. In the withdrawal/negative affect stage, subjects experience negative somatic and emotional states of drug withdrawal, which promote negative reinforcement mechanisms associated with the development of addiction3. In the preoccupation/anticipation stage, subjects reinstate drug-seeking after extinction, which could be triggered by drug-associated cues or stressors. In animal studies of drug addiction, conditioned place preference (CPP) paradigms are used to mimic the positive reinforcing effect of drugs4,5,6 and, thus, it is used to model the initial stage of the drug addiction cycle. Morphine, a member of the opioid family, produces a positive reinforcing effect by activating the opioid receptors in the brain7,8.
The CPP paradigm is based on the principle of classical (Pavlovian) conditioning and commonly used to measure the rewarding effect of drugs of abuse, such as morphine4,5,9,10, due to its easy availability across laboratories. A typical CPP paradigm consists of three phases: pre-conditioning, conditioning and test phases. During the conditioning phase, the rewarding properties of a drug (unconditioned stimulus) are paired with one or more neutral contextual stimuli, which gain rewarding properties after several conditioning pairings and serve as conditioned stimuli. During the test phase, the animals&#39; preference for drug-paired compartments are measured and reflected as their reward memory. For this reason, the CPP paradigm is also used to investigate the mechanisms of learning and memory. To establish a long-term reward memory of morphine, variables that affect the establishment of CPP have been adjusted and optimized with the below described protocol11. Contextual stimuli are a combination of visual, tactile and odor cues. Variations of contextual stimuli and testing time points are made according to different research purposes. For example in our previous study12, the somatosensory cues (different floor textures) were mainly used as contextual stimuli to investigate the role of the caudal part of the insular cortex, a brain region with somatosensory function, in the acquisition and maintenance of long-term reward memory. The design of the apparatus and the assignment of rats to preferred/non-preferred sides affect the establishment of CPP. A three-chamber apparatus instead of a two-chamber apparatus is used in the below protocol to avoid forced choices. The balance of two side chambers and the initial assignment of animals into them affect the acquisition of CPP4,13. Thus, a balanced design of the side chambers and biased assignment of animals are applied in this protocol to acquire a strong place preference. Maintenance of long-term reward memory requires more conditioning trials during the conditioning phase. To reduce the time cost during conditioning phase, two conditioning trials per day are carried out and morphine/saline injections are counterbalanced to avoid the acute withdrawal effect of morphine in the saline paired trials4.
Reinstatement animal models14 have been established to model the preoccupation/anticipation stage of drug addiction cycle. CPP is also utilized to study reinstatement that models the relapse to addictive behavior5. Relapse is one of the barriers in the treatment of drug addiction. Craving is one cause of repeated relapse. The craving for drugs increases constantly during the abstinence period, a phenomenon termed incubation of craving15,16. Incubation of craving is widely studied in the drug self-administration paradigm but not the CPP paradigm. The use of the CPP paradigm to reflect incubation of craving was first proposed by Li et al.17. Their results17 showed that the CPP scores of low doses of morphine (1 mg/kg and 3 mg/kg) but not a high dose (10 mg/kg) increased progressively during the first 14 days of abstinence. Similarly, mice injected with a low dose of cocaine showed increased CPP scores over time18. The increased CPP scores of the low doses of morphine were used to reflect incubated craving17. Both duration spent in and number of entries into the drug-associated environment are suggested to reflect the reinforcing conditioned responses in the CPP paradigm19. Most of the recent studies use CPP scores to directly or indirectly reflect time spent in drug-paired chambers while the number of entries to side chambers as a parameter is often neglected. For these reasons, a morphine dose of 10 mg/kg was used and the number of entries was measured in our study11. The results show that entrances of rats to the side chambers increased significantly during the abstinence period11. This indicates that both CPP scores and number of entries are useful parameters in assessing increased craving for drugs and the CPP paradigm is applicable to model the incubation of craving phenomenon. However, to reduce the number of animals required, a group of morphine-experienced animals is tested repeatedly; thus, the effect of extinction could not be excluded with the current within subject design. A between subject design with the same protocol can overcome this limitation.
The following describes the CPP protocol that established long-term reward memory and modeled increased craving. Briefly, after three pre-conditioning days, rats go through six conditioning days with two conditioning trials per day and then are tested at different time points after the last conditioning trial. In this CPP paradigm, a three-chamber polyvinyl chloride (PVC) box used as the CPP apparatus is constructed and the floor of the apparatus is balanced. The apparatus consists of two large black side chambers (30 cm &#215; 25 cm &#215; 30 cm, L&#215;W&#215;H) and one middle white chamber (11 cm &#215; 25 cm &#215; 30 cm, L&#215;W&#215;H) with white smooth PVC floor. The two side chambers have different floor textures as somatosensory cues: one with a grid plexiglass floor, the other with a rough PVC floor.

<table><tbody><tr><td>Morphine</td><td>Shenyang First Pharmaceutical Factory</td><td></td><td></td></tr><tr><td>CPP apparatus/Camera</td><td>Anlai Technology Company</td><td></td><td></td></tr><tr><td>SPSS</td><td>IBM Inc</td><td></td><td></td></tr></tbody></table>

a conditioned place preference protocol for measuring incubation of craving in rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

The following representative results show that a morphine CPP can be successfully established by the protocol described above and long-term morphine reward memory (Figure 2) can be maintained. The number of entries to side chambers, analogous to number of active responses in the self-administration paradigm, reflects the reinforcing property of morphine. The increased number of entries (Figure 3), indicating the incubation of craving, was observed using this morphine CPP paradigm.
CPP memory could last at least 18 days after the last conditioning session.
Twenty-one rats in the morphine group and 10 rats in the saline group were used to test the expression of the long-term rewarding memory. A mixed ANOVA revealed significant differences in the interaction between group and test (F (3,87) = 4.973, p = 0.003), test (F (3,87) = 18.237, p&#60; 0.001) and group (F (1,29) = 11.413, p = 0.002). Significant differences among tests (F (3,60) = 46.628, p &#60; 0.001) in the morphine group but not saline group(F(3,27) = 1.576, p = 0.218) were detected by a one-way repeated ANOVA. The multiple comparison analysis with Bonferroni adjustment showed significant differences between the three tests and T0 in the morphine group. As shown in Figure 2, the morphine reward memory lasted at least 18 days and decreased in a time-dependent trend. Data are obtained from a previous published study11.
Entrances to side compartments in morphine group increased progressively
Morphine group 
To test the trend of entrances, group (saline-paired and morphine-paired) as between-subjects factor and test (T0, T1, T2 and T3) as within-subjects factor were set for a mixed ANOVA. No significant differences in the interaction between group and test (F (2.311,92.449) = 1.915, p = 0.147) and group (F(1,40) = 0.898, p = 0.349), but significance in test (F(2.311,92.499)= 24.243, p &#60; 0.001), were detected. The multiple comparison analysis with least significant difference (LSD) test showed significant differences between T1 and T0 (p&#60;0.001), T2 and T0 (p &#60; 0.001) and T3 and T0 (p &#60; 0.001) in the morphine group in morphine-paired entrances. Concerning saline-paired compartments, statistical significances existed between T2 and T0 (p = 0.002) and T3 and T0 (p = 0.003) but not T1 and T0 (p = 0.246), which is similar to the pattern of incubation of heroin craving24. In general, entrances to side chambers in the morphine group increased time-dependently.
Rats entered more frequently to the morphine-paired compartment than saline-paired compartment at T1 (t(1,21) = -2.833, p = 0.010) and T2 (t(1,21) = -4.458, p = 0.0002) but not T3 (t(1,21) = -0.471, p = 0.642) (two-tailed paired t-tests).
Saline group 
Similar with the morphine group, a mixed ANOVA was applied and revealed no significant differences in the interaction between group and test (F(3,54) = 0.345, p = 0.793), test (F(3,54) = 1.793, p = 0.159) and group (F(1,18) = 0.151, p = 0.702).Thus, entrances to side chambers in the saline group were stable over time.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58384/58384fig2.jpg" /> 
Figure 2: CPP memory can last at least 18 days but declines with time. Memory retention of CPP in the morphine group (n = 21) and the saline group (n = 10) was assessed by measuring the CPP score for several time points after last conditioning (T1, 2 days; T2,10 days and T3, 18 days). All values of CPP scores are mean &#177; SEM. ### indicates p&#60;0.001 and ## means p &#60; 0.01 compared with saline group; *** indicates p &#60; 0.001 compared with T0 in the morphine group. This figure has been modified from Sun et al.,201711. <a href="https://www.jove.com/files/ftp_upload/58384/58384fig2large.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/58384/58384fig3.jpg" /> 
Figure 3: Entrances to side-chambers in morphine and saline groups. Entrances to two side chambers in the morphine group (n = 21) and the saline group (n = 10) were assessed at several time points after the last conditioning (T1, 2 days; T2,10 days and T3, 18 days) and compared with T0. All values of entrances are mean &#177; SEM. In the morphine group (A), entrances to the morphine-paired side increased very significantly from T1 to T3 while entrances of saline-paired chamber only increased significantly from T2. Paired t tests with morphine-paired compartment and saline-paired compartment showed very significant increases in both T1 and T2. In the saline group (B), there are no significant changes in entrances to both sides. This figure has been modified from Sun et al.,201711. <a href="https://www.jove.com/files/ftp_upload/58384/58384fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...

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16.9K Views.  Chinese Academy of Sciences. Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

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

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

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 ...

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

Neuroscience

Fat Preference: A Novel Model of Eating Behavior in Rats

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

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

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

A Procedure 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 ...

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

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

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

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

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

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.

This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to ...

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

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

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

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

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 ...