Name: A Conflict Model of Reward-seeking Behavior in Male Rats (Video) | JoVE Journal
Uploaded: 2019-02-20T15:00:00.000+00:00
Duration: 6 min 11 s
Description: Explore a conflict model for assessing inhibitory control impairment in male rats after drug exposure. This method evaluates reward-seeking behavior using sexual stimuli and aversive obstacles without requiring prior learning.

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This paper was supported by CAS Key Laboratory of Mental Health, Institute of Psychology (KLMH2016K01) and Evaluation and Intervention Technology Research for Post-traumatic Stress Patients Population (JCYJ20170413170301569)

This study is approved by the International Review Board (IRB) of the Institute of Psychology, Chinese Academy of Sciences, and all experiments are conducted in accordance with the National Institutes of Health (U.S.A) Guide for Care and Use of Laboratory Animals (2011).
1. Materials and Setup for the Conflict Model
<ol>
	<li>House four rats per cage (50 cm long x 22.5 cm wide x 30 cm high) in colony rooms with controlled temperature (22&#8211;25 &#176;C) and a reversed 12 h/12 h light/dark cycle (lights on at 21:00) for at least 10 days. 
	NOTE: Male and female Sprague-Dawley rats weighing 330&#8211;400 and 230&#8211;250 g at the beginning of the experiments, respectively were used.</li>
	<li>Perform all the tests under dim lighting during the dark phase.</li>
	<li>Use the opaque cartons (37 cm long x 26 cm wide x 18 cm high) with pine wood shaving bedding to transport rats from home cages to testing rooms.</li>
	<li>Handle rats 3 min each per day for 5 days prior to beginning the experiments.</li>
	<li>Prepare 120 mL of morphine for the whole experiment. Dissolve morphine hydrochloride in 0.9% NaCl (physiological saline) at a final concentration of 20 mg/mL. Store at room temperature (RT).</li>
	<li>Prepare 10 mL of the pentobarbital sodium solution for the surgery. Dissolve pentobarbital sodium in 0.9% NaCl at a concentration of 1 g/mL. Store at 4 &#176;C.</li>
	<li>Prepare 10 mL of estradiol benzoate (EB) and 10 mL of progesterone for artificial induction of estrus. Dissolve estradiol benzoate and progesterone in sesame oil at a concentration of 0.125 mg/mL and 5 mg/mL, respectively. 
	NOTE: Incubate the oil suspension of EB or progesterone in a water bath (55&#8211;60 &#176;C) for at least 1 h and then thoroughly shake it. Ensure that EB or progesterone is dissolved completely. Store at RT.</li>
	<li>For the risky reward-seeking behavior testing, use the open-field reward-proximity chambers made of black acrylic glass. At one end of the open-field arena (85 cm long x 35 cm wide x 50 cm high) mount a wire-screen stimulus cage (15 cm long x 25 cm wide x 25 cm high). Install the board with pins on the floor about 20 cm in front of the stimulus-cage. 
	NOTE: Pins are fixed on the 34.5 cm long x 13 cm wide boards (Figure 1). The boards with three types of pins are used in turn during testing (Table 2).</li>
</ol>
2. Estrus induction in Females and Mating Screening in Males
<ol>
	<li>Bilateral ovariectomy in female rats 
	NOTE: The surgical instruments and other items used in surgery are sterile. And surgery is performed using aseptic technique.
	<ol>
		<li>Prepare surgical instruments and materials such as scalpels, surgical blades, hemostatic forceps, tweezers, ophthalmic scissors, gauze, cotton swabs, suture needles and silk sutures in advance, as well as 75% alcohol, iodine, sodium pentobarbital, 0.9% NaCl, penicillin sodium.</li>
		<li>Intraperitoneally inject pentobarbital sodium (55 mg/kg), waiting for complete anesthesia of female rats. Rats respire smoothly and do not react to tail pinch. 
		NOTE: The ovariectomy is performed only when the weight of female rat reaches at least 240 g.</li>
		<li>Place the female rat in prone position, shave off the fur on the back, and disinfect the exposed skin with iodophor. Then use a scalpel to make a middle vertical incision (about 2 cm long) on the back (1 cm below the edge of the ribcage).</li>
		<li>Pull the skin to the left side, and blunt dissect subcutaneous tissue with hemostatic forceps to expose the lumbar muscles. Cut the muscle layer (incision of 1 cm) into the abdominal cavity, till the adipose tissue is reached.</li>
		<li>Pull out the adipose tissue with bent tweezers, and locate the ovary which is a flesh-pink tissue (about 0.5 cm x 0.4 cm x 0.3 cm) surrounded by adipose tissue with the twining fallopian tube on the surface.</li>
		<li>Clamp the fallopian tube with forceps and ligate it, then cut off the ovary together with the surrounding adipose tissue.</li>
		<li>Put the residual fallopian tube and adipose tissue back into the abdominal cavity after ensuring no bleeding, and suture the muscle layer.</li>
		<li>Remove the ovary on the other side by the same procedure and then suture the skin incision.</li>
		<li>Place the rat on a heated blanket till awakening and then put it back into the home cage.</li>
		<li>After at least two weeks of recovery, use the female rats as a tool for mating screening and behavioral testing. 
		NOTE: Pay attention to the state of the female rat after surgery, and supply adequate water and food. Keep the ovariectomized rats single housed for one week and then house four per cage.</li>
	</ol>
	</li>
	<li>Induction of estrus in ovariectomized female rats
	<ol>
		<li>Handle all ovariectomized female rats three times (3 min/day) before using them for mating screening.
		<ol>
			<li>Gently pick up a rat from the transport box with the left hand and hold it in the arms for a few seconds. Then put the rat back into the box and repeat these operations for 3 min.</li>
		</ol>
		</li>
		<li>Subcutaneously inject estradiol benzoate (25 &#956;g/rat) about 48&#8211;52 h before mating screening or the conflict test.</li>
		<li>Subcutaneously inject progesterone (1 mg/rat) about 4&#8211;6 h before mating screening or the conflict test. 
		NOTE: The estrogenic hormones are injected subcutaneously on the back of the neck. Since an estrus cycle lasts for ~4&#8210;5 days, the female rats are used once a week.</li>
	</ol>
	</li>
	<li>Screening male rats for mating performance 
	NOTE: Screening is conducted under dim light during the dark phase of the light/dark cycle in the housing room.
	<ol>
		<li>Place a male rat individually into the carton (60 cm long x 50 cm wide x 40 cm high) with pine wood shaving bedding and leave it to habituate for 5 min.</li>
		<li>Introduce an estrous female rat into the carton and monitor male copulatory behaviors (by experienced observers).</li>
		<li>Put the rats back to home cages after male rats complete their first ejaculation within 30 min or do not display intromission within 15 min or ejaculation within 30 min.</li>
		<li>Assign the male rats that pass the screening (successful ejaculation for three consecutive days) randomly into different groups (such as the saline- and morphine-treated groups).</li>
	</ol>
	</li>
</ol>
3. Pretreatment in Male Rats Prior to the Conflict Test
<ol>
	<li>Binge-like morphine treatment 
	NOTE: Male rats are intraperitoneally injected with saline or morphine delivered in a binge-like regimen14 (Table 1).
	<ol>
		<li>Weigh the male rats and calculate the injection volume for each rat based on the body weight (see Table 1).</li>
		<li>Prepare syringes with morphine or saline solutions.</li>
		<li>Inject one rat at a time intraperitoneally and immediately place it gently into the home cage (4 rats/cage).</li>
		<li>After at least 6 h, give the male rats the second injection in the same way.</li>
	</ol>
	</li>
	<li>Acute stress 
	NOTE: Foot shocks are delivered prior to each conflict test in four identical chambers assembled with four shock generators and controlled by professional software installed on a computer.
	<ol>
		<li>On the day of the conflict test, take the male rats to another room different from the conflict testing room.</li>
		<li>Put the male rats into the chambers (30.5 cm long x 25.4 cm wide x 30.5 cm high) to habituate for 1 min.</li>
		<li>Set up the software program in advance. The program includes the intermittent foot shocks delivered within 10 min (0.5 mA x 0.5 s x 10 min; mean inter-shock interval 40 s, range 10&#8211;70 s).</li>
		<li>Enter animal IDs and choose whether to turn on the shock generators according to the grouping (the shock group and the control group). Then press the start button.</li>
		<li>When the stress procedure is finished, bring the rats immediately to the conflict test room in transport boxes; one rat per box.</li>
	</ol>
	</li>
</ol>
4. The Conflict Test
NOTE: The test is conducted under dim light during the dark phase of the light/dark cycle in the conflict test room.
<ol>
	<li>On the day before testing, bring all rats to the conflict test room and allow them to habituate to the open-field reward-proximity chamber (without any obstacle, Figure 1) for 15 min.</li>
	<li>On the testing day, place the male rat in the chamber allowing free exploration for 10 min (under the same condition as the day before testing).</li>
	<li>Place an estrous female rat in the stimulus-cage as an incentive and allow the male subject to freely approach and investigate the incentive rat for 5 min. 
	NOTE: The male rat is randomly exposed to an estrous female, and this female is not a familiar rat.</li>
	<li>After 5 min-free approach, move the male subject from the stimulus-cage to the other end of the arena, place an obstacle (a 14 cm-wide board thick with pins), and then start the first trial of the test. 
	NOTE: The difficulty level of obstacles is varied among trials based on the types of pins and the height of the board. The grading system is shown in Table 2.</li>
	<li>Move the male rat away from the stimulus-cage about 15&#8211;20 s after each time it surmounts the obstacle.</li>
	<li>End a trial if the male subject climbs or jumps over the obstacle 3 times within 4 min and immediately start the next trial with increasing difficulty of the obstacle.</li>
	<li>If a male subject surmounts the obstacle less than three times within 4 min, end the test and record the times it surmounts the obstacle.</li>
	<li>Bring the male rat back to the home cage and scrub the open-field chamber with 0.05% glacial acetic acid.</li>
	<li>Score each surmounting (or approaching) as per Table 2. Use the sum of the scores for all of the surmountings as a total score for a male subject in this conflict test.</li>
</ol>
5. Statistical Analysis
<ol>
	<li>Present the data as mean &#177; SEM or single data points. In case that homogeneity of variance or normal distribution of the datasets is challenged, log-transform the data sets.</li>
	<li>Analyze the effects of morphine pretreatment on reward-seeking behavior displayed on day 7 and day 17 of withdrawal (Wd7 and Wd17) using t-tests with &#8220;pretreatment&#8221; as a between-subjects factor (morphine versus saline, Figure 2).</li>
	<li>Analyze the effects of morphine pretreatment on reward-seeking behavior after repeated testing using repeated-measure&#8217;s analysis of variance (ANOVA) with &#8220;withdrawal time&#8221; (Wd7 versus Wd14) as the within-subjects factor and &#8220;pretreatment&#8221; (morphine versus saline) as a between-subjects factor.</li>
	<li>In addition, use Pearson&#8217;s correlation to analyze the correlation between the scores that male subjects acquired on days 7 and 14 of abstinence (Wd7 and Wd14, Figure 3). 
	NOTE: After the logarithmic conversion of the original data, statistical analyses are performed.</li>
	<li>Analyze the effect of acute stress on reward-seeking behavior in drug-na&#239;ve rats using t-test with stress as a between-subjects factor (shock versus control, Figure 4)</li>
</ol>

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</ol>

The authors have nothing to disclose.

The inhibitory control deficits caused by drug abuse18 play a key role in promoting compulsive drug seeking/taking behaviors and relapse19,20. The conflict model presented here provides a new approach to explore the changes in inhibitory control of the individuals exposed to addictive drugs.
There are several critical steps in the protocol. First of all, the subjects (male rats) must acquire sexual experience be...

Drug addiction is a chronic brain disease which is characterized by impulsive and compulsive drug seeking and taking1. These key features of addiction have both been hypothesized to result from the impaired ability of inhibitory control2,3, i.e., failing in inhibiting the immediate pursuit of rewarding stimuli and thus developing maladaptive patterns of behavior4.
The go/no-go task and stop-signal task are the prototypical tasks used to measure the ability of response inhibition2,5. These two experimental paradigms assess one&#39;s ability to suppress actions that are inappropriate, by contrasting infrequent inhibitory responses against an implicit go baseline6,7. The response inhibition displayed in these tasks has been shown to be impaired in cocaine users8,9, opiate addicts10, and nicotine users11. Another two tasks&#8212;reversal learning and multiple choice serial reaction time tasks&#8212;also provide measurements of response inhibition/inhibitory control12,13. However, most of these paradigms performed in rodents not only require long-term training so that subjects can distinguish the response requirements represented by different signals, but also the individual differences in learning speed and learning effects may interfere with the results of subsequent inhibitory test11.
In this paper, we present a novel conflict task which can be used to measure the impairment of inhibitory control after exposure to addictive drugs. In this task, a natural rewarding stimulus (sexual stimulus) that represents a high-value reward14, and the aversive stimuli (pins) that male rats have to conquer, are concurrently presented. The male rats have to climb or jump over the obstacle full of pins to approach and investigate the sexual partner. If the animal persists in its approaching behavior regardless of the aversive stimuli, it is considered as a maladaptive or risky reward-seeking behavior. One of the rationales for establishing this task is that it is conceptually simple and does not place heavy demands on executive processes as other tasks do. Compared to other tasks which measure response inhibition, this conflict task is based on natural behavior, and rats with normal sexual function and sexual experience can be tested directly without a learning process. Another rationale is that a conflict presented in this task between approaching the reward and avoiding the aversive stimuli (or the risk of being pricked) may have a better validity, as it mimics what occurs in addicts who often place themselves in the similar conflict but persistently pursue drug reward regardless of the risk of negative consequences in real life15.
Therefore, application of this conflict model is a quick and sensitive way to discover the deficit in inhibitory control after exposure to addictive drugs, or other factors that may influence ability of inhibitory control, such as stress. It also provides a novel behavioral strategy for investigation of neural mechanisms underlying deficits in inhibitory control. Furthermore, alternative modifications can be added onto this task. For example, altering the cost/benefit ratio by replacing the sexual stimulus with the social stimulus can reveal more behavioral significances.

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a conflict model of reward-seeking behavior in male rats

A Conflict Model of Reward-seeking Behavior in Male Rats

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual stimulus) that represents a high-value reward, and the aversive stimuli (pins), are concurrently presented. The male rats have to climb or jump over the obstacle full of pins to approach and investigate the sexual partner. If the animal persists in their approaching behavior regardless of the aversive stimuli, it is considered as a maladaptive or risky reward-seeking behavior. The conflict task permits the evaluation of deficit in inhibitory control resulting from exposure to abused drug, such as morphine, or a stressful event. 
The main advantage of this model is that it provides a simple and quick way to discover the deficit in inhibitory control after exposure to opiate drugs or other stressful events. In addition to opiates, this behavioral model would also be useful for quickly discovering the inhibitory control deficits induced by other addictive drugs. However, the limitation is that the male rats' performance may be subject to exercising effects with repeated testing under this conflict task. In the future, one can hope that the individuals with the compulsive phenotype of reward-seeking behavior after exposure to opiates will be identified based on modifying this conflict model.

To explore whether this conflict model can reveal maladaptive/risky reward-seeking behavior induced by opiates, the reward-seeking behaviors displayed by the saline- and morphine-pretreated groups were compared by t-tests after short-term (Wd7) and long-term (Wd17) withdrawal from morphine respectively (Figure 2). The results show that on both day 7 and day 17 of withdrawal, the morphine-pretreated rats showed significantly more approaching behaviors than the...

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.

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

a-conflict-model-of-reward-seeking-behavior-in-male-rats

Research

JoVE Journal

Behavior

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Explore a conflict model for assessing inhibitory control impairment in male rats after drug exposure. This method evaluates reward-seeking behavior using sexual stimuli and aversive obstacles without requiring prior learning.

A Conflict Model of Reward-seeking Behavior in Male Rats

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