The authors gratefully acknowledge Dr. R. Bilyar, Resident, Urology Department, Soroka Medical Center, for his help in the laboratory as well as in the video analysis. The support of Shira Ovadia, Director of Animal Resources Unit, is also gratefully acknowledged. Many thanks to A. Alir and to the staff at the Critical Care Unit, Soroka Medical Center for their support and helpful discussions.

The following procedure was conducted according to the recommendations of the Declaration of Helsinki and Tokyo and the Guidelines for the Use of Experimental Animals of the European Community. The experiments were also approved by the Animal Care Committee at the Ben-Gurion University of the Negev.
1. Preparing Rats for the Experimental Procedure
Note: For the experimental procedure, select male Sprague-Dawley rats with no overt pathology, each weighing between 300 and 350 g.
<ol>
	<li>House three rats per cage, with chow and water available ad libitum, and allow for at least two weeks of adaptation, alternating between 12 h of daylight and 12 h of darkness.</li>
	<li>Before initiation of the experiment, test all rats for the presence of depressive-like behaviors using a 3-day sucrose preference test (refer to step 4).</li>
	<li>Exclude rats that are perceived to display depressive-like behaviors and those that are housed in the same cages as such rats, so as to avoid possible spread of depression to unaffected rats before the experiment has begun first, eliminate rats that test positive for depression; then, eliminate rats that are housed in the same cages as the depressed rats.</li>
	<li>If necessary, exclude additional rats at random to create final groups of 60 rats for the depression experiment, 30 rats for the contagion group, and 30 ratsforthe control group.</li>
	<li>Tag rats (using numbered stickers), throughout the experimental procedures. Temporarily transfer rats to single cages during the experiments (in the same room and dark cycle for the CUS, Sucrose and open field tests, and in a different room and dark cycle for the forced-swim test and for one of the stressors (refer to 2.1.7)) and return them to their original social groups at the end.</li>
</ol>
2. The Procedure for the Induction of Depression in Rats
Note: In order to induce depression, subject the 60 rats in the CUS-exposed group to several manipulations of CUS for five weeks20.
<ol>
	<li>Expose rats daily to any 2 of the following 7 stressors, in a random order; to one stressor during the day and to the other at night, for 5 consecutive weeks.
	<ol>
		<li>House the rats in groups (six rats, instead of the usual three, per cage for 18 h);</li>
		<li>Place the rats in a tilted cage (45&#176; along the vertical axis for 3 h);</li>
		<li>Deprive the rats of food (18 h);</li>
		<li>Deprive the rats of water (18 h) and expose them to empty water bottles immediately following a period of acute water deprivation (1 h);</li>
		<li>Place the rats in a wet bedding cage (with 300 mL of water spilled in the bedding) for 8 h (night cycle);</li>
		<li>Expose the rats to continuous lighting (24 h) and reversed light/dark cycle (12 h) twice per week;</li>
		<li>Place the rats in a hot environment (40&#176;C, 5 min - night cycle), with another apparatus used to increase the heat.</li>
	</ol>
	</li>
	<li>Confirm the development of depression by performing a sucrose preference test (refer to step 4). 
	Note: The protocol can be paused after each of the above steps.&#160;Additionally, if any aberrant behavior is displayed by any rat, then, eliminate the rat and any other rat that might have been affected by the erratic rat.</li>
</ol>
3. The Procedure for Establishing Depression-Contagion in Na&#239;ve Rats
<ol>
	<li>Establish 30 social groups with rats from the CUS-exposed (60) and depression-contagion (30) groups:
	<ol>
		<li>House two CUS-exposed rats per cage in 30 different cages.</li>
		<li>Add one rat from the depression-contagion group to each cage containing two CUS- exposed rats.</li>
	</ol>
	</li>
	<li>Allow cohabitation for 5 weeks , under standard conditions,with chow and water available ad libitum.</li>
	<li>After 5 weeks of cohabitation, subject all groups to the sucrose preference, open field, and forced-swim tests in the order shown in Figure 1.</li>
</ol>
4. The Sucrose preference test21
<ol>
	<li>During the dark cycle, place the rats in individual cages in the same room as the housing.</li>
	<li>Place a bottle of 100 mL 1% (w/v) sucrose solution in each cage for 24 h, and allow for adaptation.</li>
	<li>Remove the bottles and deprive the rats of food and water for 12 h.</li>
	<li>Place two bottles, one containing 100 mL of sucrose solution (1 %, w/v) and the other, 100 mL of tap water, in each cage for 4 h.</li>
	<li>Record the volume (in mL) of both the consumed sucrose solution and water, and calculate the affinity to sucrose preference as follows: 
	<img alt="Equation 1" src="/files/ftp_upload/57137/57137eq1.jpg" /></li>
</ol>
5. The Open Field Test
Note: The standard open field test is commonly used to assess locomotor, exploratory, and anxiety-like behaviors, and behavioral responses to novelty in laboratory animals22, 23. This test has also been shown to analyze depressive behaviors24. The open field test examines two opposing parameters; the rodents&#39; fear for the bright light shining at the center of the field against their expressive desire towards novelty. When anxious, rodents tend to avoid discovery and stay put by the walls (thigmotaxis). This anxiety is determined by the degree of averting the brightly lit central field. The open field, consisting of a black lusterless acrylic box (120 cm &#215; 60 cm &#215; 60 cm), is divided into the central part (25%) and the rest of the box (75%). This test takes place in the same room as the housing during the dark cycle. A video camera, suspended about 200 cm above the field, is used for recording the open field test22, 23.
<ol>
	<li>Clean the apparatus with 5% alcohol prior to the introduction of each animal. Place the rat at the corner of the open field facing the wall.</li>
	<li>Record the rat&#39;s behavior with a video camera for 5 min.</li>
	<li>Analyze post-recordings of the following parameters: total travel distance, velocity in the central part of the field, and time spent in the central part of the field.</li>
</ol>
6. The Forced-Swim Test
Note: The principle of the forced swim test is based on the fact that when rats are forced to swim in a confined space, they eventually give up and remain immobile, occasionally moving their bodies in order to avoid drowning21, 23. Due to the enormity of the already assembled apparatus, this test was carried out in the different room during the dark cycle.
<ol>
	<li>For habituation, place the rat in a glass cylinder (100 cm tall, 40 cm in diameter, and 40 cm deep) containing water at room temperature, for a 15 min swimming session.</li>
	<li>Remove the rat from the cylinder, dry with paper towels, and place in a warm cage for 15 min.</li>
	<li>Return the rat to its home (original) cage; 
	Note: The protocol can be paused here.&#160;Also, if any rat in the course of this test is drowning, pull it out immediately.</li>
	<li>On the following day, videotape a 5-min swim session and analyze post-recordings of the following parameters: Immobility, Climbing, and Defecation.</li>
</ol>

The authors have nothing to disclose.

According to the results obtained with the application of this protocol, healthy rats, like humans, were negatively affected by depressed rats when housed together over an extended period. The contagiously depressed rats were affected by their already depressed counterparts after five weeks of cohabitation, establishing a distinct animal depression-contagion model for the first time. An earlier study with pigs had also suggested shared emotional statutes between depressed and healthy pigs25.
The induction of depression in rats was achieved with the application of CUS. This method expresses features common to every day&#39;s socio-environmental stressors. Archives show that CUS is the most used method of inducing depression in animals. The results obtained with this method share strong similarities with clinical symptoms and depressive-like behaviors. If it is properly used on rats, the rats canexpress all known depressive signs26. The ability to bring out all depressive behaviors when applied on rats validates CUS as a reliable depression-inducing method with high predictability and construct validity27, 28. The CUS method is equally used as an animal depression model to analyze cellular and molecular mechanisms of depression&#39;s pathophysiology, as well as to study the mechanism of action of antidepressants29,30,31,32,33.
The sucrose preference test applied after cohabitation of depressed and na&#239;ve rats enabled the evaluation of the spread of depression, resulting in the expression of depressive behaviors in both groups of rats after 5 weeks (Figure 2, Figure 3, Figure 4). The forced-swim test20 and the open field test34,35,36,37,38,39,40 were also used to investigate depression in rats41. These methods mirror an extensive range of behavioral irregularities. The fact that there is no specific mechanism of contagion means that conscious and unconscious components could be advanced as potential hypothetical outcomes. Unconsciousness could manifest through mimicry42, by way of healthy rats copying body movements of depressed rats. Movements most likely to be reproduced are such as facial expressions and the neuron system43. Consciousness, on the other hand, could arise by means of communication methods. One such method is co-rumination44.
Although the healthy rats became depressed when sharing housing with the depressed rats, depressed rats also became less depressed, compared to their state after CUS, as a result of cohabiting with their healthy counterparts. This follows a pattern that was observed in earlier findings when depressed college students were able to contagiously become undepressed after spending time with their healthy roommates4. There is, therefore, not only a negative impact with contagious depression, but also a reciprocal effect. Depressed individuals have a negative bearing on healthy ones, while they themselves slightly recover from depression by virtue of being exposed to non-depressed colleagues. Such a finding might be a boost to psychiatry in tackling both depressed individuals and populations.
Prior to this test, animal models of depression-contagion were nonexistent, and even though a breakthrough was made here, this test has its limitations. It can be considered somewhat weak for a few reasons. The difference between the sucrose uptake and water uptake in the supposedly depressed rats, when compared to controlled rats, is not highly significant and is not demonstrative of a highly depressed state in the experimental rats. Also, the use of the forced-swim test would not be advisable if better alternative options can be found. The damages caused by this test are traumatic, permanent, and irreversible. Consequently, animals subjected to the forced-swim test cannot be recycled or used for further experiments. Regardless of the drawbacks of the method, it remains a pioneering method that produced decent results.
Much has been said and done to come to terms with depression, but it has remained a major cause for concern, with the increasing number of life-threatening situations highlighting its severity in society today. The complex nature defining the interactions between humans makes it problematic to systematically evaluate depression contagion. Thus, an advanced understanding of an animal model could be key to unlocking the mechanism of depression in the human population, subsequently establishing therapeutic answers to this problematic disease.

Tests carried out in recent times have suggested that psychiatric illnesses could be easily spread to healthy individuals via contagion1. In this case, it is termed social contagion and is spread through affect, attitude, or behavior. This only requires one depressed individual to interact with one or more healthy individuals, thereby facilitating the sharing of emotion. Social relationships are hence a very important component of mood, as they define the transfer of emotions from one individual to the other, by way of mimicry and &#34;emotional contagion&#34;. Time frames for contagion to take effect vary2, inevitably depending on the severity of the emotion and the strength of resistance of the recipient.
The significant consequences of emotional contagion have ensured that studies made in the past mainly focused on the negative aspects. The outcome of negative effects ensured contagion depression received enormous attention, with studies showing that contagion depression increases the likelihood of families and friends of a depressed individual exhibiting depressive behaviors3,4,5,6.
There are both personal and economic reasons for tackling depression. It commonly causes morbidity; and its lifetime incidence is between 13.3 and 17.1% in the United States7. The World Health Organization files show that depression is fourth on the list of global diseases with huge burdens, occurring in people of all genders, ages, social backgrounds, and is equally capable of inflicting poor health, impacting the ability to interact with others8,9,10,11, and causing excess disability12,13. 850,000 lives are estimated to be lost to depressive suicide each year14. Patients are typically prescribed antidepressant medications or advised to undergo cognitive behavioral therapy. These treatmentshelp about 60-80% of patients. However, dealing with the disease still poses a big problem; treatment is not available to all depressed patients. For those who get treatment, some suffer side effects, while others poorly comply with guidelines15. The number of patients resistant to treatment is up to about 40%14. With depression, the economy has frequently suffered in the form of expensive treatment, a decrease in the workforce, and early retirements16. An estimated annual loss of $44 billion in the United States is caused by depression, accounting for almost half of the country&#39;s lost productivity17. Expensive treatment requires careful medical attention, which incurs a variety of increased medical costs and necessitates anticipation of undesirable outcomes, as well as poor response to treatment18.
Having not come across an already proven animal model to study the depression-contagion mechanism, its prevention, and treatment, this hypothetical animal protocol was used for the first time. It suggests that through cohabitation with CUS-exposed rats, healthy rats tend to express depressive behaviors. The main objective of this experiment was to establish a laboratory procedure that highlighted the transfer of depression, via contagion, from CUS-exposed rats to healthy ones. Next, results were evaluated to determine if depression-contagion was limited only to depressive symptoms, or if it was related to other mood disorders, such as anxiety. The ultimate goal of the experiment is to draw closer to better understanding the mechanism of depression-contagion in the race to develop new therapeutic approaches19.

<table border="0" cellpadding="0" cellspacing="0" style="width:849px;" width="849">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Rat Cages</td>
			<td style="width:123px;">Techniplast</td>
			<td style="width:344px;">2000P</td>
			<td style="width:167px;">Conventional housing for rodents. Was used for housing rats throughout the experiment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Water</td>
			<td>-</td>
			<td>-</td>
			<td style="width:167px;">Common tap water used througout the experiment at different stages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Purina Chow</td>
			<td>Purina</td>
			<td>5001</td>
			<td style="width:167px;">Rodent laboratory chow given to rats, mice and hamster is a life-cycle nutrition that has been used in biomedical researc for over 5 decades. Provided to rats ad libitum in this experiment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bottles</td>
			<td>Techniplast</td>
			<td>ACBT0262SU</td>
			<td style="width:167px;">150 ml bottles filled with 100 ml of water and 100 ml 1%(w/v) sucrose solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Black lusterless perspex box</td>
			<td>-</td>
			<td>-</td>
			<td style="width:167px;">(120 cm &#215; 60 cm &#215; 60 cm), divided into a 25% central zone and the surrounding border zone</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Video Camera</td>
			<td>Canon</td>
			<td>-</td>
			<td style="width:167px;">Digital video camera for high definition recording of rat behavior under open field test</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alcohol</td>
			<td>Pharmacy</td>
			<td>-</td>
			<td style="width:167px;">99% pharmaceutical alcohol diluted to 5% and used for lceaning the open field test box before the introduction of each rat</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass cylinder</td>
			<td>-</td>
			<td>-</td>
			<td style="width:167px;">100 cm tall, 40 cm in diameter, and 40 cm deep cylinder used for carrying out the forced swim test</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paper towels</td>
			<td>Pharmacy</td>
			<td>-</td>
			<td style="width:167px;">Dry towels used for keeping rats dry after immersing them in water</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bold markers</td>
			<td>-</td>
			<td>-</td>
			<td style="width:167px;">Common bold markers used for labeling rats</td>
		</tr>
	</tbody>
</table>

a new method for inducing a depression-like behavior in rats

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is no existing format for studying the mechanism of action, prevention, containment, and treatment of contagious depression. The purpose of this study, therefore, was to establish the first animal model of contagious depression.
Healthy rats can contract depressive behaviors if exposed to depressed rats. Depression is induced in rats by subjecting them to several manipulations of chronic unpredictable stress (CUS) over 5 weeks, as described in the protocol. A successful sucrose preference test confirmed the development of depression in the rats. The CUS-exposed rats were then caged with na&#239;ve rats from the contagion group (1 na&#239;ve rat/2 depressed rats in a cage) for an additional 5 weeks. 30 social groups were created from the combination of CUS-exposed rats and na&#239;ve rats.
This proposed depression-contagion protocol in animals consists mainly of cohabiting CUS-exposed and healthy rats for 5 weeks. To ensure that this method works, a series of tests are carried out - first, the sucrose preference test upon inducing depression to rats, then, the sucrose preference test, alongside the open field and forced-swim tests at the end of the cohabitation period. Throughout the experiment, rats are given tags and are always returned to their cages after each test.
A few limitations to this method are the weak differences recorded between the experimental and control groups in the sucrose preference test and the irreversible traumatic outcome of the forced swim test. These may be worth considering for suitability before any future application of the protocol. Nonetheless, following the experiment, na&#239;ve rats developed contagion depression after 5 weeks of sharing the same cage with the CUS-exposed rats.

The sucrose preference test: After exposing rats to 5 weeks of CUS for induction of depression, and then subsequently exposing healthy rats to the CUS-exposed rats for a further 5 weeks, both sets of rats displayed depressive-like behaviorsat the end of the experiment (Figure 2). The evidence of this behavior was seen in the reduced preference for sucrose by the depressed rats, after CUS (65 &#177; 2.8%, p &#60; 0.001, Figure 2), when compared to the 30 rats in the control group (101 &#177; 7%, (Figure 2). After 10 weeks, the 60 CUS-exposed rats demonstrated a statistically significant change (72 &#177; 3.3%, p &#60; 0.001, Figure 2) when compared to the 30 rats in the control group (95 &#177; 3.4%,&#160;Figure 2), as did the 30 depression-contagion rats (76 &#177; 4.7% p &#60; 0.001, Figure 2), after 5 weeks of cohabitation with the CUS-exposed rats.
The open field test: Rats in both the CUS-exposed and depression-contagion groups had decreased total travel distances (depression-contagion - p &#60; 0.05 and CUS-exposed - p &#60; 0.01, Figure 3A), and decreased mean velocities (CUS-exposed - p &#60; 0.05,&#160;Figure 3C), compared to the control group. The time spent at the center of the open fieldby rats from the control group and the two experimental groups was non-significantly different (Figure 3B).
The forced-swim test: Results from the evaluation of immobility were as expected. Both CUS-exposed and depression-contagion rats showed extended immobility following the forced swim test. However, a significantly extended time of inactivity was registered only in the CUS-exposed rats (p &#60; 0.01) when compared with the control group (Figure 4A). In another investigated forced-swim test parameter, climbing time, both groups of CUS-exposed and contagion-depression rats exhibited highly limited climbing properties (p &#60; 0.01) when compared to the control group (Figure 4B). The assessment of the rate of defecation resulted in the registration of high quantities of feces for both experimental groups (p &#60; 0.01), compared with control (Figure 4C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57137/57137fig1.jpg" /> 
Figure 1: The experimental protocol design. Shows the order and timing of the respective experimental protocols. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57137/57137fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57137/57137fig2.jpg" /> 
Figure 2: The sucrose preference test. The percentage change in the sucrose preference test significantly decreased in the 60 CUS-exposed rats (65 &#177; 2.8% vs. 101 &#177; 7%, p &#60; 0.001) compared to the 30 rats in the control group, after 5 weeks of CUS. There was a significant percentage change in the sucrose preference test in both the 60 CUS-exposed (72 &#177; 3.3%, p &#60; 0.001) and 30 depression-contagion (76 &#177; 4.7%, p &#60; 0.001) rats after 10 weeks, compared to the 30 rats in the control group (95 &#177; 3.4%). Statistical analysis was done with one-way ANOVA with Bonferroni&#39;s post hoc test. The data are presented as percentages of the baseline and expressed as mean &#177; SEM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57137/57137fig2large.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/57137/57137fig3.jpg" /> 
Figure 3: The open field test. Both the depression-contagion (68.1 &#177; 6.5%, p &#60; 0.05,&#160;Figure 3A) and CUS-exposed rats (59.6 &#177; 5.7%, p &#60; 0.01,&#160;Figure 3A) had decreased total travel distances, compared with control rats (100 &#177; 13%, Figure 3A). No significant differences were found for time spent by all 3 groups of rats in the central part of the open field (Figure 3B). The CUS-exposed (75.4 &#177; 6%, p &#60; 0.05,&#160;Figure 3C) and depressed-contagion (88 &#177; 5.6%, p &#60; 0.005,&#160;Figure 3C) rats both had decreased mean velocities, but a significant change was recorded only in the CUS-exposed group, compared to rats in the control group. In all 3 tests, the Kruskal-Wallis, followed by Mann-Whitney test were used. The data are presented as percentages of the control group and expressed as mean &#177; SEM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57137/57137fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57137/57137fig4.jpg" /> 
Figure 4: The Forced-swim test. The CUS-exposed (151 &#177; 3.3%, p &#60;0.01,&#160;Figure 4A) and depression-contagion (107 &#177; 6.7%, Figure 4A) rats were both found to be highly immobile, but a significant change was recorded only in the CUS-exposed group, compared to the rats in the control group. The CUS-exposed (46 &#177; 5.5%, p &#60; 0.01,&#160;Figure 4B) and depression-contagion (64 &#177; 5.4% p &#60; 0.01, Figure 4B) rats both expressed reduced climbing times. The defecation rates of the 60 CUS-exposed rats (278 &#177; 32%, p &#60; 0.01,Figure 4C) and 30 depression-contagion rats (131 &#177; 37%, Figure 4C) were significantly greater compared to the defecation rate of the 30 control rats (100 &#177; 22.5%, p &#60; 0.01).&#160;Post hoc analysis found no significant difference between the depression-contagion rats and the controls, but showed a significant difference between CUS-exposed compared with control rats (p&#60;0.01 Figure 4C). Statistical analysis was done with one-way ANOVA. The data are presented as percentages of the control groupand expressed as mean &#177; SEM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57137/57137fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A New Method for Inducing a Depression-Like Behavior in Rats at JoVE.com

A New Method for Inducing a Depression-Like Behavior in Rats

This protocol describes a new model by which healthy rats could contract depression over a given time periodthrough contagion by exposure to chronic unpredictable stressed (CUS) rats.

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is ...

a-new-method-for-inducing-a-depression-like-behavior-in-rats

Nanyang Technological University

Research

JoVE Journal

Behavior

20.7K Views.  Ben-Gurion University of the Negev. This protocol describes a new model by which healthy rats could contract depression over a given time periodthrough contagion by exposure to chronic unpredictable stressed (CUS) rats.

Video: A New Method for Inducing a Depression-Like Behavior in Rats

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 ...

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 ...

Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several independent laboratories have demonstrated robust alterations in the social behavior of rodents exposed to alcohol during brain development across a wide range of exposure durations, timing, doses, and ages at the time of behavioral quantification. Prior work from this laboratory has identified reliable alterations in specific forms of social interaction following moderate prenatal alcohol exposure (PAE) in the rat that persist well into adulthood, including increased wrestling and decreased investigation. These behavioral alterations have been useful in identifying neural circuits altered by moderate PAE1, and may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior. This paper describes procedures for performing moderate PAE in which rat dams voluntarily consume ethanol or saccharin (control) throughout gestation, and measurement of social behaviors in adult offspring.

The goal of the protocol presented here is to describe procedures to expose rats to moderate levels of alcohol during prenatal brain development and to quantify resulting alterations in social behavior during adulthood.

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

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

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

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

The Knob Supination Task: A Semi-automated Method for Assessing Forelimb Function in Rats

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity largely use video analysis of reaching or food manipulation. While these tasks are easy to implement and are robust across disease models, they are subjective and laborious for the experimenter. Automating traditional tasks or creating new automated tasks can make the tasks more efficient, objective, and quantitative. Since rats are less dexterous than primates, central nervous system (CNS) injury produces more subtle deficits in dexterity, however, supination is highly affected in rodents and crucial to hand function in primates. Therefore, we designed a semi-automated task that measures forelimb supination in rats. Rats are trained to reach and grasp a knob-shaped manipulandum and turn the manipulandum in supination to receive a reward. Rats can acquire the skill within 20 &plusmn; 5 days. While the early part of training is highly supervised, much of the training is done without direct supervision. The task reliably and reproducibly captures subtle deficits after injury and shows functional recovery that accurately reflects clinical recovery curves. Analysis of data is performed by specialized software through a graphical user interface that is designed to be intuitive. We also give solutions to common problems encountered during training, and show that minor corrections to behavior early in training produce reliable acquisition of supination. Thus, the knob supination task provides efficient and quantitative evaluation of a critical movement for dexterity in rats.

This manuscript describes a semi-automated task that quantifies supination in rats. Rats reach, grasp, and supinate a spherical manipulandum. The rat is rewarded with a pellet if the turn angle exceeds a criterion set by the user. This task increases throughput, sensitivity to injury, and objectivity compared to traditional tasks.

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity ...

Tickling, a Technique for Inducing Positive Affect When Handling Rats

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

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

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

Testing for Metacognitive Responding Using an Odor-based Delayed Match-to-Sample Test in Rats

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, numerous behavioral tasks have been created, many of which utilize an option to decline memory tests. To assess metamemory in rats, we utilized this decline-test option paradigm by adapting previous visual delayed-match-to-sample tests (DMTS)1,2 developed for primate species to an odor-based test suitable for rodents. First,&#160;rats are given a sample to remember by digging in a cup of scented sand. After a delay, the rat is presented with four distinctly scented cups, one of which contains the identical scent experienced during the sample; if this matching cup is selected, then the rat obtains a preferred, larger reward. Selection of any of the other three non-matching sand-filled scented cups results in no reward. Retention intervals are individually titrated such that subjects perform between 40 and 70% correct, therefore ensuring rats sometimes remember and sometimes forget the sample. Here, the operational definition of metamemory is the ability to distinguish between the presence and absence of memory through behavioral responding. Towards this end, on two-thirds of trials, a decline option is presented in addition to the four choice cups (choice trials). If the decline-test option- an unscented colored sand cup, is selected, the subject receives a smaller less-preferred reward and avoids the memory test. On the remaining third of trials, the decline-test option is not available (forced trials), causing subjects to guess the correct cup when the sample is forgotten. On choice tests, subjects that know when they remember should select the decline option when memory is weak rather than take the test and choose incorrectly. Therefore, significantly higher performance on chosen tests as compared to forced memory tests is indicative of the adaptive use of the decline-test response and metacognitive responding.

This protocol describes a method for investigating the possibility of metamemory, or memory awareness, in rodents. The odor-based delayed-matching-to-sample paradigm is a novel, ecologically-relevant behavioral test useful for determining the extent to which rodents can adaptively respond based on cognitively monitoring the strength of their memory states.

Metamemory involves the cognitive ability to assess the strength of one&#39;s memories. To explore the possibility of metamemory in non-human animals, ...