These studies were supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) to F.L and a Canadian Graduate Scholarship (CGSD) from NSERC to AM.L.

1. Animal Care and Housing
All experiments are approved by the Animal Care Committee of the University of Guelph, and are carried out in accordance with the recommendations of the Canadian Council on Animal Care. The current study includes 22 adult male Sprague-Dawley rats (Charles River, QC) weighing 200-225 g at the beginning of the experiment.
<ol>
	<li>Single house rats and maintain them on a reverse light/dark cycle (7:00 am OFF - 7:00 pm ON) with ad libitum access to food and water except when otherwise indicated, or during behavioral testing which occurs during the active cycle.</li>
	<li>Habituate rats to the colony room and human handling by weighing daily for one week prior to surgery.</li>
</ol>
2. Intraoral Cannulation Surgery: Materials and Procedures
The intraoral (IO) surgery and materials are described in Limebeer et al.14
<ol>
	<li>To construct the IO cannula, cut a piece of polyethylene plastic tubing to approximately 130 mm in length (inner diameter of 0.86 mm and outer diameter of 1.27 mm).</li>
	<li>The tube to be secured in the oral cavity requires a flanged end (~5 mm in diameter). Create this flanged end by heating the end of the tube with a soldering iron for 2-5 sec and then promptly flatten the heated edge of the tube against a flat surface. Cut a surgical mesh disc (approximately 6-7 mm diameter to fit around the diameter of the flanged end of the cannula) and thread it onto the length of the tube until the disc rests against the back of the flanged end of the IO cannula.</li>
	<li>For each IO cannula, cut one additional surgical mesh disc (8 mm in diameter) and three squares of an elastic band (6 mm x 6 mm; see Figure 1).</li>
	<li>Sterilize the IO cannula, surgical mesh discs, and elastic squares in an antibacterial solution for a minimum of 24 hr prior to surgery.</li>
	<li>Prior to surgery, autoclave all surgical tools and disinfect the surgical field. Between surgeries, use a glass bead sterilizer to sterilize tools, and wear a hairnet, nitrile gloves, and surgical masks to maintain aseptic conditions.</li>
</ol>
Analgesia and anaesthesia
<ol start="6">
	<li>Approximately 30 min prior to surgery administer a subcutaneous (SC) injection of the antibiotic Depocillin (0.33 mg/kg) and the analgesic Meloxicam (5 mg/kg SC).</li>
	<li>Induce general anaesthesia using Isoflurane (4% induction and 3% maintenance).</li>
	<li>Once rats are unresponsive to toe pinch, and prior to making any incisions, use a cotton tipped swab to administer a topical anaesthetic along the inner left cheek of rats where the cannula is to be implanted, and inject a local anaesthetic such as lidocaine HCl at the incision site for the biopsy needle (base of neck) and head cap (scalp).</li>
	<li>Apply lubricating eye ointment to both eyes prior to the beginning of surgery.</li>
</ol>
Surgical procedures
<ol start="10">
	<li>Shave a 10 mm x 10 mm patch of hair at the base of the neck. Use sterile gauzes to swab and cleanse the shaved area in the following order: antibacterial soap, isopropyl 70% alcohol, and Betadine.</li>
	<li>Insert a 15 G stainless steel needle at the base of the neck and move it subcutaneously around the ear to the left cheek. Position the needle tip and pass it through the cheek behind the first molar inside of the oral cavity.</li>
	<li>Pass the IO cannula through the tip of the needle protruding from the oral cavity and then remove the needle leaving only the IO cannula.</li>
	<li>Thread one surgical mesh disc followed by three square elastic discs onto the exterior portion of the IO cannula and draw them to the exposed skin at the back of the neck to stabilize it.</li>
	<li>Flush the IO cannula with an antibacterial oral rinse</li>
	<li>Prior to the beginning of surgery, modify a Nylon bolt (28 mm length; 4 mm point; 8 mm head) to remove its head and carve a 2 mm groove along the length of the thread (see Figure 2).</li>
	<li>Shave a 10 mm x 20 mm strip of hair along the midline of the head. Use sterile gauzes to swab and cleanse the shaved area in the following order: antibacterial soap, isopropyl 70% alcohol, and Betadine.</li>
	<li>Use a surgical scalpel to make a 20 mm long incision along the sagittal suture. Use a hand drill to make 4 holes (2 on each side of the suture) and use a hand held screw driver to secure four jewelers screws to the skull.</li>
	<li>Place the bolt in the center of the four jewelers screws with the groove positioned towards the posterior of the animal. Mount the bolt to the skull of the rat by building a headcap with dental cement (use sufficient dental cement to cover the surface of the jewelers screws). Allow the dental cement to dry before removing the animal from the surgical field and terminate anaesthesia.</li>
</ol>
Post-operative monitoring and care
<ol start="19">
	<li>Individually house rats in bedded cages immediately following surgery and closely monitor for 24 hr.</li>
	<li>Administer a second injection of the analgesic Meloxicam 24 hr post-surgery and flush the IO cannula with an antibacterial oral rinse once/day for three days.</li>
</ol>
Give rats 3-5 days to recover from surgery prior to the beginning of the experiment.
3. IO Self-administration Procedures
Apparatus
For self-administration, Plexiglas operant conditioning chambers are used, and each chamber is enclosed in a larger sound-attenuating plywood box. Each operant conditioning chamber has a house light (28 V) and two retractable levers (active and inactive) located on opposite sides of the chamber, 8 cm above the floor. The active lever is connected to an infusion pump positioned outside the sound-insulating chamber and a white light (28 W) is located 3 cm above it to serve as a stimulus light paired with the delivery of IO infusions. The inactive lever serves to control for baseline, nonreinforced operant behavior; pressing this lever has no consequence, but all presses are recorded.
Food
<ol>
	<li>Provide rats with a standard rat chow (3.1 kcal/g) in their home cage.</li>
	<li>Dilute high fructose corn syrup (55% formula) to three different concentrations (8% = 0.026 kcal/80 &#956;l infusion; 25% = 0.08 kcal/80 &#956;l infusion; 50% = 0.17 kcal/80 &#956;l infusion) using reverse osmosis water. These concentrations are selected because each produces a viscosity that is appropriate to deliver rapid (2.5 sec) IO infusions given the experimental apparatus (i.e. diameter of syringes and tubing).</li>
</ol>
Food deprivation
In order to facilitate the acquisition of IO self-administration, but promote normal growth, rats are cycled through periods of food deprivation and ad libitum access to standard rat chow.
<ol start="3">
	<li>Weigh rats daily at 7 pm and remove chow from the feeding trough of the cage.</li>
	<li>The following day, weigh rats at 12:00 pm (approximately 18 hr later) and begin IO self-administration by 1:00 pm.</li>
	<li>Return rats to their home cages at 4:00 pm and provide ad libitum access to chow for 3 consecutive hr. Then, record the weight of the rats and the total amount of chow consumed over the three hours. Resume food deprivation as described.</li>
</ol>
General IO self-administration procedure
Operant responding for IO infusions of HFCS is evaluated on PR and continuous schedules of reinforcement. Rats are tested in IO self-administration until intake is stable as defined by: 1) a significant difference in responding between active and inactive levers for at least 2 consecutive sessions; and 2) less than 20% variation in the number of infusions earned per session for 2 consecutive sessions. Each session begins 6 hr into the dark cycle and lasts 3 hr. The acquisition phase consists of 10 consecutive IO self-administration sessions on a continuous schedule of reinforcement, and rats receive a single PR test 24 hr prior to the beginning of acquisition (PR test I) and 24 hr after the final acquisition session (PR Test II). For PR tests, the breakpoint (BP) is defined as the last infusion received prior to cessation of responding on the active lever for at least 1 hr18.
<ol start="6">
	<li>Counterbalance rats on the basis of weights taken for three days prior to the start of the experiment and assign rats to self-administer one of three concentrations of HFCS: 8%, 25%, or 50%.</li>
	<li>Weigh rats at 12:00 pm and transfer them from the colony room to the testing room in their home cages. To remove food or bedding that can accumulate in the IO cannula, pump air through the cannula using a 12 ml syringe fixed with a 16 G needle with 50 mm of polyethylene tubing (inner diameter of 1.14 mm and outer diameter of 1.57 mm) attached to the end.</li>
	<li>In the operant chamber, connect the IO cannula to Tygon tubing (inner diameter of 0.02 mm and outer diameter of 1.53 mm) by a shaft constructed of a modified 20 G needle (remove the plastic hub and beveled tip). Position the lower portion of the tubing (i.e. closer to the animal) within the grove of the bolt. The higher portion of the tubing is connected to a swivel, and protected by a spring. Connect the base of the spring to the thread of the bolt mounted on the head of the rat.</li>
	<li>Give rats five minutes to acclimatize to the operant chamber and then start the IO self-administration session. The IO self-administration session begins with the activation of the house light, entry of the two retractable levers, and illumination of the light stimulus above the active lever for 30 sec.</li>
	<li>For PR Test I, use the PR schedule described by Richardson and Roberts18 (response ratio escalation = (5e(injection number x 0.2)) - 5).</li>
	<li>For acquisition sessions 1-10, use a continuous schedule of reinforcement whereby each press on the active lever results in the delivery of an 80 &#956;l infusion of the test solution over 2.5 sec.</li>
	<li>To allow sufficient time for ingestion, impose a time-out period of 27.5 sec during which the active lever is retracted and the stimulus light is activated (i.e. rats can obtain an IO infusion every 30 sec). Do not impose a limit on the number of infusions obtainable within each IO self-administration session.</li>
	<li>If the rat does not make a response on the active lever for 20 consecutive minutes, prime the rat (open the operant chamber and facilitate the rat pressing once on the active lever). Prime the animals every 20 min up to 5x/session during the first three days of acquisition only.</li>
	<li>Administer the second PR test using the parameters described in step 3.10.</li>
	<li>Following IO self-administration sessions, remove rats from the operant chambers, flush the IO cannula with 2.0 ml water using the same materials described in 3.7, and return rats to their home cage in the colony room.</li>
	<li>Following three hours of ad libitum access to rat chow, weight rats at 7:00 pm, remove food from the feeding trough, and resume food deprivation.</li>
</ol>
Self-administration data are analyzed using two-factor repeated measures Analyses of Variance (ANOVA). Multiple comparisons using the Student-Newman-Keuls method (&#945;=0.05) were employed to explore any significant interactions or main effects.

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+transition+in+cocaine+self-administration:+Behavioral+and+neurobiological+implications.">A critical transition in cocaine self-administration: Behavioral and neurobiological implications.</a> Psychopharmacology. 192 (3), 337-146 (2007).</li><li>Stanhope, K. L., Havel, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+and+metabolic+effects+of+consuming+beverages+sweetened+with+fructose,+glucose,+sucrose,+or+high+fructose+corn+syrup+Am.">Endocrine and metabolic effects of consuming beverages sweetened with fructose, glucose, sucrose, or high fructose corn syrup Am.</a> J. Clin. Nutr. 88 (6), 17335-17375 (2008).</li><li>Deroche-Gamonet, V., Belin, B., Piazza, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+addiction-like+behaviour+in+the+rat.">Evidence for addiction-like behaviour in the rat.</a> Science. 305, 1014-1017 (2004).</li></ol>

The authors having nothing to disclose.

The current study illustrates a novel approach for evaluating the reinforcing properties of sweet solutions by combining methods traditionally employed to study behavior motivated by drugs of abuse (intravenous operant self-administration) and to assess the palatability of tastants (taste reactivity through intraoral infusions). To this end, under very brief isoflurane-induced anesthesia, rats are implanted with an IO cannula that allows for the delivery of test solutions directly into their mouth. Following recovery fro...

The study of the neurobiological and behavioral basis of addiction to food relies on the observations that, similarly to drugs of abuse, excessive consumption of palatable foods promotes behavioral dependence1-4 and induces alterations in brain reward circuits in both humans5-6 and laboratory animals7-8. But, while there are several protocols and procedures for studying the addictive properties of drugs of abuse in laboratory rats, adapting these methods to assess &#34;addictive&#34; behaviors induced by food presents unique challenges. The goal of the current study was to apply principles of operant intravenous (IV) drug self-administration to the investigation of operant self-administration of sweet solutions delivered by intraoral (IO) infusion. The sweet employed was high fructose corn syrup (HFCS) because, although controversial, there is evidence that HFCS may be linked to the modern epidemic of obesity9-12.
Traditionally, IO infusions are delivered to study the palatability of tastants in taste reactivity experiments13. Briefly, an IO cannula is surgically implanted into the cheek of rats and IO infusions of various solutions are passively delivered14. The objective is to study orofacial reactions of the animals to the tastants. However, IO cannulas have also been implanted chronically in the palate of rats to determine whether they would learn to press a lever to self-infuse solutions directly into their oral cavity; hence the term IO self-injection15-17. In the current study, we describe a procedure that utilizes minor surgery&#160;and that allows long term operant self-administration testing. This procedure has several significant advantages over traditional procedures that involve drinking a solution from a spout (a), or operant responding for solid pellets (b), or operant responding for drops of fluid delivered in a receptacle (c).
In comparison to (a), IO self-administration involves an operant response (i.e.&#160;pressing a lever) and therefore it is possible to modify the schedule regulating the relationship between response requirements and delivery of IO infusions. For example, by employing a progressive ratio (PR) schedule, whereby responses required for successive infusions increase exponentially within a session18, it is possible to assess how much an animal &#34;wants&#34; the next infusion19. This important aspect of &#34;seeking&#34; behavior cannot be assessed when animals drink a solution from a spout, unless a special apparatus that controls fluid delivery and monitors responses is employed20. Moreover, IO self-administration provides a mean to compare behaviors motivated by different reinforcing stimuli. That is, it is possible to compare operant responding maintained by sweets and other reinforcers such as drugs of abuse in so called &#34;substitution studies.&#34;
In comparison to (b), IO self-administration allows testing of any concentration and any volume of any water-soluble food additive. This is critical to study behavior motivated by sweets such as HFCS because, to the best of our knowledge, there are no commercially available solid pellets of fructose, or fructose-glucose combinations at different ratios, that would be suitable for operant chambers. Also, the importance of controlling and manipulating concentration/volume ratios is mandatory in experiments where intake can be modulated both by the caloric value of a solution (leading to nutrient-specific satiety) and by how much of that solution can be consumed within a given period of time (i.e.&#160;fullness)21. IO self-administration also shortens the delay between the operant response and the delivery of the primary reinforcer, a factor that plays an important role in the acquisition and maintenance of operant behavior16,22-23.
Finally, in comparison to (c), IO self-administration allows the delivery of passive IO infusions of controlled quantities of the test solution, and this makes it possible to measure orofacial responses of &#34;liking&#34; (objective hedonic reaction such as tongue protrusions24-25) and whether these responses change during IO self-administration. Furthermore, the ability to administer passive IO infusions has significant applications for the study of relapse to food-seeking. That is, in studies of IV drug self-administration, following periods of extinction, drug primes (i.e.&#160;administration of a small dose of the drug26) can &#34;reinstate&#34; responding27-28. Therefore, the ability to deliver IO infusions without any action on the part of the animal can be used to study &#34;food&#34; primed reinstatement, as well as potential cross reinstatement between food and drugs of abuse.
IO self-administration of sweet solutions is also preferable to IV and intra-gastric self-administration. In fact, although IV infusions of fructose in both humans and laboratory animals produce physiological consequences similar to those observed following oral consumption29-31, this is a poor model of how sweets are normally consumed (i.e.&#160;orally). Moreover, gustatory signals produced by mastication confer important information about the palatability of food, and when this stage of the digestive process is omitted, the development of maladaptive behaviors such as overeating is reduced32-33.

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a novel procedure for evaluating the reinforcing properties of tastants in laboratory rats: operant intraoral self-administration

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste reactivity with the behavioral aspects of operant self-administration of drugs. Under very brief general anaesthesia, rats are implanted with an intraoral (IO) cannula that allows delivery of test solutions directly in the oral cavity. Animals are then tested in operant self-administration chambers whereby they can press a lever to receive IO infusions of test solutions. IO self-administration has several advantages over experimental procedures that involve drinking a solution from a spout&#160;or operant responding for solid pellets or solutions delivered in a receptacle. Here, we show that IO self-administration can be employed to study self-administration of high fructose corn syrup (HFCS). Rats were first tested for self-administration on a progressive ratio (PR) schedule, which assesses the maximum amount of operant behavior that will be emitted for different concentrations of HFCS (i.e.&#160;8%, 25%, and 50%). Following this test, rats self-administered these concentrations on a continuous schedule of reinforcement (i.e.&#160;one infusion for each lever press) for 10 consecutive days (1 session/day; each lasting 3 hr), and then they were retested on the PR schedule. On the continuous reinforcement schedule, rats took fewer infusions of higher concentrations, although the lowest concentration of HFCS (8%) maintained more variable self-administration. Furthermore, the PR tests revealed that 8% had lower reinforcing value than 25% and 50%. These results indicate that IO self-administration can be employed to study acquisition and maintenance of responding for sweet solutions. The sensitivity of the operant response to differences in concentration and schedule of reinforcement makes IO self-administration an ideal procedure to investigate the neurobiology of voluntary intake of sweets.

Acquisition of IO self-administration
Figure 3 represents operant responding on the active and inactive levers during self-administration of 8%, 25%, or 50% HFCS for 10 consecutive sessions. Rats acquire IO self-administration of HFCS within the first three sessions and maintain stable active lever responding (as described above) for the remainder of acquisition. Concentration-dependent differences in active lever responses are also evident: compared to 25%, responding i...

Watch this Scientific Journal Video about A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration at JoVE.com

A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration

The current study evaluates a novel procedure for assessing the reinforcing effects of palatable solutions in laboratory rats: intraoral self-administration. To this end, operant responding (i.e.&#160;lever pressing) for intraoral infusions of sweet solutions at different concentrations was measured on continuous and progressive ratios schedules of reinforcement.

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste ...

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JoVE Journal

Behavior

12.6K vues.  University of Guelph.  La présente étude évalue une nouvelle procédure d'évaluation des effets de renforcement de solutions agréables au goût chez les rats de laboratoire: intra auto-administration. À cette fin, répondant opérant (c'est à dire en appuyant sur ​​le levier) pour perfusions intra-orale de solutions sucrées à différentes concentrations a été mesurée sur continue et progressive des ratios programmes de renforcement. 

Vidéo: A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration

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 ...

Operant Procedures for Assessing Behavioral Flexibility in Rats

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these processes is the ability to adjust behavior in response to changes in one&#8217;s environment (i.e., behavioral flexibility). These processes are essential to normal human behavior, and may be disrupted in diverse neuropsychiatric conditions, including schizophrenia, alcoholism, depression, stroke, and Alzheimer&#8217;s disease. Understanding of the neurobiology of executive functions has been greatly advanced by the availability of animal tasks for assessing discrete components of behavioral flexibility, particularly strategy shifting and reversal learning. While several types of tasks have been developed, most are non-automated, labor intensive, and allow testing of only one animal at a time. The recent development of automated, operant-based tasks for assessing behavioral flexibility streamlines testing, standardizes stimulus presentation and data recording, and dramatically improves throughput. Here, we describe automated strategy shifting and reversal tasks, using operant chambers controlled by custom written software programs. Using these tasks, we have shown that the medial prefrontal cortex governs strategy shifting but not reversal learning in the rat, similar to the dissociation observed in humans. Moreover, animals with a neonatal hippocampal lesion, a neurodevelopmental model of schizophrenia, are selectively impaired on the strategy shifting task but not the reversal task. The strategy shifting task also allows the identification of separate types of performance errors, each of which is attributable to distinct neural substrates. The availability of these automated tasks, and the evidence supporting the dissociable contributions of separate prefrontal areas, makes them particularly well-suited assays for the investigation of basic neurobiological processes as well as drug discovery and screening in disease models.

The ability to assess executive functions such as behavioral flexibility in rats is useful for investigating the neurobiology of cognition in both intact animals and disease models. Here we describe automated tasks for assessing strategy shifting and reversal learning, which are particularly sensitive to disruptions in prefrontal cortical networks.

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these ...

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 ...

A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people. There is a need for novel, effective and practical treatment strategies that are validated in animal models. Neuromodulation, including deep brain stimulation (DBS) therapy, refers to the use of electricity to influence pathological neuronal activity and has shown promise for psychiatric disorders, including drug dependence. DBS in clinical practice involves the continuous delivery of stimulation into brain structures using an implantable pacemaker-like system that is programmed externally by a physician to alleviate symptoms. This treatment will be limited in methamphetamine users due to challenging psychosocial situations. Electrical treatments that can be delivered intermittently, non-invasively and remotely from the drug-use setting will be more realistic. This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence. Methamphetamine dependence is rapidly developed in rodents using an operant paradigm of intravenous (IV) self-administration that incorporates a period of extended access to drug and demonstrates both escalation of use and high motivation to obtain drug.

This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence.

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people.  There is a need ...

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods ...

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.

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 ...

Three Laboratory Procedures for Assessing Different Manifestations of Impulsivity in Rats

The present article provides a guide for the conduction and analysis of three conditioning-based protocols to evaluate impulsivity in rats. Impulsivity is a meaningful concept because it is associated with psychiatric conditions in humans and with maladaptive behavior in non-human animals. It is believed that impulsivity is composed of separate factors. There are laboratory protocols devised to assess each of these factors using standardized automated equipment. Delay discounting is associated with the incapacity to be motivated by delayed outcomes. This factor is evaluated through intertemporal choice protocols, which consist&#160;of presenting the individual with a choice situation involving an immediate reward and a larger but delayed reward. Response inhibition deficit is associated with the incapacity to withhold prepotent responses. Differential reinforcement of low rates (DLR) and feature-negative discrimination protocols assess the response inhibition deficit factor of impulsivity. The former imposes a condition to a motivated individual in which most wait a minimum period of time for a response to be rewarded. The latter evaluates the capacity of individuals to refrain from food seeking responses when a signal of the absence of food is presented. The purpose of these protocols is to construct an objective quantitative measure of impulsivity, which serves to make cross-species comparisons, allowing the possibility of translational research. The advantages of these particular protocols include their easy set-up and application, which stems from the relatively small amount of equipment needed and the automated nature of these protocols.

We present three protocols that assess different forms of impulsivity in rats and other small mammals. Intertemporal choice procedures evaluate the tendency to discount the value of delayed outcomes. Differential reinforcement of low rates and feature-negative discrimination evaluate response inhibition capacity with and without punishment for inappropriate responses, respectively.

The present article provides a guide for the conduction and analysis of three conditioning-based protocols to evaluate impulsivity in rats. Impulsivity is ...

A Laboratory Method to Measure Contagious Yawning in Rats

Communication is an essential aspect of animal social life. Animals may influence one another and come together in schools, flocks, and herds. Communication is also the way sexes interact during courtship and how rivals settle disputes without fighting. However, there are some behavioral patterns for which it is difficult to test the existence of a communicatory function, because several types of sensory modalities are likely involved. For example, contagious yawning is a communicatory act in mammals that potentially occurs through sight, hearing, smell, or a combination of these senses depending on whether the animals are familiar to one another. Therefore, to test hypotheses about the possible communicatory role of such behaviors, a suitable method is necessary to identify the participating sensory modalities.
The method proposed here aims to obtain yawn contagion curves for familiar and unfamiliar rats and evaluate the relative participation of visual and olfactory sensory modalities. The method uses inexpensive materials, and with some minor changes, it can also be used with other rodent species such as mice. Overall, the method involves the substitution of clear dividers (with or without holes) with opaque dividers (with or without holes) that either allow or prevent communication between rats placed in adjacent cages with holes in adjoining sides. Accordingly, four conditions can be tested: olfactory communication, visual communication, both visual and olfactory communication, and neither visual nor olfactory communication. As social interaction occurs between the rats, these test conditions simulate what may occur in a natural environment. In this respect, the method proposed here is more effective than traditional methods that rely on video presentations whose biological validity can raise concerns. Nonetheless, it does not discriminate between the potential role of hearing and roles of smell and vision in yawn contagion.

The method described here aims to obtain yawn contagion curves in pairs of familiar or unfamiliar male rats. Cages with holes separated by either clear or opaque partitions with (or without) holes are used to detect whether visual, olfactory, or both types of sensory cues can stimulate yawn contagion.