This work was supported by the Swedish Research Council.

All procedures are conducted in accordance with the NIH Guide for Care and Use of Laboratory Animals.
1. Animal Care and Housing
<ol>
	<li>Upon arrival in the colony, house male Wistar rats weighing 200 - 225 g at arrival in pairs in a temperature (21 &#176;C) and humidity-controlled environment with a reversed 12 h light-dark cycle. 
	Note: Depending on the rationale of the experiments, rats may be single-housed.</li>
	<li>Allow the rats to acclimate to the vivarium and the light cycle for at least one week before starting an experiment and handle them daily. Weigh the animals once weekly.</li>
	<li>Give rats free access to chow and tap water for the duration of the experiment (water deprivation is not necessary to initiate responding12). Conduct all behavioral testing during the dark phase of the light-dark cycle in a dark testing room. 
	Note: Although we routinely use young adult male Wistar rats (postnatal days 62 - 65), this protocol can theoretically be suitable for investigating other strains of rats, as well as sex and/or age differences.</li>
</ol>
2. Operant Training
<ol>
	<li>Conduct all behavioral training and testing in identical operant chambers measuring 30.5 &#215; 29.2 &#215; 24.1 cm and containing grid floors and waste pans lined with bedding (changed in between animals to reduce odor cues), housed in sound-attenuating cubicles equipped with exhaust fans for ventilation. 
	Note: Rats are trained by operant-conditioning from day 1 of training (a response on a lever is necessary to obtain a reinforcer) between + 1 h and + 8 h of the dark cycle. There is no pre-exposure to alcohol, nor food or fluid shaping before alcohol training.</li>
	<li>Equip each operant chamber with stimulus lights, syringe pumps and two retractable levers positioned laterally to a liquid cup receptacle. Interface chambers and control by a computer using the appropriate software. 
	Note: Most vendors will provide self-administration packages containing all the necessary components as well as software and interfaces necessary to control the chambers.</li>
	<li>Write an operant-conditioning program that initiates the fixed-ratio (FR) schedule of 20% ethanol reinforcement (an operant schedule in which a response is reinforced only after a specified number of responses) according to the manufacturer&#39;s instructions. For example, in a fixed-ratio 1, rats have to produce one response to obtain a reinforcer (see the Supplemental Code Files section for an example of a FR1 program). Execute the following commands.
	<ol>
		<li>Extend two levers to mark the onset of the session and to signal alcohol availability. 
		Note: One lever press on the lever associated with ethanol (active) is rewarded by the delivery of a volume of 100 &#956;L of 20% ethanol in water in the adjacent drinking well and initiates a concomitant 5-s time-out period signaled by the illumination of the cue-light above the lever. The cue-light is introduced on day 1 of the training.</li>
		<li>During the time-out period, record responding although it has no scheduled consequences. Record responses on the other lever (inactive) although they never have behavioral consequences. Note: The inactive lever serves as a control to assess non-specific behavior.</li>
		<li>Record the number of responses on the active lever (response rates) and the number of 20% ethanol rewards earned.</li>
		<li>After 30 min have elapsed, retract the two levers to signal the end of the session and to signal the end of alcohol availability.</li>
		<li>Save and archive all data.</li>
	</ol>
	</li>
	<li>Before starting the training session, check the proper functioning of all devices in each operant chamber (retractable levers, fluid dispenser).</li>
	<li>Prepare the 20% v/v ethanol solution from 190 Proof (95%) ethanol diluted in tap water. 
	Note: Ethanol solution can be kept at room temperature. Depending on the manufacturer, stock ethanol solutions may be up to 99.98%. Also note that, while in most published manuscripts, ethanol concentrations are usually presented as volume/volume, some may present them as weight/volume.</li>
	<li>Fill the syringe with the 20% ethanol solution and make sure that there are no leaks or air bubbles in the infusion lines. Manually push a small volume of ethanol through the infusion lines to make sure that ethanol will be correctly delivered into the receptacle from the first reinforced response. Dry the receptacle with a paper towel and make sure that it is empty before starting the session.</li>
	<li>Load the software that controls the FR schedule of 20% ethanol reinforcement</li>
	<li>Train operant- and drug-naive rats under a FR1 schedule to self-administer 20% ethanol without water deprivation during 30 min sessions.
	<ol>
		<li>Transport the rats from the vivarium to the testing room using a transport cage.</li>
		<li>Assign each rat to one chamber. Take each individual rat from the transport cage and put it in the assigned self-administration chamber.</li>
		<li>Try to keep the conditions of the experiment consistent each day. Therefore, always test rats in the same self-administration chamber at roughly the same time every day.</li>
		<li>Start the software that initiates the FR1 schedule of 20% ethanol reinforcement.</li>
		<li>At the end of the session, remove the rats from the self-administration chambers and return them to the vivarium.</li>
		<li>In order to manually confirm that the alcohol solution is consumed by the animals, use a paper towel and check if the receptacle is dried. Alternatively, use a 1 or 2 mL syringe and collect the fluid remaining in the receptacle to estimate the volume that is not consumed. 
		Note: For a more precise measurement (but also more expensive), it is possible to equip each chamber with a lickometer system connected to the liquid cup receptacle that allows a precise count of the licks produced by the rats during the self-administration session.</li>
		<li>Clean the walls and grid floor of each chamber with a surface disinfectant.</li>
		<li>After use, maintain the infusion lines by cleaning them with a 70% ethanol solution. When not in use, insert the cap to avoid mold and dust.</li>
	</ol>
	</li>
	<li>After each session, collect and analyze the data generated. More particularly, calculate the mean number of active lever presses, the mean number of alcohol rewards obtained as well as the mean number of inactive lever presses. 
	Note: In most papers, alcohol consumption is also presented as ethanol intake in g/kg (bodyweight). Ethanol intake can be calculated using the following formula: 
	<img alt="Equation1" src="/files/ftp_upload/53305/53305equation1.jpg" /> 
	With volume consumed (mL) - number of rewards earned &#177; unitary volume/reward and density of ethanol at room temp - 0.789 g/mL. 
	For example, if a rat weighing 400 g earned 20 rewards of 20% v/v ethanol solution: 
	<img alt="Equation2" src="/files/ftp_upload/53305/53305equation2.jpg" /></li>
	<li>Conduct sessions 5/6 days a week until stabilization of performance (defined as a minimum of 15 sessions and no change greater than 15% in the total number of rewards earned during the last 3 sessions). 
	Note: Once rats have reached a stable baseline on FR1, the operant conditioning program can be modified according to the manufacturer&#39;s instructions to increase the number of responses necessary to obtain an alcohol reward. The fixed-ratio can for example be increased to FR2 or FR3 in order to minimize accidental reinforcement.</li>
	<li>Measure blood ethanol concentrations:
	<ol>
		<li>To confirm that the rewards obtained in a self-administration session are consumed by the rats, measure the blood ethanol concentrations following self-administration. Once rats have reached a stable baseline, collect blood from the lateral tail vein immediately following the session. Insert a 23 G needle into the blood vessel and collect 50 &#181;L blood samples using a capillary tube.</li>
		<li>Transfer the 50 &#181;L blood samples to 10 mL headspace vials and add 50 &#181;L of 1:1,000 isopropyl alcohol as internal standard. Prepare a standard curve 10 to 300 mg/dL from ethanol standards. Process samples with a headspace sampler with a chromatography column13 according to the manufacturer settings.</li>
	</ol>
	</li>
	<li>Progressive ratio
	<ol>
		<li>Use progressive ratio (PR) schedules to assess the motivation of the animals to consume a reward14. PR is an operant schedule in which the number of responses to obtain a reinforcer is increased gradually (in other terms, rats have to work harder for each successive reward). 
		Note: Contrary to FR sessions, a PR session is not timed and will only be terminated once 30 min has elapsed without a completed ratio.
		<ol>
			<li>Place rats in their assigned operant boxes and allow them to respond for 20% ethanol under a PR schedule of reinforcement: Keep all experimental conditions identical to those used in the FR schedule (see step 2.3), except increase the response requirement or cost within-session (i.e., the number of lever presses required on the active lever to receive a single ethanol reward) according to the following formula: 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 28, 32&#8230; Note: An example of the PR program is provided in the Supplemental Code Files section.</li>
			<li>Terminate the PR session once 30 min has elapsed without a reward. 
			Note: The breakpoint is defined as the last completed response requirement during the PR session.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Testing the Predictive Validity of an Operant Model of Alcohol Self-administration
Note: Once rats have acquired a stable self-administration baseline (see step 2), it is possible to assess the predictive validity of the model by testing the efficacy of naltrexone, a currently FDA-approved medication for alcohol dependence in reducing 20% ethanol self-administration. We recommend doing this testing on the highest FR achieved during training (FR2), when responses are reliably high. Once predictive validity is established, the model can be used to evaluate new drug candidates.
<ol>
	<li>Before test day, give rats subcutaneous saline injections 30 min prior to self-administration sessions to habituate them to the injection during a minimum of two consecutive self-administration sessions or until responding for ethanol is not affected by the saline injections (defined as no change greater than 15% in the total number of rewards earned during the last 2 sessions).</li>
	<li>Dissolve naltrexone in saline and adjust the pH to reach neutrality to facilitate the injections to the animals. Inject the drug at a volume of 1.0 mL/kg 30 min before a session, using the subcutaneous route of administration. Note: The literature provides good evidence for a dose choice between the ranges of 0.1 - 1mg/kg, so 0.1, 0.3 and 1 mg/kg are used in this protocol. 
	Note: Research indicates use of both subcutaneous and intraperitoneal routes of administration for naltrexone, though their potency may differ with subcutaneous injection being 30-fold more potent that intraperitoneal injections15. For this reason, we recommend using the subcutaneous route as the use of this route for the injection of naltrexone has been validated and replicated by various laboratories in a range of concentration between 0.1 and 1 mg/kg12,15,16.</li>
	<li>During test day, inject rats in a balanced/random order in a between-subjects design across one of the four naltrexone dosing cycles (0, 0.1, 0.3 and 1 mg/kg) 30 min before the self-administration session.</li>
	<li>Between each dosing cycle, allow rats to washout the drug with a minimum of two consecutive self-administration sessions or until responding for ethanol is back to baseline. Note: As a result, at the end of the test, all rats have been injected with each of the four doses.</li>
	<li>After this phase, select the most efficient dose (1 mg/kg) and test the effect of naltrexone on the motivation of the animals to consume alcohol using a progressive ratio schedule (see step 2.12).</li>
</ol>

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The authors have nothing to disclose.

With this protocol, we are presenting a new method to acquire and maintain stable oral self-administration of 20% ethanol in rats that, contrary to classic models of ethanol self-administration, does not require the use of water deprivation, extended access training, or saccharin/sucrose fading12. Furthermore, naltrexone, a currently FDA-approved medication for alcohol dependence, successfully decreases alcohol self-administration and the motivation to consume alcohol of Wistar rats trained with this protocol. This result provides an important pharmacological validation of the protocol as it is consistent with previous reports showing that naltrexone is effective in reducing alcohol consumption in non-dependent rats16-18. Naltrexone is an opiate receptor antagonist that has shown promising results in both animal models of alcohol intake and clinical trials. In animal models, naltrexone is efficient in reducing alcohol consumption and self-administration in non-dependent animals16,17,19, without affecting non-specific behavior (inactive lever presses)12. It has also proven efficient in reducing food intake. For example, it successfully decreases the rat&#39;s fluid intake of both a sweet non-caloric solution of saccharin as well as a highly palatable and caloric chocolate drink20. Naltrexone also reduces sucrose self-administration16, water intake after water deprivation21, as well as saccharin choice in a two-bottle choice setting22. All together, these results indicate that naltrexone probably does not affect alcohol responding by modulating the caloric value of liquid reinforcers, but more likely by decreasing the immediate rewarding value of alcohol.
There are several important advantages to this model. First, the acquisition period is faster than with previous paradigms. Rats reach active lever pressing rates for relevant levels of ethanol23 in one week. Sugar-fading based paradigms necessitate six to eight weeks of testing to produce reliable ethanol intake1,3, thus wasting valuable time and resources. Secondly, this model eliminates the marked decrease in ethanol consumption commonly seen following the removal of sweeteners, therefore possibly decreasing the rate of attrition in ethanol self-administration studies24. Eight different cohorts of rats amounting to a total of 239 operant- and drug-naive Wistar rats were run using this protocol and only 9 meet criteria for failing to acquire self-administration (defined as a minimum of 10 active lever presses during the last 3 stabilized fixed-ratio 1 sessions). Thus, this novel training protocol yields an impressive successful acquisition rate of 96%. With this protocol, rats reached robust responding rates in less than two weeks, resulting in an average ethanol intake of 0.86 g/kg. This level of consumption is equivalent to the ones reached by rats trained with the sucrose/saccharin fading procedures that usually achieved an average consumption between 0.5 and 0.8 g/kg1,2,23,25.
Furthermore, this model simplifies the interpretation of ethanol self-administration studies by removing confounds due to the inherently rewarding properties of saccharin and sucrose in rats. The potential addictive properties of food rewards and more particularly of palatable foods such as sugar have been a subject of interest in recent years and ultimately emphasize the need for a more specific model of ethanol self-administration. For example, there is evidence to support the idea that sucrose may itself be addictive in rats, as rats display behaviors such as bingeing, seeking, and withdrawal in response to excessive sucrose intake26-28. In addition, sucrose and saccharin are more reinforcing than drugs of abuse such as cocaine and opiates6-8,29 and these sweeteners alter Fos activation in brain regions including the nucleus accumbens in a manner similar to ethanol and other drugs of abuse9. Finally, this model is suitable to study animal models of drug relapse. We have previously shown that animals trained using this procedure were able to robustly extinguish their responses for alcohol12, similarly to what is usually obtained with classic models of ethanol self-administration25. These animals can then be reinstated by utilizing a stressor 12 or by reintroducing a cue light30.
Ethanol is an ambivalent drug, which exerts rewarding and aversive properties. It is widely believed that the primary aversive taste of ethanol is a major barrier to achieve stable self-administration in most rodents, especially at high concentrations1. However, a study investigating the taste reactivity in rats of a wide range of ethanol concentrations (from 5 to 40% v/v) surprisingly found that the number of hedonic, but not aversive, orofacial responses tend to increase with higher ethanol concentrations31. Moreover, with repeated exposure to alcohol, the number of aversive orofacial responses decreased when rats were exposed to high ethanol concentrations (20 to 40% v/v) while it remained unchanged with lower concentrations (5 to 10% v/v). Similarly, rats that had a concurrent operant access to two ethanol solutions23 responded more for the higher concentrations (20 or 40% v/v) than the lower (10% v/v, the concentration that is mostly used in sucrose/saccharin fading procedure) and self-administration for oral ethanol displays an inverted U shaped dose response curve with highest ethanol intake during self-administration at a 20% ethanol concentration11. These data indicate that higher concentrations of ethanol may be more rewarding and less aversive to rats, explaining why rats acquire self-administration of 20% ethanol in our protocol without the need of ethanol pre-exposure or sucrose/saccharin fading procedure.
However, it is important to note that this model does not produce escalated level of ethanol self-administration and high ethanol blood concentrations. Therefore, this model does not induce physical dependence in rats and should be combined with other models if the goal of the experiment is to induce excessive alcohol consumption in rodents.
As with many behavioral tasks, the first critical parameter is to keep the conditions of the experiment as consistent as possible every day. More precisely, rats should always be tested in the same self-administration chamber. The session should occur at the same time (&#177; 30 min) throughout the whole experiment. The configuration of the chamber (see step 2.2) should remained unchanged. During self-administration training, the first sessions can be critical. Particular attention should be given to the drug delivery tubing, the liquid cup receptacle, the lever and the syringe pumps. It is critical that ethanol is correctly delivered into the receptacle from the first reinforced response, as any malfunction can delay or impair acquisition of ethanol self-administration.
In summary, the new model of training rats for operant self-administration of oral alcohol described in this protocol greatly improves the efficiency by decreasing training time while increasing the acquisition rate, simplifies the overall procedure as well as the interpretation of results, and is validated as a sound model of ethanol drinking by the naltrexone experiment. This protocol is suitable for investigating the rewarding and motivational properties of alcohol in rats and can be combined with classical operant self-administration procedures such as reinstatements models to assess the neural basis of ethanol-seeking behavior.

The development of animal models to study the reinforcing effects of drugs has proven an important tool to study human drug addiction. More specifically, operant self-administration is a widely used behavioral model that is one of the most effective means for assessing the positive reinforcing effects of an orally consumed ethanol solution. An early issue with developing such a model was the primary aversive taste of high concentrations of ethanol for most rodents, a phenomenon that is also shared in humans with little or no experience with alcohol1. A standard protocol to overcome this barrier requires water deprivation and/or saccharin or sucrose fading for the acquisition and maintenance of self-administration. However, these two approaches are not advantageous. They require long periods of training to simply initiate responding for ethanol and obtain a relative success rate of acquisition. The use of sweeteners also introduces a potential bias in the interpretation of the self-administration data. These limitations don&#39;t apply to the following protocol.
Briefly, Samson and colleagues2 have shown that dissolving ethanol in a sweet solution of 20% sucrose and then fading out the sweetness over 4 weeks of training is required to initiate responding for 10% ethanol in water. Furthermore, reliable ethanol intake is usually achieved in 6 to 8 weeks1-3. This approach is highly problematic. First, it requires extended periods of training before investigators can begin to measure ethanol self-administration. In contrast, intravenous self-administration of cocaine or heroin requires 0 - 1 days of pre-drug training on a food-delivering lever in food restricted animals, and stable responding for drug is often achieved in 10 - 12 days4,5. Another limitation of this method is the fact that saccharin and sucrose are highly rewarding to rats and elicit brain activation patterns similar to drugs of abuse, thus introducing the potential for confounds in ethanol self-administration studies6-9. Finally, rats acquiring self-administration of an ethanol solution using this method show variability in acquisition and response rate1,10, with a substantial proportion of rats consistently excluded from experiments due to unsuccessful acquisition and/or insufficient response rate.
By contrast, with this protocol, we present a simple yet efficient method for acquisition and maintenance of oral self-administration of a 20% ethanol in water solution that does not necessitate water deprivation, sucrose/saccharin fading or extended access training. A recent investigation found that self-administration for oral ethanol displays an inverted U shaped dose response curve with highest ethanol intake during self-administration at a 20% ethanol concentration, thus providing a rationale for selecting 20% ethanol solution in our experimental design11.

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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.

Figure 1 shows the representative self-administration behavior of operant- and drug-naive Wistar rats (eight different cohorts amounting to a total of 239 rats) trained on a FR1 schedule to self-administer 20% ethanol without water deprivation or saccharin/sucrose fading during 30-minute sessions. With this protocol, rats initiate lever pressing to obtain an ethanol reward very quickly, already obtaining more than 10 rewards during the first sessions (Figure 1A). They reach stable responding rates on the active lever by session 10 of self-administration, obtaining a mean of 23.7 &#177; 0.7 rewards on FR1 (Figure 1B), corresponding to an average ethanol intake of 0.86 g/kg.
Figure 2 shows the effects of different doses of naltrexone on alcohol self-administration. To assess the predictive validity of an animal model of ethanol seeking, the efficacy of naltrexone, a currently FDA-approved medication for alcohol dependence, should be verified on a group of animals trained under an extended fixed-ratio-3 (FR3) reinforcement schedule. A treatment with naltrexone is expected to produce a dose-dependent decrease in alcohol-self administration (Figure 2A and 2B) as well as a reduced motivation to obtain alcohol assessed using a progressive ratio schedule (Figure 2C).
<img alt="Figure 1" src="/files/ftp_upload/53305/53305fig1.jpg" /> 
Figure 1: Wistar rats acquire and maintain self-administration of 20% ethanol. A: Mean rewards (&#177; SEM) earned during 30 min self-administration sessions (FR1, acquisition phase) of 20% ethanol (n= 239 rats). B: Mean rewards obtained during 30 min self-administration sessions (FR1, FR2, stable phase) of 20% (n= 239 rats). C: Mean active and inactive lever presses (&#177; SEM) completed during 30-minute self-administration sessions (FR1, FR2, stable phase) of 20% (n= 239 rats). D: Number of rewards earned during one 30 min self-administration session versus BEC (mg/dl) (n= 36 rats, R2= 0.67, p&#60; 0.001). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53305/53305fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53305/53305fig2.jpg" /> 
Figure 2: Naltrexone reduces both self-administration and motivation to consume alcohol. A: Mean active lever presses (&#177; SEM) completed during a 30 min self-administration session (FR3) of 20% ethanol following either saline or naltrexone treatment (0.1, 0.3 or 1 mg/kg) in Wistar rats (n=16) (** = p&#60; 0.01, *** = p&#60; 0.001). B: Mean rewards (&#177;SEM) earned during a 30-minute self-administration session (FR3) of 20% ethanol following either saline or naltrexone treatment (0.1, 0.3 or 1 mg/kg) in Wistar rats (n=16) (** = p&#60; 0.01, *** = p&#60; 0.001). C: Mean breakpoint (&#177;SEM) reached during a PR session of 20% ethanol following either saline or naltrexone treatment (1 mg/kg) in Wistar rats (n=16) (* = p&#60; 0.05). D: Number of rewards (&#177;SEM) earned during a PR session of 20% ethanol following either saline or naltrexone treatment (1 mg/kg) in Wistar rats (n= 16) (* = p&#60; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53305/53305fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training at JoVE.com

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

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-method-for-evaluating-reinforcing-properties-ethanol-rats-without

Nanyang Technological University

Research

JoVE Journal

Behavior

9.5K Views.  Linköping University. 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.

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

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 ...

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.

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 ...

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 ...

Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response

Underwater submergence produces autonomic changes that are observed in virtually all diving animals. This reflexly-induced response consists of apnea, a parasympathetically-induced bradycardia and a sympathetically-induced alteration of vascular resistance that maintains blood flow to the heart, brain and exercising muscles. While many of the metabolic and cardiorespiratory aspects of the diving response have been studied in marine animals, investigations of the central integrative aspects of this brainstem reflex have been relatively lacking. Because the physiology and neuroanatomy of the rat are well characterized, the rat can be used to help ascertain the central pathways of the mammalian diving response. Detailed instructions are provided on how to train rats to swim and voluntarily dive underwater through a 5 m long Plexiglas maze. Considerations regarding tank design and procedure room requirements are also given. The behavioral training is conducted in such a way as to reduce the stressfulness that could otherwise be associated with forced underwater submergence, thus minimizing activation of central stress pathways. The training procedures are not technically difficult, but they can be time-consuming. Since behavioral training of animals can only provide a model to be used with other experimental techniques, examples of how voluntarily diving rats have been used in conjunction with other physiological and neuroanatomical research techniques, and how the basic training procedures may need to be modified to accommodate these techniques, are also provided. These experiments show that voluntarily diving rats exhibit the same cardiorespiratory changes typically seen in other diving animals. The ease with which rats can be trained to voluntarily dive underwater, and the already available data from rats collected in other neurophysiological studies, makes voluntarily diving rats a good behavioral model to be used in studies investigating the central aspects of the mammalian diving response.

Detailed instructions are provided on how to train rats to voluntarily dive underwater through a 5 m long Plexiglas maze. Because the brains of rats have been very well characterized, voluntarily diving rats may help elucidate the central pathways of the mammalian diving response.

Underwater submergence produces autonomic changes that are observed in virtually all diving animals. This reflexly-induced response consists of apnea, a ...

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 Treatment Package without Escape Extinction to Address Food Selectivity

Feeding difficulties and feeding disorders are a commonly occurring problem for young children, particularly children with developmental delays including autism. Behavior analytic interventions for the treatment of feeding difficulties oftentimes include escape extinction as a primary component of treatment. The use of escape extinction, while effective, may be problematic as it is also associated with the emergence of challenging behavior (e.g., extinction burst). Such challenging behavior may be an acceptable side effect in treatment cases where feeding problems are severe and chronic (e.g., failure to thrive). However, in more acute cases (e.g., selective eating), the negative side effect may be unwarranted and undesired. More recent research on the behavioral treatment of food selectivity has begun to evaluate treatments for feeding difficulties that do not include escape extinction (e.g., demand fading, behavioral momentum), with some success. However, research to date reveals individual differences in responsiveness to such treatments and no clear preferable treatment has emerged. This manuscript describes a multi-component treatment package that includes shaping, sequential presentation and simultaneous presentation, for the treatment of food selectivity in four young children with developmental delays. This treatment package extends the literature on the behavioral treatment for food selectivity and offers a multi-component treatment protocol that may be clinically applicable across a range of treatment scenarios and settings.

Feeding difficulties are a common problem for children with developmental disorders, including autism, and behavioral interventions often include escape extinction. Recent research has begun to evaluate treatments that do not include escape extinction. This manuscript describes a multicomponent treatment package that does not use escape extinction to treat feeding difficulties.

Feeding difficulties and feeding disorders are a commonly occurring problem for young children, particularly children with developmental delays including ...

Pavlovian Conditioned Approach Training in Rats

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the attribution of incentive salience, or the transformation of neutral stimuli into attractive, &#34;wanted&#39; stimuli capable of motivating behavior. Pavlovian conditioned approach (PCA) develops after the response-independent presentation of a conditioned stimulus (CS; e.g., a lever) that predicts the delivery of an unconditioned stimulus (US; e.g., a food pellet) and can be used to measure incentive salience. During training, three patterns of conditioned responses (CRs) can develop: sign-tracking behavior (CS-directed CR), goal-tracking behavior (US-directed CR), and an intermediate response (both CRs). Sign-trackers attribute incentive salience to reward-related cues and are more vulnerable to cue-induced reinstatement of drug-seeking as well as other addiction-related behaviors, making PCA a potentially valuable procedure for studying addiction vulnerability. Here, we describe materials and methods used to elicit PCA behavior from rats as well as analyze and interpret PCA behavior in individual experiments.

Here, we present a protocol to elicit Pavlovian conditioned approach behavior in rats. This procedure can be used to measure individual differences in the tendency to approach and attribute incentive salience to reward-related cues and investigate addiction vulnerability.

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the ...

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 ...

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 ...

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 ...