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

9.6K Views.  Linköping University.  Dit protocol beschrijft een nieuwe en efficiënte methode om snel te starten operante reageren voor ethanol in ratten die, in tegenstelling tot standaard methoden, geen water ontbering of saccharine / sucrose vervagen te initiëren reageren vereist. 

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

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 ...

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

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

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

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

A General Method for Evaluating Incubation of Sucrose Craving in Rats

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

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

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

Neuroscience

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Murine Drinking Models in the Development of Pharmacotherapies for Alcoholism: Drinking in the Dark and Two-bottle Choice

Alcohol Use Disorder (AUD) is a major problem with more than an estimated 76 million people worldwide meeting the diagnostic criteria. Current treatments are limited to three FDA-approved medications that are largely ineffective even when combined with psychosocial intervention, as is evident by the high relapse rate. As such, the search for more novel treatments represents an important public health goal. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds: two-bottle choice (TBC) and drinking in the dark (DID). The former allows mice to voluntary drink in moderation while the latter induces mice to voluntary consume a large amount of alcohol in a short period that mimics binge drinking. The simple and high throughput nature of both of these paradigms allow for rapid screening of pharmacological agents or for identifying strains of mice that exhibit certain voluntary drinking behavior.

Alcohol Use Disorder (AUD) is a major national health problem and the development of more effective treatments is&#160;required to offset the needs of this patient population. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds.

Alcohol Use Disorder (AUD) is a major problem with more than an estimated 76 million people worldwide meeting the diagnostic criteria. Current treatments ...

Medicine

Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral studies in animal models. In this protocol, to study depression-like and anhedonic behavior in rats, the established sucrose preference and novelty-induced hypophagia tests are combined with an automated food and liquid intake monitoring system. Prior to testing, in the sucrose preference paradigm, male rats are trained for at least 2 days to consume a sucrose solution in addition to tap water. During the test, rats are again exposed to water and sucrose solution. Consumption is registered every second by the automated system. The ratio of sucrose to total water intake (sucrose preference ratio) is a surrogate parameter for anhedonia. In the novelty-induced hypophagia test, male rats undergo a training period in which they are exposed to a palatable snack. During training, rodents show a stable baseline snack intake. On test day, the animals are transferred from home cages into a fresh, empty cage representing a novel unknown environment with access to the known palatable snack. The automated system records the total intake and its underlying microstructure (e.g., latency to approaching the snack), providing insight into anhedonic and anxious behaviors. The combination of these paradigms with an automated measuring system provides more detailed information, along with higher accuracy by reducing measuring errors. However, the tests use surrogate parameters and only depict depression and anhedonia in an indirect manner.

Presented here is a protocol to study depression-like and anhedonic behavior in rats. It combines two well-established behavioral methods, the sucrose preference and novelty-induced hypophagia tests, with an automated food and liquid intake monitoring system, to indirectly investigate rodent behavior using surrogate parameters.

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral ...

Modeling Alcohol Use Disorder Using Chronic Intermittent Ethanol Vapor Exposure

Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder

Alcohol use disorder (AUD) is a chronic alcohol-related disorder that typically presents as uncontrolled drinking and preoccupation with alcohol. A key component of AUD research is using translationally relevant preclinical models. Over the past several decades, a variety of animal models have been used to study AUD. One prominent model of AUD is the chronic intermittent ethanol vapor exposure (CIE) model, which is a well-established approach for inducing alcohol dependence in rodents through repeated cycles of ethanol exposure via inhalation. To model AUD in mice, the CIE exposure is paired with a voluntary two-bottle choice (2BC) of alcohol drinking and water to measure the escalation of alcohol drinking. The 2BC/CIE procedure involves alternating weeks of 2BC drinking and CIE, which repeat until the escalation of alcohol drinking is achieved. In the present study, we outline the procedures for performing 2BC/CIE, including the daily use of the CIE vapor chamber, and provide an example of escalated alcohol drinking in C57BL/6J mice using this approach.

Presented here is a protocol for the 2BC/CIE model of alcohol dependence in mice to study alcohol use disorder.

Alcohol use disorder (AUD) is a chronic alcohol-related disorder that typically presents as uncontrolled drinking and preoccupation with alcohol. A key ...

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

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