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 wee...

<ol>
	<li>Koob, G. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+motivation+for+drinking+in+rodents+with+a+focus+on+opioid+receptor+neuropharmacology.+Recent+developments+in+alcoholism+:+an+official+publication.">Animal models of motivation for drinking in rodents with a focus on opioid receptor neuropharmacology. Recent developments in alcoholism : an official publication.</a> of the American Medical Society on Alcoholism, the Research Society on Alcoholism, and the National Council on Alcoholism. 16, 263-281 (2003).</li><li>Samson, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+ethanol+reinforcement+using+a+sucrose-substitution+procedure+in+food-+and+water-sated+rats.">Initiation of ethanol reinforcement using a sucrose-substitution procedure in food- and water-sated rats.</a> Alcoholism, clinical and experimental research. 10, 436-442 (1986).</li><li>Weiss, F., Mitchiner, M., Bloom, F. E., Koob, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-choice+responding+for+ethanol+versus+water+in+alcohol+preferring+(P)+and+unselected+Wistar+rats+is+differentially+modified+by+naloxone,+bromocriptine,+and+methysergide.">Free-choice responding for ethanol versus water in alcohol preferring (P) and unselected Wistar rats is differentially modified by naloxone, bromocriptine, and methysergide.</a> Psychopharmacology. , 178-186 (1990).</li><li>Koya, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ventral+medial+prefrontal+cortex+in+incubation+of+cocaine+craving.">Role of ventral medial prefrontal cortex in incubation of cocaine craving.</a> Neuropharmacology. 56, 177-185 (2009).</li><li>Karlsson, R. M., Kircher, D. M., Shaham, Y., O'Donnell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exaggerated+cue-induced+reinstatement+of+cocaine+seeking+but+not+incubation+of+cocaine+craving+in+a+developmental+rat+model+of+schizophrenia.">Exaggerated cue-induced reinstatement of cocaine seeking but not incubation of cocaine craving in a developmental rat model of schizophrenia.</a> Psychopharmacology. , 45-51 (2013).</li><li>Augier, E., Vouillac, C., Ahmed, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diazepam+promotes+choice+of+abstinence+in+cocaine+self-administering+rats.">Diazepam promotes choice of abstinence in cocaine self-administering rats.</a> Addiction biology. 17, 378-391 (2012).</li><li>Cantin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocaine+is+low+on+the+value+ladder+of+rats:+possible+evidence+for+resilience+to+addiction.">Cocaine is low on the value ladder of rats: possible evidence for resilience to addiction.</a> PloS one. 5, (2010).</li><li>Lenoir, M., Serre, F., Cantin, L., Ahmed, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intense+sweetness+surpasses+cocaine+reward.">Intense sweetness surpasses cocaine reward.</a> PloS one. 2, (2007).</li><li>Spangler, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opiate-like+effects+of+sugar+on+gene+expression+in+reward+areas+of+the+rat+brain.+Brain+research.">Opiate-like effects of sugar on gene expression in reward areas of the rat brain. Brain research.</a> Molecular brain research. , 134-142 (2004).</li><li>Rassnick, S., Pulvirenti, L., Koob, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDZ-205,152,+a+novel+dopamine+receptor+agonist,+reduces+oral+ethanol+self-administration+in+rats.">SDZ-205,152, a novel dopamine receptor agonist, reduces oral ethanol self-administration in rats.</a> Alcohol. 10, 127-132 (1993).</li><li>Carnicella, S., Yowell, Q. V., Ron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+operant+oral+ethanol+self-administration:+a+dose-response+curve+study+in+rats.">Regulation of operant oral ethanol self-administration: a dose-response curve study in rats.</a> Alcoholism, clinical and experimental research. 35, 116-125 (2011).</li><li>Augier, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wistar+rats+acquire+and+maintain+self-administration+of+20+%+ethanol+without+water+deprivation,+saccharin/sucrose+fading,+or+extended+access+training.">Wistar rats acquire and maintain self-administration of 20 % ethanol without water deprivation, saccharin/sucrose fading, or extended access training.</a> Psychopharmacology. , (2014).</li><li>Macchia, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+in+biological+fluids:+headspace+GC+measurement.">Ethanol in biological fluids: headspace GC measurement.</a> Journal of analytical toxicology. 19, 241-246 (1995).</li><li>Hodos, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+ratio+as+a+measure+of+reward+strength.">Progressive ratio as a measure of reward strength.</a> Science. 134, 943-944 (1961).</li><li>Williams, K. L., Broadbridge, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potency+of+naltrexone+to+reduce+ethanol+self-administration+in+rats+is+greater+for+subcutaneous+versus+intraperitoneal+injection.">Potency of naltrexone to reduce ethanol self-administration in rats is greater for subcutaneous versus intraperitoneal injection.</a> Alcohol. 43, 119-126 (2009).</li><li>Czachowski, C. L., Delory, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acamprosate+and+naltrexone+treatment+effects+on+ethanol+and+sucrose+seeking+and+intake+in+ethanol-dependent+and+nondependent+rats.">Acamprosate and naltrexone treatment effects on ethanol and sucrose seeking and intake in ethanol-dependent and nondependent rats.</a> Psychopharmacology. , 335-348 (2009).</li><li>Stromberg, M. F., Volpicelli, J. R., O'Brien, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+naltrexone+administered+repeatedly+across+30+or+60+days+on+ethanol+consumption+using+a+limited+access+procedure+in+the+rat.">Effects of naltrexone administered repeatedly across 30 or 60 days on ethanol consumption using a limited access procedure in the rat.</a> Alcoholism, clinical and experimental research. 22, 2186-2191 (1998).</li><li>Stromberg, M. F., Casale, M., Volpicelli, L., Volpicelli, J. R., O'Brien, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+effects+of+the+opioid+antagonists+naltrexone,+naltrindole,+and+beta-funaltrexamine+on+ethanol+consumption+in+the+rat.">A comparison of the effects of the opioid antagonists naltrexone, naltrindole, and beta-funaltrexamine on ethanol consumption in the rat.</a> Alcohol. 15, 281-289 (1998).</li><li>Gonzales, R. A., Weiss, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+ethanol-reinforced+behavior+by+naltrexone+is+associated+with+attenuation+of+the+ethanol-induced+increase+in+dialysate+dopamine+levels+in+the+nucleus+accumbens.">Suppression of ethanol-reinforced behavior by naltrexone is associated with attenuation of the ethanol-induced increase in dialysate dopamine levels in the nucleus accumbens.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 18, 10663-10671 (1998).</li><li>Biggs, T. A., Myers, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naltrexone+and+amperozide+modify+chocolate+and+saccharin+drinking+in+high+alcohol-preferring+P+rats.">Naltrexone and amperozide modify chocolate and saccharin drinking in high alcohol-preferring P rats.</a> Pharmacology, biochemistry, and behavior. 60, 407-413 (1998).</li><li>Beczkowska, I. W., Bowen, W. D., Bodnar, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+opioid+receptor+subtype+antagonists+differentially+alter+sucrose+and+deprivation-induced+water+intake+in+rats.">Central opioid receptor subtype antagonists differentially alter sucrose and deprivation-induced water intake in rats.</a> Brain research. 589, 291-301 (1992).</li><li>Cooper, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+opiate+agonists+and+antagonists+on+fluid+intake+and+saccharin+choice+in+the+rat.">Effects of opiate agonists and antagonists on fluid intake and saccharin choice in the rat.</a> Neuropharmacology. 22, 323-328 (1983).</li><li>Samson, H. H., Pfeffer, A. O., Tolliver, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+ethanol+self-administration+in+rats:+models+of+alcohol-seeking+behavior.">Oral ethanol self-administration in rats: models of alcohol-seeking behavior.</a> Alcoholism, clinical and experimental research. 12, 591-598 (1988).</li><li>Koob, G. F., Weiss, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+of+drug+self-administration.">Pharmacology of drug self-administration.</a> Alcohol. 7, 193-197 (1990).</li><li>Schank, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+the+Neurokinin-1+Receptor+in+Stress-Induced+Reinstatement+of+Alcohol+and+Cocaine+Seeking.">The Role of the Neurokinin-1 Receptor in Stress-Induced Reinstatement of Alcohol and Cocaine Seeking.</a> Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. , (2013).</li><li>Avena, N. M., Bocarsly, M. E., Rada, P., Kim, A., Hoebel, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=After+daily+bingeing+on+a+sucrose+solution,+food+deprivation+induces+anxiety+and+accumbens+dopamine/acetylcholine+imbalance.">After daily bingeing on a sucrose solution, food deprivation induces anxiety and accumbens dopamine/acetylcholine imbalance.</a> Physiology. 94, 309-315 (2008).</li><li>Avena, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+study+of+food+addiction+using+animal+models+of+binge+eating.">The study of food addiction using animal models of binge eating.</a> Appetite. 55, 734-737 (2010).</li><li>Morgan, D., Sizemore, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+addiction:+fat+and+sugar.">Animal models of addiction: fat and sugar.</a> Current pharmaceutical design. 17, 1168-1172 (2011).</li><li>Lenoir, M., Cantin, L., Vanhille, N., Serre, F., Ahmed, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+heroin+access+increases+heroin+choices+over+a+potent+nondrug+alternative.">Extended heroin access increases heroin choices over a potent nondrug alternative.</a> Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 38, 1209-1220 (2013).</li><li>Augier, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mGluR2+Positive+Allosteric+Modulator,+AZD8529,+and+Cue-Induced+Relapse+to+Alcohol+Seeking+in+Rats.">The mGluR2 Positive Allosteric Modulator, AZD8529, and Cue-Induced Relapse to Alcohol Seeking in Rats.</a> Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 41, 2932-2940 (2016).</li><li>Bice, P. J., Kiefer, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+reactivity+in+alcohol+preferring+and+nonpreferring+rats.">Taste reactivity in alcohol preferring and nonpreferring rats.</a> Alcoholism, clinical and experimental research. 14, 721-727 (1990).</li></ol>

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....

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</s...

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 ...

The overall goal of this protocol is to quickly initiate operant responding for alcohol in rats with a method that is convenient for experiementers and, contrary to standard methods, does not require water deprivation, or saccharin sucrose fading. So this method is a very useful tool to answer key questions about the neurobiology and neuropharmacology of alcohol reward in alcohol seeking in rats. The main advantage of this technique is that it does not require saccharin sucrose fading to initiate alcohol responding in rats.First eliminating the potentially downfall of using sweeteners in ethanol self-administration administration procedure. For this experiment, obtain rats weighing 200 to 225 grams. Wistar males are used here.House them in pairs or singly, depending on the rationale of the experiment. Keep the colony on a reverse 12-12 light-dark cycle with controlled temperature and humidity and conduct the experiments during the dark phase. Weigh the animals weekly, and permit them to acclimate for at least one week before beginning the experiment.Do not limit the rat's access to food and water. It isn't necessary for conditioning. Conduct all the behavior training in the same sized operant chamber with a flood grid and waste pan.The chamber must be fitted to a vented, sound-attenuating cubicle. Each chamber must have stimulus lights, syringe pumps, and two retractable levers. The levels should be positioned laterally to a liquid cup receptacle.Each chamber should be under automated control with a computer interface. Use this software to prepare the operant conditioning program as needed. For example, execute the following commands.Extend two levers to mark the onset of the session and to signal availability of alcohol. Have the ethanol lever, when pressed, deliver 100 microliters of 20%ethanol into the adjacent drinking well, and time-out for five seconds. Signal the time-out by illuminating a cue light above the lever.The other lever, when pressed, has no programmed consequence, and serves as a control for non-specific behavior. Even during time-outs, record how the rats use the levers, although there is no consequence to the rat. After 30 minutes, retract the two levers to signal the end of the session.Load an ethanol solution, made from 95%ethanol and tap water, into a syringe. Eject the solution without introducing air bubbles into the tap lines. Manually confirm the solution's flow.Always manually test the devices. Also, always dry out the solution receptacle before starting any session. Prior to beginning a session, load the software controls and make certain everything is operational.Move the rats to the testing room in a transport cage, and assign one rat to each chamber. Keep the testing room at the same temperature and humidity as the vivarium. Load the rats into their chambers in a consistent manner, and at the same time of day for each session.Then start the software to initiate the session. After the session ends, return the rats to the vivarium and use a disinfectant to clean the operant conditioning cages. Conduct the sessions daily, five to six times per week.After each session, calculate the mean number of active lever presses, inactive lever presses, and alcohol rewards. Performance will have stabilized when over three consecutive sessions the rewards do not vary by more than 15%Rats on a stable baseline can then be manipulated. One possible manipulation for rats on the stabilized baseline is to assess their motivation for the ethanol rewards.To do that, subject them to a progressive ratio schedule. Program the chambers to initially deliver a single volume of alcohol with one lever press, but increase the number of lever presses required after each alcohol reinforcer. Each animal will reach a break point, corresponding to the number of presses made for the last reinforcer earned.This model has been evaluated for its predictive validity using the approved alcoholics medication, naltrexone. Indeed, predictive validity was established, and therefore the model can be used to evaluate new drug candidates. Initially, inject the rats with saline 30 minutes prior to each session to habituate them to the injection.Analyze the results of the sessions and proceed when the number of rewards between the last two sessions varies by 15%or less. Then, deliver the naltrexone in saline with pH adjusted to neutral. Randomize the testing order between the different treatment groups, and untreated controls.Between drug administrations, let each rat go through two normal sessions with no treatment, exposed only to saline. This functions as the washout period, and resets the rat. Repeat the tests until ultimately each conditioned rat receives each treatment level once, and the control treatment once.After analyzing the data, the most effective dose can be selected to use on the animals during progressive ratio testing. Using the described protocol, 239 rats from eight different cohorts were trained to self-administer the alcohol reward without water deprivation or saccharin sucrose fading. Rats initiated lever pressing quickly, obtaining more than 10 rewards in the first session.Eventually, the rats obtained stable responding rates, earning about 24 rewards over each half hour session. An FDA approved drug, naltrexone, was tested at three concentrations. As expected, naltrexone treatment produced a dose-dependent decrease in alcohol self-administration, providing an important pharmacological validation for this protocol.Also as expected, naltrexone reduced the rat's motivation for alcohol under a progressive ratio schedule, as shown by the decline in the break point measure. After watching this video, you should have a good understanding on how to quickly initiate ethanol operant responding in rats without using water deprivation or saccharin sucrose fading procedure. With this protocol, rats acquire stable self-administration rates in about two weeks, that result in pharmacologically relevant blood alcohol concentrations.While attempting this procedure, it is important to keep the day-to-day condition as consistent as possible. It is also critical that the ethanol is correctly delivered into the receptacle from the first reinforced response, as any malfunction may alter or delay acquisition of ethanol self-administration. Following this procedure, other experiments can be carried out to study the neural basis of alcohol seeking and taking.

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9.3K visualizações.  Linköping University. The overall goal of this protocol is to quickly initiate operant responding for alcohol in rats with a method that is convenient for experiementers and, contrary to standard methods, does not require water deprivation, or saccharin sucrose fading. So this method is a very useful tool to answer key questions about the neurobiology and neuropharmacology of alcohol reward in alcohol seeking in rats. The main advantage of this technique is that it does not require saccharin sucrose fading to init...