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

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.