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

Moderate prenatal alcohol exposure and quantification of social behavior in adult rats. This video describes a prenatal alcohol exposure paradigm followed by quantification of social behavior in adulthood. All procedures described in this video have been approved by the Institutional Animal Care and use Committee of the Health Sciences Center and the main campus at the University of New Mexico begin by obtaining adult female rats six to seven weeks of age that average 125 to 150 grams in weight and proven male rat breeders.

Typically 12 weeks of age allow at least one week for the animals to acclimate to their new housing environment. Overall, the prenatal alcohol exposure timeline includes a two week pre-exposure period, a breeding period in which the animals do not have access to alcohol, and the prenatal exposure period, which takes place throughout gestation. Details of each stage are as follows.

In order to evaluate pre-pregnancy drinking levels, the female rats voluntarily consume saccharine water containing incrementally higher concentrations of alcohol over a two week period. The function of this evaluation is to identify rats that drink at desired levels for subsequent phases of the drinking protocol. This aspect of the alcohol exposure paradigm also ensures that all female rats have experienced drinking prior to pregnancy.

Prepare drinking tubes by gathering the following supplies. Saccharin sodium salt, hydrate, 190 proof ethanol, beaded glass drinking tubes, natural rubber white, one hole number four stoppers one inch bent tubes with a ball point and paper rulers prepare a stalk solution of 0.066%saccharin water dissolved in tap water. Use a portion of the 0.066%saccharin water to make a stock Saccharin solution containing 2.5%ethanol and a portion to make a stock solution containing 5%ethanol.

Prepare drinking tubes by taping a paper ruler onto a tube. Fill the tube to the 20 millimeter mark with the correct solution for the day. Be sure to obtain baseline body weights for each female rat drinking sessions take place for four hours per day, beginning one hour after the start of the dark cycle.

On days one and two, the females will be given access to 0.066%saccharin water. On days three and four, the females will be given access to the SAC solution containing 2.5%ethanol. Beginning on day five, the females will be given access to the SAC solution containing 5%ethanol drinking sessions involving the 5%ethanol solution.

Continue until the pre-pregnancy drinking phase is complete. Food and tap water are available during all drinking sessions. At the end of the four hour drinking session, quantify the weight of the ethanol solution consumed per kilogram of body weight.

Upon completion of the pre-pregnancy drinking phase, calculate the mean ethanol consumption and standard deviation for the entire group and remove rats for which mean consumption is one. Standard deviation or more. Above or below the group mean.

Assign the remaining rats to either the saccharin control or prenatal ethanol exposure conditions. Match the pre-pregnancy drinking levels as closely as possible. For the two groups, pair each female rat with a proven male breeder.

Female rats do not consume alcohol during the breeding phase. When pregnancy is confirmed by the presence of a vaginal plug, weigh the female rat and Hauser individually beginning on gestational day one. Provide alcohol or saccharin solutions according to the group assignment.

For four hours per day, allow access to food and tap water during drinking sessions. Continue daily drinking sessions throughout the duration of the pregnancy. Quantify drinking consumption daily and weigh rat dams Weekly.

Cease the alcohol exposure procedures when the offspring are born. Designate the day of birth as postnatal. Day zero.

Call the litter to 10 pups around postnatal day two or three and record the pup weights. Wean the animals at approximately postnatal day 21 to 24 and house in same sex pairs with an animal for the same prenatal treatment condition. Do not use more than one to two rats from each litter per experiment to limit potential litter effects.

Quantification of social behavior in adulthood. Obtain all required materials and equipment. We utilize a custom built chamber, which is 95 centimeters long, 47 centimeters wide, and 43 centimeters tall.

The chamber is constructed of composite plastics material that is easily cleaned and disinfected and has a transparent plastic front and open top. The floor and sidewalls are lined with plastic to facilitate cleaning. The back interior wall is covered with mirrored tiles to aid analysis.

Additional materials needed include the following video cameras that are capable of recording under low or no light conditions. Ideally, the camera should have a high resolution in the infrared spectrum, infrared illuminators, which are positioned around the apparatus to improve lighting of the apparatus. In the video recording laboratory grade sandy chips, a brush and dust pan for cleaning the apparatus between sessions and 70%isopropyl alcohol for eliminating rat odors between sessions.

For measurement in adulthood, rats are at least 90 days of age when this phase of the experiment begins for three consecutive days, an animal and its cage mate are placed into the apparatus for 30 minutes to acclimate to the apparatus. Prior to each session, any woodchips are removed. The apparatus is wiped down with isopropyl alcohol and fresh woodchips are provided clean and disinfect the apparatus with an appropriate cleaning agent.

As necessary, the wood chips should entirely cover the bottom of the apparatus. During the acclimation session, all room lights are turned off at the end of the third acclimation session. House the animals individually in new cages with fresh wood chips, food and water.

Social interaction is recorded on the following day, 24 hours after the animals were separated in order to evaluate social behavior, begin by cleaning the apparatus and replacing the wood chips. Position one or more cameras to record the interaction. In our laboratory, we utilize the unique features of the fur patterns as a way of identifying the individual animals alternatives for marking the animals are described in the text upon completion of filming social interaction, identify the behaviors of interest In our laboratory.

We've quantified partner directed behaviors and other behaviors directed towards the environment. In our prior studies, wrestling and other measures of social investigation such as anal, genital sniffing or alleg grooming have reliably distinguished ethanol exposed animals from sac exposed animals. In the following video clips, examples of each behavior of interest are shown.

Wrestling occurs when one or both animals are engaged in an attempt to pounce on the partner and often results in one member of the pair pinning the other animal on its side or back. Wrestling is often preceded by chasing and can also be proceeded by other behaviors such as boxing, crossing over under, as well as an genital sniffing. Coating of the behaviors is terminated when the animals separate.

Occasionally, animals will quickly engage in another bout of wrestling if the animals have clearly separated from one another. The second bout would be coated as a separate instance. Because wrestling requires participation from both animals from the pear, the measures should be equivalent for the pear when coating.

You also may wish to note which animals ended up pinning versus being pinned, as well as which animal engaged in chasing and pouncing. It may also be useful to code the behaviors that comprise bouts of wrestling separately. If a more fine grained analysis is needed in boxing, both animals rear on their hind limbs and push one another with their four paws.

In episodes of crossing over or under one animal deliberately climbs on top or dives under the partner. In this behavior, one animal sniffs the anal genital region of the partner. One function of anal genital sniffing is identification.

Body sniffing refers to other sniffing of the partner's body and is often directed toward the side or flank of the partner. Animal alle grooming is grooming of the partner alle. Grooming is typically directed toward the nape of the neck and head area.

In rearing the animal rises onto the hind limbs similar to rearing, digging and sniffing in the bedding is a common environment directed behavior that provides a measure of behavior directed toward the substrate used in the apparatus for coding and quantification of behaviors, we use a custom MATLAB program developed in our laboratory. The main features of this program are playback of video at variable speed toggle buttons associated with each behavior of interest for which the state on or off indicates the presence or absence of the behavior, automatic time coding of the onset and offset of each behavior and data output, including total duration, frequency, and latency to first instance of each behavior After coding is completed for all animals, the resulting duration, frequency, and latency data are analyzed with a statistical package. In the example shown here, you can see that the frequency, duration, and latency to first instance of wrestling, as well as the duration of anal genital sniffing, differed significantly between saccharin and ethanol exposed animals.

Other behavioral measures did not distinguish between groups. The methods presented in this video streamlined the process of analyzing social behavior, following prenatal ethanol exposure into a simple and efficient protocol. In addition to the usefulness of this approach for distinguishing rats that have been exposed to moderate levels of ethanol during prenatal development from non-exposed animals, these methods could easily be adapted for evaluation of other ethanol exposure protocols, including variations in the timing, dose, duration, and age at the time of behavioral analysis.

Further, the specific behaviors quantified during analysis can be tailored to capture the major effects of ethanol exposure at various stages of development.