Name: Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats (Video) | JoVE Journal
Uploaded: 2014-12-14T14:00:00.000+00:00
Duration: 11 min 1 s
Description: Learn a prenatal alcohol exposure protocol for quantifying social behavior changes in adult rats. This method enables researchers to study developmental effects of moderate ethanol exposure on neurobehavioral outcomes.

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5 minutes

Support provided by grant AA019462 to DAH and AA019884 to DDS.

All procedures described here and in the accompanying video have been approved by the Institutional Animal Care and Use Committees of the Health Sciences Center and the main campus of the University of New Mexico.
1. Prenatal Ethanol Exposure
<ol>
	<li>Obtain all required materials and chemicals: Saccharin sodium salt hydrate, 190 proof ethanol (95% alcohol by volume), beaded glass drinking tubes , natural rubber white #4 stopper one hole , 1&#34; bend tubes-ball point, paper rulers printed from www.vendian.org/mncharity/dir3/paper_rulers/.</li>
	<li>Obtain proven adult breeder rats from a vendor or alternative source. Use Long-Evans breeders for the methods and representative data presented in this protocol. Females weigh 125-150 g and 6-7 weeks of age upon arrival such that they are approximately 9-10 weeks old at the time of breeding (step 1.5). Ensure that all prospective dams are first-time mothers. Make sure that males are 12 weeks of age at arrival and 15 weeks old at the start of the breeding protocol.</li>
	<li>House the animals individually in plastic cages at 22 &#176;C on a reverse 12 hr light/dark schedule (lights on from 21:00 to 09:00 hr). Provide access to food and tap water ad libitum throughout the study, including during all drinking sessions. Allow at least 1 week for acclimation to the facility before proceeding to step 1.4.</li>
	<li>Obtain baseline body weights for each female rat.</li>
	<li>Evaluate pre-pregnancy drinking levels in female rats for 2 weeks. Follow steps 1.5.1&#8211;1.5.6 in order.
	<ol>
		<li>On days 1 and 2 fill a drinking tube containing 0.066% saccharin solution (no ethanol) in tap water. To quantify consumption affix a paper ruler with mm precision to the drinking tube prior to filling it. Use this method as it reduces error in measurement associated with weighing tubes to quantify ethanol consumption. Fill the tube to the 20 mm mark on the ruler. Begin four-hour drinking sessions (steps 1.5.1-1.5.6 and 1.7) 1 hr after the onset of the dark phase (10:00 hr) when activity and drinking levels are highest.</li>
		<li>At the end of each drinking session quantify the volume of the saccharin solution that was consumed. To facilitate measurement, determine the volume of solution per mm on the ruler in advance and convert mm to volumes. For the tubes recommended here each mm corresponds to 0.366 ml.</li>
		<li>On days 3 and 4 fill a tube with 2.5% ethanol (v/v) and 0.066% saccharin solution in tap water. At the end of each session quantify the amount of ethanol solution (weight per kg of body weight) that was consumed.</li>
		<li>On day 5 and thereafter fill a tube with 5% ethanol (v/v) and 0.066% saccharin solution in tap water. At the end of each session quantify the amount of ethanol solution (weight per kg of body weight) that was consumed.</li>
		<li>Upon completion of the pre-pregnancy drinking phase weigh the rats and calculate the mean ethanol consumption and standard deviation for the entire group. Remove rats for which mean consumption is greater than 1 standard deviation above or below the group mean from the study to reduce the variability in voluntary drinking during pregnancy.</li>
		<li>Assign the remaining rats to either the saccharin control or PAE conditions such that the pre-pregnancy drinking levels are matched as closely as possible for the two groups.</li>
	</ol>
	</li>
	<li>Within 1-14 days pair each female rat with a proven male breeder. Female rats do not consume ethanol during the breeding phase. Confirm pregnancy by the presence of a vaginal plug, weigh the female rat, and house her individually. This is defined as gestational day 1. Optionally, leave the female rats with a male breeder for up to 5 days, after which remove the females from the study. 
	NOTE: Because ethanol consumption during pregnancy begins at 10:00 hr on gestational day 1 inspection for the presence of a vaginal plug should be performed prior to this time.</li>
	<li>Provide ethanol or saccharin solutions for 4 hr per day (1,000 to 1,400 hr) for the duration of pregnancy.
	<ol>
		<li>Beginning on gestational day 1 provide the female rat with either 0% or 5% ethanol and 0.066% saccharin solution in tap water based on the group assignment. Ensure that the volume of 0% ethanol solution provided to saccharin control rats is matched to the mean volume of 5% ethanol solution consumed by ethanol rats. At the end of each session quantify the amount of ethanol solution (weight per kg of body weight) or saccharin that was consumed.</li>
		<li>Weigh rat dams weekly to assess maternal weight gain.</li>
		<li>Provide food and tap water at all times including the drinking sessions.</li>
		<li>Cease the ethanol exposure procedures when the offspring are born. Record the number of live pups and pup weights at birth. Designate the day of birth as postnatal day 0.</li>
		<li>Cull the litter to 10 pups around postnatal day 2-3. Attempt to maintain an equal ratio of male to female animals in each litter. If this is not possible, cull to 10 pups with an unequal number of males and females.</li>
		<li>Record pup weights 2-3 days after birth.</li>
		<li>Wean the animals at approximately postnatal day 21-24 and house in same-sex pairs with an animal from the same prenatal treatment condition. Do not use more than 1-2 rats from each litter per experiment to limit potential litter effects.</li>
	</ol>
	</li>
</ol>
2. Social Behavior
<ol>
	<li>Obtain and prepare all required materials and equipment.
	<ol>
		<li>Obtain an apparatus for social interaction. Use a chamber with an open top constructed of material that is easy to clean and sanitize. The front of the apparatus should be covered with rigid transparent plastic (~2 mm thickness) for filming. Line the interior walls and floor with transparent plastic (~2 mm thickness) to aid in cleaning and odor control. Place mirrors along the back interior wall to aid analysis. 
		NOTE : For the representative data reported here a custom chamber (95 cm long &#215; 47 cm wide &#215; 43 cm tall) with an open top and rigid transparent plastic (2 mm thickness) covering the interior sides and floor of the apparatus was used. Mirrors were placed along the back wall. The specific dimensions and materials are not critical for measurement of social behavior, however, it is recommended that the dimensions be sufficiently large to ensure that genuine social interaction can be distinguished from other behaviors. That is, the apparatus should be large enough so that animals are not always in close proximity to one another.</li>
		<li>Obtain video cameras capable of recording under low or no-light conditions as all filming is conducted with little or no ambient lighting within the visible spectrum. Ensure that the camera has a high resolution within the infrared spectrum, however, any camera capable of recording under dark conditions in its native night mode or with additional infrared illuminators should be sufficient.</li>
		<li>Position infrared illuminators around the apparatus to improve lighting of the apparatus in the video recording.</li>
		<li>Obtain laboratory grade wood chips (aspen chip).</li>
		<li>Obtain a brush, dustpan, chlorine dioxide and isopropyl alcohol (70%) for cleaning the apparatus between sessions and odor control.</li>
	</ol>
	</li>
	<li>Apparatus acclimation and social behavior
	<ol>
		<li>For this phase of the experiment, make measurements in adult rats that are at least 90 days of age.</li>
		<li>Prior to each session remove any woods chips, wipe the apparatus clean with isopropyl alcohol to control odors between sessions and provide fresh wood chips. Ensure that the wood chips entirely cover the bottom of the apparatus. Clean and sanitize the apparatus with an appropriate agent, such as chlorine dioxide, as necessary.</li>
		<li>For three consecutive days place an animal and its cage-mate into the chamber for 30 minutes to acclimate the animals to the apparatus. During the acclimation sessions all room lights are turned off.</li>
		<li>At the end of the third acclimation session house the animals individually in new cages with fresh bedding, food, and water for 24 hr to motivate social interaction.</li>
		<li>Record social interaction on the following day 24 hr after the animals were separated.
		<ol>
			<li>Remove wood chips from the apparatus, clean and sanitize with chlorine dioxide, wipe with isopropyl alcohol to control odors, and replace the wood chips prior to the session.</li>
			<li>Position one or more cameras to record the interaction. Position at least one camera in front of the apparatus so that the mirrors on the back wall of the apparatus can provide an additional perspective for analysis.</li>
			<li>Retrieve animals one at a time and hold the animal in front of the camera so that the unique features of the fur pattern can be noted. These identifying features of the fur can be used to distinguish rats during analysis rather than artificially marking the animals. 
			NOTE: Because many aspects of rodent social interaction involve olfactory signals and smelling the partner, wherever possible introducing foreign odors should be avoided. Long-Evans rats typically have some feature of the fur pattern that can be utilized to distinguish any given pair of animals. For other strains (e.g., Sprague-Dawley rats) alternative approaches such as marking the tail with an unscented dye could be used. It is important to recognize that many behaviors of interest are directed toward specific targets (e.g., anogenital sniffing directed near the base of the tail, playful attacks directed at the nape of the neck, or aggressive attacks directed toward the flanks or belly). Marking the animals closer to the tip of the tail far from these targets of interest is recommended.</li>
			<li>Record video of the social interaction for at least 12 min.</li>
			<li>Monitor animals for fighting throughout the session. If possible, watch the animals via a monitor or window so that the experimenter is not in the room during the session. 
			NOTE: Fighting has been only rarely observed in studies with adult rats, however, animals should be monitored throughout the session. The session should be ceased if there is excessive fighting or there are signs of harm or injury to an animal.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Behavioral coding and analysis.
	<ol>
		<li>Identify the following behaviors of interest as per previous work with PAE1,18. Quantify the duration, frequency and latency to first occurrence of the following behaviors; wrestling (including pinning), boxing, crawling (crossing) over/under the partner, anogenital sniffing, other sniffing of the partner&#8217;s body (body sniffing), allogrooming (grooming of the partner), rearing, and sniffing/digging in the wood chips. Examples of each behavior are illustrated in the video component of this article.</li>
		<li>Quantify the social behaviors of interest from the video. Obtain the frequency, total duration and latency to first instance for each behavior of interest. 
		NOTE: Obtaining these measures can be achieved manually, however, quantification of these measures using computerized analyses of digitized video is recommended. A Matlab (www.mathworks.com) script for playback of the video and quantification of behaviors is provided as a supplement to this article.</li>
		<li>After coding is completed for all rats the resulting duration, frequency and latency data are analyzed with a statistical package.</li>
	</ol>
	</li>
</ol>

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<li>Pellis, S. M., Pellis, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Play-fighting+differs+from+serious+fighting+in+both+target+of+attack+and+tactics+of+fighting+in+the+laboratory+rat+Rattus-Norvegicus%5BTitle%5D)+AND+%22Aggressive+Behav%22%5BJournal%5D)">Play-fighting differs from serious fighting in both target of attack and tactics of fighting in the laboratory rat Rattus-Norvegicus.</a> Aggressive Behav. 13 (4), 227-242 (1987).</li>
<li>Meaney, M. J., Stewart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+descriptive+study+of+social-development+in+the+rat+%28Rattus-Norvegicus%29%5BTitle%5D)+AND+%22Anim.+Behav%22%5BJournal%5D)">A descriptive study of social-development in the rat (Rattus-Norvegicus).</a> Anim. Behav. 29 (1), 34-45 (1981).</li>
<li>Pellis, S. M., Pellis, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+prejuvenile+onset+of+play+fighting+in+laboratory+rats+%28Rattus+norvegicus%29%5BTitle%5D)+AND+%22Dev.+Psychobiol%22%5BJournal%5D)">The prejuvenile onset of play fighting in laboratory rats (Rattus norvegicus).</a> Dev. Psychobiol. 31 (3), 193-205 (1997).</li>
<li>Himmler, B. T., Pellis, V. C., Pellis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peering+into+the+dynamics+of+social+interactions%3A+measuring+play+fighting+in+rats%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Peering into the dynamics of social interactions: measuring play fighting in rats.</a> J. Vis. Exp. (71), e4288-e4288 (2013).</li>
<li>Panksepp, J., Siviy, S., Normansell, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+psychobiology+of+play+%3A+Theoretical+and+methodological+perspectives%5BTitle%5D)+AND+%22Neurosci.+Biobehav.+Rev%22%5BJournal%5D)">The psychobiology of play : Theoretical and methodological perspectives.</a> Neurosci. Biobehav. Rev. 8 (4), 465-492 (1984).</li>
<li>Siviy, S. M., Panksepp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensory+modulation+of+juvenile+play+in+rats%5BTitle%5D)+AND+%22Dev.+Psychobiol%22%5BJournal%5D)">Sensory modulation of juvenile play in rats.</a> Dev. Psychobiol. 20 (1), 39-55 (1987).</li>
<li>Pellis, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+orbital+frontal+cortex+damage+on+the+modulation+of+defensive+responses+by+rats+in+playful+and+nonplayful+social+contexts%5BTitle%5D)+AND+%22Behav.+Neurosci%22%5BJournal%5D)">The effects of orbital frontal cortex damage on the modulation of defensive responses by rats in playful and nonplayful social contexts.</a> Behav. Neurosci. 120 (1), 72-84 (2006).</li>
<li>Blanchard, R. J., Blanchard, D. C., Takahashi, T., Kelley, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Attack+and+defensive+behavior+in+albino-rat%5BTitle%5D)+AND+%22Anim.+Behav%22%5BJournal%5D)">Attack and defensive behavior in albino-rat.</a> Anim. Behav. 25 (3), 622-634 (1977).</li>
</ol>

The authors have no conflicts of interest to disclose.

The prenatal alcohol exposure paradigm described here involves voluntary consumption of ethanol (5% v/v) by rat dams during pregnancy. There are a number of protocols for exposing non-human animals to ethanol during brain development represented in the literature, which differ with respect to the timing, dose, duration and route of ethanol administration as well as the species under investigation. Although a thorough treatment of the advantages of various exposure protocols is not provided here, several advantages of the...

An estimated 1-5% of children are diagnosed with Fetal Alcohol Spectrum Disorders (FASDs)2, which include Fetal Alcohol Syndrome (FAS), partial FAS (pFAS), and Alcohol-Related Neurodevelopmental Disorders (ARNDs)3. Deficits in social behavior and cognition are among the most common adverse outcomes observed in children with FASDs4-7. Negative consequences are not limited to heavy prenatal alcohol exposure (PAE), as moderate PAE that does not lead to the conspicuous morphological, behavioral and cognitive deficits characteristic of FAS can cause comparatively subtle, but nonetheless persistent, deficits in humans with FASDs8-10 and non-human animals exposed to ethanol during brain development11. The importance of understanding the behavioral and corresponding neurobiological consequences of moderate PAE is underscored by current estimates indicating that the large majority of FASD cases fall within the less severe range of the spectrum12.
Several independent laboratories have reported alterations in rodent social behavior related to ethanol exposure during brain development, including decreased investigation and interaction1,13-15, altered play14,16,17, increased aggressive interactions17,18, alterations in responsiveness to social stimuli19-21, and deficits in socially acquired food preferences and social recognition memory22. Social behavior deficits have been observed following exposure to heavy (blood ethanol concentrations (BECs) ~300mg/dl)22,23 or more moderate levels of ethanol (BECs ~80mg/dl)1, and across a broad range of parameters for other significant factors including exposure timing, duration of exposure, and age at the time of behavioral measurement.
Previous research has demonstrated that alterations in specific aspects of social interaction in adulthood discriminate rats exposed to moderate levels of alcohol from control animals exposed to saccharin1,18. In particular, moderate PAE has consistently been associated with robust increases in wrestling, which suggests increases in aggressive behavior, and lower levels of social investigation (e.g., sniffing of the partner) in adulthood. Because alterations in social behavior are reliable consequences of PAE, the quantification of social behavior following PAE may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior and the development of interventional approaches. The goal of this paper and the associated video is to provide instruction on the moderate PAE protocol and methods for quantification of social behavior in adult offspring that have reliably distinguished prenatal alcohol-exposed from non-exposed rat offspring.

<table><tbody><tr><td>Saccharin sodium salt hydrate</td><td>Sigma</td><td>S1002</td><td></td></tr><tr><td>190 proof ethanol</td><td>Sigma</td><td>493538</td><td></td></tr><tr><td>Beaded glass drinking tubes</td><td>Fisher</td><td>14-955K</td><td></td></tr><tr><td>Natural rubber white #4 stopper one hole</td><td>Plasticoid</td><td>LSG4M181</td><td></td></tr><tr><td>1&quot; bend tubes-ball point</td><td>Ancare</td><td>TD-199-3&quot;</td><td></td></tr><tr><td>Paper rulers</td><td>N/A</td><td>N/A</td><td>&lt;a href=&quot;http://www.vendian.org/mncharity/dir3/paper_rulers&quot;&gt;www.vendian.org/mncharity/dir3/paper_rulers&lt;/a&gt;</td></tr><tr><td>Apparatus for social interaction</td><td>Custom built</td><td>N/A</td><td>95 cm X 47 cm X 43 cm</td></tr><tr><td>Video cameras</td><td>N/A</td><td>N/A</td><td>Capable of recording low/no light conditions</td></tr><tr><td>Infrared illuminators</td><td>Vitek</td><td>VT-IR1-12</td><td></td></tr><tr><td>Teklad laboratory grade sani-chips</td><td>Harlan</td><td>7090A</td><td></td></tr><tr><td>Brush and dustpan</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>Sigma</td><td>W292907</td><td></td></tr><tr><td>Chlorine Dioxide (1.5 mg Tablets)</td><td>Quiplabs</td><td>N/A</td><td>Prepare per manufacturer&#039;s recommendation</td></tr></tbody></table>

moderate prenatal alcohol exposure and quantification of social behavior in adult rats

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.

Over the course of many breeding rounds female rats in the ethanol condition consistently drink an average of about 2.1 g/kg of ethanol per 4 hr drinking session. Rat dams consume approximately one-half of the four hr total during the first 15 to 30 min after the introduction of the drinking tubes, resulting in a peak maternal serum ethanol concentration of about 60 mg/dl, measured at the 45 min time point. Over the remaining 3.5 hr of the drinking period, they continue to consume 5% ethanol at a lower, but relatively st...

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 ...

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.

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In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1.  We then seek to find potential interventions and rapidly test them in this animal model.

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

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Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Cardiovascular exercise and stimulating experiences in a complex environment have positive benefits on multiple measures of neuroplasticity within the rodent brain. This article will discuss the implementation of these interventions as a "superintervention" which combines wheel running and environmental complexity and will address the limitations of these interventions.

Wheel Running and Environmental Complexity as a Therapeutic Intervention in an Animal Model of FASD

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Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

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Modeling Alcohol Consumption in Rodents Using Two-Bottle Choice Home Cage Drinking and Microstructural Analysis

As legislative restrictions change, resulting in increased accessibility of cannabinoid products, it is critically important to develop models to study the impact of these exposures, particularly during pregnancy. A preclinical model of moderate prenatal cannabinoid exposure through voluntary ingestion has been developed, enabling more in-depth investigations.

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Learn a prenatal alcohol exposure protocol for quantifying social behavior changes in adult rats. This method enables researchers to study developmental effects of moderate ethanol exposure on neurobehavioral outcomes.

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

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