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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 FASDs^4-7. 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 FASDs^8-10 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 interaction^1,13-15, altered play^14,16,17, increased aggressive interactions^17,18, alterations in responsiveness to social stimuli^19-21, 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 saccharin^1,18. 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.

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Protokół

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

1. 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" bend tubes-ball point, paper rulers printed from www.vendian.org/mncharity/dir3/paper_rulers/.
2. 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.
3. House the animals individually in plastic cages at 22 °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.
4. Obtain baseline body weights for each female rat.
5. Evaluate pre-pregnancy drinking levels in female rats for 2 weeks. Follow steps 1.5.1–1.5.6 in order.
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. 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.
6. 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.
7. Provide ethanol or saccharin solutions for 4 hr per day (1,000 to 1,400 hr) for the duration of pregnancy.
   1. 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.
   2. Weigh rat dams weekly to assess maternal weight gain.
   3. Provide food and tap water at all times including the drinking sessions.
   4. 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.
   5. 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.
   6. Record pup weights 2-3 days after birth.
   7. 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.

2. Social Behavior

1. Obtain and prepare all required materials and equipment.
   1. 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 × 47 cm wide × 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.
   2. 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.
   3. Position infrared illuminators around the apparatus to improve lighting of the apparatus in the video recording.
   4. Obtain laboratory grade wood chips (aspen chip).
   5. Obtain a brush, dustpan, chlorine dioxide and isopropyl alcohol (70%) for cleaning the apparatus between sessions and odor control.
2. Apparatus acclimation and social behavior
   1. For this phase of the experiment, make measurements in adult rats that are at least 90 days of age.
   2. 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.
   3. 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.
   4. 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.
   5. Record social interaction on the following day 24 hr after the animals were separated.
      1. 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.
      2. 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.
      3. 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.
      4. Record video of the social interaction for at least 12 min.
      5. 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.
3. Behavioral coding and analysis.
   1. Identify the following behaviors of interest as per previous work with PAE^1,18. 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’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.
   2. 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.
   3. After coding is completed for all rats the resulting duration, frequency and latency data are analyzed with a statistical package.

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Wyniki

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...

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Dyskusje

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...

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Materiały

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