Name: moderate prenatal alcohol exposure and quantification of social behavior in adult rats
Uploaded: 2014-12-14T14:00:00.000+00:00
Duration: 11 min 1 s
Description: Watch this Scientific Journal Video about Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats at JoVE.com

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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(71), e4288-e4288 (2013).</li><li>Panksepp, J., Siviy, S., Normansell, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+psychobiology+of+play+:+Theoretical+and+methodological+perspectives.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+modulation+of+juvenile+play+in+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+orbital+frontal+cortex+damage+on+the+modulation+of+defensive+responses+by+rats+in+playful+and+nonplayful+social+contexts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attack+and+defensive+behavior+in+albino-rat.">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

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...

Watch this Scientific Journal Video about Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats at JoVE.com

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

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-quantification-social-behavior

Research

JoVE Journal

Behavior

9.6K Views.  University of New Mexico. 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. 

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

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.

This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

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.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Medicine

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

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, such as rates of adult neurogenesis, angiogenesis, and expression of neurotrophic factors in rodents. This intervention has also been shown to mitigate behavioral and neuroanatomical aspects of the negative impacts of teratogens (i.e., developmental exposure to alcohol) and age-related neurodegeneration in rodents. Environmental complexity (EC) has been shown to produce numerous neuroplastic benefits in cortical and subcortical structures and can be coupled with wheel running to increase the proliferation and survival of new cells in the adult hippocampus. The combination of these two interventions provides a robust &#34;superintervention&#34; (WR-EC) that can be implemented in a range of rodent models of neurological disorders. We will discuss the implementation of WR/EC and its constituent interventions for use as a more powerful therapeutic intervention in rats using the animal model of prenatal exposure to alcohol in humans. We will also discuss which elements of the procedures are absolutely necessary for the interventions and which ones may be altered depending on the experimenter&#39;s question or facilities.

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 &#34;superintervention&#34; which combines wheel running and environmental complexity and will address the limitations of these interventions.

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, ...

Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use products. Based on their chemical structure and estradiol-like biological properties, they have been classified as endocrine disrupting compounds (EDC). Long-term exposure to EDCs, even at low doses, has been linked to various health defects including cancer, behavioral disorders and infertility, with greater vulnerability indicated during early developmental periods. Cellular and molecular studies with the genetically tractable nematode model Caenorhabditis elegans have demonstrated that exposure to BPA causes apoptosis, embryonic lethality and disruption in the DNA repair mechanisms. We have previously reported that exposure of C. elegans embryos to low doses of different bisphenols decreases fecundity. In addition, we have shown that the effects of exposure during the very early stages of development persist into adulthood as assayed by quantifying habituation behavior, a form of non-associative learning. Here, we provide detailed protocols for embryonic exposure to low-dose EDCs as well as the associated fecundity and anterior touch habituation assays, along with representative results.

Exposure to environmental toxicants can acutely impact development with long-term effects. Detailed protocols are provided to illustrate a strategy using an effective lab model to study the effect of early embryonic exposure to bisphenol A. We provide fecundity and behavioral assays to monitor the effectiveness of our toxicant exposure bio-assays.

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use ...

Developmental Biology

Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This is mostly due to the difficulty of microdissection of fetal brains and their sectioning for determination of apoptotic cells caused by prenatal exposure to alcohol. The experiments described here provide visualized techniques from mice breeding to the identification of cell death in fetal brain tissue. This study used C57BL/6 mice as the animal model for studying fetal alcohol exposure and the role of trophic peptide against alcohol-induced apoptosis. The breeding consists of a 2-hr matting window to determine the exact stage of embryonic age. An established fetal alcohol exposure model has been used in this study to determine the effects of prenatal alcohol exposure in fetal brains. This involves free access to alcohol or pair-fed liquid diets as the sole source of nutrients for the pregnant mice.
The techniques involving dissection of fetuses and microdissection of fetal brains are described carefully, since the latter can be challenging. Microdissection requires a stereomicroscope and ultra-fine forceps. Step-by-step procedures for dissecting the fetal brains are provided visually. The fetal brains are dissected from the base of the primordium olfactory bulb to the base of the metencephalon.
For investigating apoptosis, fetal brains are first embedded in gelatin using a peel-away mold to facilitate their sectioning with a vibratome apparatus. Fetal brains embedded and fixed in paraformaldehyde are easily sectioned, and the free floating sections can be mounted in superfrost plus slides for determination of apoptosis or cell death.
TUNEL (TdT-mediated dUTP Nick End Labeling; TdT: terminal deoxynucleotidyl transferase) assay has been used to identify cell death or apoptotic cells. It is noteworthy that apoptosis and cell-mediated cytotoxicity are characterized by DNA fragmentation. Thus, the visualized TUNEL-positive cells are indicative of cell death or apoptotic cells.
The experimental designs here provide information about the use of an established liquid diet for studying the effects of alcohol and the role of neurotrophic peptides during pregnancy in fetal brains. This involves breeding and feeding pregnant mice, microdissecting fetal brains, and determining apoptosis. Together, these visual and textual techniques might be a source for investigating prenatal exposure of harmful agents in fetal brains.

The experimental designs proposed here focus on studying the effects of alcohol exposure in apoptosis and the application of neurotrophic peptide during pregnancy in fetal brain. A detailed description from the breeding to the collection of fetal brains is described. Techniques for determination of apoptosis are also described in detail.

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This ...

Neuroscience

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

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

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

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, and cognitive abnormalities are associated with FASD. Social difficulties are common behavioral abnormalities associated with FASD and often result in serious health issues. Animal models are critical to understanding the mechanisms responsible for ethanol-induced social defects. Zebrafish are social vertebrates that produce externally fertilized transparent eggs; these characteristics provide researchers with a precise yet simple procedure for creating the FASD phenotype and an innate behavior that can be leveraged to model the social deficits associated with FASD. Thus, zebrafish are ideal for characterizing the social deficits of FASD. The goal of the current protocol is to provide the user with a simple behavioral assay that can be used to characterize the consequences of a negative environment early during development and the effects it can have on social behavior in adulthood. The protocol can be used to characterize the effect mutations or teratogens have on adult social behavior. The protocol outlined here demonstrates how to characterize the social behavior of individual fish during a 20-min social assay. Furthermore, the data obtained using the current protocol provides evidence that the protocol can be used to characterize the effects of embryonic ethanol-induced social defects in adult zebrafish.

The goal of the current protocol is to outline the steps necessary to establish and use a social preference assay for adult zebrafish and demonstrate that it can be used to characterize ethanol-induced social defects.

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Modeling Alcohol Use Disorder Using Chronic Intermittent Ethanol Vapor Exposure

Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder

Alcohol use disorder (AUD) is a chronic alcohol-related disorder that typically presents as uncontrolled drinking and preoccupation with alcohol. A key component of AUD research is using translationally relevant preclinical models. Over the past several decades, a variety of animal models have been used to study AUD. One prominent model of AUD is the chronic intermittent ethanol vapor exposure (CIE) model, which is a well-established approach for inducing alcohol dependence in rodents through repeated cycles of ethanol exposure via inhalation. To model AUD in mice, the CIE exposure is paired with a voluntary two-bottle choice (2BC) of alcohol drinking and water to measure the escalation of alcohol drinking. The 2BC/CIE procedure involves alternating weeks of 2BC drinking and CIE, which repeat until the escalation of alcohol drinking is achieved. In the present study, we outline the procedures for performing 2BC/CIE, including the daily use of the CIE vapor chamber, and provide an example of escalated alcohol drinking in C57BL/6J mice using this approach.

Presented here is a protocol for the 2BC/CIE model of alcohol dependence in mice to study alcohol use disorder.

Alcohol use disorder (AUD) is a chronic alcohol-related disorder that typically presents as uncontrolled drinking and preoccupation with alcohol. A key ...