Supported by National Institutes of Health grants R01-AA015614, R01-AA014973, T32-AA014127 and K12-GM088021. The authors thank Samantha L. Blomquist for technical assistance and Drs. Kevin Caldwell and Donald Partridge for critically evaluating the manuscript and video.

All Animal procedures were approved by the University of New Mexico-Health Sciences Center Institutional Animal Care and Use Committee.
1. Vapor Chamber Assembly
<ol>
	<li>Cut polycarbonate sheets with a circular saw or jigsaw to the dimensions provided in the video for the top, bottom, front, back, sides, and door (Figure 1 and Table 1).</li>
	<li>With a circular saw or jigsaw, cut an opening 8 inches high by 16 inches wide in the middle of the front panel.</li>
	<li>Measure and mark the holes for the piano hinge onto the 18 inch by 10 inch polycarbonate sheet that will become the door of the chamber.</li>
	<li>In the door, drill counter sink holes with a 5/16 inch drill bit, and with a 3/16 inch drill bit, drill the hole for the screws. Make sure to drill counter sink holes on the inside of the door for the screw heads.</li>
	<li>Prepare the polycarbonate sheets for welding.</li>
	<li>Assemble front, back, and side panels onto the bottom panel using Weld-on #16.</li>
	<li>Seal any gaps between panels with Weld-on #16.</li>
	<li>Attach the top panel with Weld-on #16.</li>
	<li>Use bar clamps or heavy text books to hold everything in place while the welds cure.</li>
	<li>Attach a 1 inch by a 12 inch piece of polycarbonate onto the front panel 1 inch below the opening for the attachment of the door hinge.</li>
	<li>Allow at least 24 hr&#160;for Weld-on to cure.</li>
	<li>Cut 2 pieces of PEX tubing 12 inches long and 1 piece of 1 inch long PEX tubing.</li>
	<li>With a 5/16 drill bit, drill a hole in each of the 12 inch PEX tubing approximately 1 to 2 inches from one end.</li>
	<li>Attach 2 x 12 inch PEX tubing to a 3/8 inch T-connector with the holes located away from the connector.</li>
	<li>Cap the open ends of the PEX tubing with 3/8 inch plugs.</li>
	<li>Attach the piano hinge to the front panel and door using 4-40 machine screws and nuts.</li>
	<li>Using the door as a guide, mark the holes needed for the piano hinge onto the 1 inch spacer on the front panel of the chamber.</li>
	<li>With a 3/16 inch drill bit, drill the holes into the front panel for the piano hinge.</li>
	<li>Attach the door and piano hinge to the front panel using 4-40 machine screws and nuts.</li>
	<li>Assemble the toggle clamps as shown in the video with a washer and nut on either side of the side arm.</li>
	<li>Mark and drill 3/16 inch holes for the toggle clamps on the front panel and attach toggle clamps with 4-40 machine screws and nuts.</li>
	<li>Add 3/8 inch rubber bulb seals to the inside of the door.</li>
	<li>With a 5/8 inch boring drill bit, drill a hole in the center of the top panel for the inlet port.</li>
	<li>Drill a 1/2 inch hole in bottom / center of the back panel for the exit port.</li>
	<li>Assemble the exit port by pushing the threaded part of the 3/8 inch through-wall adaptors through the back wall of the vapor chamber in the exit port hole. Attach the nut from the inside of the chamber to hold in place.</li>
	<li>Remove the protective covering from polycarbonate sheets.</li>
	<li>Attach the 1 inch piece of PEX tubing to the T-connector and force the PEX tubing through the hole in the top of the chamber from the inside.</li>
	<li>Attach a 3/8 inch 90 elbow to the top of the 1 inch PEX tubing from the outside. 
	NOTE: Drill a 1/2 inch hole in the door and insert a 1/2 inch seal septa into the hole.</li>
	<li>Repeat steps 1-22 for the air only control chamber.</li>
</ol>
2. Rack and Air Delivery Assembly
<ol>
	<li>If placing the chambers on the cart/rack mentioned in the material list, assemble the cart according to the manufacturer&#8217;s instructions.</li>
	<li>With spare nuts and bolts, attach a scrap piece of polycarbonate to the rack to hold the airflow regulators.</li>
	<li>Anywhere on the scrap piece of polycarbonate, mark and drill 3/4 inch holes for the airflow regulators and attach regulators with the nuts provided.</li>
	<li>Attach 3/8 inch through-wall adapter to the inlet and outlet ports of the air flow regulators.</li>
	<li>Assemble the ethanol flask with the aeration stone, #8 stopper and quick release in-line connectors.</li>
	<li>Using 3/8 inch TYGON tubing, connect the flow regulators to the air pump and ethanol flask as shown in Figure 1.</li>
	<li>For the air only chamber, attach the exit port of the airflow regulator to the inlet 3/8 inch 90 elbow with 3/8 inch TYGON tubing as shown in Figure 1.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51839/51839fig1highres.jpg" width="500" /> 
Figure 1. Schematic representation of the configuration of the vapor chambers. A T-connector is attached to the low noise air pump. One side of the T-connector is directly connected to an airflow regulator for the air only vapor chamber. The other side is split again and attached to 2 different airflow regulators, one for air and one for ethanol. The air chamber is directly connected to the regulator as shown. For the ethanol vapor chamber, one airflow regulator is connected to the aerator stone immersed in liquid ethanol in the filter flask. The side arm port of the filter flask is connected with the output of the air regulator as indicated. The merged ethanol vapor and air is then connected to the inlet of the ethanol vapor chamber. The outlet tubes (not shown) are connected to a ventilation outlet in the room.
3. Test Vapor Chambers and Adjust Ethanol Levels
<ol>
	<li>Add 600 ml&#160;of 190 proof ethanol to the filter flask, insert the aeration stone, and hook up to inlet tube to the side arm of the flask.</li>
	<li>Close chamber doors and turn on air pump.</li>
	<li>Adjust airflow regulators so that approximately half as much air flows through the liquid alcohol as the air it is mixed with. Adjust the air only airflow to the combined flow of the alcohol and air of the ethanol chamber.</li>
	<li>Allow chambers to equilibrate for at least 30 min&#160;before measuring the air ethanol concentration.</li>
	<li>Measure air ethanol concentration by extracting 5 ml&#160;of air with an 18G&#160;needle and a 60 ml&#160;syringe through the septum. Dilute that sample with room air by drawing the plunger back to 60 ml&#160;(1:12 dilution). Measure air ethanol level using a breathalyzer in accordance to manufacturer&#8217;s directions. The dilution of the chamber air is needed to achieve alcohol vapor levels within the detection range of the breathalyzer. 
	NOTE: If using different levels of alcohol exposure the dilution may need to be adjusted.</li>
	<li>As a starting point, adjust the airflow regulators as needed to achieve an air alcohol concentration of approximately 4.5-5 g/dl&#160;(g of vaporized alcohol per dl&#160;of air).</li>
</ol>
4. Animal Breeding
<ol>
	<li>Group house females C578BL/6 mice (2-3 months old) for at least 1 week to synchronize ovarian cycles.</li>
	<li>Individually house male C57BL/6 mice (2-5 months old) for at least 2 weeks.</li>
	<li>After synchronizing, put a single female in with a single male for 5 days to allow for mating.</li>
	<li>Following mating, remove males and individually house females and place them into chambers.</li>
</ol>
5. Pre- and Post-natal Ethanol Vapor Exposure
<ol>
	<li>Expose pregnant dams to ethanol vapor for 4 hr&#160;per day starting at 10 am during the light cycle (lights on from 6 am-6 pm) except the day of birth to prevent pup death.</li>
	<li>Weigh dams on gestational day (G) 5, G13-G14, and G18-G20 to monitor pregnancies; bedding was changed on days that females were weighed to minimize handling.</li>
	<li>Each day replace the food for the ethanol exposed groups to avoid consumption of pellets with any ethanol absorbed into them.</li>
	<li>On the day of birth do not expose animals. After birth expose the dams and pups for 4 hr&#160;per day starting at 10 am from P1-P12.</li>
	<li>Weigh pups on P2, P8, P12, and P25; and change bedding on P8 and P12 to minimize additional handling.</li>
	<li>Immediately after the last exposure (P12), transfer the cages to a standard animal housing room.</li>
</ol>

The authors have nothing to disclose.

Here, we describe in detail the methods for the construction of vapor inhalation chambers. The materials and tools required to build the chambers are readily available from a number of commercial suppliers and the steps for the construction of the chambers are relatively straightforward. The system that we describe here does not contain any in-line check valves to prevent back-flow and mixing. We were unable to measure any detectable ethanol in the air only chambers suggesting that we do not have any mixing or back flow ...

Drinking during pregnancy can harm the fetus, causing persistent alterations in many organs and systems that significantly decrease the quality of life for the affected individuals and their families. It is estimated that approximately 10-30% of women drink during pregnancy in the U.S., with 1-8% drinking in a binge pattern1,2. The range of effects produced by ethanol exposure during fetal development is collectively known as fetal alcohol spectrum disorders (FASDs). Recent estimates indicate that FASDs are a major public health problem with a prevalence as high as 2-5% in the U.S.3. The more severe manifestation of FASDs is Fetal Alcohol Syndrome (FAS), which is characterized by growth retardation, craniofacial abnormalities, and neurobehavioral deficits, including learning disabilities. The prevalence of FAS has been estimated to be 0.2-0.7% in the U.S.3. The currently available treatments for FASDs are only partially effective and development of more effective treatments is limited by the poor understanding of the cellular and molecular underpinnings of this complex spectrum of disorders.
Data from the National Birth Defects Prevention Study (NBDPS) indicate that pregnant women most commonly drink during the 1st trimester, before pregnancy has been detected, followed by abstinence during later stages of gestation 2. The NBDPS also found that the second most common pattern of ethanol consumption during gestation involves drinking throughout all trimesters of pregnancy2. The reasons for this include lack of awareness about the potentially harmful effects of fetal ethanol exposure (even at low doses), limited access to prenatal care, positive history for neuropsychiatric disorders, and abuse of or dependence on ethanol4. Interestingly, the NBDPS reported that the third most common pattern of consumption involved abstinence during the 1st and 2nd trimesters followed by consumption during the 3rd trimester, when it is often assumed that drinking is safe because organogenesis has been mostly completed. However, the 3rd trimester is a period of high susceptibility to ethanol-induced nervous system damage because this is a period when neuronal circuits undergo profound refinement2. The NBDPS also identified other, less frequent patterns of alcohol consumption that occur during pregnancy, including consumption throughout the 1st and 2nd trimesters followed by abstinence during the 3rd trimester2.
In an attempt to model the different patterns of ethanol consumption observed in pregnant women, a number of developmental ethanol exposure paradigms have been established using diverse animal species, with rats and mice being most common5,6. The duration of pregnancy in these animals typically lasts approximately 3 weeks, which corresponds to the 1st and 2nd trimesters of human pregnancy. Many rodent studies have assessed the impact of various doses and patterns of ethanol exposure during this period. Examples of the methods frequently used to administer ethanol to pregnant mice and rats include administration via liquid diets7,8, addition of ethanol to the drinking water9,10, voluntary drinking of saccharin-sweetened solutions11, gastric gavage12, vapor inhalation13, and subcutaneous or intraperitoneal injection14. Results of these studies have recapitulated several of the deficits observed in humans with FASDs, demonstrating that exposure during early stages of pregnancy is sufficient to damage neuronal circuits across the brain (reviewed in 6,15).
Experiments with rodents have also demonstrated that exposure during the equivalent to the 3rd trimester of human pregnancy, which approximately corresponds to the first 1-2 weeks of postnatal life in rats and mice, can significantly impair brain development. Exposure during this period has been modeled by administering ethanol to neonatal rats or mice. Ethanol has been administered to these animals using a variety of methods, including feeding via gastrostomy in artificially-reared animals16-18, intragastric intubation19, subcutaneous injection20, and vapor inhalation21,22. These studies have convincingly demonstrated that the brain growth spurt is a period of high vulnerability to the developmental effects of ethanol6.
As mentioned above, drinking during all trimesters of pregnancy is a common pattern of ethanol consumption in women2. However, comparatively few studies have assessed the impact of this pattern of exposure using animal models. Some of these studies have taken advantage of large animals where the 3rd trimester-equivalent occurs in utero rather than the neonatal period as in the case of rats and mice. These animal models include non-human primates23,24 and sheep25-27. However, these animal models have not been widely used in FASDs research, in part, because of high cost and the need for specialized care facilities. Rodents have been more commonly used to characterize the effect of all-trimester ethanol exposure on fetal development5. Guinea pigs have been particularly advantageous in this regard given their extensive prenatal development and similarities in brain maturation to that of humans28,29. With guinea pigs, it has been possible to characterize the effect of ethanol exposure in utero that includes the equivalent development period of the human 3rd trimester. The comparatively high cost of these animals, as well as the relatively long duration of their pregnancy (~67 days), has limited their use to a few laboratories working on FASDs research.
Because of their cost-effectiveness and wide use in biomedical research, investigators have used rats to model exposure to ethanol during all trimesters of pregnancy. In initial studies, rats were exposed during pregnancy via liquid diets followed by administration of ethanol via gastrostomy to artificially reared neonates (postnatal days (P) 1-10) resulting in peak blood ethanol levels (BEC) in the dams of 0.08 g/dl&#160;and in the pups 0.16 g/dl. This paradigm caused long-lasting alterations in optic nerve myelination and reduced the number of Bergmann glia fibers in the cerebellum30-32. Similarly, Maier and collaborators using artificial rearing conditions administered ethanol to pregnant rat dams in a binge-like manner via intragastric intubation followed by neonatal administration during part of the 3rd trimester equivalent (P4-9)33,34. Peak maternal and pup BECs were 0.3 g/dl at both gestational day 20 and P6. This all-trimester exposure paradigm resulted in growth retardation that was significantly greater than that observed in pups exposed during selected periods of gestation33. In addition, rats exposed to ethanol during the equivalent to all trimesters exhibited a reduction in the number of cerebellar Purkinje and granule cells that was greater than that observed in animals exposed during other periods34. Reductions in hippocampal cell numbers were also reported with this paradigm, but these effects appear to be primarily a consequence of exposure during the 3rd trimester-equivalent35. A method that involves ethanol administration via intragastric gavage to both pregnant rats and neonatal mice has also been used to model all trimester exposure36. This method, which yielded BECs of 0.13 g/dl in the dams (gestational day 17) and 0.24 g/dl in P6 pups, induced long-lasting alterations in monoamine neurotransmitter levels in the hippocampus and hypothalamus, and increased expression of DNA methyltransferases and methyl CpG binding protein 2 in the hippocampus37,38. Using a similar exposure paradigm (BEC = 0.14-0.2 g/dl in dams and 0.2 g/dl in pups), Gil-Mohapel et al.39 detected an increase in the number of new immature neurons in the dentate gyrus of adult rats that may represent a compensatory mechanism to ethanol-induced neuronal damage or an alteration in the maturation of adult-born neurons. Investigators have also attempted to model all trimester ethanol exposure by exposing dams via liquid diets or drinking water during both pregnancy and lactation9,40. However, the utility of exposing the pups via their mother&#8217;s milk is limited because it typically results in low pup BECs (e.g., 0.002-0.05 g/dl; 41,42).
Mice have also been used extensively to characterize the effects of developmental ethanol exposure. This animal model shares many of the strengths described above for the rat animal model, with the additional advantage that many genetically modified mouse strains are available5. Mice have been successfully used to characterize the effects of ethanol during the 1st, 2nd or 3rd trimesters of pregnancy43,44. However, the impact of all trimester exposure on these animals has not been well characterized because it is technically more difficult to expose mice during the equivalent to all trimesters of human pregnancy. For instance, artificial rearing and gastric gavage, which have been used successfully in rats, require more specialized procedures in mice45. To the best of our knowledge, only one study to date has attempted to study the effect of all trimester ethanol exposure using mice; these animals were exposed to ethanol solution in drinking water during pregnancy and lactation46. Maternal BECs were 0.07 g/dl and pup BECs were not determined, but expected to be a fraction of those in dams.
Here, we describe a new model for all-trimester ethanol exposure of mice where alcohol is administered to both pregnant dams and neonates via vapor inhalation chambers. Vapor chambers were built based on a previous design47. We provide detailed instructions on how to build the inhalation chambers and carry out the exposure procedures. We also provide information on the BECs that can be achieved and the impact of exposure on pup survival and growth.

<table><tbody><tr><td>Polycarbonate 1/4&quot; clear 48&quot; x 24&quot;</td><td>McMaster-Carr</td><td>8574K23</td><td>10</td></tr><tr><td>Foam Rubber bulb seal 3/8&quot;w x 7/32&quot;h</td><td>McMaster-Carr</td><td>93085K67</td><td>10 ft</td></tr><tr><td>Weld-on #16</td><td>McMaster-Carr</td><td>7515A11</td><td>3</td></tr><tr><td>Piano hinge 12&quot; long</td><td>McMaster-Carr</td><td>1658A11</td><td>2 x 1 ft</td></tr><tr><td>Hold-down toggle clamps standard</td><td>McMaster-Carr</td><td>5126A26</td><td>8</td></tr><tr><td>PEX tubing 1/2&quot;</td><td>McMaster-Carr</td><td>51275K88</td><td>10 ft</td></tr><tr><td>Barbed Tee tube fitting (Black) Pkg 10</td><td>McMaster-Carr</td><td>5463K608</td><td>1</td></tr><tr><td>Barbed plug fitting (Black) Pkg 10</td><td>McMaster-Carr</td><td>5462K79</td><td>1</td></tr><tr><td>Barbed Elbow tube fitting (Black) Pkg 10</td><td>McMaster-Carr</td><td>5463K596</td><td>1</td></tr><tr><td>3/8&quot; Through-Wall Adapters, Tube to Threaded Pipe</td><td>McMaster-Carr</td><td>5463K851</td><td>1</td></tr><tr><td>Phillips Machine screw 4-40</td><td>McMaster-Carr</td><td>91772A112</td><td>1</td></tr><tr><td>Machine screw hex nut 4-40</td><td>McMaster-Carr</td><td>90480A005</td><td>1</td></tr><tr><td>Panel-mount flowmeter 2-20</td><td>McMaster-Carr</td><td>41945K76</td><td>3</td></tr><tr><td>FLASK, FILTER 1,000 ml 6/PACK</td><td>VWR</td><td>89001-800</td><td>2</td></tr><tr><td>Precision Seal Septa</td><td>VWR</td><td>89084-490</td><td>1</td></tr><tr><td>VWR Black Rubber Stopper #8 1-hole</td><td>VWR</td><td>59581-367</td><td>1</td></tr><tr><td>TUBE TYGON R3603 3/8X9/16 50&#039;</td><td>VWR</td><td>89068-556</td><td>1</td></tr><tr><td>TUBE TYGON R3603 1/4X11/16 50&#039;</td><td>VWR</td><td>89068-502</td><td>1</td></tr><tr><td>Aerator Stone P2120</td><td>VWR</td><td>32573-007</td><td>1</td></tr><tr><td>3/8&quot; T-connectors Pk of 20</td><td>VWR</td><td>46600-060</td><td>1</td></tr><tr><td>VWR Disconnectors tapered Pk of 10</td><td>VWR</td><td>46600-110</td><td>1</td></tr><tr><td>3/8 Hose Barb valved in-line coupling</td><td>Colder Products Company</td><td>HFCD17612</td><td>1</td></tr><tr><td>Air pump medium capacity</td><td>LMI Manufacturers</td><td>DB60L</td><td>1</td></tr><tr><td>Nexelate Wire Shelving 36&quot;W X 24&quot;D X 63&quot;H</td><td>Global industrial</td><td>T9A990135</td><td>1</td></tr><tr><td>Stem Casters Set of (4) 5&quot; Polyurethane Wheel</td><td>Global industrial</td><td>T9A500591</td><td>1</td></tr></tbody></table>

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.

Figure 2A shows that both pregnant mice and neonatal offspring were exposed to relatively stable ethanol vapor concentrations in the chambers. These ranged between 4-6 g/dl. Figure 2B shows the BECs achieved in the pregnant mice as a function of time. BECs were measured using a standard alcohol dehydrogenase based assay48. At G5, BECs quickly rose to ~60 mM 2 hr after the start of exposure and peaked at the end of the 4 hr exposure period. BECs gradually decreased to ~12 mM af...

Watch this Scientific Journal Video about Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development at JoVE.com

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

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 ...

construction-vapor-chambers-used-to-expose-mice-to-alcohol-during

Research

JoVE Journal

Medicine

14.6K Views.  University of New Mexico Health Sciences Center. 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 trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

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

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Developmental Biology

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.

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 ...

Behavior

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, ...

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

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

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 ...

Alcohol Diminishes Wasabi Reactions: Behavioral and AI Analysis in Mice

Integration of Animal Behavioral Assessment and Convolutional Neural Network to Study Wasabi-Alcohol Taste-Smell Interaction

The commercial wasabi pastes commonly used for food preparation contain a homologous compound of chemosensory isothiocyanates (ITCs) that elicit an irritating sensation upon consumption. The impact of sniffing dietary alcoholic beverages on the sensation of wasabi spiciness has never been studied. While most sensory evaluation studies focus on individual food and beverages separately, there is a lack of research on the olfactory study of sniffing liquor while consuming wasabi. Here, a methodology is developed that combines the use of an animal behavioral study and a convolutional neural network to analyze the facial expressions of mice when they simultaneously sniff liquor and consume wasabi. The results demonstrate that the trained and validated deep learning model recognizes 29% of the images depicting co-treatment of wasabi and alcohol belonging to the class of the wasabi-negative liquor-positive group without the need for prior training materials filtering. Statistical analysis of mouse grimace scale scores obtained from the selected video frame images reveals a significant difference (P &lt; 0.01) between the presence and absence of liquor. This finding suggests that dietary alcoholic beverages might have a diminishing effect on the wasabi-elicited reactions in mice. This combinatory methodology holds potential for individual ITC compound screening and sensory analyses of spirit components in the future. However, further study is required to investigate the underlying mechanism of alcohol-induced suppression of wasabi pungency.

This article describes a set of methods for measuring the suppressive ability of sniffing alcoholic beverages on the wasabi-elicited stinging sensation.

The commercial wasabi pastes commonly used for food preparation contain a homologous compound of chemosensory isothiocyanates (ITCs) that elicit an ...

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 ...

Murine Drinking Models in the Development of Pharmacotherapies for Alcoholism: Drinking in the Dark and Two-bottle Choice

Alcohol Use Disorder (AUD) is a major problem with more than an estimated 76 million people worldwide meeting the diagnostic criteria. Current treatments are limited to three FDA-approved medications that are largely ineffective even when combined with psychosocial intervention, as is evident by the high relapse rate. As such, the search for more novel treatments represents an important public health goal. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds: two-bottle choice (TBC) and drinking in the dark (DID). The former allows mice to voluntary drink in moderation while the latter induces mice to voluntary consume a large amount of alcohol in a short period that mimics binge drinking. The simple and high throughput nature of both of these paradigms allow for rapid screening of pharmacological agents or for identifying strains of mice that exhibit certain voluntary drinking behavior.

Alcohol Use Disorder (AUD) is a major national health problem and the development of more effective treatments is&#160;required to offset the needs of this patient population. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds.

Alcohol Use Disorder (AUD) is a major problem with more than an estimated 76 million people worldwide meeting the diagnostic criteria. Current treatments ...

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 ...

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

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Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

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

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

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

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

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

Integration of Animal Behavioral Assessment and Convolutional Neural Network to Study Wasabi-Alcohol Taste-Smell Interaction

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

Murine Drinking Models in the Development of Pharmacotherapies for Alcoholism: Drinking in the Dark and Two-bottle Choice

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