This work was supported, in part, by Research grants SC CTSI NIH/NCRR/ NCATS -- UL1TR000130 (D.L.D.), AA022448 (D.L.D.), and the USC School of Pharmacy.

All procedures described here have been approved by the Institutional Animal Care and Use Committees of the University of Southern California Health Sciences Campus.
1. Experimental Setup and Assembly
<ol>
	<li>Acquire all of the following supplies and chemicals before the start of the study: mice, cages/metal cage tops, bedding, food, water, ethanol, pipets, sipper tops, shrink wrap, utility knife, zip ties, tape, Bunsen burner, scale, headlamp.</li>
	<li>Obtain C57BL/6J mice, either from a commercial source or an in-house colony, keeping in mind that mice can be group housed until time of testing. 
	&#8203;NOTE: The total number of mice procured depends on the complexity of the experimental design. Plan to accommodate roughly 12 - 15 mice per group, with pilot studies no smaller than 5 - 7 mice per group. In the representative results shown below we utilized a simple two-group set-up to assess the cause-effect relationship using a single dose (5 mg/kg) of the drug (MOX).</li>
	<li>Follow the steps below to assemble the bottles18.
	<ol>
		<li>Heat a utility knife using a Bunsen burner.</li>
		<li>Using this knife, cut roughly an inch off the top and bottom ends of a plastic 18 mL serological pipet.
		<ol>
			<li>Note that smaller volume pipets (i.e., 10 mL) can also be used to increase the precision of the measurement.</li>
		</ol>
		</li>
		<li>Warm the pipet under a heat gun.</li>
		<li>Insert the ball bearing sipper tube into the &#34;bottom&#34; end of the pipet (in other words, the opening nearest to the 18 mL dash-line).</li>
		<li>Seal the sipper tube in place with shrink wrap using a commercially available shrink wrap gun.</li>
		<li>Cap off the other opening with a silicone stopper.</li>
	</ol>
	</li>
</ol>
2. Animal Habituation
<ol>
	<li>Beginning at least 1 week prior to the start date of the experiment, transfer the mice to the room where the experimental procedures are to be carried out so that they may acclimate to the husbandry conditions (including the ambient temperature (21 &#177; 1 &#176;C) and 12-h reverse light/dark cycle, with lights off at 12 noon). Be sure to follow institutional guidelines and notify the appropriate channels before moving animals from one location to another.
	<ol>
		<li>If mice are being transferred from a standard light/dark cycle allow 2 weeks of additional habituation time.</li>
	</ol>
	</li>
	<li>Fill the newly made bottles to the brim with water. Make sure that the cap is closed securely and devoid of any air bubbles or leaks from the spout. If solution is leaking, re-secure the cap. Remove any air bubbles by simply tapping on the bottle so that the air can escape the tube.</li>
	<li>Upon arrival, single house each mouse in standard polycarbonate/polysulfone cages with bedding and a metal grid cage top; remove the cage lid as it will no longer be used.
	<ol>
		<li>Provide access to food and water bottle(s) ad libitum.</li>
		<li>Secure each bottle to the cage top by wrapping a plastic zip tie around each bottle to hold it in place. Trim any excess plastic from the zip tie to ensure that it does not protrude into the cage. 
		&#8203;NOTE: For the drinking in the dark (DID) procedure, only a single bottle of water is required. Habituation to the two bottle choice (TBC) paradigm, however, requires that the cage set-up include two bottles of water. If the metal grid hopper provided is designed to hold only a single bottle of solution, gently bend apart its bars to create space for an additional plate to accommodate the second bottle for TBC.</li>
	</ol>
	</li>
	<li>Set up at least 3 mouse free control cages. This will allow for the monitoring of any fluid loss caused by evaporation or spillage from the bottles, which is simply a natural occurrence that happens as cages are placed on and off the cage rack (see step 2.6.3, 3.6.1, and 4.6.1 for equations).</li>
	<li>Beginning on day 4 of the 1-week single housing acclimation, measure and record the daily body and food weights of each mouse, using a scale, (in grams) as well as the water intake, using the etchings alongside the inverted bottle to record the highest point of the meniscus (in mL).
	<ol>
		<li>While it is standard scientific practice to read the lowest point of the concave meniscus, because the bottles remain in an inverted position during the measurement, record the highest point of the meniscus.</li>
	</ol>
	</li>
	<li>Assess the parameters of 2.5 using the equations below:
	<ol>
		<li>Measure the Body Weight Change (g): weight of current day (g) - weight of previous day (g).</li>
		<li>Measure the Food Intake (g): weight of food on previous day (g) - weight of food on current day (g).</li>
		<li>Measure the Water Intake (mL): [volume of water on current day (mL) - volume of water on previous day (mL)] - average water loss from all control cages (mL).</li>
	</ol>
	</li>
	<li>Repeat step 2.5 consecutively, on days 5 - 7, to allow for the determination of a baseline for the three days immediately prior to the introduction of ethanol. If a consecutive recording cannot be collected, extend the acclimation period to allow for the evaluation of baseline measurements.</li>
	<li>Once water intake has stabilized to &#177; 10% variability from the mean of the last 3 days, begin ethanol access with either TBC (unlimited access) or DID (limited access). 
	NOTE: On rare occasion, one to two additional days may be needed for subjects to attain this stability; don&#39;t be alarmed if additional time is needed for the values to display &#177;10% variability from the mean of the last 3 days.</li>
</ol>
3. 24-Hour Two Bottle Choice (TBC)
Note: A schematic is prepared in Figure 1.
<ol>
	<li>Prepare a 10% (v/v) ethanol solution at a 500 mL volume by adding 52.65&#160;mL of 190 proof grain ethanol (~95% ethanol) to 447.35&#160;mL of H2O; be sure to shake thoroughly. Given that ethanol evaporates quickly, replace the solution in every 3-4 day&#39;s interval. 
	NOTE: Other concentrations of ethanol can be used as well, but the authors recommend a 10% concentration for this model.</li>
	<li>On the first day of TBC, (day 8 at the earliest) empty 1 of the 2 water bottles, in each cage, and fill it to the brim with the freshly prepared ethanol solution. Given that ethanol and water are difficult to be distinguished visually, clearly label the bottles with their corresponding contents. Simply apply a piece of masking tape to the bottle and labeling it with a marker or by writing directly on the bottle.</li>
	<li>Add more solution to the water bottle, as needed.</li>
	<li>Place bottles back on the cage, making sure that all caps are closed securely and devoid of any air bubbles or leaks from the spout. If the solution is leaking, re-secure the cap. Remove any air bubbles by simply tapping on the bottle so that the air can escape the bottle.</li>
	<li>Alternate the position of the bottles every other day as to correct for conditioned place preference related influences in drinking activity (see Discussion for more).</li>
	<li>In addition to the daily measurements from 2.5 and 2.6, which have been ongoing, begin to read and record ethanol intake levels as well. Analyze the 10% ethanol intake and preference ratio using the following equations:
	<ol>
		<li>Measure the 10% Ethanol Intake (mL): [volume of ethanol on current day (mL) - volume of ethanol on previous day (mL))] - average ethanol loss from all control cages (mL).</li>
		<li>Measure the 10% Ethanol Intake (g/kg): [10% ethanol Intake (mL) x 0.07893 g/mL] / body weight (kg).</li>
		<li>Measure the Preference %: [10% Ethanol Intake (mL) / Water (mL)] x 100.</li>
	</ol>
	</li>
	<li>As soon as ethanol intake has become stable, administer a single control (saline) intraperitoneal (i.p.) injection (0.01 mL/g of body weight) to each mouse during the daily measurement routine. In this way, subjects become accustomed to the injection itself.
	<ol>
		<li>Stability is defined as&#160;&#177; 10% variability from the mean of the last 3 days (same as in section 2.8). 
		&#8203;NOTE: It can take up to 1 week for ethanol levels to stabilize. This is especially true if mice are being re-used from a previous experiment and have had previous exposure to ethanol. The control is simply the solvent used to dissolve the drug.</li>
	</ol>
	</li>
	<li>Once an ethanol baseline with low variability is re-established, split the mice into dosing groups using the ethanol intake values so that all groups have roughly similar average ethanol intake values.
	<ol>
		<li>Designate one group as the control (continuing to receive saline) and the other as the experimental (i.p. injection of the investigated drug at 0.01 mL/g of body weight). Begin daily drug dosing, either for an acute or multi-day duration. Subsequently, the control can be re-introduced to test the post-drug effects (optional). 
		NOTE: Because drinking is monitored across a 24-h long period, the time of dosage administration is not dependent on the dark cycle.</li>
	</ol>
	</li>
</ol>
4. Drinking in the Dark (DID)
NOTE: A schematic is prepared in Figure 3.
<ol>
	<li>On each day of scheduled ethanol access (Days 1 - 4) record the measurements, for water volume, food intake, and body weight, and perform drug dosing. Do this during a preselected time during the light cycle that is chosen in accordance with the pharmacokinetics of the drug so that the compound is at/or approaching the maximum brain concentration during the drinking period. 
	&#8203;NOTE: Remember days 1 - 3 are meant to simply acclimate the mice to drinking copious levels of ethanol in a short period of time. While mice do not reach BEC levels comparable to the human 0.08; these &#34;training&#34; days ensure that on day 4, during the slightly longer drinking session, they will in fact drink to that level.
	<ol>
		<li>Give all mice the control (saline) on days 1 - 3, and either the control or drug on day 4. This DID procedure occurs over the span of 3 weeks to include a pre-drug (week 1), drug (week 2), and post-drug (week 4) drinking session.</li>
		<li>NOTE: Note that during the drug-dosing week, day 3 ethanol intake levels are used to assign mice to either the control or drug group in a way where the ethanol intake levels of both groups have the least variability. This is unlike TBC, which assigns groups based on a 3 day average.</li>
	</ol>
	</li>
	<li>Prepare a 20% (v/v) ethanol solution (20E) at a 500 mL volume by adding 105.25 mL of 190 proof grain ethanol (~95% ethanol) to 394.75 mL of H2O; be sure to shake thoroughly.</li>
	<li>Fill the ethanol bottles before the start of the drinking session so that as soon as the DID begins the water bottles can simply be replaced with the alcohol bottles.</li>
	<li>During the entire DID session (steps 4.5 - 4.8), use a red-light headlamp as to not disturb the animals.</li>
	<li>At the start of the DID drinking session, scheduled to begin 3 hours into the dark cycle, record the volume of water for each mouse. Then, replace each water bottle with a bottle of the 20E solution and record the starting ethanol volume.</li>
	<li>Read and record the final ethanol volume 2 hours later, at the end of the drinking session on Days 1 - 3 and 4 hours later on Day 4. Analyze the 20% ethanol intake using the following equations.
	<ol>
		<li>Measure the 20% Ethanol Intake (mL): [volume of ethanol at end of drinking session (mL) - volume of ethanol at start of drinking session (mL)] - average ethanol loss from all control cages (mL).</li>
		<li>Measure the 20% Ethanol Intake (g/kg): [20% Ethanol Intake (mL) x 0.15786 g/mL] / body weight (kg).</li>
	</ol>
	</li>
	<li>On Day 4 only, immediately after recording ethanol volumes, and before re-introducing access to water, collect blood to assess the blood ethanol concentration of each mouse (optional). 
	&#8203;NOTE: Any non-terminal blood collection method may be used, such as retro-orbital sinus blood collection, or saphenous vein blood collection. For useful protocols see references for Parasuraman et al.21 and Yardley et al.20.
	<ol>
		<li>Perform analysis using various methods including LCMS, or commercially available machinery (see Table of Materials).</li>
	</ol>
	</li>
	<li>Replace all ethanol bottles with water bottles and record water volume.</li>
</ol>

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Z., 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=Medications+development+to+treat+alcohol+dependence:+a+vision+for+the+next+decade.">Medications development to treat alcohol dependence: a vision for the next decade.</a> Addiction Biology. 17 (3), 513-527 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Johnson+Medication+Treatment+of+Different+Types+of+Alcoholism.">Johnson Medication Treatment of Different Types of Alcoholism.</a> American Journal of Psychiatry. 167 (6), 630-639 (2010).</li><li>Litten, R. Z., Wilford, B. B., Falk, D. E., Ryan, M. L., Fertig, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+medications+for+the+treatment+of+alcohol+use+disorder:+An+evaluation+of+clinical+efficacy+and+safety.">Potential medications for the treatment of alcohol use disorder: An evaluation of clinical efficacy and safety.</a> Substance Abuse. 37 (2), 286-298 (2016).</li><li>Litten, R. Z., Ryan, M. L., Falk, D. E., Reilly, M., Fertig, J. B., Koob, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+Alcohol+Use+Disorder:+Understanding+Mechanisms+to+Advance+Personalized+Treatment.+Alcoholism:+Clinical+and+Experimental.">Heterogeneity of Alcohol Use Disorder: Understanding Mechanisms to Advance Personalized Treatment. Alcoholism: Clinical and Experimental.</a> Research. 39 (4), 579-584 (2015).</li><li>Schuckit, M. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+simple+model+of+ethanol+drinking+to+intoxication+in+C57BL/6J+mice.">Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice.</a> Physiology &amp; Behavior. 84 (1), 53-63 (2005).</li><li>Thiele, T. E., Navarro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Drinking+in+the+dark"+(DID)+procedures:+A+model+of+binge-like+ethanol+drinking+in+non-dependent+mice.">"Drinking in the dark" (DID) procedures: A model of binge-like ethanol drinking in non-dependent mice.</a> Alcohol. 48 (3), 235-241 (2014).</li><li>Crabbe, J. C., Spence, S. E., Brown, L. L., Metten, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+preference+drinking+in+a+mouse+line+selectively+bred+for+high+drinking+in+the+dark.">Alcohol preference drinking in a mouse line selectively bred for high drinking in the dark.</a> Alcohol. 45 (5), 427-440 (2011).</li><li>Sprow, G. M., Thiele, T. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+an+Orally+Dissolving+Strip+for+Pharmacological+and+Toxicological+Studies:+A+Simple+and+Humane+Alternative+to+Oral+Gavage+for+Animals.">Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals.</a> Journal of Visualized Experiments. (109), (2016).</li><li>Yardley, M. 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DLD and LA are inventors on a patent for the repurposing of ivermectin and related avermectins for the treatment of alcohol-use disorders. The authors have no other conflicts of interest and are entirely responsible for the scientific content of the paper.

Worldwide estimates indicate that as many as 76 million people meet the criteria to warrant a diagnosis for Alcohol Use Disorder (AUD). Unfortunately, pharmaceutical treatments currently available are largely ineffective and further development is necessary to offset the needs of this clinical population20. To this end, the following protocol aims to facilitate this endeavor by exemplifying two of the most basic rodent drinking paradigms: two-bottle-choice (TBC) and drinking in the dark (DID). Bot...

For the past 25 plus years, significant effort has been put towards developing medications for the treatment of Alcohol Use Disorder (AUD)1.Although many advances have been made, AUD still remains a major public health problem, affecting over 18 million Americans, and costing over $220 billion annually2,3. Currently there are only three FDA-approved medications, disulfiram, naltrexone, and acamprosate, all of which have yielded inconsistent results in clinical trials and limited success even when combined with psychosocial intervention in the clinic settings4,5,6,7.
A primary reason for failures of current AUD therapy is linked to the heterogenous nature of AUD8. While both environmental and genetic factors contribute to the development of AUD, heritability accounts for an estimated 50 - 60% of the risk of onset9. Similar to the treatment of depression, it is widely accepted that patients suffering from AUD will need a variety of medications that are tailored to meet the needs of each patient10.
Clearly, there is an urgent need for more effective treatments that would be facilitated if the already arduous and time consuming process of drug discovery were streamlined3. To this end, the following protocol demonstrates the preclinical applicability of two rodent drinking models widely used to examine the neurobiological basis of AUD11. More specifically, the method introduced herein can assess the efficacy of candidate compounds at reducing alcohol consumption in both &#34;moderate&#34; and &#34;binge drinking&#34; scenarios utilizing the two-bottle choice (TBC) and drinking in the dark (DID) paradigms, respectively. Both paradigms examine non-operant ethanol self-administration, whereby mice ingest ethanol orally and at will, and therefore illustrate high face and construct validity as a model of human alcoholism11.
In TBC drinking, also known as free choice drinking, preference drinking, or social drinking, two bottles of solution are continuously available in the home cage. One bottle contains water, and the other contains a diluted solution of ethanol, whereby the concentration of ethanol can be varied (e.g., 5 - 30% v/v)11,12. The mice have constant access to both bottles, and therefore, can choose how much to drink from each bottle.
This model assesses the ethanol consumption of each mouse (g/kg), as well as the ethanol preference ratio (volume of ethanol consumed &#247; total volume liquid consumed). It is routinely used to compare drinking levels across different strains of mice, or after a specific genetic manipulation (e.g., gene knockout or knockdown) and results in blood ethanol concentrations (BECs) similar to what is found in humans when drinking in moderation13,14.
In the DID procedure, 3 h after the start of the dark cycle, the home cage bottle of water is exchanged with a bottle of 20% (v/v) ethanol solution for a limited access drinking session. The drinking sessions occur as a consecutive 4-day cycle, lasting 2 h on days 1 - 3 and 4 h on day 4. Days 1-3 serve as an alcohol-habituation period before the testing on day 4. Consequently, mice will reliably consume enough ethanol to attain BECs &#62;100 mg/dL and as a result, exhibit the behavioral effects of intoxication found in humans that are binge drinking13,14,15. Water access is available at all times other than the drinking session.
There are several variations of limited access drinking. For example, in the intermittent access model, mice receive two bottles (one containing water and the other containing 20% (v/v) ethanol) only on Monday, Wednesday, and Friday with a 24 h and 48 h withdrawal period on weekdays and weekends, respectively16. After several weeks of intermittent access, the mice will gradually and voluntarily escalate drinking levels, eventually attaining BECS similar to what is observed in the DID model. The DID, however, appears to be the most commonly used model to assess binge-like drinking behavior. Other models of intermittent drinking exist, but they rely on restricting access to food or vapor chamber induced increases in voluntary self-administration, which makes them less representative of voluntary human alcohol consumption16.

<table border="0" cellpadding="0" cellspacing="0" style="width:1617px;" width="1616">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:483px;">1 L&#160; graduated cylinder</td>
			<td style="width:315px;">VWR</td>
			<td style="width:232px;">https://us.vwr.com/store/product/20935285/marisco-single-scale-cylinder-graduates-john-m-maris-co</td>
			<td style="width:588px;">To prepare ethanol solution.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1 L glass bottle</td>
			<td>Pyrex (Fisher Scientific)</td>
			<td>https://www.fishersci.com/shop/products/pyrex-reusable-media-storage-bottles-12/p-42752</td>
			<td>To prepare ethanol solution.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">100 mL graduated cylinder</td>
			<td>Fisher Scientific</td>
			<td>https://www.fishersci.com/shop/products/kimble-chase-kimax-class-a-to-contain-graduated-cylinders-8/p-4369311</td>
			<td>To prepare ethanol solution.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Analox</td>
			<td></td>
			<td></td>
			<td>One potential method of analyzing DID blood samples is by using the analox machine</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ball-bearing sipper tubes</td>
			<td>Ancare Corp.</td>
			<td>http://www.ancare.com/products/watering-equipment/open-drinking-tubes/straight-tubes-ball-point</td>
			<td>Length: 2.5 inches, Diameter: 5/16 inches, Model: TD100</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">C57BL/6J Mice</td>
			<td>Jackson lab</td>
			<td>https://www.jax.org/strain/000664</td>
			<td>May also come from internal breeding colony</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">disposable serological pipets</td>
			<td>VWR International (VWR)</td>
			<td>https://us.vwr.com/store/product/4760455/vwr-disposable-serological-pipets-polystyrene-sterile-plugged</td>
			<td>10 mL, 18 mL, or 25 mL&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ethanol, pure, 190 proof (95%), USP, KOPTEC</td>
			<td>Decon Labs (VWR)</td>
			<td>https://us.vwr.com/store/product/4542412/ethanol-pure-190-proof-95-usp-koptec</td>
			<td>---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">heat gun&#160;</td>
			<td>Master Appliances Corp.</td>
			<td colspan="2">http://www.masterappliance.com/master-heat-guns-kits/</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">heat shrink tubing</td>
			<td>---</td>
			<td>---</td>
			<td>Diameter: 3/8 inches</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">industrial knife/blade</td>
			<td>---</td>
			<td>---</td>
			<td>---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">metal cage plate</td>
			<td>---</td>
			<td>---</td>
			<td>Should be available through the university/institutional vivarium</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">mouse RO water</td>
			<td>---</td>
			<td>---</td>
			<td>Should be available through the university/institutional vivarium</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">portable electronic scale</td>
			<td>Ohaus (VWR)</td>
			<td>https://us.vwr.com/store/product/4789377/portable-electronic-cs-series-scales-ohaus</td>
			<td>---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">red light headlamp</td>
			<td>nyteBright (Amazon)</td>
			<td>https://www.amazon.com/LED-Headlamp-Flashlight-Red-Light/dp/B00R0LMMF8/ref=sr_1_1?ie=UTF8&#38;qid=1499591137&#38;sr=8-1-spons&#38;keywords=red+lamp+headlamp&#38;psc=1</td>
			<td>---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">silicone stoppers</td>
			<td>Fisher</td>
			<td>---</td>
			<td>---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">thermometer</td>
			<td>Fisher Scientific</td>
			<td>https://www.fishersci.com/shop/products/fisher-scientific-hygro-thermometer-clock-large-display-2/p-4077232</td>
			<td>---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">weigh boat</td>
			<td>VWR International (VWR)</td>
			<td>https://us.vwr.com/store/product/16773534/vwr-pour-boat-weighing-dishes</td>
			<td>The lid from a pipete tip box is an appropriate alternative</td>
		</tr>
	</tbody>
</table>

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.

In the following representative investigations, social drinking was modeled using the two-bottle choice (TBC) paradigm. Briefly, mice had access to two bottles of solution, one of which contained water, and the other a 10% (v/v) ethanol solution. Subjects were subsequently split and evenly assigned to drug treatment groups, moxidectin (MOX) vs. saline control, so that each group would have averaged ethanol intake levels with the least amount of variation.
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Watch this Scientific Journal Video about Murine Drinking Models in the Development of Pharmacotherapies for Alcoholism: Drinking in the Dark and Two-bottle Choice at JoVE.com

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 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 ...

murine-drinking-models-development-pharmacotherapies-for-alcoholism

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Drug Abuse and Addiction

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7.7K Views.  University of Southern California. Alcohol Use Disorder (AUD) is a major national health problem and the development of more effective treatments is 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.

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

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

A Real-World High-Intensity Interval Training Protocol for Cardiorespiratory Fitness Improvement

High-Intensity Interval Training (HIIT) has emerged as an interesting time-efficient approach to increase exercise adherence and improve health. However, few studies have tested the efficiency of HIIT protocols in a &#34;real world&#34; setting, e.g., HIIT protocols designed for outdoor spaces without specialized equipment. This study presents a &#34;real world&#34; training protocol, named &#34;beep training&#34;, and compares the efficiency of a HIIT regiment versus a traditional long-duration Moderate-Intensity Continuous Training (MICT) regiment using this beep training protocol on VO2 max of overweight untrained men. Twenty-two subjects performed outdoor running with MICT (n = 11) or HIIT (n = 11). Cardiorespiratory fitness was assessed before and after training protocols using a metabolic analyzer. Both training protocols were performed 3 days a week for 8 weeks using the Beep Test results. The MICT group performed the exercise program at 60%-75% of the maximum speed of the 20 m shuttle test (Vmax) and with a progression of the distance of 3,500-5,000 m. The HIIT group performed the interval exercise with 7-10 bouts of 200 m at 85%-100% of the maximum speed of the 20 m shuttle test (Vmax), interspersed with 1 min of passive recovery. Although the HIIT group presented a significantly lower training volume than the MICT group (p &#60; 0.05) after 8 weeks of beep training, HIIT was superior to MICT in improving VO2 max (MICT: ~4.1%; HIIT: ~7.3%; p &#60; 0.05). The &#34;real world&#34; HIIT regiment based on beep training protocol is a time-efficient, low-cost, and easy-to-implement protocol for overweight untrained men.

This study presents a low-cost and easy-to-implement "real world" high-intensity interval training (HIIT) protocol for scientific research and discusses its efficiency for cardiorespiratory fitness.

High-Intensity Interval Training (HIIT) has emerged as an interesting time-efficient approach to increase exercise adherence and improve health. However, ...