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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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><tbody><tr><td>1 L&amp;nbsp; graduated cylinder</td><td>VWR</td><td>https://us.vwr.com/store/product/20935285/marisco-single-scale-cylinder-graduates-john-m-maris-co</td><td>To prepare ethanol solution.</td></tr><tr><td>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><td>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><td>Analox</td><td></td><td></td><td>One potential method of analyzing DID blood samples is by using the analox machine</td></tr><tr><td>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><td>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><td>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&amp;nbsp;</td></tr><tr><td>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><td>heat gun&amp;nbsp;</td><td>Master Appliances Corp.</td><td>http://www.masterappliance.com/master-heat-guns-kits/</td><td></td></tr><tr><td>heat shrink tubing</td><td>---</td><td>---</td><td>Diameter: 3/8 inches</td></tr><tr><td>industrial knife/blade</td><td>---</td><td>---</td><td>---</td></tr><tr><td>metal cage plate</td><td>---</td><td>---</td><td>Should be available through the university/institutional vivarium</td></tr><tr><td>mouse RO water</td><td>---</td><td>---</td><td>Should be available through the university/institutional vivarium</td></tr><tr><td>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><td>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&amp;amp;qid=1499591137&amp;amp;sr=8-1-spons&amp;amp;keywords=red+lamp+headlamp&amp;amp;psc=1</td><td>---</td></tr><tr><td>silicone stoppers</td><td>Fisher</td><td>---</td><td>---</td></tr><tr><td>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><td>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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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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Unit 1

Drug Abuse and Addiction

SCIENTISTS IN ACTION

7.9K Views.  University of Southern California. Het algemene doel van dit paradigma is om de effectiviteit van een potentiële anti-alcohol drug met behulp van knaagdier modellen van alcoholgebruik stoornis te beoordelen. Alcoholgebruik stoornis vormt een grote volksgezondheid crisis. De huidige behandelingen zijn niet effectief en lopend preklinisch onderzoek is nodig.Ons protocol stelt ons in staat om de veiligheid en het nut van een potentiële behandeling te evalueren en om een kijkje te nemen op de onderliggende neurobiologie v...

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

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods ...

Behavior

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 ...

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

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

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

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

The Motivation for Alcohol Reward: Predictors of Progressive-Ratio Intravenous Alcohol Self-Administration in Humans

The Progressive Ratio (PR) self-administration paradigm is a common pre-clinical method used to examine the motivation for a drug attributed to a craving, reward, or the relief of negative affect. The Computer-assisted Alcohol Infusion System (CAIS) enables intravenous alcohol self-administration behavior in humans. This system provides the investigator with control over the trajectory of each incremental breath alcohol concentration (BrAC) reward and the maximum BrAC allowed in a session. This paradigm allows participants to earn these alcohol rewards using a sequence of button presses specified by the investigator. The system employs a physiologically-based pharmacokinetic model-based algorithm to achieve the same incremental BrAC exposure in every participant. Participants (n = 11) took part in two identical sessions to examine test-retest reliability, and an additional group (n = 73) completed a single session. Sessions began with a 25 min priming phase: participants were instructed to press a button an increasing number of times per reward, accumulating four standardized incremental BrAC trajectories. The second phase comprised an ad-lib, PR paradigm lasting 125 min. Each reward required an increasing number of button presses. Measures of self-administration included: average and peak BrAC, total rewards earned, total grams of ethanol consumed per unit of total body water, the total number of button presses, and the average rate of button pressing. Self-administration measures were highly correlated both between and within sessions, demonstrating test-retest reliability and internal consistency. Recent drinking history was strongly associated with self-administration measures; heavier drinkers chose greater alcohol self-administration. These results indicate the reliability and sensitivity of this progressive-ratio intravenous alcohol self-administration method for assessing the motivational properties of alcohol, with the potential for improved testing of the efficacy of new medications thought to reduce consumption of alcohol. This method can be used to understand the genetic and environmental determinants of alcohol self-administration in humans.

This study aims to show that the Progressive-Ratio Computer-assisted Alcohol-Infusion System (CAIS) paradigm is a reliable and sensitive method that can be used to examine the motivating properties associated with alcohol self-administration in humans.

The Progressive Ratio (PR) self-administration paradigm is a common pre-clinical method used to examine the motivation for a drug attributed to a craving, ...

Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is required. In most cases, the preferred route of administration is via oral delivery. At the present time, oral delivery is mostly accomplished using an oral gavage procedure, in part, because it can achieve consistent and precise dosing in the animal model. Although this method is well established it does have complications that can result in a high rate of animal attrition. To this end, the procedure introduced here describes an alternative to the oral gavage method in which the desired drug is incorporated into a tastant, orally dissolving strip (ODS) that can simply be presented to the test animal where it is then rapidly taken up with minimal manipulation of the test subject. Herein, we demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

The following protocol describes a novel method for chronic oral drug administration using an orally dissolving strip (ODS) in lieu of the more commonly used oral gavage method. We demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is ...

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 ...

Neuroscience

Intermittent-Access Two-Bottle Choice Drinking with Lick Detection in Rats

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

Two-bottle choice home cage drinking is one of the most widely used paradigms to study ethanol consumption in rodents. In its simplest form, animals are provided with access to two drinking bottles, one of which contains regular tap water and the other ethanol, with daily intake measured by change in bottle weight over the 24 h period. Consequently, this approach requires no specialized laboratory equipment. While such ease of implementation is likely the greatest contributor to its widespread adoption by preclinical alcohol researchers, the resolution of drinking data acquired using this approach is limited by the number of times the researcher measures bottle weight (e.g., once daily). However, the desire to examine drinking patterns in the context of overall intake, pharmacological interventions, and neuronal manipulations has prompted the development of home cage lick detection systems that can acquire data at the level of individual licks. Although a number of these systems have been developed recently, the open-source system, LIQ HD (Lick Instance Quantifier Home cage Device), has garnered significant attention in the field for its affordability and user friendliness. Although exciting, this system was designed for use in mice. Here, we review appropriate procedures for standard and lickometer-equipped two-bottle choice home cage drinking. We also introduce methods for adapting the LIQ HD system to rats including hardware modifications to accommodate larger cage size and a redesigned 3D printed bottle holder compatible with standard off-the-shelf drinking bottles. Using this approach, researchers can examine daily drinking patterns in addition to levels of intake in many rats in parallel thereby increasing the resolution of acquired data with minimal investment in additional resources. These methods provide researchers with the flexibility to use either standard bottles or a lickometer-equipped apparatus to interrogate the neurobiological mechanisms underlying alcohol drinking depending on their precise experimental needs.

This protocol describes a standard intermittent-access two-bottle choice home cage drinking paradigm to model alcohol consumption in rats. In addition, it provides step-by-step instructions to augment the standard protocol with a DIY lick detection system that enables microstructural analysis of drinking behavior.

Two-bottle choice home cage drinking is one of the most widely used paradigms to study ethanol consumption in rodents. In its simplest form, animals are ...

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

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