Funding for this research was provided by a Program in Neuroscience Award from the Florida State University College of Medicine and support from the Department of Biological Science at FSU.&#160; Additional Funding was provided by a Grant-in-Aid from the Alcohol Beverage Manufacturer&#8217;s Research Fund.

1. Assembly of the FlyBar
Rationale and overview: The system is designed to administer controlled percentages of alcohol vapor to flies. Note: Figure 1 provides a schematic overview of the FlyBar set-up as described below in three stages (assembly of the air flow, set-up of the alcohol and water bottles, and assembly of the observation vials). In short, a steady airflow is split into two fractions that are bubbled through alcohol and water, respectively, mixed, and administered to 4 observation vials.
<ol>
	<li>Assembly of the Airflow
		<ol>
			<li>Connect a short piece of flexible silicone tubing to either building air or an aquarium aerator to generate a consistent air flow and split using a y-connector. Connect the first branch to the air flow regulator that controls the total amount of air through the system (typically 1,000 ml/min for 4 observation vials).</li>
			<li>Insert a quick connector in the second tube so that the air stream can be interrupted at the beginning of the experiment without affecting the calibrated airflow. Add a y-connector and connect each branching tube to the airflow regulator.</li>
			<li>Connect tubing to the airflow regulators and then connect two airflow meters.</li>
		</ol>
	</li>
	<li>Set-up of the Alcohol and Water Bottles
		<ol>
			<li>Add flexible tubing to the exit of the airflow meters and insert a thin glass tube (sections of 1 ml glass pipettes) with a 90&#176; bend into the end of each length of tubing. This will serve as the inlet air flow into the water and alcohol bottles.</li>
			<li>Place rubber stoppers with 2 holes through them into the bottles filled with alcohol and water. Keep both bottles at a constant temperature 2&#176; C higher than ambient air temperature using a water bath. In our experiments, the environmental room is maintained at 25 &#176;C, while the water bath is at 27 &#176;C.</li>
			<li>Insert the straight glass pipette for the air inlet through the rubber stopper and extend into the fluid till approximately 1 cm from the bottom of the bottle.</li>
			<li>Insert an elbow section of glass pipette into the remaining hole in the rubber stopper until the end of the glass is flush with the bottom of the stopper inside the bottle. Insert this air outlet in another length of tubing.</li>
			<li>Reunite the air streams using a y-connector and use another length of silicone tubing to direct the air flow through an empty mixing flask or bottle with bent glass pipette sections inserted through a two-holed rubber stopper. Use another length of silicone tubing for the outlet mixed airstream.</li>
		</ol>
	</li>
	<li>Assembly of the Observation Vial
		<ol>
			<li>Split the outlet airstream emerging from the mixing flask 2-3x to obtain 4 or 8 smaller streams of air so that multiple observation vials can be used in each experiment. Connect the flexible silicone tubing to the observation vials.</li>
			<li>Set up the observation vials using empty vials sealed with a rubber stopper containing two holes through which glass tubes provide an inlet and an outlet for the alcohol vapor.</li>
			<li>Cover the end of the first glass tube with netting and keep the netting in place using a small piece of flexible plastic tubing. Insert this tube through the first hole until it extends to approximately half the length of the vial. If needed, use Teflon tape to obtain a snug fit.</li>
			<li>Insert the second glass tube also with the end covered by netting until it is flush with the inside edge of the rubber stopper.</li>
			<li>Place the vials horizontally on a white piece of paper to maximize contrast with the flies under dim red light conditions.</li>
			<li>Mix appropriate fractions of the airstream bubbled through alcohol and the airstream bubbled through water. Monitor the air pressure continuously and make adjustments as needed to maintain desired mixing of the air streams. 
				Note: The continuous running of several Fly Bar assays in parallel or even a single assay in a small room can lead to a noticeable accumulation of alcohol vapor. To avoid continual release of alcohol vapor that potentially can affect the researcher in a closed room, an appropriate system needs to be put in place that adequately removes alcohol vapor generated during the experiment. To remove alcohol vapors, connect a 6-12 inch piece of tubing onto the second glass tube protruding from each vial, bundle them and direct to a funnel-vacuum system. Researchers should also ensure that the experimental testing room is adequately ventilated.</li>
		</ol>
	</li>
</ol>
2. Preparation of Experimental Animals
Rationale and overview: Proper culture and housing of the flies will reduce variability in the data. This is achieved through standardization and minimization of stress experienced by the flies. For that reason, no anesthesia (CO2 or alternatives) is used during any of the following steps of the protocol. Furthermore, flies should be age matched across experiments and time points to minimize variability as is standard for other behavioral analyses including learning and memory experiments17.
Different light:dark conditions can be used to probe the function of the circadian clock in the behavioral response to ethanol. To determine if a diurnal rhythm exists, experiments can be performed under a defined LD cycle to measure performance at specific Zeitgeber Times (ZT). ZT 0 represents dawn and is defined as the time of lights on under LD cycles, while ZT 12 is the time lights are turned off with a 12:12 hr LD cycle. Under constant conditions, the Circadian time (CT) measures time for the animal in the absence of environmental signals, i.e. free-running time, and is related to the previous LD entrainment cycle. In wild-type Drosophila, CT reflects the previous ZT for the first several days in constant conditions as the free-running circadian period and rhythms are ~ 24 hr. To measure circadian modulation and eliminate acute light effects on behavior, flies are entrained to light:dark cycles and then transferred to constant dark conditions (DD) prior to experiments. Circadian experiments are performed on the second day of DD to measure performance at specific Circadian Times (CT).
In Drosophila, continuous light (LL) conditions result in circadian dysfunction with dampened or abolished molecular oscillations of core circadian genes and disruption of behavioral circadian rhythms as evidenced by arrhythmic locomotor activity18-21 and arrhythmic short-term memory17. The protocol is optimized for circadian studies and can be simplified for other experiments. All circadian experiments are conducted using dim red light (ambient overhead red light &#60; 1 lux on bench top; small red lights used at 12 inches from tubes ~ 1 lux light).
<ol>
	<li>Rear the flies at 25 &#176;C under 12:12 hr light:dark entrainment conditions (LD).</li>
	<li>Collect freshly eclosed flies at the end of the day-light period on day 1 and store them for 24 hr under LD conditions in holding vials containing a small amount of high agar concentration food to minimize food stickiness. Note: To ensure healthy, normally developed flies are collected, only use flies collected within the first days after eclosion starts in a culture bottle.</li>
	<li>Collect batches of approximately 30 (25-35) flies using an aspirator on day 2 towards the end of the light period, and transfer to fresh holding vials.</li>
	<li>Use a strong light source to direct flies to the far end of the vial. Within this range, the exact number of flies in each vial is not critical as behavioral observations are reported in percentages with total number of flies counted at the end of each experiment.</li>
	<li>Maintain flies under DD conditions at 25 &#176;C for two days.</li>
	<li>On the day of the experiment, place all flies housed in different conditions or incubators other than the experimental behavior room in the room for at least 1 hr prior to the experiment. Acclimation reduces variability due to changes in temperature or humidity.</li>
	<li>Make observations at six time points a day (CT 1, 5, 9, 13, 17 and 21) in order to test for circadian modulation of behavior.</li>
	<li>Compare multiple time points within a single set of behavioral experiments to increase the robustness of the experimental design and minimize variability specific to a single experiment. For example, observations of CT 1 and CT 13 can be obtained simultaneously if two incubators with opposite light-dark schedules are used for entrainment. 
		Note: The above procedure describes the preparation of experimental animals for assays performed in circadian condition of constant darkness. Different light:dark conditions can be used to probe the function of the clock in the behavioral responses to alcohol. To determine if a diurnal rhythm exists the experiments can be performed under an LD cycle to measure performance at specific Zeitgeber Times (ZT). Additionally, the protocol may be used with flies raised under constant light conditions for experiments testing circadian dysfunction. For alternate protocols that test flies housed in light conditions, behavioral assays should still be performed under dark conditions. Flies should be transferred into the dark for 1 hr prior to the experiment to minimize behavioral variability due to the acute effects of light on behavior.</li>
</ol>
3. Behavioral Observations
Rationale and overview: The following alcohol administration protocol is optimized for observations under dim red light condition. The two behavioral measures LoRR and sedation represent two distinct points of fly inebriation. LoRR represents a late point of inebriation incorporating the loss of motor and postural control, whereas sedation measures a very late end point of intoxication. Genotype or circadian modulation may affect these two measures differently; hence one may wish to examine both. In short, flies are loaded in the vials, the number of flies displaying LoRR or sedation are scored every 5 min during alcohol vapor exposure, and the total number of flies counted at the end of the experiment.
<ol>
	<li>Before starting the experiment, run the air through the system (air bubbled through water and alcohol bottles) for at least 10 min and use that time to calibrate the airflows.</li>
	<li>Disconnect the quick release to stop the airflow. Load the flies in the vials, and reconnect the airflow and start the timers. Note: If unresponsive flies or dead flies are left in the holding vials, this could be indicative of stress conditions. In general, these conditions may be alleviated by housing fewer flies in the holding vials or decreasing food stickiness using a slightly higher agar concentration during food preparation. For optimal behavioral analyses and minimal variability between experiments, flies should be healthy prior to experiments.</li>
	<li>For accurate time keeping, use one timer to keep track of the total time of alcohol exposure and use a second count-back timer to mark the 5-min intervals.</li>
	<li>Place a piece of white paper under the vials to increase contrast and fly visibility, especially under dim red light conditions.</li>
	<li>Check the airflow regularly during an experiment to maintain constant levels. Generally, once airflows have stabilized, they remain stable during the length of the experiment.</li>
	<li>Count the number of flies that have lost their righting reflex once every 5 min for a 1 hr period. As the alcohol sensitivity varies between genotypes and genetic backgrounds, it may be desirable to perform more frequent assessments or to conduct the experiment for a longer period of time.</li>
	<li>Lift the vial slightly from the surface, and direct the light from a red-light flashlight toward the paper behind the vial. Keep the hand-held red flashlights at a distance of at least 12 inches to the experimental vial to maintain light levels no greater than 1 lux for all experiments under dim red light conditions.</li>
	<li>Measure light levels using a light meter to establish standards for all experiments.</li>
	<li>Determine the number of flies that have lost their righting reflex by applying a firm tap to the vial and count how many flies fail to right themselves within approximately 4 sec. Flies that display LoRR may still move their legs and wings, but cannot turn themselves upright.</li>
	<li>At the end of the session, count the total number of flies in each vial. 
		Note: Occasionally, a single fly may be caught between the stopper and the side when loading flies into the experimental vials. As this is done in the dark, it may not be readily noticed so it is necessary to count the total number of flies at the end of the experiment to correctly calculate percentages.</li>
</ol>
Additionally, this procedure also may be used to measure sedation of flies, which represents a different behavioral endpoint. While sedated flies have lost their right reflex, sedation requires greater alcohol exposure. Behaviorally, sedation may be characterized by the complete lack of apparent motor activity with flies remaining motionless in the vial following a a firm tap to the vial. For sedation, count the number of flies that remain motionless with no leg waving following delivery of a firm tap of the vial. Additionally, the vial can be rolled side to side to determine whether individual flies still retain their grabbing reflex.
4. Data Analysis
<ol>
	<li>Determine the percentage LoRR at each time assessed based upon the total number of flies in each vial.</li>
	<li>Estimate differences between circadian time points or strains by calculating the 50% LoRR for each sample, which falls within the linear portion of the sigmoid curve (See Figure 2 below).</li>
	<li>Alternative statistics:
		<ol>
			<li>If comparisons between genotypes are planned, it is desirable to analyze the whole time course using repeated measures ANOVAand to determine the range of time points that the differences are significant with post-hoc tests (Figure 3). For these tests, we prefer to use an a value of 0.001. This allows differences at individual exposure times to be assessed as well as differences between genotypes in the slope of the curve.</li>
			<li>Differences in sensitivity for can be determined for specific times of alcohol exposure for a particular response such as sedation from the linear portion of the graph (Figure 4).</li>
			<li>Differences between strains or circadian time points can be estimated using standard F-statistics and post-hoc tests.</li>
		</ol>
	</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+Drosophila+circadian+control+circuit.">Organization of the Drosophila circadian control circuit.</a> Current Biology. 18, 84-93 (2008).</li><li>Sheeba, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+melanogaster+circadian+pacemaker+circuit.">The Drosophila melanogaster circadian pacemaker circuit.</a> J. Genet. 87, 485-493 (2008).</li><li>Cohan, F. M., Graf, J. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Latitudinal+cline+in+Drosophila+melanogaster+for+knockdown+resistance+to+ethanol+fumes+and+for+rates+of+response+to+selection+for+further+resistance.">Latitudinal cline in Drosophila melanogaster for knockdown resistance to ethanol fumes and for rates of response to selection for further resistance.</a> Evolution. , 278-293 (1985).</li><li>Moore, M. S., 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=Ethanol+intoxication+in+Drosophila:+Genetic+and+pharmacological+evidence+for+regulation+by+the+cAMP+signaling+pathway.">Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway.</a> Cell. 93, 997-1007 (1998).</li><li>Berger, K. H., 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=Ethanol+sensitivity+and+tolerance+in+long-term+memory+mutants+of+Drosophila+melanogaster.">Ethanol sensitivity and tolerance in long-term memory mutants of Drosophila melanogaster.</a> Alcohol Clin Exp Res. 32, 895-908 (2008).</li><li>Pohl, J. B., 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=Circadian+Genes+Differentially+Affect+Tolerance+to+Ethanol.">Circadian Genes Differentially Affect Tolerance to Ethanol.</a> in Drosophila. Alcoholism: Clinical and Experimental Research. , (2013).</li><li>Bhandari, P., Kendler, K. S., Bettinger, J. C., Davies, A. G., Grotewiel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+for+evoked+locomotor+behavior+in+Drosophila+reveals+a+role+for+integrins+in+ethanol+sensitivity+and+rapid+ethanol+tolerance.">An assay for evoked locomotor behavior in Drosophila reveals a role for integrins in ethanol sensitivity and rapid ethanol tolerance.</a> Alcoholism: Clinical and Experimental Research. 33, 1794-1805 (2009).</li><li>Rothenfluh, A., 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=Distinct+behavioral+responses+to+ethanol+are+regulated+by+alternate+RhoGAP18B+isoforms.">Distinct behavioral responses to ethanol are regulated by alternate RhoGAP18B isoforms.</a> Cell. 127, (1016).</li></ol>

The authors declare that they have no competing financial interests.&#160;

The costs of alcohol abuse and alcoholism for society is tremendous, both in terms of human29 and economic costs30,31. Drosophila as a model offers a fast and versatile system to quickly examine the behavioral responses of a large number of individuals and as such has been extensively used for both alcohol5,7,32-34 and circadian research35-37.
Here, we described a straightforward protocol for the controlled administration of alcohol vapor to...

Drosophila melanogaster demonstrate biphasic behavioral responses to alcohol1 that are analogous to human responses to this drug2,3. Upon initial exposure to low concentrations of alcohol, flies exhibit increased locomotor activity, replaced by a lack of motor coordination, the loss of postural control and righting reflexes (Loss of Righting Reflex: LoRR), and sedation (complete lack of motor activity in response to mechanical stimulation) as exposure to alcohol progresses4-9. The endogenous circadian clock is a strong modulator of alcohol sensitivity and toxicity as observed in mice10,11, rats12, and humans13. Recent advances in Drosophila research have shown the circadian clock modulates acute alcohol sensitivity but not alcohol tolerance1. The powerful genetic approaches available in Drosophila through mutant studies and transgenic manipulations of spatial and temporal gene expression provide a system that allows rapid progress in identifying the underlying cellular and molecular mechanisms for complex behaviors. The use of Drosophila as an investigative tool has permitted substantive advances in understanding alcohol neurobiology that can be rapidly translated to mammals14-16. In order to facilitate understanding of the molecular mechanisms through which the circadian clock modulates alcohol sensitivity and to uniformly measure behavioral responses across circadian time points, an alcohol administration protocol suitable for use in dim red light conditions is required. For Drosophila, alcohol may be administered through food supplementation for chronic exposure or reliably through administrating alcohol in the form of vapor for acute exposures. Here, we describe an alcohol administration protocol suitable for the assessment of the circadian modulation of Loss-of-Righting Reflex (LoRR)1 as well as sedation.

Flies are entrained with 12 hr:12 hr LD cycles at constant temperature and then transferred to a controlled light regime for 2-5&#160;days depending on the experimental question. Flies are exposed to ethanol vapor in a device known as the FlyBar.&#160; In this device, controlled amounts of air are bubbled through water and alcohol; the vapors are then mixed and directed into a vial housing the flies. Every 5&#160;min&#160;the flies are scored for the number that fails to display righting reflexes or have become sedated. LoRR percentages for each time point are calculated and compared among circadian time points or between strains of flies. The simplicity and reliability of alcohol delivery using the FlyBar alcohol delivery combined with behavioral analysis options provides a significant benefit for circadian experiments conducted under dark conditions.

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the flybar: administering alcohol to flies

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g., CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr&#160;to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes.

Circadian Modulation of Alcohol Sensitivity using the 50% LoRR as a Marker.
A representative example showing circadian modulation in alcohol sensitivity during the day is presented in Figure 2.&#160; LoRR was measured at six time points during the 2nd day of DD in Canton-S and the 50% LoRR was determined for each time point. Analysis showed a significant effect of time of day (ANOVA: F5,45=7.39, p&#60;0.001, N=6-10 per time point). The Fisher LSD...

Watch this Scientific Journal Video about The FlyBar: Administering Alcohol to Flies at JoVE.com

The FlyBar: Administering Alcohol to Flies

Drosophila has emerged as a significant model system for dissecting the cellular and molecular underpinnings of behavioral responses to alcohol. Here we present a protocol for the collection of alcohol sensitivity data in a circadian context that can be easily applied to other experiments and is well-suited for undergraduate research.

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian ...

the-flybar-administering-alcohol-to-flies

Research

JoVE Journal

Neuroscience

10.6K Views.  Florida State University. Drosophila has emerged as a significant model system for dissecting the cellular and molecular underpinnings of behavioral responses to alcohol. Here we present a protocol for the collection of alcohol sensitivity data in a circadian context that can be easily applied to other experiments and is well-suited for undergraduate research.

Video: The FlyBar: Administering Alcohol to Flies

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

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

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

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

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

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...