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 ...

<ol>
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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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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Alcohol 190 proof</td>
			<td style="width:115px;">Various</td>
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			<td style="width:296px;">We use Whisper 60</td>
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			<td height="21" style="height:21px;width:285px;">Silicone tubing 1/8&#8221;</td>
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			<td style="width:296px;">Either this or previous</td>
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			<td style="width:119px;">1722A43</td>
			<td style="width:296px;">To hold flow meters</td>
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			<td height="21" style="height:21px;width:285px;">Vial</td>
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			<td height="21" style="height:21px;width:285px;">5 mm Pyrex Glass tubes</td>
			<td style="width:115px;">Trikinetics</td>
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			<td style="width:296px;">Fits best in previous stopper.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Teflon tape</td>
			<td style="width:115px;">Hardware store</td>
			<td style="width:119px;"></td>
			<td style="width:296px;">To achieve snug fit in stoppers if necessary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Rubber stopper with two holes</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">59582-122</td>
			<td style="width:296px;">Fits our bottles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Disposable glass pipets</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">53283-768</td>
			<td style="width:296px;">Cut to length and bend by heating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Very fine nylon netting</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">Various</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">15 watt bulbs</td>
			<td style="width:115px;">Hardware store</td>
			<td style="width:119px;"></td>
			<td style="width:296px;">Overhead red light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Photographic red safe light filters</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td style="width:296px;">Overhead red light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Mini Flashlights with red filters</td>
			<td style="width:115px;">Mag-light</td>
			<td style="width:119px;"></td>
			<td style="width:296px;"></td>
		</tr>
	</tbody>
</table>

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 ビュー.  Florida State University.  ショウジョウバエは、アルコールに対する行動反応の細胞および分子基盤を解剖するための重要なモデル系として浮上している。ここでは、簡単に他の実験に適用することができ、学部、研究に適してい概日文脈におけるアルコール感度データの収集のためのプロトコルを提示する。 

ビデオ: 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...

a simple way to measure ethanol sensitivity in flies

A Simple Way to Measure Ethanol Sensitivity in Flies

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

the motivation for alcohol reward: predictors of progressive-ratio intravenous alcohol self-administration in humans

The Motivation for Alcohol Reward: Predictors of Progressive-Ratio Intravenous 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...

sieving fruit pulp to detect immature tephritid fruit flies in the field

Author Spotlight: Sieving Fruit Pulp to Detect Immature Tephritid Fruit Flies in the Field  

Increasing the detection of immature tephritid fruit flies in the field can trigger timely efforts to eliminate populations of these destructive pests. Detecting late instar larvae is faster and more accurate when mushing host fruit in a bag and passing the pulp through a series of sieves than hand cutting and visual...

Sieving Fruit Pulp to Detect Immature Tephritid Fruit Flies in the Field

an in vivo assay to quantify bacterial phagocytosis in adult drosophila flies

This video demonstrates an in vivo assay designed to measure bacterial phagocytosis in adult Drosophila flies. Following the injection of fluorescently labeled bacteria into the abdomen of adult flies, hemocytes associated with the dorsal vessel engage in phagocytosis. The introduction of trypan blue dye serves to quench the fluorescence emitted by extracellular, non-phagocytosed bacteria. This quenching process enhances the identification of phagocytosed bacteria when observed under a...

An In Vivo Assay to Quantify Bacterial Phagocytosis in Adult Drosophila Flies

the polyvinyl alcohol sponge model implantation

The Polyvinyl Alcohol Sponge Model Implantation

A useful tool to analyze the effects of drugs, growth factors, and/or manipulated cells in an animal model of wound repair is described. This technique utilizes the properties of a polyvinyl alcohol (PVA) sponge to deliver and contain the desired treatment and also provide a platform to be excised and...

construction of vapor chambers used to expose mice to alcohol during the equivalent of all three trimesters of human development

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

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

tracking freely walking flies: a method to assess locomotion in drosophila

This video describes how to track freely walking flies to assess their general locomotion ability. The featured protocol demonstrates the assay&#39;s setup and data analysis using video tracking...

Tracking Freely Walking Flies: A Method to Assess Locomotion in Drosophila

intrathoracic viral nano-injection to study host-virus interactions in adult flies

Use a syringe equipped with a 30-gauge needle to fill the glass needle with mineral oil and place the needle through the collar. Place the larger O-ring around the base of the collar, about 1 millimeter from the blunt end of the needle, and mount the needle onto the plunger of the...

Intrathoracic Viral Nano-Injection to Study Host-Virus Interactions in Adult Flies

simple homemade tools to handle fruit flies&#8212;drosophila melanogaster

Simple Homemade Tools to Handle Fruit Flies&amp;#8212;&lt;em&gt;Drosophila melanogaster&lt;/em&gt;

Described here is the use of several homemade tools to transfer, chill, and kill adult Drosophila, as well as to clean glass culture vials and collect eggs. These tools are easy to make and are rather efficient in handling...

Simple Homemade Tools to Handle Fruit Flies&#8212;Drosophila melanogaster

a septic pinprick technique to introduce systemic bacterial infection in flies

Anesthetize flies with carbon dioxide, and prick the flies in the sternal pleural plate of the thorax with the needle avoiding the attachment sites of the wings and the legs. Remove the flies from the minutien pin using soft forceps. Make sure to dip the pin into the bacterial suspension between pricking each...

A Septic Pinprick Technique to Introduce Systemic Bacterial Infection in Flies

topical application bioassay to quantify insecticide toxicity for mosquitoes and fruit flies

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

In this video, we describe a procedure to perform a nano-injection of a positive-sense, single-stranded RNA virus into adult male Drosophila flies and subsequently study the antiviral responses initiated due to the infection. Polymerase chain reaction analysis identifies the upregulation of specific genes in the infected flies that indicate the activation of immune response pathways including RNA interference and JAK/STAT...

drosophila y-maze assay: a method to assess olfactory responses in flies

This video describes a method to test olfactory exploratory behavior in the fruit fly, called the Y-maze. The featured protocol clip demonstrates how to set up and conduct the...

Drosophila Y-maze Assay: A Method to Assess Olfactory Responses in Flies

studying aggression in drosophila (fruit flies)

Studying Aggression in Drosophila fruit flies

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

Studying Aggression in Drosophila (fruit flies)

This video demonstrates a technique to infect Drosophila by introducing a pathogenic bacteria directly into their body cavity. This method bypasses epithelial barriers and other passive defense mechanisms, allowing for the induction of systemic infection. Upon infection, the resulting immune response is assessed by monitoring fly mortality and performing downstream...

laboratory rearing of stable flies and other muscoid diptera

Laboratory Rearing of Stable Flies and Other Muscoid Diptera

A procedure for rearing stable flies (Stomoxys calcitrans) is presented. The procedure uses locally available materials for diet components, equipment and...

drosophila aggression assay: a method to test behavioral plasticity in flies

Aggression is an innate behavior and previous fighting experience affects fighting strategy and outcome of later contests in flies. This video describes an assay used to study Drosophila&#39;s aggressive behavior. The example protocol features the assay performed in a setup that eliminates fly handling and therefore reduces stress for the animals, which would otherwise impact the subjects&#39;...

Drosophila Aggression Assay: A Method to Test Behavioral Plasticity in Flies

chronic intermittent ethanol vapor exposure paired with two-bottle choice to model alcohol use disorder

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

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

Modeling Alcohol Use Disorder Using Chronic Intermittent Ethanol Vapor Exposure

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inkjet-printed polyvinyl alcohol multilayers

Inkjet-printed Polyvinyl Alcohol Multilayers

An inkjet printer was used to manufacture polyvinyl alcohol multilayers. Polyvinyl alcohol water-based ink was formulated, and the main physical properties were investigated.

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 ...

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 ...