These studies were supported by grants from the National Institutes of Health, National Institute for Alcoholism and Alcohol Abuse to M.G. (P20AA017828, R01AA020634, P50 AA022537). The authors thank Jill Bettinger for helpful discussions and Jacqueline DeLoyht for technical assistance.

Tahlil önce 1. Gün <ol><li> Kısa (1-5 dk) CO 2 altında 11 (tek seks) gruplar halinde taze gıda şişeleri içine sinekler toplayın. </li><li> Sinekler, çevre şartları kontrol alanı gıda şişeler içinde (tipik olarak 25 ° C,% 60 nispi nem, 12 saat aydınlık / karanlık döngüsü) O / N kurtarmak için izin verin. </li><li> Planlanan deney için uygun nihai konsantrasyon (lar) saflaştırılarak (≥18 MΩ) su içinde (% 100), etanol seyrelterek etanol çözeltisi (ler) hazırlayın. Çözüm (ler) RT O / N dönmek için izin verin. Not: etanol seyreltilmesi ekzotermiktir. </li></ol> Deney 2. Gün <ol><li> Sineklerin her flakon test edilebilmesi için, temiz, boş gıda flakon (i) hazırlamak; yani, test şişe, (ii), yeni bir selüloz asetat fiş, (iii) silikon durdurucu ve (iv) etanol çözeltisi (bakınız Tablo 3), 1 mi. </li><li> 55-65% 20-25 ° C sıcaklık ve bağıl nem ayarlayarak test odası hazırlayın. </li><li> Başka bir işçi varflakon her grup için benzersiz bir kod atamak ve sonrası için kod kaydedin. Bir kaç dakika ACCLIMATE test odasında sinekler ile kodlanmış şişeleri yerleştirin. </li><li> 2.3 den sinek şişeleri kodları maç boş test şişeleri etiketleyin. </li><li> Tablo 1 benzer bir elektronik sütunların içine kodları (tek sütun / şişe) girerek sert bir kopya test günlüğü oluşturun. </li><li> Bir kılavuz olarak test günlüğünü kullanarak, test odasında dizileri eşleşen içine sinek ve boş şişeleri test ile kodlanmış gıda şişeleri düzenlemek. Not: sınamak için flakon makul sayısı 24 olan (yani, 4 flakon her 6 setleri). </li><li> Aktarım eşleşti / etiketli boş test şişeleri içine gıda şişeleri uçar ve selüloz asetat fişler flakon üstleri altında 2 cm kadar hemen test şişeleri içine selüloz asetat fişlerini takın. </li><li> Bundan sonra, sendeleyerek 1 dakika aralıklarla bir dizi olarak dört flakon her satırı ele. </li><li> Zaman 0 değerlendirme için, ayrı ayrı w her şişeyi kavramaki başparmak ve işaret parmağı, şişenin dibine vurmak sinekler 30 saniye bekleyin ve sonra ölü / hareketsiz olan sinekler sayısını saymak için masaya hafifçe üç kez dokunun. Zaman basılı test günlüğüne 0 dakika, her flakon için hareketsiz / ölü sinekler numarasını kaydedin. </li><li> Zamanlayıcı 0 zamanında sürekli olarak saymaya başlar ve hemen ardından 4 şişeleri birinci satır / grubu için, ampuller içindeki selüloz asetat fişleri etanol, 1 ml ilave başlar. Bu test edilecektir amacıyla 5 saniye aralıklarla şişeler içinde selüloz asetat fişleri etanol 1 ml ilave edilir. Etanol selüloz asetat fişleri boyunca eşit emilir ve böylece bir dairesel hareketlerle selüloz asetat fişleri etanol ekleyin. Etanol kümesindeki tüm 4 test şişelerine eklendi, bunu kapatmak için her bir tüp içinde bir silikon fiş yerleştirin. </li><li> Kez 1, 2, 3, 4 ve 5 dakika içinde, sırasıyla, 4 şişeleri ikinci, üçüncü, dördüncü, beşinci ve altıncı kümelerine etanol 1 ml. Sonra silikon tapalar ekleyerek devam4 şişelere her bir grubu için etanol ilave edilmesi. </li><li> Süresi 6 dk, başparmak ve işaret parmağı ile her flakon açgözlü şişenin dibine sinekler devirmek için masaya hafifçe üç kez dokunarak, 30 sn bekledikten sonra sayma ve toplam sayısını kaydederek 4 flakon ilk seti testi sedasyon olan sinekler. Skor onlar (i) şişenin yere durmak fakat ya kanatlarını çırparak olmadan sırtlarında yürümek veya (ii) yalan yoksa sedasyon gibi uçar. </li><li> Tablo 2&#39;de zamanlama ile 5 sn aralıklarla kümesi içinde her şişeyi tutun. </li><li> Ilk kümesi için yapıldığı gibi kat 7, 8, 9, 10 ve 11 dakika içinde, sırasıyla küçük şişeler, ikinci, üçüncü, dördüncü, beşinci ve altıncı kümeleri test edin. </li><li> Süresi 12 dakika içinde 2.12 tarif edildiği gibi, tekrar 4 şişeleri ilk seti test sırasıyla, 13, 14, 15, 16 ve 17 dakikada şişeleri ikinci, üçüncü, dördüncü, beşinci ve altıncı kümeleri test etmektedir. </li><li> Tüm sinekler s kadar 2.12 açıklandığı gibi test sinekler Devamedated. </li><li> Basılı test günlüğüne her şişe içinde sinekler toplam sayısını girin. Sineklerin sayısı zaman 0 hareketsiz / ölü sinekler sansür. </li><li> Zaman (x-ekseni) karşı% aktif sinekler (y-ekseni) ve her değerlendirme noktasında yüzde aktif sinekler ve arsa verileri hesaplar. Polinom veya Sigmoidal eğrisi uyan üçüncü sırasından Sedasyon Süresi 50 değerleri (ST50,% 50 sedasyon süresi) interpolating veya eğri altında kalan alan hesaplanarak etanol sedasyon nicelleştirmektedir. </li><li> Çözülmüş şişeleri verileri derlemek ve istatistiksel analizler gerçekleştirmek (örn, tek yönlü ANOVA Bonferroni çoklu karşılaştırma testi ile) deneysel tasarım için uygun. </li></ol>

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The authors have nothing to disclose.

Tekrarlanabilir anlamlı fenotipleri ölçmek basit testler davranış analizi için büyük değer taşımaktadır. Burada anlatılan çalışmalar Drosophila etanol sedasyon hassasiyet ve hızlı tolerans ölçmek için bir tahlil birkaç pratik yönlerini ele almaktadır. Bu çalışmanın bir odak rağmen, davranışsal analizler bir çalışma içinde denek için çevre ve genetik arka plan sabit sürdürerek kolaylaştırılmıştır. Ayrıca, karşılaştırma tipik haliyle yetiştirilen ve yan yana test...

Alkol kullanım bozukluğu (AKB) dünya çapında (1 gözden) büyük bir sağlık sorunudur. AUD gelişimini sürüş mekanizmaları karmaşık olmasına rağmen, bu bozukluklar önemli bir genetik bileşeni var (örneğin, 2). AUD ve korunmuş davranışsal tepkilerin büyük kalıtım daha moleküler temelini anlamaya yönelik etanol ilgili davranışlar belirli genleri araştırmak için genetik model organizmalar kullanarak güçlü bir ilgi yarattı (3,4 gözden) birçok türler arasında etanole AUD. meyve sineği Drosophila melanogaster, (3,4 gözden) etanol ile ilgili davranışlar moleküler genetik mekanizmaları keşfetmek için bir lider model organizma olarak ortaya çıkmıştır. Sinekler Çalışmaları (5 gözden) etanol davranışsal tepkilerin çeşitli sinyal yolları için roller sermiştir. Ilginç genlerin ve yolların bazı davranışsal RESPONS etkilediğisinekler etanol için es de kemirgen etanol ilgili davranışlar ve / veya insan AUD (örneğin, 6-14) suçlanmıştır. Drosophila modeli sisteminde mevcut genetik araçları paketi ile birleştiğinde türler arasında etanol ilgili davranışları, sürüş mekanizmalarının korunması, etanol davranışsal tepkilerin genetik araştırmak için meyve sineği modelinin programı altını çiziyor. Hassasiyet 15,16 ve insanlarda etanol (17 gözden) tolerans AUD gelişimine bağlıdır. Etanol bu davranışsal tepkilerin her ikisi de (3,4 gözden) laboratuar deneyleri çeşitli yoluyla sinekler örnek alınabilir. Yazarlar tarafından bilinen uçucu deneylerinin her etanol sakinleştirici ya da zamana-bağlı etanol kaynaklı sedasyon / koordinasyon bozukluğu veya zamana bağımlı geri dayanmaktadır. Etanol duyarlılık ve r genetiği bizim gruptan bir önceki makaledeDrosophila apid toleransı, sineklerin etanol buharı kaynaklı sedasyon dayalı bir davranış tahlil 18 kullanılmıştır. Bu tahlilde Test canlı yetişkin aktararak başlatılan selüloz asetat fişinin üst (yani olmayan sinek tarafı) etanol ilave bir selüloz asetat fişi ile şişeler içinde sinekler yakalama, gıda şişeleri boşaltmak için anestezi olmadan uçar, ve Bir silikon tıpa ile sinekler, selüloz asetat fişi ve etanol içeren flakon sızdırmazlık (18 referans, Şekil S3 şematik bakınız). Sinekler farklı grupları temsil Çok sayıdaki vial, bu tahlilin arttırarak, paralel olarak değerlendirildi. Şişeler anonim kod verildi ve deneyci sedasyon değerlendirilmesinde istenmeyen önyargı önlemek için tedavi grubuna kör edildi. Standart bir deneyde, şişeler içinde, 30 saniyelik bir geri alındıktan sonra her şişeye sedasyon sinekler sayısı sayılmış ve Converte edildi, 6 dakika aralıklarla yavaşça akıtıldı, daha edildi ve sineklerYüzde aktif sinekler d. Sinekler iç etanol 18 ve sedasyon (bu raporda cf referans 18 ve Şekil 1A ve 1B) ilerici artışa neden bir zaman-bağımlı bir şekilde selüloz asetat prizinden etanol buharı absorbe. Bu tahlilde Sedasyon operasyonel yürüyüş veya (ii) veya kanatlarını çırparak olmadan sırtlarında yatan yokluğunda sinekler (i) ayakta olarak tanımlandı. Burada, bu etanol sedasyon deney ayrıntılı olarak tarif edilmiştir, bunu kullanarak ilgili daha fazla işlevsel optimizasyonu sağlanır ve deney uçucu sedasyon duyarlılık gıda takviyesi seçenekleri katkı adreslemek için kullanılır.

<table border="0" cellpadding="0" cellspacing="0">
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			<td>food vials</td>
			<td>VWR</td>
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			<td>Genesee/flystuff.com</td>
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			<td>Fisher Scientific</td>
			<td>09-704-1l</td>
			<td>#4</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Pharmaco-Aaper</td>
			<td>111000200</td>
			<td>200 proof</td>
		</tr>
	</tbody>
</table>

an inexpensive, scalable behavioral assay for measuring ethanol sedation sensitivity and rapid tolerance in drosophila

Alkol kullanım bozukluğu (AKB) ciddi bir sağlık sorundur. TL büyük bir kalıtsal bileşeni olmasına rağmen, birkaç gen açıkça etiolojilerine implike edilmiştir. meyve sineği Drosophila melanogaster, alkolle ilişkili davranışları altında yatan moleküler genetik mekanizmaları keşfetmek için güçlü bir modeldir ve bu nedenle belirlenmesi ve TL etkileyen genlerin fonksiyonunu anlamak için büyük umut vaat ediyor. çalışmaların bu tür Drosophila modelinin kullanılması güvenilir etanol davranışsal tepkilerini ölçmek tahlillerin bulunmasına bağlıdır. Bu rapor sinekler etanol duyarlılığı ve hızlı tolerans değerlendirmek için uygun bir deneyi anlatır. Bu deneyde ölçüldüğü Etanol duyarlılık sinekler testten hemen önce gıda olmadan muhafaza edilir seviyesi ve kullanılan etanol konsantrasyonu, daha önce bildirilen genetik manipülasyonlar, bir dizi ve zamanın da uzunluğu ile etkilenir. Bunun aksine, etanol sBu deneyde ölçüldüğü ensitivity uçucu işleme, sinekler cinsiyeti ve antibiyotik veya canlı maya büyüme ortamı takviyesi gücü etkilenmez. Nicelleştirme etanol hassasiyeti için üç farklı yöntem esas itibarı ile ayırt etanol duyarlılık sonucu yol açıklanmıştır. genel sadeliği ile birlikte bu testin ölçeklenebilir yapısı, ve nispeten düşük gider set-up Drosophila etanol duyarlılığı ve hızlı hoşgörü küçük ve büyük ölçekli genetik analiz için uygun hale getirmek için.

Bu etanol sedasyon testte elde edilen ham veriler, etanol buhar maruz kalma süresinin bir fonksiyonu olarak sedasyon edilir sinek sayısıdır. Ham veri zamanın bir fonksiyonu olarak yüzde aktif sinekler dönüştürülür (birincil veri 1A Şekil, B, D - F). Eğri uygun birincil veri etanol sedasyon duyarlılık interpolasyon yoluyla, Sedasyon Süresi 50 (ST50) olarak eğri (AUC) altında sedasyon veya aea olmak için sinek% 50 için gerekli zamanı miktarı belirlenebilir. Daha ön...

Watch this Scientific Journal Video about An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila at JoVE.com

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.

Etanol sedasyon Hassasiyet ve hızlı Hoşgörü Ölçüm Ucuz bir, Ölçeklenebilir Davranış Deneyi<em&gt; Drosophila</em

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

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Research

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Neuroscience

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

10.2K Görüntüleme.  Virginia Commonwealth University. 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.

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

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Davranış Endokrinoloji ve Deneysel İktisat birleştiren: Testosteron ve Sosyal Karar Verme

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Nörobilim

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.

Sinekler Etanol Hassasiyet Tedbir Basit Yolu

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 ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Sıçanlarda Sakkaroz Craving Kuluçka değerlendirilmesi için Genel Bir Yöntem

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Fareler Soğuk Hassasiyeti ve Uyum belirlenmesi için Basit ve ucuz Yöntem

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

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.

Locomotion Speed ​​Etanol Etkilerinin Ölçülmesi için bir Testi<em&gt; Caenorhabditis elegans</em

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

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Arasında Mechanosensation için bir davranış tahlilinde klonlarda MARCM tabanlı<em&gt; Drosophila melanogaster</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Recording Hücre içi Gerilim Yanıtları için Elektrofizyolojik Yöntem<em&gt; Drosophila</em&gt; Işık Uyaranların için Fotoreseptörler ve internöronlar<em&gt; İn Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

Ölçme ve Male Değiştirme Çiftleşme Sürücü<em&gt; Drosophila melanogaster</em

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.