우리는 중요한 원고의 읽기, 도움이 토론에 대한 실험실의 구성원에 대한 Aylin Rodan 감사합니다. 이 연구는 NIH, R01AA019526에 의해 지원됩니다.

1. 노출 챔버 만들기 <ol><li> 면도날을 사용하여 반으로 병을 플러그 컷. 또는, 면화 공을 사용합니다. </li><li> 파리가 인치 끼지는 것이다 부드러운 표면을 만들어 식품 약병의 하단에, 유리병 플러그 절반 또는 목화 공을 밀어 </li><li> 다중 노출 챔버 스를 만드는 경우, 유리병 플러그 식품 각 챔버에서 동일한 볼륨을 받아 있는지 확인하십시오. 유리병의 측면에 마크는 이것을 용이하게하실 수 있습니다. </li></ol> 2. 파리를 노출 <ol><li> 하나의 성별, 나이 1~5일, 누룩을 넣지 않은 일반 식품 유리병에 노출 전날 8 파리를 수집합니다. </li><li> 코드에서 실험자 사용 실제 유전자형 / 변형에 멀게됩니다 그러한 그들을 분류. </li><li> 감청에 의해 노출 실에 8 파리를 전송합니다. 마취하지 마십시오. </li><li> 그것이 쉽게 볼 수 있도록 200 증거 에탄올 음식 염료, 예 : 산성 블루 9의 작은 금액을 추가합니다. </li><li> 0.5 ML의 에탄올과 코팅은 유리 플러그의 바닥이나 면화 공. </li><li> 노출 약병에 에탄올 플러그를 삽입합니다. 알코올은 유리병에, 아니라 유리병의 벽을 향해 마주보고 있는지 확인하십시오. </li><li> 세는 타이머를 시작합니다. </li></ol> 3. 기록 에탄올 유도 중독증 <ol><li> 타이머 후 각 분 시작은, 파리를 놀라게하고 10 초 동안 그들을 관찰하기 위해 벤치에 노출 챔버를 누릅니다. </li><li> 각 분간 정지 파리의 번호를 기록합니다. 파리는 그들이 귀찮게 안할 수 없다면 한 위치에 남아, 또는 빠르게 전체 10 초 관찰 - 기간 내내 자신의 날개를 진동 정지됩니다. 이러한 파리의 대부분 빠른 날개 운동의 기간 사이에 고정되기 때문에 후자 파리는 고정 간주됩니다. 파리는 그들이 귀찮게하고 거리 및 / 또는 유리 벽까지 걸을 수있다면 고정되지 않습니다. </li><li> 4 개 이상 날아가 정지로 기록하는 경우 노출 재판이 완료됩니다. </li><li> 파리를 취소하고 나중에 재사용을위한 노출 챔버 공기를 내보 내줘. </li></ol> 4. 50 % 진정 작용 시간을 결정 50 %의 진정 작용 (ST50)하는 데 걸리는 시간은 8 파리 그 중 넷은 고정 남아있는 에탄올의 노출의 시작 후 시간입니다. 이것은 다음과 같이 결정됩니다 : 어느 : 정확히 네 파리 고정하는 첫번째 측정 분. 또는 : 이하보다 4 파리 (Y1)가 한 번에 포인트 (분 X), 그리고 다음 측정 시점에 개 이상 (Y2) (X 일분) 공식을 사용하여 다음 선형 보간 ST50에 정지하는 경우 : ST50 = (4 - Y1) / (Y2 - Y1) + X 5. 공차를 결정하는 노출을 반복 파리를 노출 <ol><li> 실험에 사용된 야생 형 제어 변형에 대한 ST50 (WT)을 결정합니다. </li><li> 위에서 설명한 에탄올로 파리의 실험 그룹을 쉽게받을 수 있습니다. </li><li> 그들 ST50 (1) 측정하지만, 당신이 시간을 2 배 ST50 (WT)를 도달할 때까지 노출로 진행합니다. </li><li> 빈 식품 약병에 다시 파리를 전송하고, 4 시간 동안 기다려. </li><li> 파리를 다시 노출하고, 노출 2 ST50 (2) 결정됩니다. </li></ol> 허용 오차를 결정 x100 - (1 ST50 (2) / ST50 (1))로 %의 허용 오차를 계산합니다. 6. 대표 결과 : 여기에 나와 우리가 유전자형과 복용량 당 파리 6-8 유리병과 함께 얻은 몇 가지 대표적인 결과입니다. 당신의 표준 오차가 크게 확대되면 체계적인 문제는 가능성이 발생했습니다. 에탄올은 챔버가 같은 볼륨되는 노광 실에 얼굴을하고 있는지 확인하십시오. <img src="/files/ftp_upload/2541/2541fig1.jpg" alt="그림 1" /> 야생 유형의 그림 1. 선량 반응 곡선은 에탄올의 증가 비율에 노출 날아. 파리의 여덟 병은 복용과 성별에 따라 노출되었습니다. 복용에 걸쳐 ST50가 증가 에탄올 선량 (P는 &lt;0.001)로 크게 감소하면서 남성과 여성의 파리는 각 복용량 (P&gt; 0.22, 2 방향 ANOVA)에서 동일한 ST50을 보여줍니다. 오버 혼잡 병에서 파리 참고 건강한 병에서보다 대폭 작은 수 있으며,이 크기의 차이가 ST50에 영향을 줄 수 있습니다. 15-20 여성과 시드 각 병 병마다 최적의 애벌레 밀도를 얻을 1~2일위한 누워. <img src="/files/ftp_upload/2541/2541fig2.jpg" alt="그림 2" /> 그림 2. 중 90 %, 또는 백퍼센트 에탄올로 파리를 노출은 흰색 rabbit1 mutation2 (P &lt;0.001, 2 방향 ANOVA)에 의한 이전에 설명한 에탄올 저항을 재현. <img src="/files/ftp_upload/2541/2541fig3.jpg" alt="그림 3" /> 그림 3. 4 시간 복구 간격에 의해 간격, 100 % 에탄올에 노출을 반복, 두 번째 노출 (P &lt;0.001, 이점 t 후 ST50에서 상당한 증가 원인테스트). 파리가 첫 번째 노출 (두 번 자신의 나의 적 ST50에 해당하는) 동안 24분에 대한 노출다고합니다. 시간 (12 분)의 짧은 금액에 대한 노출을 다시 노출 (데이터가 표시되지 않음)에 따라 ST50에 상당한 증가를 일으키지 않았다.

<ol>
	<li>Bainton, R. J., Tsai, L. T. -. Y., Singh, C. S., Moore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Curr. Biol. 10, 187-194 (2000).</li><li>Rothenfluh, A., Threlkeld, R. J., Bainton, R. J., Tsai, L. T. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 127, 199-211 (2006).</li><li>Devineni, A. V., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Curr. Biol. 19, 2126-2132 (2009).</li><li>Scholz, H., Ramond, J., Singh, C. S., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 28, 261-271 (2000).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, M. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Semin. Cell Dev. Biol. 17, 31-41 (2006).</li><li>Feany, M. B., Bender, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 404, 394-398 (2000).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 프레 젠 테이션에서, 우리는 베인튼 외 의해 설명되어 이전 assays에 따라 에탄올 유발 중독증을​​ 결정하는 간단한 방법을 설명합니다. 2000 1 및 Rothenfluh 외. 2006 2. 알코올 유발 진정 작용은 알코올 음주 환경 설정 3 상관 있기 때문에,이 분석은 알코올 사용 장애 연구 관련입니다. 설명 분석은 놀라게 유발 부정 geotaxis에 따라, 어떻게 알코올 노출이 타고난 응답을 방해합니다. 노출를 시작하기 전에, 모든 파리는 음식 약병에서 파리를 감청 후, 그들이 모두 10초 관측 시간 내에 아래에서 위로 이동합니다 즉, 같은 놀라게 정상적인 반응을 보여주 있는지 확인하십시오. 감소 부정 geotaxis하는 데 파리의 예는 다음과 플레이에 민감한 발작 돌연변 5, 도파민 뉴런 6 변성에 의한 운동 - 결함 파리를 포함합니다. 설명 분석은 비교적 간단하다하더라도, 두 변수가 재현성 결과를 보장하기 위해 가장 주목 만하다. 첫째, 모든 노출 튜브는 동일한 볼륨해야합니다. 이것은 유리병의 바닥 같은 음식 + 유리병 플러그 / 목화 공의 볼륨뿐만 아니라 에탄올이 포함된 플러그인은 모든 병에 대해 동일한 거리를 삽입을 포함하고 있는지 확인이 포함됩니다. 완전히 빈 유리병이 분석에 사용하지만, 그것은 분석의 처리량을 줄이는 더 이상 ST50 가치로 연결하실 수 있습니다. 이 분석은 또한 반복 노출시 에탄올 개발 내성 파리의 양을 결정하기 위해 적합합니다. 반복 노출을 할 때 변수의 수를 고려되어야합니다. 첫째, 남북 노출 간격은 여기 4시간로 설명되어 있습니다. 이 변수는 변경하지만, 우리는 그것이 첫 번째 노출 에탄올의 허가를 위해 2 시간보다 낮을 수 없습니다 권장 수 있습니다. 슐츠 외를 참조하십시오. 에탄올 허용 개발 및 부패의 반응 속도론 2000 4. 고려해야 할 두 번째 변수는 첫 번째 노출의 기간입니다. 이것은 분명 제어 및 실험 파리에 대해 동일합니다. 그러나,이 이러한 플라이 계통 모두에 전달 에탄올 같은 금액을 초래하지 않을 수 있습니다. 진정 파리 일찍 나중 - sedating 변형보다 높은 복용량가 나타날 수 있습니다 진정제 변형 따라서 Active 파리 2 이상의 에탄올을 흡수하고, 따라서 더 높은 초기 복용으로 인해 더 많은 관용을 개발. 초기 ST50 (1)의 차이를 보여 종자의 내성 개발을 비교할 때 우리는 강력하게 독자를주의하십시오. 우리가 여기서 설명하는 분석 쉽고 reproducibly 알코올에 파리 행동 반응을 결정하는 유용한 도구입니다.

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<td>Exposure vials</td>
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a simple way to measure ethanol sensitivity in flies

에탄올 원인의 낮은 복용량이 높은 양이 sedating있는 동안, hyperactive 될 날아. 주어진 플라이 변형의 에탄올 유발 진정 작용에 감도는 그 변형의 에탄올 환경과 상호, 따라서 진정 작용은 알코올 반응과 음주의 유전을 연구하기 위해 관련성이 조치입니다. 우리는 환상적 효과를 에탄올과 측정 파리 노출하는 간단한 방법을 보여줍니다. 우리가 설명하는 분석은 반복 노출에 의해 유도된 급성 감도뿐만 아니라 에탄올 내성을 확인할 수 있습니다. 그것은 기술적으로 관련된 설정을 필요하지 않습니다, 따라서 기본적인 비행 문화 도구로 어떤 실험에 적용할 수 있습니다.

Watch this Scientific Journal Video about A Simple Way to Measure Ethanol Sensitivity in Flies at JoVE.com

A Simple Way to Measure Ethanol Sensitivity in Flies

에 대한 에탄올의 sedating 효과를 측정하는 간단한 분석<em&gt; Drosophila</em반사를 righting의 손실에 따라&gt; 파리는, 설명합니다.

파리에서 에탄올 민감도를 측정하는 간단한 방법

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-simple-way-to-measure-ethanol-sensitivity-in-flies

Research

JoVE Journal

Neuroscience

23.1K 조회수.  University of Texas Southwestern Medical Center. 에 대한 에탄올의 sedating 효과를 측정하는 간단한 분석 Drosophila 파리는, 설명합니다.

비디오: A Simple Way to Measure Ethanol Sensitivity in Flies

Hyponeophagia: A Measure of Anxiety in the Mouse

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often hampered by a phenomenon known as "bait shyness". Mice and rats cannot vomit, due to the tightness of the cardiac sphincter of the stomach, so to overcome the problem of potential food toxicity they have evolved a strategy of first ingesting only very small amounts of novel substances. The amounts ingested then gradually increase until the animal has determined whether the substance is safe and nutritious. So the old rat-catchers would first put a palatable substance such as oatmeal, which was to be the vehicle for the toxin, in the infested area. Only when large amounts were being readily consumed would they then add the poison, in amounts calculated not to affect the taste of the vehicle. The poisoned bait, which the animals were now readily eating in large amounts, would then swiftly perform its function.
Bait shyness is now used in the behavioural laboratory as a way of measuring anxiety. A highly palatable but novel substance, such as sweet corn, nuts or sweetened condensed milk, is offered to the mice (or rats) in a novel situation, such as a new cage. The latency to consume a defined amount of the new food is then measured.
Robert M.J. Deacon can be reach at <a href="mailto:robert.deacon@psy.ox.ac.uk">robert.deacon@psy.ox.ac.uk</a>

Mice and rats, due to their innate cautiousness, are initially slow in consuming a novel food, particularly in a novel place. This hyponeophagia can readily be measured in the laboratory, even though laboratory animals are much less anxious than their wild counterparts

Hyponeophagia : 마우스에서 불안의 측정

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often ...

연구

신경과학

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

가스 크로마토 그래피에 의한 operant 에탄올 자기 관리 및 에탄올 결정시 에탄올의 Microdialysis

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

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.

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.

의 flybar : 파리에 주류 관리

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

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

뇌 슬라이스 바이오 티 닐화 :<em&gt; 전의 VIVO</em성인 뉴런에서 지역 고유의 플라즈마 막 단백질 인신 매매를 측정하는&gt; 접근

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

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.

마우스에 콜드 민감도 및 적응을 결정하기위한 간단하고 저렴한 방법

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

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.

에탄올 진정 감도 및 신속한 공차 측정을위한 저렴하고 확장 가능한 행동 분석<em&gt; 초파리</em

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

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

miRNA의 뇌의 특정 최저 대한 정위 주입하는 간단한 대안

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

사용 보상 추구 행동의 변화를 측정하는 간단한 방법<em&gt; 노랑 초파리</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

간단한 한 단계 성인의 전체 마운트 준비에 대한 해부 프로토콜<em&gt; 초파리</em&gt; 두뇌

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).