Aylin Rodan yazının kritik okuma ve yararlı bir tartışma için laboratuar üyeleri için teşekkür ederiz. Bu araştırma, NIH, R01AA019526 tarafından desteklenmektedir.

1. Pozlama odasına oluşturma <ol><li> Yarım jilet kullanarak bir şişe tapası kesin. Alternatif olarak, pamuk topları kullanın. </li><li> Sinekler içeri takılıp almazsınız pürüzsüz bir yüzey yapmak için bir gıda flakonun alt flakon fiş yarısı veya pamuk itin </li><li> Eğer birden fazla maruz odaları, flakon fiş ve gıda her odasında aynı hacimde sürebilir emin olun. Flakonun tarafında bir işareti bu kolaylaştırabilir. </li></ol> 2. Sinekler merceğin <ol><li> Bir cinsiyet, yaş, maya olmadan düzenli gıda şişeleri içine maruz önce 1-5 gün gün 8 sinekleri toplayın. </li><li> Deneyci kullanılan gerçek genotip / zorlanma kör olduğu gibi, kod onları Etiket. </li><li> Pozlama odaları dokunarak içine sekiz sinekler aktarın. Uyutmak değil. </li><li> Kolayca görünür hale getirmek için 200 kanıtı etanol küçük bir miktar, örneğin asit mavi 9, gıda boyası ekleyin. </li><li> Coat 0.5 ml etanol ile bir şişe tapası altında, ya da pamuk topağı,. </li><li> Pozlama şişenin içine etanol takın. Alkol şişenin içine değil, flakon duvara doğru baktığından emin olun. </li><li> Sayan bir sayaç başlayın. </li></ol> 3. Kayıt etanol-indüklenen zehirlenme <ol><li> Zamanlayıcı sonra her dakika, sinekler irkilme ve on saniye için onları gözlemlemek için poz odasında bankta dokunun. </li><li> Her dakika için sabit sineklerin numarasını kaydedin. Sinekler arkalarını kurtulmak edemiyorsa, tek bir yerde kalır, ya da tüm on saniyelik bir gözlem süresi boyunca kanatlarını hızla titreşmeye sabit. Ikinci sinekler, bu sineklerin büyük çoğunluğu hızlı bir kanat hareketi dönemleri arasında sabit çünkü sabit olarak kabul edilir. Sinekler arkalarını kurtulmak ve uzakta ve / veya flakon duvar kadar yürümek durumunda sabit DEĞİLDİR. </li><li> 4 veya daha fazla sinek sabit olarak kaydedilmektedir bir poz deneme sona erdi. </li><li> Sinek atın ve daha sonra yeniden kullanmak için poz odası havayı boşaltıyorum. </li></ol> 4. % 50 sedasyon zamanı belirleyin Sekiz sinek dört sabit kalır etanol maruz kalma başladıktan sonra zaman zaman% 50 sedasyon (ST50). Bu aşağıdaki şekilde belirlenir: Ya tam dört sinekler sabit olan ilk ölçüm dakika. Veya: az dört sinekler (Y1) bir kez noktası (X dakika), ve sonraki ölçüm süresi noktada fazla dört (Y2) (X 1 dakika), sonra aşağıdaki formül kullanılarak doğrusal enterpolasyon ST50 sabit: ST50 = (4 - Y1) / (Y2-Y1) + X 5. Hoşgörü belirlemek için poz tekrarlayın. Sinekler merceğin <ol><li> Deneyde kullanılan yabani tip kontrol suşu için ST50 (ağırlık) belirleyin. </li><li> Yukarıda açıklandığı gibi etanol sinek deneysel gruplar Açığa. </li><li> Kendi ST50 (1) Ölçü, ama 2x ST50 (wt) ulaşana kadar maruz devam. </li><li> Sineklerin boş bir gıda şişenin içine geri aktarın ve 4 saat boyunca basılı tutun. </li><li> Sinekler yeniden ortaya çıkarmak, ve maruz kalma 2 ST50 (2) belirlemek. </li></ol> Hoşgörü belirleyin X100 - (1 ST50 (2) / ST50 (1))% tolerans hesaplayın. 6. Temsilcisi Sonuçlar: Burada gösterilen, genotip ve doz başına düşen sinek 6-8 şişe ile elde edilen bazı temsilcisi sonuçları. Standart hata ölçüde büyükse, daha sonra sistematik bir sorun muhtemelen meydana. Etanol odaları aynı hacimde olduğunu maruz kalma odalarına karşı karşıyadır ve bu emin olun. <img src="/files/ftp_upload/2541/2541fig1.jpg" alt="Şekil 1" /> Vahşi tip Şekil 1 doz yanıt eğrisi artan etanol yüzdeleri maruz uçar. Sinek Sekiz şişe doz ve cinsiyet başına maruz bırakıldı. Erkek ve dişi sinekler, her doz (p&gt; 0.22, 2-yönlü ANOVA) aynı ST50 ST50 dozlarda çapında artan etanol doz (p &lt;0,001) ile önemli ölçüde azalır. Aşırı kalabalık şişe uçar sağlıklı şişelerden oldukça küçük olabilir ve bu boyut farkı ST50 etkileyebilir. Tohum 15-20 kadın ile her şişe şişe başına optimal larva yoğunluğu elde etmek için 1-2 gün için döşeme. <img src="/files/ftp_upload/2541/2541fig2.jpg" alt="Şekil 2" /> Şekil 2 veya% 90 veya% 100 etanol sinekler merceğin beyaz rabbit1 mutation2 (p &lt;0.001, 2-yönlü ANOVA) neden daha önce açıklanan etanol-direnç üretir. <img src="/files/ftp_upload/2541/2541fig3.jpg" alt="Şekil 3" /> Şekil 3, 4 saat kurtarma aralığı aralıklı,% 100 etanol maruz tekrarlayın, ikinci maruz kalma (p &lt;0.001, eşleştirilmiş t sonra ST50 önemli bir artışa neden olur-Testi). Sinekler ilk pozlama (iki katına na ve ST50 tekabül eden) sırasında 24 dakika boyunca maruz kaldığı unutmayın. Yeniden pozlama (veriler gösterilmemiştir) üzerine ST50, kısa bir süre (12 dakika) miktarı için maruz kalma önemli bir artışa neden olmadı.

<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>

Bu sunumda, Bainton ve arkadaşları tarafından açıklanan önceki denemelere göre etanol bağlı zehirlenme belirlemek için basit bir şekilde özetlenmiştir var. 2000 1 ve Rothenfluh ve ark. 2006 2. Alkol tercih 3 alkole bağlı sedasyon ile ilişkili olduğundan, bu testte alkol kullanım bozuklukları çalışma için ilgili. Açıklanan tahlil irkilme-kaynaklı olumsuz geotaxis dayanarak ve nasıl bir alkol maruz kalma, bu doğuştan gelen bir tepki ile müdahale. Pozlama başlamadan önce, tüm sinekler sinekler yiyecek flakon aşağı düğmesine dokunduktan sonra, hepsi bir 10 saniye gözlem süre içinde aşağıdan yukarıya hareket etmelidir, yani böyle bir irkilme normal bir tepki gösterir emin olun. Örnekler azalır olumsuz geotaxis olan sinekler bang-duyarlı nöbet mutantlar 5 ve 6 dopamin nöron dejenerasyonu ile lokomosyon eksikliği sinekler neden sayılabilir. Açıklanan tahlil nispeten basit olsa da, iki değişken tekrarlanabilir sonuçlar sağlamak için en ilgi duymaktadırlar. İlk olarak, tüm maruz şişeleri aynı hacimde olmalıdır. Bu flakon alt aynı gıda + flakon fiş / pamuk hacminin yanı sıra her flakon için de aynı mesafe takmadan etanol içeren fiş içerdiğinden emin içerir. Bu testte tamamen boş bir flakon için kullanılan, ancak testinin verim azalmakta, uzun ST50 değerlere neden olabilir. Bu testte de tekrar etanol maruz kalma üzerine geliştirmek tolerans sinek miktarını belirlemek için uygundur. Tekrar pozlama yaparken değişkenlerin sayısı göz önüne alınmalıdır. İlk olarak, inter-pozlama aralığı burada 4 saat olarak tarif edilir. Bu değişken değişti, ama biz ilk pozlama etanol temizlenmesi sağlamak için, 2 saatten daha düşük olmamalıdır. Scholz et al. 2000 4 etanol tolerans geliştirme ve çürüme kinetiği. İkinci değişken dikkate alınması gereken ilk maruz kalma süresi. Bu tabii ki, kontrol ve deney sinekler için aynı olmak zorundadır. Ancak, bu sinek suşları hem de teslim etanol aynı miktarda olmayabilir. Bu nedenle, daha önce daha yüksek bir doz daha sonra sedatif gerginlik alabilirsiniz sedates büyük bir yük sedasyon sinekler aktif sinek 2&#39;den daha fazla etanol emer ve böylece daha yüksek bir başlangıç ​​dozu nedeniyle daha fazla tolerans geliştirmek. Önemle okuyucu ilk ST50 (1) farklılıklar göstermektedir suşları tolerans geliştirme karşılaştırırken dikkatli olun. Biz burada açıklamak tahlil, kolay ve tekrarlanabilir alkol sinekler davranışsal tepkilerin belirlenmesinde yararlı bir araçtır.

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a simple way to measure ethanol sensitivity in flies

Düşük dozlarda etanol neden yüksek dozda sedatif ise, hiperaktif olmak için uçar. Verilen bir sinek suşu etanol bağlı sedasyon hassasiyet o gerginlik etanol tercihi ile ilişkilidir ve bu nedenle sedasyon alkol yanıtları ve içme genetik çalışma son derece alakalı bir ölçüsüdür. Biz onun sarhoş edici etkilerinin etanol ve ölçmek için sinekler ortaya çıkarmak için basit bir yol göstermektedir. Tarif testi tekrar maruz tarafından oluşturulan akut bir hassasiyet, etanolun yanı sıra hoşgörü belirleyebilirsiniz. Teknik olarak katılan bir kurulum gerektirmez ve bu nedenle temel sinek kültür araçları ile, herhangi bir laboratuvar uygulanabilir.

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

Etanol sedatif etkilerini ölçmek için basit bir tahlil<em&gt; Drosophila</em&gt; Refleks düzeltilmesi kaybı dayalı sinekler, açıklanmıştır.

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 ...

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Research

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Neuroscience

23.1K Görüntüleme.  University of Texas Southwestern Medical Center. Etanol sedatif etkilerini ölçmek için basit bir tahlil Drosophila Refleks düzeltilmesi kaybı dayalı sinekler, açıklanmıştır.

Video: 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: Fare Anksiyete ölçün

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 ...

Nörobilim

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.

Gaz kromatografisi ile operant etanol kendi kendine uygulama ve etanol belirleme sırasında etanol mikrodivaliz

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.

Flybar: Sineklerin için Alkol yönetme

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.

Beyin Dilim Biyotinilasyonu: An<em&gt; Ex Vivo</emYetişkin Nöronlar Bölge özgü Plazma Membran Protein Ticaretiyle Tedbir&gt; Yaklaşım

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.

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 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.

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 ...

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.

MiRNA Beyin Özgül demonte için Stereotaktik Injection Basit Alternatif

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.

Kullanımı Ödül arayan davranış değişiklikleri Tedbir Basit Yolu<em&gt; Drosophila melanogaster</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.

Basit Bir adım Yetişkin Tüm montaj Hazırlama Diseksiyon Protokolü<em&gt; Drosophila</em&gt; Brain

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).