Biz mıR resim için bu film ve M. Nicolescu başlangıç ​​aşamasında katkıda K. Mikic, kabul. C. Huber Universtitätsklinikum Tübingen Fortune program tarafından Universtitätsklinikum Tübingen IZKF, A. Alwin Prem Anand bir burs ile desteklenmiştir.

1.. In ovo Elektroporasyonla için gereksinimler <ol><li> Vektör yapısı hazırlanması: miR sens ve antisens primerleri, Ambion talimatlarından sonra tasarlanmıştır tavlanmış ve Apal ya lige edilmiştir / EcoRI ile sindirilmiş vektör pSilencer U6.1. Başarılı dönüşüm sonra elde edilen klonlardan biri büyütüldü ve plazmid pSilencer bir Quiagen midi hazırlık kiti kullanılarak alkalin lisiz yöntemi ile izole edildi. </li><li> Elektrotlar: Elektrotlar satın alınabilir ya da kolayca 13 inşa edilecek. Biz platin tel kendine yapılan elektrotlar (Φ = çapı 0.2, 0.25 veya 0.5 mm) kullanmak veya bazı durumlarda da küçük bir tungsten tel elektrot (Φ = 0.1 mm) 8 biz electroporate istediğiniz alana bağlı. Doku yanmazlar için, doku, elektrotların ince için yakın bir kural olarak kullanın. Ortabeyin geniş elektroporasyon için, bir pirinç / paslanmaz çelik boru lehimli 0.5 mm platin telleri kullanmakmili ve 3-5 mm&#39;lik bir mesafe (Şekil 1) ile bir elektrot tutucu sabitlenir. Belirli bölgeleri elektroporasyonu için, 0.25 mm platin tel anot ve 0.2 mm platin teli ya da 1 mm veya daha az yaklaşık bir mesafe ile katot için 0.1 mm tungsten kullanın. </li><li> Çözümler: Aşağıdaki çözümler hazır: <ol><li> Otoklavda tavuk Ringer, pH 7.5 (9.0 g NaCl, 0.42 g KCl, 0.24 g CaCl2, 1 L damıtılmış su içinde çözülür.) </li><li> PBS (NaCl 8 g, 0.2 g KCI, 0.24 g KH 2 PO4, 1.44 g 2 Na HPO 4, 1l damıtık H 2 O içinde çözülmüştür.) </li><li> Hızlı Yeşil: stok çözelti 10 mg / ml distile H 2 O </li><li> Yavaş kuruyan mürekkep (az gomalak ve embriyo için böylece daha az zehirlenmesi). Not: Canto-Soler ve Adler 14 anlatıldığı gibi bir fiber optik lamba bağlı bir mavi dikroik filtre kontrastı ve mürekkep enjekte etmek ihtiyacını ortadan kaldırabilir. </li></ol></li></ol><img src="/files/ftp_upload/50024/50024fig1.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50024/50024fig1highres.jpg"/> Şekil 1. Elektrotlar ve elektrot tutucu şematik. (A, B, C) ​​platin tel, bir paslanmaz çelik boru şekilli milinin bir tarafında lehimlenmiştir edildi. Bir delik böylece bu fişler (A) Kırmızı ve mavi renkli teller pin fişler bağlı idi içeri sığabilecek boru şeklindeki milinin diğer tarafına açılmış. Karşı ucunda tel elektroporatörü elektrik prizleri ile çalışmak muz konnektörleri takıldı. (A, C) platin tel 90 ° lik bir melek olarak bükülmüştür. Tırnak cilasının tabakası büklümünün mili izole edilmiştir. 2. Doğrudan In ovo Elektroporasyonla önce Hazırlıklar <ol><li> Aşağıdaki malzemeyi hazırlayın: <ol><li> Tavuk Ringer 01:06 ile Ink sulandırmak GIBCO gelen Antibiyotik-Antimikotik solüsyonu (100x eklemek) 1:100 o. </li><li> Vektör çözelti hazırlayın: H 2 O spesifik DNA başına 1-2 mg / ml Fast Green (01:20) ile takviye edilmiştir. </li><li> % 70 etanol ile forseps, makas, iğne ve tungsten elektrotları sterilize edin. </li><li> Borosilikat cam kılcal kısımların (100 mm x 0.9 mm, dış çap uzunluğu) ikinci bir nükleik asit, enjeksiyon için mikropipetler çekin. Biz Tablo 1&#39;de gösterilen ayarları ile basit bir çektirmenin PUL-1 (TEFE, Berlin) kullanın. </li><li> Tablo 2&#39;de gösterildiği gibi elektroporatörü parametrelerini ayarlayın. </li></ol></li></ol><table border="1" ><tbody><tr><td fo:width="1in"> Parametreler </td><td fo:width="1in"> Değerler </td></tr><tr><td> Isı </td><td> 350 </td></tr><tr><td> Çekme </td><td> 100 </td></tr><tr><td> Hız </td&gt; <td> 50 </td></tr><tr><td> Gecikme </td><td> 200 </td></tr></tbody></table> Tablo 1. PUL-1 için Ayarlar <table border="1"><tbody><tr><td fo:width="1.5in"> Parametreler </td><td fo:width="1.5in"> Değerler </td></tr><tr><td> Frekans </td><td> 50 Hz </td></tr><tr><td> Gecikme </td><td> 20 - 50 msn * </td></tr><tr><td> Genişlik </td><td> 20 milisaniye </td></tr><tr><td> Gerilim </td><td> 8-25 V * </td></tr><tr><td> Nabız </td><td> 3-5 defa </td></tr><tr><td colspan="2"> * Elektrot çapı ve mesafeye bağlı. Lütfenparametrelerden birini değiştirirken bunu dikkate alır. </td></tr></tbody></table> Tablo 2. Darbe Stimulator için Ayarlar <ol start="2"><li> Elektroporasyon için civciv embriyo hazırlanması: <ol><li> Hamburger ve Hamilton (HH) arasında embriyolar elde etmek için 39 saat boyunca% 65 nem ile 37 ° C &#39;de, daha uzun tarafında fertilize tavuk yumurtası inkübe 15 9-11 aşamaları. Embriyo yumurtanın üst kısmında yerleşir, böylece bir kalem hattı ile işaretlemeniz gerekir. </li><li> % 70 etanol ile yumurta dezenfekte edin. </li><li> Hava boşluğu olması gereken yerde, daha geniş tarafında yumurta delmek. </li><li> Yumurta beyazı 1-2 ml çıkarın. Bu kabuğu gelen embriyo ayırmak olacaktır. </li><li> Viyolonsel-bant embriyo üzerine düşen kabuk parçalanmak önlemek için yumurta kabuğu. Kalem çizgisi etrafında yumurta kabuğu içine küçük bir pencere kesmek için bir makas kullanın. </li><li> Mürekkep biraz enjekteembriyo görselleştirmek için alan Opaca altında. </li><li> Nükleik asit Enjeksiyon ve elektroporasyon için embriyo daha iyi bir erişim elde etmek için, bir iğne ile keskinleştirilmiş tungsten vitellin membran içine bir kesim yapın. </li></ol></li></ol> 3. Elektroporasyon ve Kuluçka <ol><li> Embriyoları üzerine sıcak Ringer solüsyonu birkaç damla ekleyin. Bu, embriyoların daha düşük aşırı ısınmasını önlemek için, dokuya elektrotların yapışmasını ve iki elektrot arasındaki akımı için bir ortam sağlar. </li><li> Orta beyin düzeyinde nöral tüpün lümenine vektör solüsyonu enjekte edilir. DNA sentezleme vektörü herhangi bir raportör gen (örn. EGFP) varsa, DNA solüsyonuna konsantre bir biraz daha düşük bir raportör geni (1 ug / ul miR 0.9 ug / ul raportör gen vektörü ifade vektörü) ekleyin. Bu transfekte hücreler daha sonra 8 tanımasını sağlayacaktır. Biz sağa sola sözde pCAX vektörü, bir tür hediye kullanınm C. Krull 13. </li><li> Ortabeyin geniş bir transfeksiyon için, orta beyin yükseklikte sol elektrotlar ve embriyo hakkı yerleştirin. Elektrotlar arasındaki mesafe, yaklaşık 3-5 mm olmalıdır. Diğer ayarlar 15V, 20 msn bakliyat ve HH 10 50 msn gecikme vardır. Hafif orta beyin ön-ventral (DV) ekseni boyunca iki elektrot konumunu değiştirme hücre, daha geniş bir transfeksiyon DV eksen transfeksiyon sağlar. </li><li> Ventral Transfeksiyon için, sarısı zarından baş yükseltmek ve ventral orta beyin altında anodu yerleştirmek için bir Zil ekleyin. Kafa nöral tüp yanmazlar sarısı ve embriyo ayıran zarı altında elektrot itmek çıkmıyorsa. Katot orta beyin ön-yanal olarak zıt bir anot, yukarıda yerleştirilmelidir. Nöral tüp dokunmayın. Biz kalp gelişimi ile herhangi bir sorun yaşamamak için orta beynin sol ventral yarısını transfect. Elektrotlar arasındaki mesafeetrafında 1-0,5 mm, ayarları geri kalanı olan 10V, 20 msn genişlik ve 50 msn gecikme olduğunu. </li><li> Soğumasını ve hava kabarcıklarını çıkarmak için embriyoların üstüne Salin başka bir damla uygulayın. </li><li> Bant ile yumurta Seal ve en az 4 saat süreyle inkübe edilir; vektör ifadesi için gerekli olan minimum süre. </li></ol> 4. Tavuk Embriyolar Fixation <ol><li> Yumurtalarından embriyolar çıkarın. </li><li> Stereomicroscope altında ve bir floresan stereo mikroskop ile transfeksiyonun ölçüde değerlendirmek temiz embriyolar. </li><li> 4 ° C &#39;de% 4 PFA içinde gece boyunca embriyolar saptamak Transfekte alanında değişiklikleri analiz etmek için (in situ hibridizasyon, immünohistokimya) kesit ve / veya boyama ile devam edin. </li></ol>

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Biz ifşa hiçbir şey yok.

Bu video civciv Ortabeyin belirli alanların nöroepitel hücrelere plazmid transfect için etkili bir yöntemi gösterir. Düşük voltaj dikdörtgen elektrik darbeleri 6,16 ovo civciv nöral tüpün hücrelerine DNA &#39;nın neden olabilir. Bununla birlikte, DNA hedefleme doğruluğu genellikle nispeten büyük elektrotlar (Φ = 0.5 mm) üzerinden yükselir geniş elektrik alanı tarafından engellenmektedir. Biz Momose vd. 8 yönergeleri izleyerek küçük çaplı elektrotlar ...

Gen ifadesi için ek oynatıcılar gibi küçük kodlayıcı olmayan RNA&#39;lar tanınması genomik programlama / gen düzenlemesi için yeni bir karmaşıklığı başlattı. Kodlayıcı olmayan RNA&#39;lar, küçük farklı türlerin kodlayıcı olmayan RNA&#39;lar 1-4 de dahil olmak üzere sinir hücreleri, fonksiyonel öneme sahiptir. Örneğin MicroRNA (miR veya MiRNA), gelişmekte olan beyin 5 ayrı ve değişen ekspresyon profillerini göstermektedir. Gelişim sırasında gen ifadesinin ve sessizliğini zamansal ve mekansal kontrolü için eşsiz bir fırsat civciv embriyo ovo elektroporasyon sağlar Hedefli. Bu video elektroporasyon 6-10 ovo kullanarak civciv Ortabeyin belirli alanlarda Mirs ektopik ifadesi yerine farklı adımları gösterir. Hücrelerde bu küçük kodlayıcı olmayan RNA&#39;lar, uzun süreli bir etki sağlamak için, Mirs DNA dizisidir mono-ya da bi-sistronik vektörlerine klonlanmıştır. Ovo elektroporasyon için, miR v içerenector yumurta kabuğu içinde küçük bir pencere yaptıktan sonra embriyo maruz bırakılması ile orta beyin nöral tüp içine enjekte edilir. Ortabeyin küçük artı (anot) ve eksi (katot) belirli alanlarda transfect platin elektrot belirli pozisyonlarda yerleştirilir. Ventral orta beyin transfeksiyon için, anot sol ventral orta beyinde ve bir akım uygulamadan önce Ortabeyin sağ yarısı üzerinde katot altına yerleştirilir. Kabuğu içindeki açma bandı ile kapatılır ve embriyo sürece herhangi bir analiz için gerekli olan süre ile inkübe edilir. Bu yöntem, ilk Muramatsu, et al. 6 ile tarif edilen ve belirli bir alan transfeksiyonu için Momose et al. 8 ile geliştirilmiştir. <img src="/files/ftp_upload/50024/50024schematic1.jpg" fo:content-width-="6in" fo:src="/files/ftp_upload/50024/50024schematic1highres.jpg"/> Şematik bir bakış. <ol><li> Yumurtanın embriyo th içine küçük bir pencere keserek maruz kalmaktadıre kabuğu. </li><li> Çözünmüş vektör (ler) bir mikro kapiller kullanılarak orta beyin içine enjekte edilir. </li><li> Iki elektrot - paralel veya embriyo altına ve üstüne yerleştirilir - darbeli bir elektrik alanı oluşturur. </li><li> Elektrik alanı geçici olarak anot 11,12 çekiliyor negatif yüklü DNA (veya RNA) ile hücre içine girişini kolaylaştırmak hücre zarında gözenekler oluşturur. </li></ol>

<table border="1">
<tbody>
 <tr>
 <td>Name </td>
 <td>Company</td>
 <td></td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Borosillicate glass capillaries </td>
 <td>Hartenstein</td>
 <td></td>
 <td>Model: 0.9 mm</td>
 </tr>
 <tr>
 <td>Microcapillary puller</td>
 <td>WPI, Berlin</td>
 <td></td>
 <td>Model: Pul1-E</td>
 </tr>
 <tr>
 <td>Electroporator</td>
 <td>Intracel</td>
 <td></td>
 <td>Model: TSSIC</td>
 </tr>
 <tr>
 <td>Stereomicroscope - fluorescence </td>
 <td>LEICA</td>
 <td></td>
 <td>Model: MZFLIII</td>
 </tr>
 <tr>
 <td>Stereomicroscope</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Stemi</td>
 </tr>
 <tr>
 <td>Camera and software</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Axiocam MRc/ Axiovision Re. 4.8</td>
 </tr>
 </tbody>
</table>

in ovo expression of microrna in ventral chick midbrain

Kodlayıcı olmayan RNA&#39;lar gen ekspresyonunu düzenleyen ek oyuncular. Belirli alanlarda ovo elektroporasyon Hedefli ektopik mıkroRNA ifade mekansal ve zamansal kontrolü için eşsiz bir araç sağlar. Ancak, ventral orta beyindeki gibi ventral beyin yapıları herhangi manipülasyonlar için ulaşmak oldukça zordur. Burada, biz ince platin elektrotlar kullanılarak ventral orta beyindeki içine miRNA electroporate için etkili bir yol göstermektedir. Bu yöntem, orta beyin ve in vivo çalışmalar için yararlı bir araç içinde belirli alanlarda transfekte etmek için güvenilir bir yol sunar.

MiR ile transfekte orta beyin alanının ölçüde miR ifade vektörü (Şekil 2) ile birlikte enjekte edilen bir raportör vektöründen GFP ekspresyonu görülebilir. 0.5 mm bir çapı olan platin elektrotları kullanarak, orta beyin ve arka beyin ve bazen diensefalon (Şekil 2A ve B de dahil) DV-ve AP-ekseni boyunca geniş bir alanda transfeksiyonu, normal olarak orta beyin potansiyel AP eksenine paralel olarak yerleştirilmiştir. Geniş bir elektrik alanında elektrotlar sonuç bü...

Watch this Scientific Journal Video about In ovo Expression of MicroRNA in Ventral Chick Midbrain at JoVE.com

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Dış ifade beyin gelişiminde microRNA rolünü izah etmek için bir tekniktir. Ancak, ovo elektroporasyon kullanarak belirli alanları hedefleyen zordur. Burada, biz verimli bir şekilde seçici electroporate ventral ve dorsal orta beyin bölgeleri göstermek.

Ventral Chick orta beyin içinde MikroRNA ovo İfade

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

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Neuroscience

In ovo Expression of MicroRNA in Ventral Chick Midbrain

10.4K Görüntüleme.  University of Tübingen.  Dış ifade beyin gelişiminde microRNA rolünü izah etmek için bir tekniktir. Ancak, ovo elektroporasyon kullanarak belirli alanları hedefleyen zordur. Burada, biz verimli bir şekilde seçici electroporate ventral ve dorsal orta beyin bölgeleri göstermek. 

Video: In ovo Expression of MicroRNA in Ventral Chick Midbrain

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Embriyonik ventral orta beyin Organotipik Dilim Kültürler: dopaminerjik Nöronal Gelişim Eğitim Bir Sistem In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Nörobilim

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Gelişmekte Neuroepithelium Hücre Davranış Yüksek çözünürlüklü Canlı Görüntüleme

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminerjik Nöron Geliştirme Gen Fonksiyonunun Çalışılması için Chick orta beyin içinde ovo Elektroporasyon yılında

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Substantia nigra ve ventral Tegmental Bölgesi Dopamin Yönetmelik Kapsamlı Profilleme

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Akut Hipokampal Dilimler içinde Synaptically-uyarılmış astroglial ve Nöronal Cevaplarının İkili Elektrofizyolojik Kayıtlar

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

Electroporated Chick Embriyonik Omurilik Diferansiyel Gen İfade RNA Sıra Analizi

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Yetişkin Farelerde Orta beyin dopamin Nöronlar Sayısının Çevre Modülleri

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

RNA sıralaması ve TH-organizatörü odaklı eGFP İfade Kullanımı orta beyin Kültürlerde Yaşayan dopaminerjik nöronlar güvenilir Kimlik

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

İnsülin mimetic bileşikler test etmek için bir Ovo içinde modeli

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

Hipokampustaki Ventral Tegmental Alan Glutamat Terminallerinin Kombine In Vivo Anatomik ve Fonksiyonel İzlenmesi

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...