Biz yönteminin kurulması sırasında yararlı tartışmalar için Manfred Heckmann teşekkür ederim. Ayrıca, biz video üreten destek için teknik yardım ve Frank Schubert Claudia Sommer teşekkür ederim. 01EO1002 ve Jena Üniversitesi Hastanesi Klinik Araştırma Disiplinlerarası Merkezi (IZKF): iş Federal Eğitim ve Araştırma Bakanlığı (BMBF), Almanya, FKZ tarafından desteklenmiştir.

Aşağıdaki protokolde belirtilen tüm deneysel prosedürleri Thüringen devlet yetkilileri (Thüringer Landesamt für Verbraucherschutz, Reg.-Nr. 02-044/12) tarafından onaylanmıştır. 1.. Deney için hazırlıklar <ol><li> Emme elektrot İmalatı <ol><li> Örneğin bir standart yama elektrot bir mikropipet çektirmesi ile standart borosilikat cam kılcal kullanarak bir mikropipet çekin. </li><li> Fren bir elmas dosyasını kullanarak (dorsal kökler çapından biraz daha büyük) 0.5-1 mm çaplı ucuna elektrot. </li><li> Isı lehçe bu standart bir laboratuvar meşale içeri emilir zaman dorsal kök zarar olmayacak şekilde uç uygun olacaktır. </li><li> Negatif basınç uygulanabilir, üzerinden bir şırıngaya bağlanmış bir elektrod tutucuya cam filament monte edin. </li></ol></li><li> Çözeltilerin hazırlanması <ol><li> Fareler için enjeksiyon anestezi: 0.75 ml ketamin 10% karıştırın, 0.24 ml% 2 ksilazin and 5 ml% 0.9 tuzlu su. Çözeltinin 10 ul / g vücut ağırlığı (BW) (ip), periton içinden enjekte edilecektir. </li><li> Yapay beyin omurilik sıvısı (CSF), KCI 3, KH 2 PO 4 1.25, MgSO 4 • H2O 2, 3 25 NaHCO; CaCl2 2; D-NaCl 134: iki kere damıtılmış su içinde (mM olarak) şu tuzları seyreltin 10, glikoz. % 0.003 &#39;lik bir son konsantrasyona kadar, H 2 O 2 ekleyin. Her zaman taze hazırlanan solüsyonu kullanın. </li></ol></li><li> Kayıt kurulum hazırlanması (Şekil 2) <ol><li> Veri sayısallaştırma için bir PC arayüzü amplifikatörü bağlayın. </li><li> PC sabit disk üzerinde bir kare darbe uyarıcı ve veri toplama tetiklemek için bir PC-tabanlı bir program veya bir analog zamanlayıcı kullanın. </li><li> Stimülasyon ve kayıt için standart cam elektrot sahiplerine bağlanan Tekrar klorid silverwires kullanın. </li><li> Bir stere etrafında sırasıyla uyarım, kayıt ve referans elektrot için üç manipülatörler arayafareler için otactic çerçeve, tüm elektrotlar daha sonra hazırlanan omuriliğe erişimi böylece. </li><li> Negatif basınç uygulamak için elma olmak üzere elektrot sahiplerine borulama ve şırınga bağlayın. </li></ol></li></ol> 2. Hayvan Deneyleri ve Kayıt Prosedürü için Hayvan Hazırlanması için Genel Yorumlar <ol><li> Ilgili kurumsal hayvan bakımı ve kullanımı komitesi kurallarına göre fareler kullanarak tüm deneyler. Hayvan acı minimize olmasını sağlamak derin anestezi altında ameliyat ve kayıtları gerçekleştirin. </li><li> (, Yukarıda tarif edildiği gibi hazırlanan solüsyonu 10 ul / g BW sırasıyla 125 mg ve ketamin ve ksilazin, g başına BW ila 8 mg), ketamin / ksilazin arasında ip enjeksiyonu ile hayvan anestezisi. Uzun süreli kayıtlar sırasında Gerekirse ilave enjeksiyonlar ip veya im yapılabilir Not: 0,05-0,1 arasında tekrarlayan im enjekte üst uyluk ketamin / ksilazin solüsyonu uygun olduğu kanıtlanmıştırkadar, 3 saat boyunca derin anestezi hayvanı korumak için. </li><li> Anestezi altında iken kuruluğunu önlemek için gözleri veteriner merhem kullanın. </li><li> Bir stereotaktik çerçeve içinde hayvanın baş düzeltmek ve deney sırasında hayvanın vücut sıcaklığını kontrol etmek için rektal prob ve refleks halkası olan bir ısıtma yastığı kullanın. Bir stereotaksik çerçeveye omurilik sabitleme gerekli değildir. </li><li> Hazırlık başlamadan önce, farelerin ayak parmakları arasında eyeblink refleks ve seğirmesi tarafından narkoz derinliğini kontrol edin. Refleksler ortadan kaldırılmalıdır. </li><li> Bir neşter kullanılarak açık bir operasyonel alanını elde etmek için bel bölgeleri alt üst torakal seviyeden omurilik üzerinde orta hat boyunca cildi açın. Cilt kesiler yapmak için makas kullanmayınız. Dikkatlice altta uzanan doku arasındaki gevşek deri. Sonraki adımlar% 0,9 tuz ile nemlendirilmiş yara tutmak sırasında. </li><li> Neşter ve makas kullanırken tendonları ve torakal seviyelere lomber vertebra iki tarafında bağ dokusu kesti. </li&gt; <li> Küçük bir cımbız kullanarak omurları etrafında spinöz süreçleri ve bağ dokusu dayanağını çıkarın. </li><li> Dikkatlice kıskaç bir mikroskop altında lomber düzeylerde (L4/L5) başlayarak ile vertebra çatlak. Vertebra ve omurilik arasındaki boşlukta çenenin ucu kıpırdamak ve dışında kemik parçaları kaldırın. Dura zarar ve omuriliğe basınç önlemek etmeyin. Hem başarısı için kritik öneme sahiptir. Bütün işlem sırasında ıslatılmış omurilik tutun. Orta torakal seviyelere devam. </li></ol> 3. Sırt Kökler ve DRP Kaydı ayıran (Şekil 2) <ol><li> Dikkatli bir bükülmüş ucu ile ince bir iğne (30 G) kullanılarak dura mater açın. Artık nemlendirilmesi için aCSF kullanın. </li><li> Ölçer kullanarak mümkün olduğunca dorsal kökleri ayırmak ve mümkün olduğunca uzak iki komşu kökleri kesmek. Güçlü ayrılması sırasında kökleri üzerinde çekme deneyi başarısını etkiler. </li><li> Do emme elektrot aşağı indirinkökleri rsal. Sıvı dorsal köklerde emmek olur mümkün olduğunca omuriliğe kadar aCSF ekleyin. </li><li> , Bir şırınga aracılığıyla negatif basınç uygulanarak bir cam pipet içine bir dorsal kök, kesilen ucu em. Gerekirse, ince bir iğne kullanılarak elektrot açıklığın önünde dorsal kök hareket ettirin. </li><li> Dorsal kök emme elektrodu içinde kuru seyrederse pipet yeterince doldurulur aCSF kadar dikkatlice emerken, ucu için bir aCSF ekleyin. </li><li> Dorsal kök emilir sonra, omurilikten elektrot ucunu yükseltmek. Özen pipet ve omurilik kısa devre kayıt / stimülasyon elektrot arasında &quot;su köprüsü&quot;. </li><li> Tekrar bir taraf bitişik kök için 3,3-3,6 adımları. </li><li> Dorsal köklerine mümkün olduğunca yakın referans elektrodu yerleştirin ve aCSF uygulayarak nemli tutmak. </li><li> Kayıt kurulum kurarak, bir kök önceden uyarılması için seçilir,Kayıt için. Ikinci dorsal kök kayıt mevcut kelepçe modunda yapılır iken gerilim adım adım artırın. Bir DRP temsil yavaş, uzun süreli yukarı sapma tarafından takip edilir kısa aşağı doğru eğilmeye fark edebilirsiniz. </li><li> (- 0.1 Hz&#39;de 30 süpürür / dorsal kök, 0,3 Hz-3 kHz arasında filtre en az 20) düzeyleri supramaksimal ve kaç silme kaydetmek için uyarıcı voltajını ayarlayın. </li><li> DRP zaman-bağımlı toplamı hakkında bilgi almak için üç uyaranlara (100 Hz) kısa trenler kaydedin. </li><li> Ek taraf kayıtlar için kayıt ve stimülasyon siteyi açın. </li><li> Hayvanlar prosedür hayatta kalmak için amaçlanmamıştır. Iken hala derin anestezi altında (ketamin / ksilizin, yukarıda bakınız 2.2 adım) başı kesilerek son kayıttan sonra hayvanlar kurban. </li></ol> 4. Veri Analizi <ol><li> Analiz programı veri aktarmak (örneğin Sigma Plot, Igor Pro veya MATLAB). </li><li> Ortalama izleri fr hesaplayınom 20-30 süpürür. </li><li> Daha fazla analiz için (Şekil 3), (DRP tepe genlik başlangıca) Voltaj sapma maksimum genliği al. </li><li> Daha sonra potansiyellerinin zamana bağlı olarak toplanır bir ölçü elde etmek için üç bakliyat ve tek bir akım sonrasında DRP zirve amplitüdünün oranını hesaplar. </li></ol>

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Yazarlar herhangi bir mali çıkarlarını beyan.

In vivo nöronal aktivite ve sinaptik potansiyellerin Ekstra ve intraselüler elektrofizyolojik kayıtlar MSS nöronal fonksiyonları ve fizyopatolojisi araştıran sanat teknikleri devlet vardır. Spinal entegrasyon motor işlev, örneğin bacak hareketi ve multimodal duyusal algı için çok önemlidir. Presinaptik inhibisyon duyusal girdilerin uygun tepkiler sağlanması bu hesap işleminde bir kritik mekanizmadır. Ia lifinden üzerinde GABAerjik sinapslar PAD motonöronlann uyarılmasını engelle...

Presinaptik inhibisyon omurilik en güçlü önleyici mekanizmalarından biridir. Bu postsinaptik zar potansiyeli ve motor nöronlar 1-3 uyarılabilirliğini değiştirmeden monosynaptically heyecan motonöronları uyancı postsinaptik potansiyeller (EPSP) inhibe eder. Duyusal presinaptik liflerin üzerine GABAerjik axo-aksonal sinaps tarafından uyarılan primer afferent depolarization (PAD) altta yatan mekanizma 4-7 (ayrıca Figure1A bakınız). Bu sinapsların GABA A ve GABA B-reseptörleri (GABA A R ve GABA B R) içerir. GABA A R aktivitesi nedeniyle lokal iyon dağılımına PAD ortaya çıkarır klorür iletkenlik bir artışa yol açar. Bu depolarizasyon bloklar akson terminalleri içine aksiyon potansiyelleri yayılma ve azalmış Ca 2 lider güçlerini azaltır +-akını ve verici serbest bir azalma. GABA B reseptörlerinin aktivasyonu yok not PAD katkıda ama bu şekilde presinaptik olarak önlenmesini artırmak Ca 2 +-akışı bir azalmaya yol açar. GABA A R&#39;nin kısa süreli aktivasyon inhibisyonu dahil olmak olmakla birlikte, GABA B R, uzun vadeli modülasyonu 8-10 katılmaktadırlar. PAD ve presinaptik inhibisyonunun önemli bir bölümünü oluşturur GABA, ek olarak, diğer vericiler sistemleri de modüle olabilir ve bu mekanizma 11,12 katkıda bulunur. Presinaptik inhibisyon patolojik değişiklikler, kusurlu GABAerjik transmisyon 17 tarafından aracılık edilen motorlu hiper-ile-çevresel iltihap ve nöropatik ağrıya 13,14 yanı sıra anormal merkezi ağrı işlem 15, omurilik yaralanması 16, ve merkezi sinir sistemi hastalıkları, örneğin, birkaç hastalık durumu çok önemli görünmektedir 18. Böylece, presinaptik inhibisyonu tahmin in vivo omurilik düzeyinde deneysel patolojik koşulları araştırmak için faydalıdır </em&gt;. PAD, omurilikte presinaptik inhibisyon doğrudan bir ölçümünü sağlayan birim gerçekleştirilen potansiyellerine neden olur. Bu potansiyelleri dorsal kök potansiyelleri (DRP) olarak adlandırılır ve komşu dorsal kökler 7 uyarılmasından sonra omurilik dorsal köklerinden ölçülebilir. DRP ilk ölçümleri kedi ve kurbağalar 19 rapor edilmiştir ve yoğun 1970&#39;lerin başında 3,4,20,21 yılında Eccles, Schmidt ve diğerleri tarafından kedilerde çalışıldı. Kedi ve 22 farelerde 23 DRP in vivo kayıtları yaygın olarak kullanılmış olsa da, farelerde ölçümleri hemen hemen tamamen izole edilmiş ex vivo omurilik preparasyonlar 15,24 gerçekleştirilmiştir. Burada, sağlam organizmada presinaptik inhibisyon doğrudan bir ölçümünü sağlayan in vivo olarak, anestezi uygulanmış farelerde DRP kaydetmek için bir yöntem tarif eder.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass tubing (inner diameter 1.16 mm)</td>
			<td>Science Products (Hofheim, Germany)</td>
			<td>GB200F-10</td>
			<td>Other glass tubing might also be suitable</td>
		</tr>
		<tr>
			<td>Superfusion solution (sterile, 0,9% NaCl)</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>3570350</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Germany)</td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazine)</td>
			<td>Bayer Animal Health GmbH (Leverkusen, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamin 10%</td>
			<td>Medistar GmbH (Ascheberg, Germany)</td>
			<td>KETAMIN 10%</td>
			<td></td>
		</tr>
		<tr>
			<td>30G micro needle/ Sterican</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>4656300</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Geramny)</td>
		</tr>
		<tr>
			<td>Salts for aCSF</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Diverse</td>
			<td></td>
		</tr>
		<tr>
			<td>S88 Dual Output Square Pulse</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>S88X</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
		</tr>
		<tr>
			<td>SIU5 RF Transformer Isolation Unit</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>SIU-V</td>
			<td></td>
		</tr>
		<tr>
			<td>InstruTECH LIH 8+8</td>
			<td>HEKA (Lambrecht, Deutschland)</td>
			<td>LIH 8+8 + Patchmaster software</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition&#160;</td>
		</tr>
		<tr>
			<td>Universal amplifier</td>
			<td>npi (Tamm, Deutschland)</td>
			<td>ELC-03X</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus (Tokyo, Japan)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>MPC-200/MPC-325</td>
			<td>Mechanical micromanipulators also possible</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket System</td>
			<td>Stoelting (Wood Dale, USA)</td>
			<td>50300V</td>
			<td></td>
		</tr>
		<tr>
			<td>Intra-/extracellular recording electrode holder</td>
			<td>Harvard Apparatus (Holliston, USA)</td>
			<td>641227</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring spinal presynaptic inhibition in mice by dorsal root potential recording in vivo

Presinaptik inhibisyon omurilik en güçlü önleyici mekanizmalarından biridir. Bunun altında yatan fizyolojik mekanizma GABAerjik axo-aksonal sinaps (birincil aferent depolarizasyon) tarafından aracılık edilen primer afferent liflerin bir depolarizasyon. Birincil aferent depolarizasyon gücü dorsal kök (dorsal kök potansiyelleri, DRP), hacimli-yürütülen potansiyelleri kayıt ile ölçülebilir. Presinaptik inhibisyon patolojik değişiklikler, bazı ağrı koşullarının anormal merkezi işleme ve motor hipereksitabilitenin bazı hastalıklarda çok önemlidir. Burada, farelerde in vivo olarak kayıt DRP için bir yöntem tarif eder. , Anestezi verilen hayvanın ve emme elektrodu kullanarak kayıt prosedürü omurilik dorsal kök preparasyonu açıklanmıştır. Bu yöntem, GABAerjik DRP ölçme ve böylece canlı fare omurilik presinaptik olarak önlenmesini tahmin sağlar. Transgenik fare modelleri ile birlikte, DRP kayıt olabilir sehastalıkla ilişkili spinal patofizyolojiye araştırmak için güçlü bir araç olarak rve. in vivo kayıt örneğin eşzamanlı kayıt ya da supraspinal ağları ve periferik sinirlerin uyarılması ile DRP indüksiyon manipülasyon olasılığı ex vivo izole spinal kord hazırlıkları kıyasla birçok avantajı vardır.

Tipik DRP izleri, Şekil 3 &#39;de gösterilmiştir. Belirgin stimülasyon dışlayıcı genellikle kısa aşağıya doğru bükülmesiyle takip edilmektedir. Daha sonra DRP temsil yavaş, uzun süreli yukarı sapma, açık bir şekilde ayırt edilebilir. Kayıtların bir altkümesinde, dorsal kök refleksleri DRP üstünde küçük sivri olarak görebilir. Stimülasyon voltaj fazla olduğu zaman, normal, vahşi tip farelerde, dorsal kök reflekslerin en çok görünür. Dorsal kök refleksleri bu hazı...

Watch this Scientific Journal Video about Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo at JoVE.com

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAerjik presinaptik inhibisyonu motor ve omurilik ağlarında duyusal sinyal entegrasyon için önemli omurilikteki güçlü bir önleyici mekanizmadır. Altta yatan primer afferent depolarizasyon dorsal kök potansiyelleri (DRP) kaydı ile ölçülebilir. Burada farelerde DRP in vivo kayıt için bir yöntem ortaya koymaktadır.

Dorsal Kök Potansiyel Kayıt By Farelerde Spinal Presinaptik Engellenme Ölçme<em&gt; In Vivo</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

measuring-spinal-presynaptic-inhibition-mice-dorsal-root-potential

Research

JoVE Journal

Neuroscience

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

13.9K Görüntüleme.  Jena University Hospital, Jena, Germany.  GABAerjik presinaptik inhibisyonu motor ve omurilik ağlarında duyusal sinyal entegrasyon için önemli omurilikteki güçlü bir önleyici mekanizmadır. Altta yatan primer afferent depolarizasyon dorsal kök potansiyelleri (DRP) kaydı ile ölçülebilir. Burada farelerde DRP in vivo kayıt için bir yöntem ortaya koymaktadır. 

Video: Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Fare Retina Ganglion Hücreleri Transfeksiyon tarafından In vivo Elektroporasyon

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Nörobilim

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograd Floresan Etiketleme Belirlenen Nöronlar gelen Hedefli Hücre dışı Tek ünitesi Kayıt izin verir<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Eşzamanlı<em&gt; Ex vivo</emİki retina&gt; Fonksiyonel Test tarafından<em&gt; In vivo</em&gt; Elektroretinogram Sistemi

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Polygraphic Recording Procedure for Measuring Sleep in Mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Farelerde Uyku Ölçüm Basım Kayıt Prosedürü

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Kemirgenler dorsal kök gangliyon Omurilik ve İzolasyon hidrolik eldesi

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Sırt kök ganglion Enjeksiyon ve Dorsal Kök Duyusal Axon Rejenerasyon Modeli Olarak Ezilme Yaralanması

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Lomber Dorsal kök gangliyon erişim ve domuz yerleştirmeye laminotomy

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Soruşturma memeli Axon rejenerasyon: Vivo Elektroporasyon yetişkin fare Dorsal kök gangliyon

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

İletim Yetmezliği Mekanizmasını İncelemek Için Bağlı Siyatik Sinir ile In Vivo Tek Lifli Kayıt ve Bozulmamış Dorsal Kök Ganglion Kullanımı

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

Dorsal Kök Ganglion Makrofajlarının Hızlı İzolasyon

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...