<p class="jove_content"&gt; Bu çalışma, Ulusal Nörolojik Bozukluklar Enstitüleri ve Ulusal Sağlık Enstitüleri Strok intramural programı tarafından desteklenmiştir. Ayrıca yöntem ve Şekil 3&#39;te verilere daha önceki katkıları için Dr George Mentis teşekkür etmek istiyorum.</p

Floresan dekstranlar anatomik araçları olarak ve görüntüleme nöronal aktivitenin 1-4 için kullanılmıştır. Alanlar ve ark., (2009) 4 model sistem olarak omurilik odaklanarak aksonal yolları için iyon ve voltaj duyarlı boya uygulamak için bir protokol yayınlamıştır. Burada in-vitro optophysiological ve morfolojik çalışmalar için kesim ventral kök, dorsal kök ve omuriliğin herhangi bir nöronal yollarını floresan ve / veya iyon duyarlı boya uygulamak için daha ayrıntılı bir prosedür tanımlamaktır. 1. Tip I ve Tip-II Pipetler Sivri uçları olan pipetler üretmek için bir alkol lambası 7 ateşte polietilen boru iki kısa bölümler (PE90, Clay Adams Marka) çekerek başlayın. Bir pipet (Tip-I) sıkıca hedef kök veya sistem (:; İç Çapı 0.2-0.4 mm: 0.1-0.3 mm Dış Çap) tutun küçük bir uç ile (3-7 mm) kısa olmalıdır. Sonra ikinci bir pipet (Tip çekin-II) daha uzun (8-12 cm) ince, şaft ve çok ince bir uç (Dış Çap: 0.2-0.3 mm, iç çapı: eklenebilen 0.1-0.2 mm) Tip-I kadarıyla Pipeti ucu. İkinci ince pipetle Tip I-pipetle sırasıyla yapay serebrospinal akışkan (aCSF) ve boya çözeltisi aspire ve tanıtmak üzere kullanılacaktır. 2. Konumlandırılması Tip-I Pipeti Inceleyin ve omurilik 8,9 yalıtmak ve yükleme süreci boyunca soğutulmuş aCSF (~ 16 ° C) ile superfused bir banyo, içine koyun. Tip-I sırtını açılış kolay, esnek bir tüp aracılığıyla bir şırınga (1ml U-100 insülin şırınga, Becton Dickenson veya eşdeğeri) bağlanabilir böylece bir elektrot tutucu (H1/12 elektrot tutucu, Narishige) üzerine Pipeti (PharMed yerleştirin BPT, Cole-Parmer, # AY242002; 10 cm) (Fig.1A-B). 3. Boya Uygulama <ol><li> Şırınga kullanarak, ilk çizmekTip I-pipetle sınırlandırılır (Şekil 1b; 2A) içine aCSF akson yolu veya (Şek. 2B) doldurulması için kök izledi. Esnek tüp daha sonra geri açılması çıkarılmalıdır. Tip-II pipet başka bir enjektöre bağlanmış ve Tip-I aksonal yolu veya kök tutarak Pipeti eklenir. Bu yüzden artık aCSF sadece aksonal yolu (; 2C-D Fig.1C) kapsar tip-II pipet, aspirat aCSF bağlı şırınga kullanma. Sonra tip-I pipetle tip-II pipet çekebilirsiniz. Aksonal yolu ya da sinirler üzerinde iyi bir sızdırmazlık doğrulamak için birkaç dakika aCSF seviyesi (&quot;iyi bir sızdırmazlık&quot; için Sorun Giderme bölümünde bakınız Bölüm 3) izleyin. </li><li> ACSF içinde boya veya çift distile su içinde% 0.2 Triton X-100 eritin ve hava kabarcıkları dahil etmemeye dikkat ederken Type-II pipet içine çekmek. Tip-I pipet içine ve artık aCSF çözüm (Şekil 2E) içine boya içeren Tip-II pipet yerleştirin. Yavaş yavaş serbest bırakınhafif pozitif basınçlı (Şekil 2F) kullanılarak aCSF içine boya. Herhangi bir hava kabarcığı tanıtmak veya ucun içinden hedef dokuda ötelemeye neden için dikkatli olun. Boya yeterli miktarda (boya çözeltisi 3-5 uL) yayımlanmıştır sonra tip-II pipet çekin. </li></ol> 4. Kuluçka Deney tipine bağlı olarak 6-20 saat arasında karanlıkta doku inkübe. Bir kez yeterli zaman doldurmak için izin verildi, hafifçe görüntüleme veya histoloji için doku hazır bırakarak doku elektrot acılır. Sorun Giderme <ol><li> Hedef dokuda Tip-I pipet ucu kolayca girmek veya bir zamanlar içinde gevşek değilse, o zaman uç çapı yanlıştır. Bu durumda, çekin ve farklı bir pipet kullanın. </li><li> Tip-I pipet içinde aCSF için boya eklemeden önce, yeterince aCSF bu ulaşılabileceği böylece kalır emin olunTip-II pipet ucu (Şekil 2B). Boya eklenir kez Aksi takdirde, bir hava boşluğu var olacaktır. Bu durumda, boya ekleyin ve pipetle aksonal yolu kaldırın ve Type-II pipet ile ulaşılabilir yeterli aCSF ile geri çekmek değildir. </li><li> Tip I-pipetle artar aCSF seviyesi, Tip II-pipetle aspire edildi sonra, bu doku ile conta iyi olmadığı anlamına gelir. Farklı bir elektrot kullanmak, aksi boya etiketleme prosedürü sırasında seyreltilmiş edilecektir. </li><li> Boya enjekte ederken bir hava kabarcığı Tip-I pipet girmiş ise, kabarcık Type-II pipet yakın ilerleyen ve yavaşça ekli şırınga kullanarak zayıf bir negatif basınç uygulayarak balonu boşaltın. </li></ol> 5.. Temsilcisi Sonuçlar Yöntemin bir uygulama göstermek için, aynı anda yükleme motonöronlar, duyu afferentleri ve üç farklı Dext ile spinal internöronlardanfloresan boyalar (Şekil 3A)-konjuge koştu. Gibi ölçülmüştür. Nöron 3B, üç sınıf etiketli; duyusal aferentlerde (kırmızı), ventral kordon (yeşil) ve motonöronlar (mavi) içine uzayan internöronlardan. <img alt="Şekil 1" src="/files/ftp_upload/4008/4008fig1.jpg" /> Şekil 1.. Şematik Pipet gösteren Tip I ve Tip-II montaj. (A) Tip I bu açılma kolayca esnek bir elastomer boru (B) bağlanabilir ve içine bir Tip II pipetle (C) eklenebilir geri var ki pipetle (açık mavi), bir elektrot tutucu üzerine yerleştirilir. <img alt="Şekil 2" src="/files/ftp_upload/4008/4008fig2.jpg" /> Şekil 2. Şematik floresan boyalarla motonöronlar ve yükleme süreci gösteriyor. (A) Tip-I pipet doldurulacak ventral kök yakın yerleştirilir. (B) Emme kök takiben pipetle içine aCSF çizmek için pipetle uygulanır. (C-D) Tip-I pipet end&#39;deki esnek elastomer tüp ayırın ve Tip-II pipet için emme uygulayarak aCSF miktarını azaltır. (E) Tip I-pipetle içinde kalan aCSF içine eritildi boya içeren Tip II-pipet ucu yerleştirin. (EF) Yavaşça boya ile Tip-I pipet doldurmak ve ardından Tip-II pipet kaldırın. <img alt="Şekil 3" src="/files/ftp_upload/4008/4008fig3.jpg" /> Şekil 3. İzole fare omuriliğe dolum işlemi uygulanması. (A) şematik gösteren üç Tip-I omurilik farklı nöronal sınıfları doldurmak için kullanılan pipetler. Motonöronlar (mavi; Cascade Mavi dekstran) ve sakral duyusal aferentlerde (kırmızı; Texas-Kırmızı dekstran) sırasıyla, ventral ve dorsal kökleri aracılığıyla backfilled edildi. Floresein dekstran olan aksonlar ventral kordon (VF) aracılığıyla yükselmek spinal internöronlardan (yeşil) yüklemek için kullanılır. Her bir boya dekstran 1 mg (10.000 MW; Molecular Probes)6 uL distile su içeren% 0.2 içinde eritildi Triton X-100 (B). Etiketli duyusal aferentlerde birkaç kontralateral projeksiyonları esas ipsilateral olduğunu A. Not açıklandığı gibi nöronlar arka etiketli olduğu bir omuriliğin sakral kord bir bölümünün Konfokal görüntü. Etiketli internöronlardan dolgu yan (ok başı) kontralateral iken etiketli motonöronlar dolu ventral kök ipsilateral vardır. 200 mikron Kalibrasyon bar.

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	<li>Nance, D. M., Burns, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dextrans+as+sensitive+anterograde+neuroanatomical+tracers:+applications+and+pitfalls.">Fluorescent dextrans as sensitive anterograde neuroanatomical tracers: applications and pitfalls.</a> Brain Res. Bull. 25, 139-145 (1990).</li><li>Glover, J. C., Petursdottir, G., Jansen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dextran-amines+used+as+axonal+tracers+in+the+nervous+system+of+the+chicken+embryo.">Fluorescent dextran-amines used as axonal tracers in the nervous system of the chicken embryo.</a> J. Neurosci. Methods. 18, 243-254 (1986).</li><li>McPherson, D. R., McClellan, A. D., O'Donovan, M. 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<p class="jove_content"&gt; Biz ifşa hiçbir şey yok.</p

<p class="jove_content"&gt; Biz burada nöronlar, sinirler ve spinal yolları içine boyalar tanıtılması için basit ve düşük maliyetli bir protokolü tanımlar. Bu yöntem, yükleme işlemi süresi için bir yüksek oranda konsantre boya çözeltisine tanımlanan anatomik yollar açığa içerir. Biraz arka plan bilgisi ile hedef sitenin retrograd etiketleme Bu sonuçlar microinjections, banyo uygulama ve elektroporasyon tekniklere nazaran. Bununla birlikte, bu yöntem, bir akson izole edilebilir yollarının sinir sisteminde siteleri il...

<table border="1"&gt;<tbody&gt;<tr&gt;<td colspan="2"<strong&gt; Malzeme Adı</strong</td&gt;<td<strong&gt; Şirket</strong</td&gt;<td<strong&gt; Katalog numarası</strong</td&gt;<td<strong&gt; Yorumlar</strong</td&gt;</tr&gt;<tr&gt;<td rowspan="2"&gt; Boru</td&gt;<td&gt; PE90 (IDxOD: 0.034 &quot;x0.050&quot;; Duvar Kalınlığı: 0.008 &quot;)</td&gt;<td&gt; Clay Adams Marka Intermedic</td&gt;<td&gt; 427.421</td&gt;<td&gt; Tip I ve Tip II pipetler için</td&gt;</tr&gt;<tr&gt;<td&gt; NSF-51 (IDxOD: 1/16x1/8; Duvar Kalınlığı: 1/32)</td&gt;<td&gt; PharMed BPT, Cole-Parmer</td&gt;<td&gt; AY242002</td&gt;<td&gt; Esnek borular için</td&gt;</tr&gt;<tr&gt;<td&gt; Şırınga</td&gt;<td&gt; 1ml insülin şırınga, U-100</td&gt;<td&gt; Becton Dickenson</td&gt;<td&gt; 329.650</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; İğne</td&gt;<td&gt; 19G x1-1/2 &quot;Metal Hub İğne</td&gt;<td&gt; MONOJECT</td&gt;<td&gt; 200.136</td&gt;<td&gt; Şırıngaya Tip II pipet Bağlanması</td&gt;</tr&gt;<tr&gt;<td&gt;</td&gt;<td&gt; Alkol Lamba</td&gt;<td&gt;</td&gt;<td&gt;</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Musluk</td&gt;<tdErkek Luer Fişi Adaptörü ile&gt; Üç yollu Musluk</td&gt;<td&gt; Baxter Healthcare Corp</td&gt;<td&gt; 2C6241</td&gt;<td&gt; Şırınga ile kullanın</td&gt;</tr&gt;<tr&gt;<td rowspan="3"&gt; Holder</td&gt;<td&gt; H-1 elektrot tutucu</td&gt;<td&gt; Narishige</td&gt;<td&gt; H-1/12</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Mıknatıs standı</td&gt;<td&gt; Narishige</td&gt;<td&gt; GJ-8</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Mikromanipülatör</td&gt;<td&gt; Narishige</td&gt;<td&gt; M-3333</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Cımbızlar</td&gt;<td&gt; S &amp; T DUMONT İsviçre 00.632-11</td&gt;<td&gt; DUMONT</td&gt;<td&gt; JF-5 TC</td&gt;<td&gt;</td&gt;</tr&gt;</tbody&gt;</table&gt;<p class="jove_content"&gt; * Bu önerilen malzemelerdir. Herhangi bir uyumlu ürünler ile değiştirilebilir</p

retrograde loading of nerves, tracts, and spinal roots with fluorescent dyes

Nöronların Retrograd etiketleme de nöronlar 3-6, kalsiyum ve voltaj duyarlı boyalar yüklemek için kullanılan bir standart anatomik yöntemi 1,2. Genel olarak, boyalar katı kristaller halinde ya da cam pipet kullanılarak lokal baskısı enjeksiyon yoluyla uygulanır. Bununla birlikte, bu birkaç saat boya difüzyonu için gerekli olan, özellikle boya ve azaltılmış etiketleme yoğunluğunun seyreltme ile sonuçlanabilir. Burada boya çözeltisi ile doldurulmuş bir polietilen emiş pipet kullanılarak nöronlar içine flüoresan ve iyon karşı hassas boyaların sokulması için basit ve düşük maliyetli bir teknikle göstermektedir. Bu yöntem, yükleme işlemi boyunca akson ile temas halinde boya yüksek bir konsantrasyonunun muhafaza için güvenilir bir yol sunar.

Watch this Scientific Journal Video about Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes at JoVE.com

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Biz nöron veya polietilen emme pipet kullanarak herhangi bir nöronal yoluna floresan ve kalsiyum duyarlı boyalar yüksek konsantrasyon tanıtmak için basit ve düşük maliyetli tekniği tanımlandı.

Sinirler, yolları ve Floresan Boyalar ile Spinal Roots Retrograd Yükleniyor

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

retrograde-loading-nerves-tracts-spinal-roots-with-fluorescent

Research

JoVE Journal

Neuroscience

12.2K Görüntüleme.  National Institute of Neurological Disorders and Stroke, National Institutes of Health. Biz nöron veya polietilen emme pipet kullanarak herhangi bir nöronal yoluna floresan ve kalsiyum duyarlı boyalar yüksek konsantrasyon tanıtmak için basit ve düşük maliyetli tekniği tanımlandı.

Video: Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

Styryl Boyaların Fotoçevrim kullanarak Synaptic veziküllü Havuzları incelenmesi

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

Nörobilim

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Gerilim duyarlı Boyalar ile Yengeç Stomatogastric Ganglion Nöronlar Optik Görüntüleme

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

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 ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

Şiddetli Spinal Kord Yaralanması sonra fibrin ve Büyüme Faktörü kokteyller ile Nöral Kök Hücre Survival ve Farklılaşma Tanıtım

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

Floresan Boyalar Yetişkin Zebra balığı Retina Ganglion Hücreleri retrograd Etiketleme

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Fare Siyatik Sinir Vivo elektrofizyolojik ölçümlerde

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Genetik Kodlanmış Floresan Gerilim Sensörleri İki Türleri ile Potansiyel görüntüleme Membran

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

Farelerde frenik Motor nöronların retrograd nöroanatomik izleme

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Etkinlik tabanlı eğitim omurilik ile bir koşu bandı üzerinde Wistar fareler yaralı

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.