NIH (NS-18.205) tarafından finanse edilmektedir.

1. Eşleştirilmiş Kayıtlar Balık hazırlanması <ol><li> Yordama başlamadan önce, dahil beş anahtar teslim çözümler hazırlamak: Banyo Çözüm Tricaine (MS222), Formamid, Nöron İç Çözüm ve kas İç Çözüm Çözüm Banyo Banyo Çözüm. </li><li> Daha sonra, 72 ila 96 saat arasında yaş ve Banyo Çözüm Tricaine ile yer arasında bir tek larva Zebra balığı toplamak. Anestezi maruz kalma bir dakika içinde, balık, dokunmaya karşı cevap vermez. </li><li> Plastik bir tabak balık üstüne yerleştirin ve bir stereomikroskopta kullanarak, çift kenar jilet ile başını kesmek. </li><li> Sylgard ile kaplı ve Banyo Çözümü ile dolu bir cam kayıt odasına balık aktarın. Notokord ile elektrolitik bilenmiş tungsten pimleri takarak her iki ucunda sabitleyin. </li><li> Rostral sonuna yakın, ince forseps bir çifti için yeterli bir kavrama sağlayacak bir iğne ile balık flep oluşturun. Bir çift ince forseps ile rostral pin yakınında sürükleyici örten deri çıkarın. Kaudal yönde yavaş yavaş soyun cilt kaldırmak için. </li><li> Kayıtları ile etkileşebilir kas kasılmaları engellemek için 3-5 dakika Formamid Banyo Çözüm 3 ml ile balık davranın. Normal Banyo Çözümü ile 5 kez yıkayın. 5 ila 6 segmentleri üzerinde yüzeysel kas tabakasının parçası hafifçe tungsten pin tarafı hurdaya çıkarın. </li><li> Elektriksel gürültü kalkan bir Faraday kafesi bulunan dik bir mikroskop balık aktarın. Mikroskop, uzun 4x mesafe 40x objektif ve zoom 0,5, sabit bir sahne ile donatılmıştır. Mikroskop yüksek güç altında sinir ve kas arasındaki hazırlık taşımak için motorize XY çeviri aşamasında üzerine monte edilmiştir. Diferansiyel girişim kontrast optik nöron görselleştirmek için yardımcı olur. </li><li> 0,5 x zoom örten kas altında, kaldırmak ve omurilik ortaya çıkarmak için geniş bir çap 15 mikron pipet kullanın. Negatif basınç, 3 cc tek kullanımlık şırınga ve kas hücreleri ile uygulanan tek tek çıkarılır. Bu prosedür, iskelet kası 5 ila 6 dorsal segmentleri için tekrarlanır. Aynı geniş delik pipet de hedef hızlı iskelet kas elde etmek için karın kas üst iki kat kaldırmak için kullanılır. </li><li> Bu eşleştirilmiş kayıtları için hazırlık tamamlar ve mikroskop zum yaklaşık 1.6x &#39;e yükselmiştir. </li></ol> 2. Eşleştirilmiş CaP Motor nöron Kayıtlar ve Hedef kas hücreleri <ol><li> Temizlenir segmentleri CaP nöronlar bulmak için taranır. Her omurilik hemi-segment, büyük bir, 10 mikron çapında, PCa motor nöron içerir. Bu gözyaşı şeklindeki soma segmental sınır kaudal en sonuna yakın spinal dura altında yüzeysel olarak yer almaktadır. Bir CaP nöron kayıt için seçilir ve komşu kaudal segmentinde ventral hedef kas bütünlüğünü kontrol etmek için taranır. </li><li> İki kayıt, nöron ve kas için ikinci bir yama elektrotlar yerleştirilir. Üst elektrodun sert spinal dura ve yaklaşık 2 mikron ucu açılış penetrasyon için sığ bir konik vardır. Alt elektrot iskelet kas düşük direnç gerilim kelepçe kayıt için dik konik </li><li> Nöron elektrot spinal dura nüfuz yaklaşık 30 derecelik bir açıyla yerleştirin, yaklaşık bir yarım seçilen CaP nöron rostral-segment. Avans yaklaşık 60 milimetre cıva nazik bir sürekli pozitif basınç uygulayarak eksenel bir sürücü manipülatör kullanarak elektrot. Elektrot dura delerek geçer gibi, CaP nöron odak yeniden düzenlenmesi gerektiren elektrottan perfüzyon yerinden olabilir. </li><li> Elektrot değdiğinde CaP nöron soma pozitif basınç yayınlandı ve gigaohm mühür hemen oluşumunda genellikle sonuçları. Ancak, bazı durumlarda bir mühür formu başarısız, nazik bir negatif basınç uygulamak için gerekli olur. Mühür kurulmasından sonra, elektrot potansiyeli to-80mV ayarlanabilir ve negatif basınç uygulaması hücre zarı rüptür. CaP nöron 140-180 Mega ohm ve aksiyon potansiyeli, bu artım +40 milivolt bir giriş direnci ile karakterizedir. Bu aksiyon potansiyeli ortaya çıkardı kadar akım pensi altında depolarizan adımları aşamalı olarak artan enjekte edilerek test edilir. </li><li> Daha sonra, mikroskop altında XY motorlu tercümecisi yüksek güç kullanarak ventral kas taşındı. Hızlı bir kas hücre hedef alan seçilir. </li><li> Serbest bırakıldığında, gigaohm mühür formları pozitif basınç altında hedef kas hücresi, kas elektrot doğru ilerletilir. Elektrot potansiyeli -50 milivolt sodyum kanallarını inaktive ayarlanır ve yumuşak emmek altında hücre zarının delinmiş veya yırtılmış. 100 ile 200 Mega-ohm direnç ve büyük kapasitif transientler sinyalleri giriş aniden ortaya bir damla. </li><li> Test etmek içinbir aksiyon potansiyeli ortaya çıkardı kadar eşleştirilmiş kayıt için, motor nöron aşamalı olarak daha büyük akım enjeksiyonu depolarize. Bu noktada bir uç plakası akımı kas anlaşılabilir olmalıdır. Arızasız son plağın akımları 1 Hertz sonuçları motorneuron Devamı stimülasyonu. 100 Hertz uyaran frekansı arttıkça uç plakası akımları genlik dalgalanma ve stimülasyon 10 saniye içinde fonksiyonel tükenmesi tabi. </li></ol> 3. Temsilcisi Sonuçlar Tipik olarak, nöron kayıt için bir saat kadar istikrarlı ama kas hücreleri seri direnç bir artış olarak yansır bozulabilir. Bu oluştuğunda deney ya iptal edilir ya da başka bir kas hücresi yama kelepçeli. Tüm kayıtlar bir saat içinde tamamlanmalıdır. <img src="/files/ftp_upload/2351/2351fig1.jpg" alt="Şekil 1" /> Şekil 1 motorneuron aksiyon potansiyeli (AP, üst) ve ilgili kas uç plakası mevcut (EPC, alt) Kayıtlar. CaP nöron için aksiyon potansiyeli aşmayı +40 mV ve EPC 1 msn altında sabit bir süre ile üstel bir zaman seyri boyunca 300 μsec ve çürüme içinde yükselmeye etmelidir. <table border="1"><tbody><tr><td> Isim </td><td> Reçete </td></tr><tr><td> Banyo Çözümü (mM) </td><td> 134 NaCl, 2.9 KCl, 2.1 CaCl 2, 1.2 MgCl 2, 10 Glikoz, 10 Na-Hepes, pH 7.8 </td></tr><tr><td> Tricaine ile Banyo Çözüm </td><td> Banyo Çözüm içeren% 0.02 tricaine </td></tr><tr><td> Banyo Formamid ile Çözüm </td><td> 2M formamid içeren Banyo Çözüm </td></tr><tr><td> Nöron İç Çözümü (mM) </td><td> 115 K-glukonat, 15 KCl, 2 MgCl 2, 10 K-HEPES, 5 K-EGTA, 4 Mg-ATP, pH 7.2 </td></tr><tr><td> Kas İç Çözüm (mM) </td><td> 120 KCl, 10 K-HEPES, 5 BAPTA, pH 7.4 </td></tr></tbody></table> Tablo 1. Çözümler.

<ol>
	<li>Fetcho, J. R., O'Malley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+networks+in+behaving+animals.">Imaging neuronal networks in behaving animals.</a> Current Opinion in Neurobiology. 7, 832-838 (1997).</li><li>Fetcho, J. R., S, H. i. g. a. s. h. i. j. i. m. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+and+genetic+approaches+toward+understanding+neuronal+circuits+in+zebrafish.">Optical and genetic approaches toward understanding neuronal circuits in zebrafish.</a> Integr Comp Biol. 44, 57-70 (2004).</li><li>Iba&#241;ez-Tallon, I., Wen, H., Miwa, J., Xing, J., Aslantas-Tekinay, A., Ono, F., Brehm, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tethering+naturally+occurring+peptide+toxins+for+cell+autonomous+modulation+of+ion+channels+and+receptors+in+vivo.">Tethering naturally occurring peptide toxins for cell autonomous modulation of ion channels and receptors in vivo.</a> Neuron. 43, 305-311 (2004).</li><li>Li, W., Ono, F., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurements+of+presynaptic+release+in+mutant+zebrafish+lacking+postsynaptic+receptors.">Optical measurements of presynaptic release in mutant zebrafish lacking postsynaptic receptors.</a> J. Neuroscience. , 23-10467 (2003).</li><li>Ono, F., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+receptors+direct+rapsyn+clusters+to+the+neuromuscular+synapse+in+zebrafish.">Acetylcholine receptors direct rapsyn clusters to the neuromuscular synapse in zebrafish.</a> J. Neuroscience. 24, 475-5481 (2004).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Fetcho, J., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paralytic+zebrafish+lacking+ACh+receptors+fail+to+localize+rapsyn+clusters+to+the+synapse.">Paralytic zebrafish lacking ACh receptors fail to localize rapsyn clusters to the synapse.</a> J. Neuroscience. 21, 5439-5448 (2001).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+motility+mutant+twitch+once+reveals+new+roles+for+rapsyn+in+synaptic+function.">The zebrafish motility mutant twitch once reveals new roles for rapsyn in synaptic function.</a> J. Neuroscience. 22, 6491-6498 (2002).</li><li>Wang, M., Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+neuromuscular+synapses+in+the+zebrafish+choline-acetyltransferase+mutant+bajan.">Function of neuromuscular synapses in the zebrafish choline-acetyltransferase mutant bajan.</a> J Neurophysiol. , 100-104 (2008).</li><li>Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+motor-neuron+muscle+recordings+in+zebrafish+test+the+receptor+blockade+model+for+shaping+synaptic+current.">Paired motor-neuron muscle recordings in zebrafish test the receptor blockade model for shaping synaptic current.</a> J. Neuroscience. 25, 8104-8111 (2005).</li></ol>

Biz esas olarak yüksek frekanslı stimülasyon sırasında vezikül serbest bırakılması ve geri dönüşüm süreçlerini incelemek için zebrafish eşleştirilmiş kayıtları (Wen ve Brehm, 2005) ile olmuştur. Bu süreç, temel sinaptik bileşenler, nokta mutasyonları nedeniyle tehlikeye motilite mutantlar neyin normal sinaptik iletimi bozulur. Örneğin, mutasyonlar postsinaptik reseptör agrega (Ono ve arkadaşları, 2001, 2002, 2004) ve presinaptik verici sentezi (Wang ve ark, 2008) un koordine yüzme sonucu bozabilir. Eşleştirilmiş kayıtları böylece davranış, genetik ve fizyoloji bağlama, fonksiyonel defekti kaynak kesin bilgi verir. Şimdi daha akıllıca rehberleri kullanarak CaP motor nöronların sinaptik iletim yoluyla geçici ifade yatan süreçleri incelemek mümkün olmalıdır. Bu şekilde dominant negatif ya da mutasyona uğramış genlerin, özellikle bu nöronların ifade ve hem de davranışsal ve elektrofizyolojik sonuçları belirlenen olabilir. CaP soma ajanları ile potansiyel vezikül geri dönüşüm, Clostrodiol toksinler ve kalsiyum tamponlar gibi değiştirebilir diyaliz tüm hücre yapılandırması tarafından sunulan ek bir avantaj sağlar. Soma ve kas arasındaki kısa mesafe nedeniyle, difüzyon kayıtların zaman çerçevesi içinde yapılması gerekir. Bu hazırlık potansiyeli sadece dokundunuz başladı. Ayrıca, bu yaş ve yüzeysel sinaps yerde balık şeffaflık, optik aletler kullanımı (Fetcho ve Higashijima, 2004 Fetcho ve O&#39;Malley, 1997) kolaylaştırır. FM boyalar ve Synaptophluorin (Li ve ark, 2003) gibi genetik göstergeleri kullanarak yaşayan balık endo ve ekzositoz ölçme çok başarılı olmuştur. Ayrıca, floresan konjuge toksinler canlı balık (Ibanez-Tallon ve ark, 2004) görüntüleme için sinaptik proteinlerin etiket kullanılabilir. Bu preparatın basitliği göz önüne alındığında gelecekteki çalışma için büyük umut vaat.

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		<th>Catalogue Number</th>
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<td>Pneumatic transducer tester</td>
<td>&nbsp;</td>
<td>Fluke Biomedical Instruments</td>
<td>DPM1B</td>
<td>&nbsp;</td>
<tr>
<td>0.002 x 3 inch tungsten rod</td>
<td>&nbsp;</td>
<td>A-M Systems Inc</td>
<td>715000</td>
<td>&nbsp;</td>
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<td>Sylgard 184 Elastomer</td>
<td>&nbsp;</td>
<td>Dow Corning Corp.</td>
<td>&nbsp;</td>
<td>The thickness of application will affect the DIC optics</td>
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paired patch clamp recordings from motor-neuron and target skeletal muscle in zebrafish

Larvaların zebrafish spinal motor nöron ve hedef kas aynı anda yama kelepçe kayıt izin vermek için ilk omurgalı model sistemi temsil eder. Bu her iki hücre tipleri ve yeteneği, görsel olarak morfolojisi ve lokasyon bazında tek segmental CaP motor nöron ayırt etmek için erişilebilirlik doğrudan bir sonucudur. Bu video bir CaP motor nöron ve hedef kas hücrelerinin yanı sıra, her bir hücre tipi kayıt için metodolojiler tanımlamak için kullanılır mikroskobik yöntemler göstermektedir. CaP motor nöron tipi Kimlik boya dolum birinin veya biyofiziksel aksiyon potansiyeli dalga şekli ve hücre giriş direnci gibi özellikleri ile teyit edilir. Motor nöron kayıtları, bir saat, birden fazla farklı hedef kas hücreleri uzun vadeli kayıtları izin rutin son. Motor nöron ateşleme deseni üzerinde kontrol nöromüsküler kavşakta sinaptik iletim frekans bağımlılığı ölçümleri sağlar. Üniter olaylar büyük bir quantal boyutu ve tüm hücre gerilimi kelepçe tarafından sağlanan düşük gürültü seviyesi sayesinde, tüm kas çözülebilir. Bu özellik sürüm özellikleri, vezikül yanı sıra geri dönüşüm, sinaptik depresyon ve kolaylaştırılması gibi temel sinaptik özellikleri çalışma izin verir. Motor nöron genellikle ekstrasellüler elektrotlar tarafından teşvik eden bu in vivo hazırlık eclipse önceki nöromüsküler model sistemler tarafından sunulan avantajları okudu ve kaslar tüm hücre yama kelepçe için çok büyük. Zebra balığı hazırlık yaklaşımların transgenik hatları, morfolino demonte, farmakolojik müdahale ve in vivo görüntüleme dahil olmak üzere geniş bir yelpazede elektrofizyolojik analizi birleştirerek için uygun. Bu yaklaşımlar, giderek artan sayıda zebrafish mutant hatları tarafından sağlanan nöromüsküler hastalık modelleri ile birleştiğinde, insan nöromüsküler bozukluklar yeni bir anlayış için kapıyı açmak.

Watch this Scientific Journal Video about Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish at JoVE.com

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larvaların zebrafish spinal motor nöron ve hedef iskelet kası aynı anda yama kelepçe kayıt izin vermek için ilk omurgalı model sistemi temsil eder. Bu video segmental CaP motor nöron ve hedef kas hücrelerinin yanı sıra, her bir hücre tipi kayıt için metodolojiler tanımlamak için kullanılır mikroskobik yöntemler göstermektedir.

Motor-nöron Paired Patch Kelepçe Kayıtlar ve Zebra balığı Hedef İskelet Kası

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

paired-patch-clamp-recordings-from-motor-neuron-target-skeletal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.1K Görüntüleme.  Oregon Health and Sciences University. Larvaların zebrafish spinal motor nöron ve hedef iskelet kası aynı anda yama kelepçe kayıt izin vermek için ilk omurgalı model sistemi temsil eder. Bu video segmental CaP motor nöron ve hedef kas hücrelerinin yanı sıra, her bir hücre tipi kayıt için metodolojiler tanımlamak için kullanılır mikroskobik yöntemler göstermektedir.

Video: Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Patch Kelepçe bir Karanlık adapte Dilim Hazırlık Fare Retina Nöronlar Kayıtlar

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Nörobilim

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

İç Kulak Saç Hücreleri Patch Clamp Kayıtlar İzole Zebrafish gelen

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<sub&gt; 1</sub&gt; F<sub&gt; O</subSubmitochondrial veziküllü Membranların Patch Kelepçe Kayıtlar Sahne&gt; ATPaz veziküllü Hazırlık ve Tekniği

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Duyusal nöronlar izolasyonu<em&gt; Aplysia californica</em&gt; Glutamaterjik Akımlar Patch Kelepçe Kayıtlar için

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Embriyonik Zebra balığı Mauthner Hücreler Patch Kelepçe Kayıtlar

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically- ve nörokimyasal tanımlanan hipokampal internöronlardan gelen tüm hücre Patch-kelepçe Kayıtlar

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Organotipik Hipokampal Dilimleri eşleştirilmiş Tüm Hücre Kayıtlar

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

Nöromusküler Kavşak: Ölçme Synapse Boyut, Konfokal Floresan Mikroskobu kullanılarak sinaptik Protein Yoğunluk Parçalanma ve Değişiklikler

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

Akut Beyin Dilimleri nöronal Mikro şemalar Elektrofizyolojik ve Morfolojik Karakterizasyonu Eşleştirilmiş Patch-Kelepçe Kayıtlar Kullanma

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Beyin Dilimleri tüm hücre Patch-kelepçe Kayıtlar

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...