Bu çalışma, Ulusal Sağlık Enstitüsü, Hibe EY17606 (APS) tarafından desteklenmiştir.

1. Hazırlanması Elektrotlar <ol><li> 1M NaCl hem daldırma ve aralarında sürüş 15V ~ 20 sn için 1 Hz sinüs dalga kullanarak kayıt ve referans elektrotlar hazırlayın </li><li> Mikropipet çektirmesi (Sutter Aletleri P-97) kullanarak kayıt elektrotları olun. Patch-pipetler, dış çapı 1.2 mm ve iç çapı 0.69 mm (Sutter Instruments, Kedi # B120-69-10) bir ile borosilikat cam kullanın. Elektrodun ucu ile seri direnci ölçün. M ~ 12 ~ 5 hücreli organları için M ~ (5) mm çaplı kullanımının cep organları için ~ 20 mm çaplı kullanımı yapısı bir pipet ucu çapı seçin hedef hücrelerin büyüklüğüne göre. </li><li> / Referans elektrot zemin hazırlayın. 1M NaCl; bir şırınga kullanarak (Cat # A7002 Sigma),% 3.5 Agar 1 mM NaCl ile küçük bir bölümü polietilen boru (Intramedic PE205) doldurmak. Ag / AgCl referans elektrot üzerinden bu agar köprüsü yerleştirin. </li></ol> 2. Hazırlanması Çözümler <ol><li> Harici banyo çözümü, Ames Bikarbonat (1 L) içeren medya: Ames 1 şişe &#39;Orta (Sigma; Kedi # A1420), 1,9 g NaHCO 3, Penisilin-Streptomisin 7 mL (Sigma; Kedi # P0781) bakteriyel önlemek için büyüme. ~ 280 mOsm ozmolarite ayarlayın. </li><li> Çözüm Dilimleme Ames pH tampon Hepes (1 L) içeren ortam: 1 şişe Ames &#39;Orta, 0.887 g NaCl, 2.38 g Hepes, 7 ml Penisilin-Streptomisin. 1M NaOH ve / veya 1M HCl ~ 7.4 pH ayarlayın. ~ 280 mOsm ozmolarite ayarlayın. </li><li> Potasyum Aspartat (K-Asp) pipetle iç çözeltisi (mM): 125 K-Aspartat, 10 KCl, 10 Hepes, 5 NMG HEDTA, 0.5 CaCl 1, 2 ATP-Mg, 0.2 GTP-Mg; pH ayarlanır. ~ 7.3 NMG-OH ve ozmolarite ~ 280 mOsm ayarlanır. </li><li> Retina içeren jelleşme sıcaklığı düşük agaroz (ağırlıkça% 5), 7.5 g Agar (Sigma; Cat # A7002) stabilize Agar döndürmez kilit dH 2 O, 150 mL erimiş </li></ol> 3. Deney günü <ol><li> Çözülme ~ 1.5 ml K-Asp İç Çözüm ve dondurucu seçtiğiniz fluorofor. Ampirik olarak tespit edilmelidir uygun konsantrasyon, fluorofor sulandırınız. </li><li> Ames (30-32 ° C) su banyosu içine ışık geçirmez saklama kabı ve yerine içine NaHCO 3 (~ 200 ml) içeren medya dökün ve% 5 CO 2 /% 95 O 2 gaz dengelenmesi </li><li> Ames &#39;medya Hepes yer buz içeren yaklaşık 110 mL şişe doldurun ve% 100 O 2 ile dengelenmesi. </li><li> Faraday kafesinin üstüne ısıtılmış bir rezervuar NaHCO 3 içeren diğer Ames &#39;medya yerleştirin ve% 5 CO 2 /% 95 O 2 gazı ile dengelenmesi. Parafilm çözüm yukarıdaki boşluk gaz yakalamak için kullanılabilir. </li><li> Jelleşme sıcaklığı düşük agaroz (% 3); (Cat # A0701 Sigma) 0.75 g, Hepes ve ısı ~ 115 ° C agaroz eritmek için çözüm ile Ames &#39;medya 25 ml ekleyerek hazırlayın. Kabarcıkları olmadan net, bir su banyosu ve mağaza çözümü kadar karıştırın (~ 42 ° C). </li><li> K-Asp iç solüsyonu ile doldurun ve bir kayıt pipet ucu direnci kontrol edin. </li></ol> 4. Deney başlayın <ol><li> Fare Euthanize ve hızlı bir şekilde gözler kavisli makas kullanarak kaldırın. Kızılötesi gözlük görsel pigment beyazlatma önlemek için tüm işlemleri için kullanılmalıdır. </li><li> Forseps ile göz tutarken gözleri filtre kağıdı, haddeleme, ve ince bir çift taraflı bıçak kullanarak onları önlemek için bir parça yerleştirin, kornea bir yarık. </li><li> Göz küresi delinmiş olduğu gibi, bunlar göz Ames &#39;medya ile dolu bir çanak içine aktarın. </li><li> Bir giriş noktası olarak kornea yarık, küçük bir makas, kornea, lens ve vitreus silinebilir sonra geri kalan kısımları kesip kullanılır. Retina Göz yuvası takılı kalır ve 32 karanlıkta Göz yuvası saklamak ° C,% 5 CO 2 /% 95 O 2 ile dengelenmiş Ames medya dikkatli olun. </li></ol> 5. Dilimleme <ol><li> GÖZ KAPAKLARI Hemisect retina ve retina pigment epiteli ayrıştırmaları. Doku düz yalan izin retinanın kenarlarını çıkarın. </li><li> Erimiş agar ile 35 mm Petri kabı doldurmak için küçük bir bardak Pasteur pipeti lateks bir ampul kullanın ve tutarlılığını test etmek için forseps agar yoluyla boyunca sürükleyin. </li><li> Agar katılaşmaya başlar. <ol class="lalpha"><li> Agar üst retina aktarın. </li><li> Retina çevresinde aşırı bir çözüm emmek için çarpık bir Kimwipe parçası kullanın. </li><li> Yeri 2-3 Agar doğrudan retinanın üzerine düşer ve hızlı bir şekilde etrafına bir duvar oluşturmak için retina üzerinde plastik bir silindir. Agar içinde retinanın merkezleme Sonra plastik silindir tarafı aşağı ek erimiş agar damla. </li><li> Petri kabı t transfero serin bir buzlu su banyosu. </li></ol></li><li> ~ 45 saniye buzlu su banyosu doku kaldırmak ve agar retinanın en uzak tarafında iterek, plastik silindir bloğu a&#39;ya bir dalgıç kullandıktan sonra. </li><li> Agar içeren retinanın küçük bir blok kesme ve numune disk agar Geri döndürmez kilit karşı blok superglue tek taraflı jileti kullanın. </li><li> Titreşimli mikrotom tepsi örnek diske aktarın ve buz Ames Hepes retina ile blok tam olarak temas etmesini sağlayan tepsiye dökün. </li><li> Retina dilim ~ 200 mikron kalınlığında kesilmiş ve maksimum bıçak titreşimli hızında ileri bir bıçak hızı retina her dilim kesmek için 4 ila 5 saniye sürer gibi ayarlanabilir. </li><li> No: 11 neşter bıçak, her bir seferde kullanılabilir dilim kaldırmak için kullanın. </li><li> İyi dilimleri içeren retina kayıt odaları yerleştirilir ve onları geçiş naylon konuları dilim çapa ile ağırlıklı. Naylon konuları kayıtlar için engelsiz retina bırakarak, retina çevreleyen agar basılı tutun. </li><li> Sahneye dik mikroskop Transfer kayıt odası, perde kapatmak ve hassas bir kızılötesi kamera dilim görüntülemek için kızılötesi ışık kaynağı açın. </li></ol> 6. Kayıt <ol><li> Bir iyi bir retina dilim (sağlam fotoreseptör ve uygun kesme açısı ile) hızında retina perfüze başlamak tanımlanan 35 37 ısıtılır ısıtılır Ames &#39;medya ile daha fazla 5 ml / dak ° C ve% 5 CO 2 ile dengelenmiş /% 95 O 2. </li><li> Bazı yüzeyine zarar dilimleme işlemi nedeniyle ortaya çıkabilir. Bu genellikle ince bir tabaka hücreleri kırık bir ucu ile bir pipet kullanarak kaldırılır ve nazikçe ölü hücre gövdeleri uzak emme olabilir. Bu genellikle canlı hücreler altında ortaya koyacaktır. Pozisyonu ilgi katmanlarda bulunan sağlıklı bir hücre gövdesi belirleyin. </li><li> Mikropipet KAsp iç solüsyonu ile doldurun ve pipet ucu ile pozitif (dışa doğru) basınç uygular ve bir micromanipulator kullanarak hücre düzeyine kayıt odasına pipet ucu düşük. </li><li> Hücre zarı gamzeler gibi pipet hareket ettirin ve yüksek bir direnç mühür yapmak için nazik negatif (içe) basınç uygulayın. Tüm hücre modunda hücre içine kırmak için elektrot negatif basınç uygulayarak elde edilebilir. </li><li> LED&#39;leri kullanarak retina dokusunun Uyarıcı ışık tepkiler uyandırabilir. </li><li> Pipetle iç çözüm fluorofor floresan çağrıştıran kayıt yapıldığı hücre Fotoğraf. </li></ol> 7. Temsilcisi Sonuçlar <img src="/files/ftp_upload/2107/2107fig1.jpg" alt="Şekil 1" /> Şekil 1 Burada, karanlık bir adapte artan gücü ışık çakmaları retina nöron gösterilmiştir potansiyelleri ışık uyarılmış. <img src="/files/ftp_upload/2107/2107fig2.jpg" alt="Şekil 2" /> Şekil 2 florofor Lucifer Sarı hücreleri ile dolu olduğu bir retina dilim Floresan resim. Uyarılmış floresan yama kayıtları yapıldığı hücrelerinin morfolojisi gösterir.

<ol>
	<li>Weblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+along+and+between+rods+in+the+tiger+salamander+retina.">Transmission along and between rods in the tiger salamander retina.</a> J Physiol. 280, 449-470 (1978).</li><li>Wu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+connections+among+retina+neurons+in+living+slices.">Synaptic connections among retina neurons in living slices.</a> J Neurosci Methods. 20, 139-149 (1987).</li><li>Rieke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+contrast+adaptation+in+salamander+bipolar+cell.">Temporal contrast adaptation in salamander bipolar cell.</a> J Neurosci. 21, 9445-9454 (2001).</li><li>Armstrong-Gold, C., &amp;amp, R. i. e. k. e., F, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bandpass+filtering+at+the+rod+to+second-order+cell+synapse+in+salamander+(Ambystoma+tigrinum)+retina.">Bandpass filtering at the rod to second-order cell synapse in salamander (Ambystoma tigrinum) retina.</a> J Neurosci. 23, 3796-3806 (2003).</li><li>Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+plan+of+the+retina.">The fundamental plan of the retina.</a> Nat Neurosci. 4, 877-886 (2001).</li><li>Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+in+the+mammalian+retina.">Parallel processing in the mammalian retina.</a> Nat Rev Neurosci. 5, 747-757 (2004).</li></ol>

Omurgalı retina Dilim kayıt onlarca yıl 1,2 için yapılmış ve retina devre olay ışık kodlar nasıl daha derin bir anlayış sağlama konusunda oldukça başarılı olmuştur. Titreşen bir mikrotom kesim yerine doğrudan daha bir jilet ile avantajları, retina dilimler yüzeyinde daha az hasar tabi ki. Emme pipet ile sadece hücre tipleri en çok 5,6 tespit edilmiştir retina devre, bağlantı korumak yüzeyinin altında bu hasar ve hedef sağlıklı hücreleri çıkarmak için kolay ve bu...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>B120-69-10</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylene tubing</td>
<td>&nbsp;</td>
<td>Intramedic</td>
<td>PE205</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7002</td>
<td>&nbsp;</td>
<tr>
<td>Low gelling temp agarose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A0701</td>
<td>&nbsp;</td>
<tr>
<td>Ames' Medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1420</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P0781</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

patch clamp recordings from mouse retinal neurons in a dark-adapted slice preparation

Bizim görsel deneyim retinal fotoreseptör görsel pigment fotonlar, ışık enerjisini emer ve hücre zarı siklik nükleotid kapılı kanallar kapatılmasına yol açan hücre içi olaylar zincirini başlatır zaman başlatılır. Membran potansiyeli ortaya çıkan değişiklik çubuk ve koni sinaptik terminalleri hem de nörotransmitter serbest miktarda azalma sırayla açar. Fotoreseptör membran potansiyeli ışık uyarılmış değişiklik retinada aşağı aktiviteye yol açar nasıl ölçmek için, bilim adamları onlarca yıl 1,2 retina dilim hazırlıkları elektrofizyolojik kayıtlar yaptık. Geçmişte bu dilimleri elle filtre kağıdı bağlı retina dokusunun bir jiletle kesilmiş; son yıllarda dilimleme başka bir yöntem, retina dokusunun düşük jelleşme sıcaklığı agar sayede gömülü geliştirilmiştir ve soğuk çözüm dilimlenmiş mikrotom titreşimli 3,4. Bu preparat ile retina dilimleri daha az yüzey hasarı ve çok sağlam bir ışık uyarılmış yanıtlar üretir. Burada bu yordamı görsel pigment beyazlatma önlemek için kızıl ötesi ışık altında nasıl yapılacağı belge.

Watch this Scientific Journal Video about Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation at JoVE.com

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

İşte biz elektrofizyolojik kayıtlar için fare retina karanlık adapte dilim üretmek için bir prosedür açıklanmaktadır.

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 ...

patch-clamp-recordings-from-mouse-retinal-neurons-dark-adapted-slice

Research

JoVE Journal

Neuroscience

14.3K Görüntüleme.  University of Southern California. İşte biz elektrofizyolojik kayıtlar için fare retina karanlık adapte dilim üretmek için bir prosedür açıklanmaktadır.

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

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

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

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.  ...

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 ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

Bir Amfibi Retina Dilim hazırlanması Fotoreseptörler ve İkinci dereceden nöronlar eş zamanlı tam hücreli Kayıtlar

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

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 ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Kayıt Patch Kelepçe Teknikleri Kullanarak Koyu adapte Fare Retina Dilim Hazırlıkları Nöronlar içinde postsinaptik Yanıtları Işık-uyarılmış

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

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 ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Nigral Dopamin Nöronlar somatodendritik Domain hücre içi Patch-kelepçe Kayıtlar

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...