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 görüntü sağlam yanıtları ışık. Bu nedenle koyu adapte retina dilim yama kelepçe kayıtları, sinir hesaplamaları tespit hücre sınıflar tarafından oynanan roller belirlemek için bir araştırmacı izin verebilirsiniz.

<table><tbody><tr><td>Micropipette Puller</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>Borosilicate glass</td><td>Sutter Instrument Co.</td><td>B120-69-10</td><td></td></tr><tr><td>Polyethylene tubing</td><td>Intramedic</td><td>PE205</td><td></td></tr><tr><td>Agar</td><td>Sigma-Aldrich</td><td>A7002</td><td></td></tr><tr><td>Low gelling temp agarose</td><td>Sigma-Aldrich</td><td>A0701</td><td></td></tr><tr><td>Ames&amp;rsquo; Medium</td><td>Sigma-Aldrich</td><td>A1420</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P0781</td><td></td></tr></tbody></table>

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

14.4K 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

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Genetik Etiketli Retina Ganglion Hücreleri Işık Tepkisi Record İzole Retina Hazırlık

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Nörobilim

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

Işık tam hücreli Kayıtlar Retina Dilim Uyarıcı Synaptic Akımlar Uyarılmış

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Biyoloji

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Patch-kelepçe Kapasitans Ölçümler ve Ca 2 + Retina Dilimleri Tek Sinir Terminalleri Görüntüleme

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

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 ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Fare Retina Ganglion Hücreleri Synaptic ve Yapay iletkenlikleri Dinamik Kelepçe Uygulama

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

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. ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

Deafferentated Fare Retina bir düz montaj Hazırlık patlaması amacrine hücreleri Yama Kelepçe Kaydı

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Elektrofizyolojik Çalışmalar Yetişkin Fare Retina Yatay Dilimleri hazırlanması

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Murin bölünmüş retinada bipolar ve yatay nöronal hücrelerin incelenmesi

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.

Yazar Spotlight: Gelişmiş Erişim ve Hızlandırılmış Deneyler için Bölünmüş Retina Tekniğini Kullanma 

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.

Patch Kelepçe bir Karanlık adapte Dilim Hazırlık Fare Retina Nöronlar Kayıtlar

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