ソリューション<ul><li>神経節細胞の細胞内溶液（mMで）：100 K -グルコン酸、8のKCl、1のMgCl 2、1 EGTA、10 HEPES、4 ATP、0.5 GTP、90μMスルホローダミンBの（染色用）、でpH = 7.4に調整KOH </li><li> 90 K -グルコン酸、8のKCl、1のMgCl 2、10 BAPTA、10 HEPES、4 ATP、0.5でpH = 7.4に調整GTP、90μMスルホローダミンB（染色用）、：双極細胞（mMで）のための細胞内液KOH </li><li>細胞外リンゲル溶液（mMで）：112塩化ナトリウム、5グルコース、5 HEPES、NaOHでpH = 7.75に調整2のKCl、2のCaCl 2、1のMgCl 2、。ソリューションの酸素化は必要ありません。 </li></ul>録音室を準備する私たちの記録室は、よく別の接着剤から切り出された小さなシリコンブロックは組織切片のために安定性を提供するために配置されるだけでなく接着剤付き顕微鏡のスライドで構成されています。 網膜スライスの準備光受容体の漂白を避けるために、郭清は、IRまたは暗赤色灯の光で行われる。一方が棒の経路からの応答を記録に興味を持っている場合は、唯一の赤外光は、（IRは、顕微鏡となく、部屋の明かりを解剖。追加の赤外線ゴーグルが顕微鏡下で行われない手続きのために使用される）を使用する必要があります。一、主に記録のコーンの光の応答に興味を持っている場合は、薄暗い赤い部屋の光または赤色閃光灯を使用することができます。顕微鏡は、しかし、それでもIR接眼レンズが必要です。 光受容体の入力が実験には無関係である場合、プロシージャは通常の顕微鏡を使用して、通常の室内光で行うことができる。 <ol><li>よく接着剤、シリコーンを持つ2つの顕微鏡スライドを準備してください（フィルタをプッシュ各スライド上にシリコーングリースの二つの平行なバンド（約0.5センチメートル離れて）を作成し、スライドの片側にフィルター長方形の紙を置いて、両方のグリースバンド交差それがうまくスライドにアタッチされるようにカミソリの刃の部分と紙） </li><li>動物はその後、断頭し、ダブルpithed、30分間氷浴中に置かれている：確立されたプロトコルに従ってサンショウウオを生け贄に捧げる。 </li><li>サンショウウオの頭から目を削除し、氷冷リンゲル液で湿らせたティッシュの紙の上に解剖顕微鏡下に置く</li><li>目から結合組織を取り除く</li><li>角膜に小さなカットをするためにカミソリの刃を使用して、 </li><li>目にその添付ファイルに沿ってハサミでそれを除去しながら鉗子で角膜を切るホールド</li><li>鉗子でレンズを取り外す</li><li>鉗子で虹彩を持ち、虹彩を削除するには、鋸状縁に沿って切断</li><li>二つの長方形の部分にアイカップを切る</li><li> （非常に慎重に押し下げながら、ピースを平らに確認してください）​​作品の一つをピックアップし、ろ紙上強膜側を上に置きます</li><li>静かに今ファイラ紙に添付すべき、網膜から強膜を持ち上げ</li><li>すぐに良くするために、氷冷リンゲル液を追加する</li><li>番目の部分に同じことを繰り返します。 </li><li> 200 -300μmの広いスライスをカットする特注のスライサーに配置されたカミソリの刃を使用してください。 </li><li>フィルター紙の側でスライスをピックアップしてピンセットを使用して、網膜の層が見えるようになるので、それを裏返し、真空グリースバンドの上に置きます</li><li>無傷に見える一は、いくつかのレイヤーを認識できる場合（倍率が充分ではないとして層は解剖顕微鏡下で確認することが困難である、しかし、スライスは通常、十分である網膜の層を示す水平バンドを表示するスライスを選択してください。それは）正立顕微鏡下で観察されると、スライスを使用するかどうかの最終決定は、しかし、行われる。つのスライドの間に呼び出し音のブリッジを構築することにより、記録のチャンバースライドに移動</li><li>スライスが傾いていないようにろ紙が録音室でゴムブロックに押し付けていることを確認してください（神経節細胞と光受容器が同時に焦点が合うはず） </li></ol>レコーディング機器で組織を配置する<ol><li>記録のスライドは、顕微鏡下に置き、重力の灌流systeを通じてリンゲルソリューションで灌流され</li><li>準備は10倍と40倍の水浸対物レンズと正立顕微鏡で可視化です。 </li><li>再び、我々は唯一の光受容体の漂白を防ぐために赤外光を使用してください。赤外線カメラは、テレビ画面に接続されている顕微鏡のサイドポートに接続されている。 </li></ol>視覚刺激視覚刺激はPsychtoolboxを使用して、MATLABでプログラミングされており、顕微鏡画像インジェクタ（MBFバイオサイエンス）を用いて顕微鏡の最上位ポートを介して網膜上に投影されています。ビット+ +デジタルビデオプロセッサ（ケンブリッジリサーチシステム）は、14ビット輝度のスケールを取得してsynchrにするために使用されますデータ収集と刺激プレゼンテーションをonize。このビデオで使用される視覚刺激は、安定した一様な背景（上2秒のために提示された100％のコントラストの明るいまたは暗いバー（幅=460μm）であり、輝度= 8 * 10 4光子/μm/ sの、サイズ：1.84 X 1.38ミリメートル）。 電気生理学<ul><li>マイクロピペットプラーで：パッチ電極は、ホウケイ酸ガラス（1.5mmのID 84ミリメ​​ートル。。OD）から抜き出されます。神経節細胞の場合、抵抗値は2〜4 Mohmをです。双極細胞の場合、4と8 Mohmを間に高い抵抗値が使用されます。 </li><li>電圧クランプ記録はMulticlamp 700A（Molecular Devices社）、アンプとLabVIEWでプログラミングされた研究室内部のソフトウェアを使用して実行されます。 </li><li>薬は、8チャンネルの灌流システムを介して灌流されています。 microperfusion電極は、スライス（あなたが血流をオンとオフ、1つのニーズが、プレッシャーはスライスがろ紙から切り離さになることができるように強すぎではないことに注意するときに組織が目に見えて移動する必要があります）の近くに配置されています。実験中にmicroperfusionは絶えず記録は血流のオンとオフを切り替えることによって邪魔されないように（何の薬が必要ない場合は、コントロールのソリューションを灌流する）オンにする必要があります。 </li></ul>

<ol>
	<li>Cook, P. B., Lukasiewicz, P. D., McReynnolds, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA(C)+receptors+control+adaptive+changes+in+a+glycinergic+inhibitory+pathway+in+salamander+retina.">GABA(C) receptors control adaptive changes in a glycinergic inhibitory pathway in salamander retina.</a> Journal of Neuroscience. 20, 806-812 (2000).</li><li>Heflin, S. J., Cook, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narrow+and+wide+field+amacrine+cells+fire+action+potentials+in+response+to+depolarization+and+light+stimulation.">Narrow and wide field amacrine cells fire action potentials in response to depolarization and light stimulation.</a> Visual Neuroscience. 24, 197-206 (2007).</li><li>Ichinose, T., Shields, C. R., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+Channels+in+the+Transient+Retinal+Bipolar+Cells+Enhance+Visual+Responses+in+Ganglion+Cells.">Sodium Channels in the Transient Retinal Bipolar Cells Enhance Visual Responses in Ganglion Cells.</a> J Neurosci.. 25, 1856-1865 (2005).</li></ol>

利点： <ol><li>すべての細胞型は、アクセス可能です </li><li>細胞型の簡単な識別は、特に、蛍光色素は、電極の溶液に添加されている場合 </li><li>薬理学的作用物質は、容易に標的細胞に到達することができます </li><li>パッチクランプ法は、網膜の計算では異なるイオンチャネルの役割を調査することができます。同じ情報が鋭い電極と細胞外スパイクの録音または録画を介して取得することが...

<table><tbody><tr><td>Multiclamp 700A</td><td>Patch Clamp Amplifier</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>SZX9</td><td>Dissection Micropscope</td><td>Olympus Corporation</td><td></td><td></td></tr><tr><td>BX50WI</td><td>Upright Microscope</td><td>Olympus Corporation</td><td></td><td>Equipped with 40x, 10x water immersion objective, fluorescent filters and mercury lamp</td></tr><tr><td>P-97</td><td>Micropipette Puller</td><td>Sutter Instrument Co.</td><td></td><td></td></tr><tr><td>Lucivid</td><td>Microscope image injector</td><td>MBF Bioscience</td><td></td><td></td></tr><tr><td>8-channel perfusion system</td><td>Microperfusion</td><td>Parker Hannifin Corporation</td><td></td><td></td></tr><tr><td>Bits++</td><td>Digital Video Processor</td><td>Cambridge Research Systems</td><td></td><td></td></tr><tr><td>Infrared Oculars</td><td>Other</td><td>ITT Visual Information Solutions</td><td></td><td></td></tr><tr><td>Adhesive silicone wells</td><td>Other</td><td>Molecular Probes, Life Technologies</td><td></td><td>20mm diameter, 1.0mm deep</td></tr><tr><td>Membrane Filters</td><td>Filter</td><td>EMD Millipore</td><td>HAWPO1300</td><td>.45&amp;#x03BC;m HA</td></tr><tr><td>Borosilicate Glass Capillaries</td><td>Electrode glass</td><td>World Precision Instruments, Inc.</td><td>1B150-4</td><td></td></tr><tr><td>Syringe Filters</td><td>Filter</td><td>Whatman, GE Healthcare</td><td>6789-0402</td><td>4mm filters, .2um Nylon Membrane, Polypropylene housing</td></tr><tr><td>IR camera</td><td>Micropscope mounted camera</td><td>Sony Corporation</td><td>SPT-M324</td><td>Extrawave HAD, B7W video camera</td></tr><tr><td>Picrotoxin</td><td>Reagent</td><td>Sigma-Aldrich</td><td>P1675</td><td></td></tr><tr><td>Strychnine</td><td>Reagent</td><td>Sigma-Aldrich</td><td>S0532</td><td></td></tr><tr><td>Imidazole-4-acetic acid sodium salt</td><td>Reagent</td><td>Sigma-Aldrich</td><td>I7013</td><td></td></tr><tr><td>Larval tiger salamanders</td><td>Animal</td><td>Charles E. Sullivan</td><td></td><td></td></tr></tbody></table>

whole-cell recordings of light evoked excitatory synaptic currents in the retinal slice

我々は、シナプス回路が網膜の基礎となる視覚情報の処理を研究するためにホールセルパッチクランプ法を使用してください。このビデオでは、我々は、網膜のスライス標本における個々のセルの光誘発電流の全細胞記録を実行するプロセスをガイドします。我々は、動物モデルとして水生トラフサンショウウオを使用してください。我々は目の解剖を記述することで開始し、スライスを電気生理学的記録のためにマウントする方法を示します。スライスが録音室に配置されると、我々は、ホールセル電圧クランプ記録を実行する方法を示します。我々は、その後、光誘発電流応答を引き出すためにスライスに光受容体の上に視覚刺激を投影。録音中に我々は、8チャンネルの灌流システムは、私たちはすぐに別のエージェントを切り替えることができることにより、薬理学的薬剤とスライスを灌流。網膜スライス標本は広く、特にホールマウントの準備にはアクセスできないアマクリンまたは双​​極細胞を、研究するために、網膜でのパッチクランプ記録のために使用されます。

Watch this Scientific Journal Video about Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice at JoVE.com

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

このビデオでは、水生トラフサンショウウオの網膜スライスにおけるホールセル電圧クランプ記録のプロセスを示しています。網膜の視覚刺激時のパッチクランプ記録を実行する方法を我々は、だけでなく、スライスの準備を示しています。

光の全細胞録音は網膜スライスにおける興奮性シナプス電流を誘発

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 ...

whole-cell-recordings-light-evoked-excitatory-synaptic-currents

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.7K ビュー.  Boston University. このビデオでは、水生トラフサンショウウオの網膜スライスにおけるホールセル電圧クランプ記録のプロセスを示しています。網膜の視覚刺激時のパッチクランプ記録を実行する方法を我々は、だけでなく、スライスの準備を示しています。

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

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.

暗順応スライス標本におけるマウス網膜神経細胞からパッチクランプ記録

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

神経科学

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.

パッチクランプキャパシタンス測定とCa 2 +</sup網膜スライスにおける単一神経終末で>イメージング

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.

両生類網膜スライス標本における感光体と2次ニューロンからの同時ホールセル録音

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

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.

パッチクランプ技術を用いたダーク適応、マウス網膜スライス標本におけるニューロンにおける記録光誘発シナプス後応答

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

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

イメージングカルシウム<sup&gt; 2+</supマウス網膜の光受容コーン軸索末端で&gt;ダイナミクス

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

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.

電気生理学的研究のための成体マウスの網膜の水平スライスの調製

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

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

網膜原生殖器およびコルチコゲン化シナプス機能の電気生理学的調査

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

マウス分裂網膜における双極性および水平神経細胞の研究

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.

著者スポットライト:Split Retina技術を使用してアクセスを強化し、実験を加速する 

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.

光の全細胞録音は網膜スライスにおける興奮性シナプス電流を誘発

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この動画の章

暗順応スライス標本におけるマウス網膜神経細胞からパッチクランプ記録

パッチクランプキャパシタンス測定とCa^{2 +イメージング}

両生類網膜スライス標本における感光体と2次ニューロンからの同時ホールセル録音

パッチクランプ技術を用いたダーク適応、マウス網膜スライス標本におけるニューロンにおける記録光誘発シナプス後応答

イメージングカルシウム

電気生理学的研究のための成体マウスの網膜の水平スライスの調製

網膜原生殖器およびコルチコゲン化シナプス機能の電気生理学的調査

脊椎動物の網膜の内核層ニューロンを研究するための改良されたフラットマウント調製としてのスプリット網膜

脊椎動物の網膜の内核層ニューロンを研究するための改良されたフラットマウント調製としてのスプリット網膜

脊椎動物の網膜の内核層ニューロンを研究するための改良されたフラットマウント調製としてのスプリット網膜

光の全細胞録音は網膜スライスにおける興奮性シナプス電流を誘発

要約

さらに動画を探す

この動画の章

暗順応スライス標本におけるマウス網膜神経細胞からパッチクランプ記録

パッチクランプキャパシタンス測定とCa 2 +イメージング

両生類網膜スライス標本における感光体と2次ニューロンからの同時ホールセル録音

パッチクランプ技術を用いたダーク適応、マウス網膜スライス標本におけるニューロンにおける記録光誘発シナプス後応答

イメージングカルシウム

電気生理学的研究のための成体マウスの網膜の水平スライスの調製

網膜原生殖器およびコルチコゲン化シナプス機能の電気生理学的調査

脊椎動物の網膜の内核層ニューロンを研究するための改良されたフラットマウント調製としてのスプリット網膜

脊椎動物の網膜の内核層ニューロンを研究するための改良されたフラットマウント調製としてのスプリット網膜

脊椎動物の網膜の内核層ニューロンを研究するための改良されたフラットマウント調製としてのスプリット網膜

パッチクランプキャパシタンス測定とCa^{2 +イメージング}