私たちがラボでプロトコルを使用して起動するときに我々は、寒天包埋網膜スライス標本の彼の種類の説明については、博士フレッドリーケに感謝。我々はまた、回路図の概要と博士の図については、ローリVaskalisに感謝。テキストとビデオでの有益なコ​​メントVeeramuthuバラクリシュナンとSoyounチョ。この作品は、NEI - NIH RO1の助成金によって支えられて、そしてまた、部分的に韓国政府の資金による韓国学術振興財団の助成金によって支えられている[KRF - 2008 - 357 - E00032]。

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U.S.A. 98, 5116-5121 (2001).</li><li>Bergstrom, L., Christensson, I., Folkesson, R., Stenstrom, B., Terenius, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ion+exchange+chromatography+and+radioimmunoassay+procedure+for+measuring+opioid+peptides+and+substance+P.">An ion exchange chromatography and radioimmunoassay procedure for measuring opioid peptides and substance P.</a> Life. Sci. 33, 1613-1619 (1983).</li><li>Falth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptidomics+strategies+for+specific+and+sensitive+identification+of+endogenous+peptides.">Neuropeptidomics strategies for specific and sensitive identification of endogenous peptides.</a> Mol. Cell. 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我々は、開示することは競合する利害関係はありません。

我々のプロトコルで重要かつ困難なステップは、寒天溶液（プロトコル3.4）への網膜の一部の移転です。それは慎重に網膜の部分から硝子体液と残留スライスの溶液を除去し、歪みや曲がりなく、それを転送する必要があります。これを達成するために、我々は先端が網膜の位置を斜めに先端の鉗子（11251〜35、ファイン科学のツール）、と一緒に、90 °の角度を形成するために?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>低ゲル化温度寒天</td><td>シグマ</td><td> A0701 </td><td>アガロース型VII - </td></tr><tr><td>パッチピペット</td><td>世界の精密機器</td><td> 1B150F - 4 </td><td>肉厚（1.5ミリメートル外径）ホウケイ酸ガラス</td></tr><tr><td>垂直プラー</td><td>ナリシゲ</td><td> PP830 </td><td></td></tr><tr><td>歯科用ワックス</td><td> Cavex </td><td></td><td></td></tr><tr><td>春のはさみ</td><td>ファイン科学ツール</td><td> 15003〜08 </td><td></td></tr><tr><td> 45 °傾斜微細な先端の鉗子</td><td>ファイン科学ツール</td><td> 11251〜35 </td><td></td></tr><tr><td>かみそりの刃</td><td> Personna </td><td></td><td>両刃、70％エタノールとH 2 Oで洗浄</td></tr><tr><td>円筒管</td><td> Fisherbrand </td><td> 03から338 - 1B </td><td>ポリエチレンのサンプルバイアル2.5ミリリットル</td></tr><tr><td>ヒアルロニダーゼ</td><td>シグマ</td><td> H6254 </td><td></td></tr><tr><td>ビブラトームスライサー</td><td>ライカ</td><td> VT1000SまたはVT1200S </td><td></td></tr><tr><td>正立顕微鏡</td><td>オリンポス</td><td> BX51WI </td><td></td></tr><tr><td> 60倍の水浸対物レンズ</td><td>オリンポス</td><td> LUMPlanFl </td><td> NA 0.90 </td></tr><tr><td> CCDカメラ</td><td>ソニー</td><td> XC - 75 </td><td></td></tr><tr><td>カメラコントローラ</td><td>浜松市</td><td> C2400 </td><td></td></tr><tr><td>モニター&lt;/ TD&gt; <td>ソニー</td><td></td><td> 13&quot;白と黒のモニタ</td></tr><tr><td>シリンジフィルタ</td><td> Nalgene </td><td></td><td> 0.2μmの</td></tr><tr><td>マニピュレーター</td><td>サッターインストゥルメント</td><td> MPC - 200 </td><td></td></tr><tr><td>ロックインアンプ</td><td> HEKA </td><td></td><td> EPC-9/10アンプは、ソフトウェアエミュレーションを持っている</td></tr><tr><td>回転するディスクのレーザー共焦点顕微鏡</td><td>横河電機</td><td> CSU - X1 </td><td>パッチクランプホールセル記録後に細胞イメージングを生きる</td></tr><tr><td> Slidebookソフトウェア</td><td>インテリジェントイメージングインスツルメンツ（3iの） </td><td></td><td>イメージングデータ収集と分析</td></tr><tr><td>パラホルムアルデヒド</td><td>シグマ</td><td> P6148 </td><td></td></tr><tr><td>リン酸緩衝液</td><td> GIBCO </td><td> 70013 </td><td></td></tr><tr><td> Superfrostスライド</td><td>フィッシャーサイエンティフィック</td><td></td><td>スライドガラス</td></tr><tr><td>退色防止剤</td><td> Biomedaコープ。 </td><td></td><td></td></tr><tr><td>共焦点レーザー走査顕微鏡</td><td>カールツァイス</td><td> LSM 710 </td><td>固定組織のイメージング</td></tr><tr><td>へら</td><td> Fisherbrand </td><td> 21から401 - 25B </td><td></td></tr><tr><td>マヌエル垂直スライサー</td><td>ナリシゲ</td><td> ST - 20 </td><td></td></tr><tr><td>オレゴングリーン488 BAPTA - 1 </td><td>インビトロジェン</td><td> O - 6806 </td><td>のCa 2 +感受性蛍光色素</td></tr><tr><td>のAlexa Fluor ® 555ヒドラジド</td><td>インビトロジェン</td><td> - 20501MP </td><td>蛍光染料</td></tr></tbody></table>

patch-clamp capacitance measurements and ca2+ imaging at single nerve terminals in retinal slices

視覚刺激は、脊椎動物の網膜の専門ニューロンによる光の強度と周波数の変化の広いダイナミックレンジにわたって検出され、搬送される。網膜神経細胞、光受容体と双極細胞の2つのクラスは、長期間にわたって持続的かつ高スループットの神経伝達物質の放出を可能にするリボン型アクティブゾーンを、使用することによってこれを実現する。光刺激に対する脱分極との混合ロッドと錐体視の入力を受け取る金魚の網膜のON型の混合双極細胞（Mb）の端末は、サイズが大きいため、両方のリボン型シナプス（〜10-12の研究に適していますμmの直径）とアマクリン細胞の樹状突起との数多くの側面と相互シナプスの接続に。パッチクランプ法と金魚網膜スライスにおけるMbの双極細胞の端末へのダイレクトアクセスは、シナプス前のCa 2 +電流、膜容量の変化、およびGABAによって仲介相互シナプスフィードバック阻害の測定を可能にする&lt;サブ&gt; AおよびGABA C受容体は、端末上で表明した。エキソサイトーシスのシナプス前膜の容量測定は1つが興奮性神経伝達物質の放出14,15の短期可塑性を調べることができます。さらに、アマクリン細胞から抑制性神経伝達物質放出の短期的および長期的な可塑性はまた、Mbの端子21に到達する相互フィードバック阻害の記録によって調査することができます。時間の短い期間（例えば〜10秒）以上、アマクリン細胞からのGABA作動性相互フィードバック阻害は、GABA小胞プールの枯渇11を介してペアパルスうつ病を受ける。網膜の内網状層における網膜マイクロ回路のシナプスダイナミクスは、このように直接研究することができる。 脳スライスの技術は40年以上前に導入したが、神経細胞の電気的特性の調査のためにまだ非常に便利です、両方とも単一の細胞体、単一の樹状突起や軸索、およびmにしたicrocircuitシナプスのレベル19。スライスチャンバーに直接接着するには小さすぎる組織は、多くの場合、最初寒天に埋め込 ​​む（またはろ紙上に配置）し、20、23、18、9スライスされています。このビデオでは、我々は金魚の網膜を使用してプリ埋め込み寒天法を採用しています。金魚の網膜の私達のスライスに巨大な双極細胞の一部の端子は、スライス手順の間に（軸索カット）axotomizedされています。 axotomized端子から録音が相馬樹のコンパートメントからの信号を除外するため、これは、私たちは、単一のシナプス前神経終末の入力を分離することができます。別の方法として、また、スライス作業中に切断されていない軸索に接続された端末から記録することで、無傷のMbの双極細胞から記録することができます。全体的に、この実験的なプロトコルの使用は、網膜シナプス生理学、マイクロ回路機能解析、およびリボンシナプスにおけるシナプス伝達の研究に役立ちます。

Watch this Scientific Journal Video about Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices at JoVE.com

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

ここでは、電気生理学とCa2 +イメージングに適した寒天包埋網膜スライスを調製するためのプロトコルについて説明します。このメソッドは1つが、単一のシナプス前神経末端の直接パッチクランプ記録を用いて網膜マイクロ回路のリボン型シナプスを研究することができます。

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 ...

patch-clamp-capacitance-measurements-ca2-imaging-at-single-nerve

Research

JoVE Journal

Neuroscience

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

18.1K ビュー.  Oregon Health and Science University. ここでは、電気生理学とCa2 +イメージングに適した寒天包埋網膜スライスを調製するためのプロトコルについて説明します。このメソッドは1つが、単一のシナプス前神経末端の直接パッチクランプ記録を用いて網膜マイクロ回路のリボン型シナプスを研究することができます。

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

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

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

4次元蛍光イメージングと住んでいる神経末端におけるエンドソームダイナミクスの可視化

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

マウス副腎クロマフィン細胞における細胞 - 添付容量測定のための方法

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

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

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

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

急性脳スライスにおけるニューロンマイクロ回路の電気生理学的および形態学的特徴は、ペアパッチクランプ記録を使用して、

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

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 ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

網膜リボンシナプスにおける膜受容体の高分解能定量的イムノゴールド分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

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.

脳スライスにおける全細胞パッチクランプ記録

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

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

スライス パッチ ・ クランプの技術学習によって誘発される可塑性を分析するため

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

脳スライスにおけるニューロン研究のための自動画像誘導パッチクランプの応用

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

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

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}