Biz laboratuarımızda protokolü kullanarak başladığı agar gömülü retina dilim hazırlık yaptığı tür bir açıklama için teşekkür ederim Dr. Fred Rieke. Ayrıca resimde şematik bakış ve Dr Lori Vaskalis teşekkür ederim. Metin ve video hakkında faydalı yorumlar için Veeramuthu Balakrishnan ve Soyoun Cho. Bu çalışma bir NEI-NIH RO1 hibe ile desteklenen ve aynı zamanda kısmen Kore Hükümeti tarafından finanse edilen bir Kore Araştırma Vakfı Hibe Programı tarafından desteklenen [KRF-2008-357-E00032].

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Biz hayır, ifşa etmek için yarışıyor.

Protokolünde kritik ve zor bir adım agar solüsyonu (protokol 3.4) retina parça transferi . Retina parça vitreus mizah ve artık dilim çözüm dikkatlice çıkarın ve bozulma veya bükmeden aktarmak için gereklidir. Bunu başarmak için, biz, bir ucu retina pozisyonu için açılı ucu forseps (11.251-35, Güzel Bilim Araçları) ile birlikte, 90 ° açı oluşturacak şekilde eğildi küçük bir spatula (21-401-25b, Fisherbrand) dilim çözüm transfer süreci boyunca parçası. Retina dilim ve...

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Düşük sıcaklık jelleşme agar </td><td> Sigma </td><td> A0701 </td><td> Agaroz tip VII-A </td></tr><tr><td> Patch pipet </td><td> Dünya Hassas Aletler </td><td> 1B150F-4 </td><td> Kalın duvarlı (1,5 mm dış çapı) borosilikat cam </td></tr><tr><td> Dikey çektirmesi </td><td> Narishige </td><td> PP830 </td><td></td></tr><tr><td> Diş balmumu </td><td> Cavex </td><td></td><td></td></tr><tr><td> Bahar makas </td><td> Güzel Bilim Araçları </td><td> 15003-08 </td><td></td></tr><tr><td> 45 ° açılı ince uçlu forseps </td><td> Güzel Bilim Araçları </td><td> 11251-35 </td><td></td></tr><tr><td> Jilet </td><td> Personna </td><td></td><td> Çift-kenarlı,% 70 etanol ve H 2 O ile temizlenmelidir </td></tr><tr><td> Silindirik tüp </td><td> Fisherbrand </td><td> 03-338-1B </td><td> Polietilen numune şişeleri 2.5 ml </td></tr><tr><td> Hyaluronidase </td><td> Sigma </td><td> H6254 </td><td></td></tr><tr><td> Vibratome dilimleme makinesi </td><td> Leica </td><td> VT1000S veya VT1200S </td><td></td></tr><tr><td> Dik mikroskop </td><td> Olympus </td><td> BX51WI </td><td></td></tr><tr><td> 60x su daldırma hedefi </td><td> Olympus </td><td> LUMPlanFl </td><td> NA 0.90 </td></tr><tr><td> CCD kamera </td><td> Sony </td><td> XC-75 </td><td></td></tr><tr><td> Kamera denetleyicisi </td><td> Hamamatsu </td><td> C2400 </td><td></td></tr><tr><td> Monitörü &lt;/ Td&gt; <td> Sony </td><td></td><td> 13 &quot;siyah ve beyaz monitör </td></tr><tr><td> Şırınga filtre </td><td> Nalgene </td><td></td><td> 0.2 mikron </td></tr><tr><td> Mikromanipülatör </td><td> Sutter Enstrüman </td><td> MPC-200 </td><td></td></tr><tr><td> Lock-amplifikatör </td><td> Heka </td><td></td><td> EPC-9/10 amplifikatörler yazılım emülasyonu </td></tr><tr><td> Dönen disk lazer konfokal mikroskop </td><td> Yokogawa </td><td> CSU-X1 </td><td> Yamadan sonra canlı hücre görüntüleme tam hücre kayıt kelepçe </td></tr><tr><td> Slidebook yazılımı </td><td> Akıllı Görüntüleme Cihazları (3i) </td><td></td><td> Görüntüleme veri toplama ve analiz </td></tr><tr><td> Paraformaldehit </td><td> Sigma </td><td> P6148 </td><td></td></tr><tr><td> Fosfat tampon çözeltisi </td><td> GIKAM </td><td> 70013 </td><td></td></tr><tr><td> SuperFrost slayt </td><td> Fisher Scientific </td><td></td><td> Slayt cam </td></tr><tr><td> Anti-solmaya ajanlar </td><td> Biomeda corp. </td><td></td><td></td></tr><tr><td> Konfokal lazer tarama mikroskobu </td><td> Carl Zeiss </td><td> EKK 710 </td><td> Sabit doku görüntülemesi </td></tr><tr><td> Spatula </td><td> Fisherbrand </td><td> 21-401-25b </td><td></td></tr><tr><td> Manuel dikey dilimleme makinesi </td><td> Narishige </td><td> ST-20 </td><td></td></tr><tr><td> Oregon Green 488 BAPTA-1 </td><td> Invitrogen </td><td> O-6806 </td><td> Ca 2 + hassas floresan boya </td></tr><tr><td> Alexa Fluor 555 Hydrazide </td><td> Invitrogen </td><td> A-20501MP </td><td> Floresan boya </td></tr></tbody></table>

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

Görsel uyaranlar, gözdeki uzman nöronların ışık yoğunluğu ve frekans değişimleri, geniş bir dinamik aralık üzerinden tespit edilir ve iletilir. Retina nöronlar, fotoreseptör ve bipolar hücreleri iki sınıf, uzun süreler boyunca sürekli ve yüksek verimli nörotransmitter sürümü sağlayan şerit tip aktif bölgeleri, kullanarak gerçekleştirmek. Goldfish retina ışık uyaranlara depolarize ve karma çubuk ve koni fotoreseptör giriş ON-tipi karma bipolar hücre (Mb) terminalleri, büyük boyutları nedeniyle her iki şerit tip sinaps (~ 10-12 çalışma için uygundur mikron çaplı) ve amacrine hücre dendritler ile çok sayıda yanal ve karşılıklı sinaptik bağlantıları. Goldfish yama klemp tekniği ile retina dilim Mb bipolar hücre terminalleri doğrudan erişim presinaptik Ca 2 ölçümünü sağlar + akımları, membran kapasitans değişiklikleri ve GABA aracılı karşılıklı sinaptik geribildirimle engelleme &lt;alt&gt; A ve GABA C reseptörleri terminallerinde dile getirdi. Ekzositoz presinaptik membran kapasitans ölçümleri bir eksitatör nörotransmitter sürümü 14,15 kısa vadeli plastisite çalışma izin verir. Buna ek olarak, kısa vadeli ve uzun vadeli plastisite amacrine hücrelerinden inhibitör nörotransmitter salınımı Mb terminali 21 gelen karşılıklı geribildirimle engelleme kayıtları incelenmiştir. Amacrine hücreleri GABAerjik karşılıklı geribildirimle engelleme süresi (örneğin ~ 10 sn) kısa süre içinde, GABA vezikül havuzu tükenmesi 11 ile eşleştirilmiş nabız depresyon uğrar. Retinanın iç pleksiform tabakası retina mikrodevreler sinaptik dinamikleri dolayısıyla doğrudan incelenebilir. Beyin-dilim tekniği, 40 yılı aşkın önce tanıtıldı ama hala çok nöronların elektriksel özellikleri, hem tek hücreli soma, tek dendrit ve akson soruşturma için yararlı ve microcircuit sinaptik düzeyde 19. Doğrudan dilimleme odasının üzerine yapıştırılmış olması çok küçük dokular genellikle ilk agar gömülü (ya da bir filtre kağıdı yerleştirilir) ve sonra 20, 23, 18, ​​9 dilimlenmiş. Bu video, goldfish retina kullanarak ön gömme agar tekniği kullanmaktayız. Bazı goldfish retinanın dilim dev bipolar hücre terminalleri dilimleme işlemi sırasında axotomized (akson-cut). Bu axotomized terminallerinden kayıt soma-dendritik bölmesi sinyalleri hariç tutması nedeniyle, tek presinaptik sinir terminal girişleri izole etmek için izin verir. Alternatif olarak, bir de dilimleme işlemi sırasında kesilmiş aksonların bağlı terminallerinden kaydederek, sağlam Mb bipolar hücrelerin kaydedebilirsiniz. Genel olarak, bu deneysel protokol kullanımı sinaptik fizyolojisi, retina mikrodevreler fonksiyonel analiz, ve şerit sinaps sinaptik iletim çalışmaları yardımcı olacaktır.

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

Burada elektrofizyoloji ve Ca2 + görüntüleme için uygun agar-gömülü retina dilim hazırlanması için bir protokol. Bu yöntem, tek presinaptik sinir uçları doğrudan patch-klemp kayıtları kullanarak retina mikrodevreler şerit-tip sinaps bir çalışma sağlar.

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 ...

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 Görüntüleme.  Oregon Health and Science University. Burada elektrofizyoloji ve Ca2 + görüntüleme için uygun agar-gömülü retina dilim hazırlanması için bir protokol. Bu yöntem, tek presinaptik sinir uçları doğrudan patch-klemp kayıtları kullanarak retina mikrodevreler şerit-tip sinaps bir çalışma sağlar.

Video: 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.

Patch Kelepçe bir Karanlık adapte Dilim Hazırlık Fare Retina Nöronlar Kayıtlar

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

Nörobilim

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.

Dört boyutlu Floresans Görüntüleme ile Sinir Terminalleri Yaşayan endozom Dinamiği Görselleştirme

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.

Fare Adrenal Kromafin Cell Hücre bağlı Kapasitans Ölçümler Yöntemleri

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.

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

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

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. ...

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.

Görüntüleme Ca<sup&gt; 2+</supFare Retina Koni fotoreseptör Akson Terminalleri içinde&gt; Dinamikleri

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.

Retina Şerit Sinapslar at Membran Reseptör Yüksek Çözünürlüklü Sayısal immunogold Analizi

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.

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 ...

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.

Dilim yama klemp tekniği öğrenme kaynaklı plastisite analiz etmek için

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.

Beyin dilimlerinde nöronların çalışması için otomatik görüntü yönlendirmeli Yama Kelepçesi Uygulaması

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 ...