Ringraziamo il Dr. Fred Rieke per la sua spiegazione del tipo di agar-embedded preparazione fetta della retina quando abbiamo iniziato con il protocollo nel nostro laboratorio. Ringraziamo anche Lori Vaskalis per l&#39;illustrazione del schematica panoramica e Drs. Veeramuthu Balakrishnan e Soyoun Cho per gli utili commenti sul testo e video. Questo lavoro è stato supportato da una NEI-NIH RO1 concessione, ed è stato anche parzialmente sostenuto da una ricerca Korea Foundation Grant finanziato dal governo coreano [KRF-2008-357-E00032].

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L., Chaurand, P., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI+imaging+mass+spectrometry:+molecular+snapshots+of+biochemical+systems.">MALDI imaging mass spectrometry: molecular snapshots of biochemical systems.</a> Nat. Methods. 4, 828-833 (2007).</li><li>Ljungdahl, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Mass+Spectrometry+Reveals+Elevated+Nigral+Levels+of+Dynorphin+Neuropeptides+in+L-DOPA-Induced+Dyskinesia+in+Rat+Model+of+Parkinson's+Disease.">Imaging Mass Spectrometry Reveals Elevated Nigral Levels of Dynorphin Neuropeptides in L-DOPA-Induced Dyskinesia in Rat Model of Parkinson's Disease.</a> PLoS ONE. 6, e25653-e25653 (2011).</li><li>Groseclose, M. R., Andersson, M., Hardesty, W. M., Caprioli, R. 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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. Proteomics. 6, 1188-1197 (2007).</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=a+database+designed+for+endogenous+peptides+and+mass+spectrometry.">a database designed for endogenous peptides and mass spectrometry.</a> Mol. Cell. Proteomics. 5, 998-1005 (2006).</li></ol>

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Un passo importante e difficile nel nostro protocollo è il trasferimento del pezzo di retina nella soluzione di agar (protocollo 3.4). E &#39;necessario rimuovere con attenzione il vitreo e la soluzione fetta residua dal pezzo della retina e trasferirlo senza distorsioni o piegature. Per fare questo, usiamo una piccola spatola (21-401-25B, Fisherbrand) con una punta piegata a formare un angolo di 90 °, insieme con una pinza punta angolata (11251-35, Strumenti Scienza Fine), di posizionare la retina pe...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Bassa temperatura gelificante agar </td><td> Sigma </td><td> A0701 </td><td> Agarosio tipo VII-A </td></tr><tr><td> Patch pipetta </td><td> Mondo Strumenti di precisione </td><td> 1B150F-4 </td><td> Pareti spesse (1,5 mm di diametro esterno) in vetro borosilicato </td></tr><tr><td> Estrattore verticale </td><td> Narishige </td><td> PP830 </td><td></td></tr><tr><td> Impronte dentarie </td><td> Cavex </td><td></td><td></td></tr><tr><td> Primavera forbici </td><td> Strumenti Scienza multa </td><td> 15003-08 </td><td></td></tr><tr><td> 45 ° angolato pinze punta fine </td><td> Strumenti Scienza multa </td><td> 11251-35 </td><td></td></tr><tr><td> Lametta </td><td> Personna </td><td></td><td> Doppio taglio, puliti con etanolo al 70% e H 2 O </td></tr><tr><td> Tubo cilindrico </td><td> Fisherbrand </td><td> 03-338-1B </td><td> Polietilene fiale campione 2,5 ml </td></tr><tr><td> Ialuronidasi </td><td> Sigma </td><td> H6254 </td><td></td></tr><tr><td> Vibratome affettatrice </td><td> Leica </td><td> VT1000S o VT1200S </td><td></td></tr><tr><td> Microscopio verticale </td><td> Olimpo </td><td> BX51WI </td><td></td></tr><tr><td> 60x acqua-immersion obiettivo </td><td> Olimpo </td><td> LUMPlanFl </td><td> NA 0,90 </td></tr><tr><td> Telecamera CCD </td><td> Sony </td><td> XC-75 </td><td></td></tr><tr><td> Telecamera di controllo </td><td> Hamamatsu </td><td> C2400 </td><td></td></tr><tr><td> Monitor &lt;/ Td&gt; <td> Sony </td><td></td><td> 13 &quot;Monitor in bianco e nero </td></tr><tr><td> Siringa filtro </td><td> Nalgene </td><td></td><td> 0,2 micron </td></tr><tr><td> Micromanipolatore </td><td> Sutter Instrument </td><td> MPC-200 </td><td></td></tr><tr><td> Amplificatore lock-in </td><td> HEKA </td><td></td><td> EPC-9/10 amplificatori hanno software di emulazione </td></tr><tr><td> Spinning microscopio laser confocale disco </td><td> Yokogawa </td><td> CSU-X1 </td><td> Imaging cellulare vivere dopo di patch clamp registrazione a cellula intera </td></tr><tr><td> Slidebook software </td><td> Intelligent Imaging Instruments (3i) </td><td></td><td> Imaging di acquisizione dati e analisi </td></tr><tr><td> Paraformaldeide </td><td> Sigma </td><td> P6148 </td><td></td></tr><tr><td> Soluzione tampone fosfato </td><td> GIBCO </td><td> 70013 </td><td></td></tr><tr><td> Superfrost scivolo </td><td> Fisher Scientific </td><td></td><td> Vetrino </td></tr><tr><td> Agenti anti-sbiadimento </td><td> Biomeda corp. </td><td></td><td></td></tr><tr><td> Confocale a scansione laser microscopio </td><td> Carl Zeiss </td><td> LSM 710 </td><td> Imaging del tessuto fisso </td></tr><tr><td> Spatola </td><td> Fisherbrand </td><td> 21-401-25B </td><td></td></tr><tr><td> Manuel verticale Affettatrice </td><td> Narishige </td><td> ST-20 </td><td></td></tr><tr><td> Oregon verde 488 Bapta-1 </td><td> Invitrogen </td><td> O-6806 </td><td> Ca 2 + colorante fluorescente sensibile </td></tr><tr><td> Alexa Fluor 555 IDRAZIDE </td><td> Invitrogen </td><td> A-20501MP </td><td> Colorante fluorescente </td></tr></tbody></table>

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

Stimoli visivi vengono rilevati e inoltrato su una ampia gamma dinamica di intensità di luce e variazioni di frequenza dai neuroni specializzati nella retina dei vertebrati. Due classi di neuroni della retina, fotorecettori e cellule bipolari, ottenere questo risultato utilizzando zone attive nastro tipo, che consentono il rilascio dei neurotrasmettitori sostenuta e high-throughput per periodi di tempo lunghi. ON-cellula bipolare di tipo misto (Mb) terminali nella retina pesci rossi, che depolarizzare agli stimoli luminosi e ricevere asta mista e di ingresso dei fotorecettori cono, sono adatti per lo studio delle sinapsi, sia a causa delle loro grandi dimensioni nastro di tipo (~ 10-12 micron di diametro) e per le loro numerose connessioni laterali e reciproca sinaptica con dendriti delle cellule amacrine. Accesso diretto ai terminali Mb cellule bipolari a fette pesci rossi della retina con la patch-clamp consente la misurazione di Ca 2 + presinaptico correnti, variazioni di capacità della membrana, e reciproca inibizione di feedback sinaptica mediata dal GABA &lt;sub&gt; recettori GABA A e C espresso sui terminali. Presinaptico misure membrana capacità di esocitosi permettono di studiare a breve termine plasticità del rilascio di neurotrasmettitore eccitatorio 14,15. Inoltre, la plasticità a breve termine ea lungo termine del rilascio di neurotrasmettitore inibitorio da parte delle cellule amacrine possono anche essere indagato da registrazioni di inibizione reciproca di feedback che arrivano al terminal Mb 21. In brevi periodi di tempo (ad esempio ~ 10 s), GABAergici inibizione di feedback reciproco a partire da cellule amacrine subisce accoppiato impulsi depressione attraverso l&#39;esaurimento GABA piscina vescicola 11. La dinamica sinaptica di microcircuiti retina nello strato plessiforme interno della retina può quindi essere direttamente studiato. Il cervello-fetta tecnica è stata introdotta più di 40 anni fa ma è ancora molto utile per lo studio delle proprietà elettriche dei neuroni, sia a singola cellula soma, dendriti singolo o assone, e microcircuit livello sinaptico 19. Tessuti che sono troppo piccole per essere incollato direttamente sulla camera di taglio sono spesso incorporati in prima agar (o la collocazione in una carta da filtro) e poi a fette 20, 23, 18, ​​9. In questo video, ci avvaliamo di pre-embedding tecnica di agar con retina pesci rossi. Alcuni dei giganti terminali delle cellule bipolari nelle nostre fette di retina pesci rossi sono axotomized (assone-cut) durante la procedura di taglio. Questo ci permette di isolare singoli ingressi terminale nervoso presinaptico, perché la registrazione da terminali axotomized esclude i segnali dal soma-dendritiche vano. In alternativa, si può anche registrare da intatto Mb cellule bipolari, registrando dai terminali collegati a assoni che non sono stati tagliati durante la procedura di taglio. Nel complesso, l&#39;uso di questo protocollo sperimentale sarà di aiuto negli studi di fisiologia sinaptica della retina, l&#39;analisi funzionale microcircuito, e la trasmissione sinaptica di sinapsi nastro.

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

Qui descriviamo un protocollo per la preparazione di agar-embedded fette della retina che sono adatti per elettrofisiologia e Ca2 + imaging. Questo metodo permette di studiare nastro di tipo sinapsi in microcircuiti della retina mediante diretta patch-clamp registrazioni dei singoli terminali nervosi presinaptici.

Patch-clamp Misure di capacità e Ca 2 + Imaging terminazioni nervose della retina singola in Fette

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

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Research

JoVE Journal

Neuroscience

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

18.1K Views.  Oregon Health and Science University. Qui descriviamo un protocollo per la preparazione di agar-embedded fette della retina che sono adatti per elettrofisiologia e Ca2 + imaging. Questo metodo permette di studiare nastro di tipo sinapsi in microcircuiti della retina mediante diretta patch-clamp registrazioni dei singoli terminali nervosi presinaptici.

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.

Bloccare Registrazioni patch da neuroni della retina del mouse in un Dark-adattato Preparazione Slice

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

Ricerca

Neuroscienze

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

Coltura ed Elettrofisiologia di Celle NRCC patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

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.

Visualizzazione del endosome Dynamics in Vivere terminazioni nervose con quattro-dimensionale imaging di fluorescenza

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.

Metodi per cellulari-attached Misure di capacità in mouse surrenale cromaffini cellulare

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.

Elettrofisiologici e morfologica Caratterizzazione neuronali Microcircuiti in Acute Fette cervello Utilizzando accoppiati di patch-clamp Recordings

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.

Registrazione Light-evocato Risposte postsinaptica nei neuroni in Dark-adattati, i preparativi della retina del mouse Slice Utilizzando tecniche di patch clamp

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.

Imaging Ca<sup&gt; 2+</sup&gt; Dinamica a cono fotorecettore Axon Terminali della retina di topo

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.

Risoluzione alta analisi quantitativa Immunogold di Recettori di membrana a retina nastro Sinapsi

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.

Registrazioni Whole-cell patch-clamp nel cervello fette

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

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.

Applicazione di morsetto di patch automatico guidato per immagini per lo studio dei neuroni in fettine di cervello

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 ...