We would like to thank all members of &#8216;Function of Neuronal Microcircuits&#8217; Group at Institute of Neuroscience and Medicine, INM-2, Research Centre J&#252;lich and the &#8216;Function of Cortical Microcircuits&#8217; Group in the Dept. of Psychiatry, Psychotherapy and Psychosomatics, Medical School, JARA, RWTH Aachen University for fruitful discussions. This work was supported by the DFG research group on Barrel Cortex Function (BaCoFun).

The authors declare no conflict of interest.

Enregistrements appariés de excitateur synaptiquement couplé et / ou neurones inhibiteurs sont une approche très polyvalent pour l&#39;étude de microcircuits neuronaux. Non seulement cette approche permet d&#39;estimer la connectivité synaptique entre les types de neurones, mais permet également de déterminer les caractéristiques fonctionnelles de la connexion et de la morphologie des neurones pré- et post-synaptiques. En outre, un agoniste et / ou antagoniste peuvent être facilement appliqués à des neurones...

Microcircuits neuronaux entre deux neurones synaptiquement couplés sont les blocs de construction de réseaux à grande échelle dans le cerveau et sont les unités fondamentales de synaptique traitement de l&#39;information. Une condition préalable pour la caractérisation de ces microcircuits neuronaux est de connaître la morphologie et les propriétés fonctionnelles des deux partenaires neurones pré- et post-synaptiques, le type de la connexion synaptique (s) et sa structure et son mécanisme fonctionnel. Cependant, dans de nombreuses études de connexions synaptiques au moins l&#39;un des neurones dans un microcircuit ne est pas bien caractérisé. Il en résulte des protocoles de stimulation relativement non spécifiques souvent utilisées dans les études de la connectivité synaptique. Par conséquent, les propriétés structurelles et fonctionnelles du neurone présynaptique sont identifiées soit pas du tout ou seulement dans une mesure relativement faible (par exemple, l&#39;expression des protéines marqueurs, etc.). Enregistrements appariés en combinaison avec coloration intracellulaire de marqueurs sUCH que biocytine, neurobiotin ou colorants fluorescents sont mieux adaptés pour l&#39;étude de petites microcircuits neuronaux. Cette technique permet d&#39;enquêter sur de nombreux paramètres structurels et fonctionnels d&#39;une connexion synaptique identifiée morphologiquement en même temps. Les soi-disant connexions monosynaptiques «unitaire» entre deux neurones ont été étudiés dans les deux régions corticales et sous-corticales du cerveau 1-10 utilisant des préparations de tranche aigus. Initialement, microélectrodes tranchants ont été utilisés dans ces expériences; plus tard, l&#39;enregistrement patch clamp a été utilisée pour obtenir des enregistrements de signaux synaptiques avec un niveau de bruit inférieur et une résolution temporelle améliorée. Un progrès technique important est l&#39;utilisation de contraste d&#39;interférence différentiel à infrarouges (IR-DIC) 14.11 optique, une technique microscopique qui améliore considérablement la visibilité et l&#39;identification des neurones dans la tranche de cerveau de sorte qu&#39;il devient possible to obtenir des enregistrements de connexions synaptiques identifiés visuellement 15-17. En général, les enregistrements appariés sont effectuées dans des préparations de tranche aigus; que très peu de publications sont disponibles enregistrements de rapports de neurones reliés par des synapses in vivo 18-20. L&#39;avantage le plus important d&#39;enregistrements appariés est le fait qu&#39;une caractérisation fonctionnelle peut être combiné avec une analyse morphologique à la fois la lumière et de l&#39;électron niveau microscopique (voir, par exemple., 7,16,21). Après le traitement histochimique, la morphologie dendritique et axonale de la paire de neurones reliés par des synapses est tracée. Par la suite, il est possible de quantifier les caractéristiques morphologiques telles que la longueur, la densité spatiale, l&#39;orientation, patron de ramification, etc. Ces paramètres peuvent alors fournir une base pour une classification objective d&#39;une connexion synaptique spécifique. En outre, contrairement à la plupart des autres techniques utilisées pour l&#39;étude de rappro neuronalestivité, jumelé enregistrements permettent également l&#39;identification des contacts synaptiques pour les connexions synaptiques unitaires. Cela peut être fait directement à l&#39;aide d&#39;une combinaison de microscopie optique et électronique 16,21-27 ou en utilisant l&#39;imagerie calcique 28,29 des épines dendritiques. Toutefois, cette dernière approche ne excitateur mais pas des connexions inhibitrices peuvent être étudiés car elle nécessite l&#39;influx de calcium par les canaux des récepteurs post-synaptiques. En plus d&#39;une analyse détaillée de la transmission synaptique chez un microcircuit couplé enregistrements neuronaux définies permettent également l&#39;étude des règles de plasticité synaptique 30,31 ou - en combinaison avec une application agoniste / antagoniste - la modulation de la transmission synaptique par les neurotransmetteurs tels que l&#39;acétylcholine et de l&#39;adénosine 32 33.

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			<td height="21" style="height:21px;width:199px;">Name</td>
			<td style="width:199px;">Company</td>
			<td style="width:199px;">Catalog Number</td>
			<td style="width:199px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Amplifier</td>
			<td style="width:199px;">HEKA</td>
			<td style="width:199px;">EPC 10 USB Triple</td>
			<td style="width:199px;">with 2-3 preamplifiers</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:199px;">Microscope</td>
			<td style="width:199px;">Olympus</td>
			<td style="width:199px;">BX51WI</td>
			<td style="width:199px;">with 2 camera ports and a 4&#215; objective, a 40&#215; water-immersion objective</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Camera</td>
			<td style="width:199px;">TILL Photonics</td>
			<td style="width:199px;">VX55</td>
			<td style="width:199px;">infrared CCD camera</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:199px;">Workstation</td>
			<td style="width:199px;">Luigs &#38; Neumann</td>
			<td style="width:199px;">Infrapatch 240</td>
			<td style="width:199px;">with a motorized x-y stage and a motorized focus axis for the microscope</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Micromanipulator</td>
			<td style="width:199px;">Luigs &#38; Neumann</td>
			<td style="width:199px;">SM-5</td>
			<td style="width:199px;">x-y-z manipulators for 2-3 preamplifiers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Faraday cage</td>
			<td style="width:199px;">Luigs &#38; Neumann</td>
			<td style="width:199px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Anti-vibration table</td>
			<td style="width:199px;">Newport Spectra-Physics</td>
			<td style="width:199px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Patchmaster</td>
			<td style="width:199px;">HEKA</td>
			<td style="width:199px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Microtome</td>
			<td style="width:199px;">Microm International</td>
			<td style="width:199px;">HM650V</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Micropipette puller</td>
			<td style="width:199px;">HEKA</td>
			<td style="width:199px;">Sutter P-97</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Neurolucida system</td>
			<td style="width:199px;">Microbrightfield</td>
			<td style="width:199px;"></td>
			<td style="width:199px;">with Neurolucida and Neuroexplorer softwares</td>
		</tr>
	</tbody>
</table>

electrophysiological and morphological characterization of neuronal microcircuits in acute brain slices using paired patch-clamp recordings

La combinaison d&#39;enregistrements patch clamp de deux (ou plus) des synapses des neurones couplés (enregistrements appariés) dans aiguës préparations de tranches de cerveau avec remplissage simultané de la biocytine intracellulaire permet une analyse corrélée de leurs propriétés structurales et fonctionnelles. Avec cette méthode, il est possible d&#39;identifier et de caractériser les neurones pré- et post-synaptiques par leur morphologie et leur profil de réponse électrophysiologique. Enregistrements appariés permettent l&#39;étude des modes de connectivité entre ces neurones ainsi que les propriétés à la fois la transmission synaptique chimique et électrique. Ici, nous donnons une description étape par étape des procédures requises pour obtenir des enregistrements appariés fiables avec une récupération optimale de la morphologie des neurones. Nous allons décrire comment paires de neurones connectés via les synapses chimiques ou de jonctions lacunaires sont identifiés dans les préparations de tranches de cerveau. Nous allons exposer comment les neurones sont reconstruits pour obtenir leur morphologie 3D de la Dendritic et le domaine axonale et comment contacts synaptique sont identifiés et localisés. Nous discuterons également les mises en garde et les limites de la technique d&#39;enregistrement couplé, en particulier ceux associés à troncatures dendritiques et axonales lors de la préparation des tranches de cerveau parce que ces affectent fortement les estimations de connectivité. Toutefois, en raison de la polyvalence de l&#39;approche d&#39;enregistrement jumelé il restera un outil précieux pour caractériser les différents aspects de la transmission synaptique au microcircuits neuronaux identifiés dans le cerveau.

Enregistrements liés sont la méthode de choix pour une caractérisation approfondie des connexions synaptiques uni ou bidirectionnels morphologiquement identifiées ainsi que jonctions lacunaires connexions (électriques) (Figure 1). Un exemple d&#39;un enregistrement couplé à la couche 4 du cortex somato-sensoriel du canon est représentée sur la Figure 1A. Les deux excitateurs unidirectionnel et connexions synaptiques inhibiteurs peuvent être caractérisées (figure 1B, ...

Watch this Scientific Journal Video about Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings at JoVE.com

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

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.

Électrophysiologiques et Caractérisation morphologique des neurones Les circuits dans aiguë tranches cerveau en utilisant jumelés patch-clamp enregistrements

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

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Neuroscience

17.1K vues.  Research Centre Jülich. 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.

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

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.

Enregistrements Patch Clamp à partir de neurones rétiniens souris dans une préparation adaptés à l&#39;obscurité Slice

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

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Neurosciences

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

Jumelés Recordings Patch Clamp du motoneurone et de muscle ciblé squelettiques chez le poisson zèbre

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Les enregistrements de patch-clamp en cellules ciliées de l&#39;oreille Isolé du poisson zèbre

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Deux enregistrements électrophysiologiques des synapses-réponses évoquées astrogliales et neuronales aiguës dans des coupes d&#39;hippocampe

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Patch-clamp enregistrements de cellules embryonnaires de poisson zèbre Mauthner

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

Un multi-électrode système de patch-clamp assistée par ordinateur

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

De cellules entières de patch-clamp enregistrements de Morphologically- et interneurones hippocampiques neurochemically identifiés

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Appariés enregistrements de cellules entières dans organotypiques coupes d&#39;hippocampe

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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.

Recordings cellules entières Patch-clamp en tranches cérébrales

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

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Subcellulaires Recordings Patch-clamp du Domaine somato de nigrales Dopamine Neurones

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...