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.

הקלטות לזווג ממעורר בשילוב synaptically ו / או נוירונים מעכבים הן גישה מאוד תכליתית ללימוד שבבים עצביים. לא רק בגישה זו אינה מאפשרת אחד כדי להעריך קישוריות הסינפטי בין סוגי תאי עצב, אלא גם מאפשרת קביעת המאפיינים הפונקציונליים של החיבור והמורפולוגיה של תאי עצב טרום postsynaptic. ?...

שבבים עצביים בין שני תאי עצב בשילוב synaptically הם אבני הבניין של רשתות בקנה מידה גדולה במוח והם היחידות הבסיסיות של עיבוד מידע הסינפטי. תנאי מוקדם לאפיון של שבבים עצביים כזו הוא לדעת את המורפולוגיה ותכונות פונקציונליות של שני נוירונים השותף טרום postsynaptic, הסוג של החיבור הסינפטי (ים) ואת המבנה שלה ומנגנון תפקודי. עם זאת, במחקרים רבים של קשרים סינפטיים לפחות אחד מתאי העצב בmicrocircuit אינו מאופיין היטב. זה נובע מפרוטוקולי הגירוי יחסית הנוקבים המשמשים לעתים קרובות במחקרים של קישוריות הסינפטי. לכן, התכונות המבניות ותפקודיות של נוירון presynaptic הם גם לא זיהו כלל או רק במידה קטנה למדי (כלומר, הביטוי של חלבוני סמן וכו &#39;). הקלטות מותאמים בשילוב עם מכתים תאיים על ידי סמנים שלuch כמו biocytin, Neurobiotin או צבעי ניאון מתאים יותר ללימוד שבבים עצביים קטנים. טכניקה זו מאפשרת לחקור פרמטרים רבים מבניים ותפקודיים של חיבור הסינפטי זיהה מורפולוגית באותו הזמן. מה שנקרא חיבורים &#39;אחידים&#39; monosynaptic בין שני תאי עצב נחקרו בשני אזורים במוח בקליפת המוח וקורטיקליים 1-10 באמצעות הכנות פרוסה חריפות. בתחילה, microelectrodes החדה שימשה בניסויים אלה; מאוחר יותר, הקלטת מהדק התיקון הועסקה כדי להשיג הקלטות של אותות הסינפטי עם רמת רעש נמוכה ורזולוציה של זמן השתפרה. מראש טכניים משמעותיים היה השימוש בניגוד להפרעות ההפרש אינפרא אדום (IR-DIC) אופטיקה 11-14, טכניקה מיקרוסקופית ששיפרה באופן משמעותי את הנראות וזיהוי של תאי עצב במוח הפרוסה כך שהוא הפך t האפשריo להשיג הקלטות מקשרים סינפטיים מזוהים מבחינה ויזואלית 15-17. באופן כללי, הקלטות לזווג נעשות בהכנות פרוסה חריפות; רק מעט מאוד פרסומי הקלטות דיווח זמינות מהנוירונים מחוברים synaptically in vivo 18-20. היתרון החשוב ביותר של הקלטות לזווג הוא העובדה שאפיון פונקציונלי יכול להיות משולב עם ניתוח מורפולוגי בשני האור ורמה מיקרוסקופית אלקטרונים (ראה, למשל., 7,16,21). לאחר עיבוד histochemical, המורפולוגיה הדנדריטים וaxonal של זוג נוירון מחובר synaptically היא לייחס. בהמשך לכך, ניתן לכמת תכונות מורפולוגיות כגון אורך, צפיפות מרחבית, אורינטציה, הסתעפות דפוס וכו &#39;פרמטרים אלה עשויים לאחר מכן לספק בסיס לסיווג אובייקטיבי של קשר הסינפטי ספציפי. יתר על כן, בניגוד לרוב הטכניקות אחרות המשמשות ללימוד connecti העצביהקלטות vity, לזווג גם לאפשר זיהוי של קשרים סינפטיים לקשרים סינפטיים יחידתי. ניתן לעשות זאת ישירות באמצעות שילוב של אור ומיקרוסקופ אלקטרונים 16,21-27 או באמצעות ההדמיה סידן 28,29 של קוצים הדנדריטים. עם זאת, עם הגישה השנייה רק ​​מעורר אבל ניתן ללמוד לא קשרים מעכבים כפי שהוא דורש זרימת סידן באמצעות ערוצי קולט postsynaptic. בנוסף לניתוח מפורט של העברת הסינפטית בהקלטות microcircuit לזווג עצביות מוגדרות גם לאפשר המחקר של כללי פלסטיות הסינפטית 30,31 או - בשילוב עם יישום אגוניסט / אנטגוניסט - האפנון של שידור סינפטי על ידי נוירוטרנסמיטרים כגון אצטילכולין 32 ואדנוזין 33.

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			<td style="width:199px;">Comments</td>
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			<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>
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		<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>
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		<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>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Patchmaster</td>
			<td style="width:199px;">HEKA</td>
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			<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>
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		<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>
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	</tbody>
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electrophysiological and morphological characterization of neuronal microcircuits in acute brain slices using paired patch-clamp recordings

השילוב של הקלטות מהדק תיקון משני (או יותר) נוירונים בשילוב synaptically (הקלטות לזווג) בהכנות פרוסה מוח חריפות עם מילוי biocytin תאיים בו זמנית מאפשר ניתוח מתואם של התכונות המבניות והתפקודיות שלהם. בשיטה זו אפשר לזהות ולאפיין את שני נוירונים טרום postsynaptic על ידי המורפולוגיה שלהם ודפוס תגובת אלקטרו. הקלטות לזווג מאפשרות ללמוד את דפוסי הקישוריות בין תאי העצב האלה, כמו גם את המאפיינים של שני שידור סינפטי הכימי וחשמלי. הנה, אנחנו נותנים תיאור צעד-אחר-צעד של ההליכים הנדרשים להשגת הקלטות לזווג אמינות יחד עם התאוששות אופטימלית של מורפולוגיה תא העצב. אנו מתארים כיצד זוגות של הנוירונים המחוברים דרך סינפסות כימיות או צמתים פער מזוהים בהכנות פרוסה מוח. אנו מתארים כיצד תאי עצב משוחזרים להשיג מורפולוגיה 3D של dendritic ותחום axonal ואיך הסינפטי קשר מזוהה ומקומי. נדונו גם באזהרות ומגבלות של טכניקת ההקלטה לזווג, בפרט אלה הקשורים לtruncations הדנדריטים וaxonal במהלך תקופת ההכנה של פרוסות המוח כי אלה חזקים משפיעים הערכות קישוריות. עם זאת, בגלל הרבגוניות של גישת ההקלטה לזווג הוא יישאר כלי רב ערך באפיון היבטים שונים של העברת הסינפטית בשבבים עצביים מזוהים במוח.

הקלטות לזווג הן את שיטת הבחירה לאפיון מעמיק של קשרים סינפטיים חד-או דו כיוונית זיהו מורפולוגית כמו גם צומת פער חיבורים (חשמל) (איור 1). דוגמא של הקלטה לזווג בשכבת 4 של קליפת החבית החושית מוצגת באיור 1 א. שניהם מעורר חד-כיווני וקשרים סינפטיים מעכבים ניתן...

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.

אלקטרו ומורפולוגי אפיון של שבבים עצביים במוח חריף Slices שימוש מותאם Patch-קלאמפ הקלטות

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

electrophysiological-morphological-characterization-neuronal

Research

JoVE Journal

Neuroscience

17.1K Views.  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.

Video: 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.

הצמד תיקון הקלטות מתא עצב עכבר רשתית בהכנת Dark המותאמות פרוסה

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

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.

מותאמים הצמד תיקון הקלטות מ-מוטור נוירון שרירים יעד השלד דג הזברה

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

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.

Culturing ו Electrophysiology של תאים על Patch-clamp צ&#39;יפס NRCC

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 ...

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.

הקלטות קלאמפ תיקון בתאי שיער באוזן פנימיים מבודדות מדג הזברה

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.

קלטות אלקטרו כפולות של תגובות Astroglial ועצביות עוררו-synaptically בפרוסות ביפוקמפוס חריפות

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.

תיקון הקלטות קלאמפ מתאים עוברי דג הזברה מאוטנר

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.

מערכת תיקון מהדק Multi-אלקטרודה בסיוע מחשב

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.

הקלטות תיקון מהדק כל התא מMorphologically- וinterneurons היפוקמפוס זיהה Neurochemically

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.

הקלטות תא כל לזווג בפרוסות בהיפוקמפוס Organotypic

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.

הקלטות תיקון מהדק כל תא פרוסות המוח

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