אנו רוצים להודות לגלעד זילברברג, מישל Pignatelli, תומאס ק ברגר, לוקה Gambazzi, וסוניה גרסיה לייעוץ רב ערך על שיפורים לאוטומציה הליך תיקון מהדק. אנו מודים Rajnish ראנג&#39;אן לקבלת ייעוץ וסיוע ביישום תוכנה יקרים. עבודה זו מומנה בחלקו על ידי האיחוד האירופי בפרויקט סינפסה ובחלקו על ידי התכנית למדע הגבולות האנושי.

<ol>
	<li>Perin, R., Berger, T. K., Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synaptic+organizing+principle+for+cortical+neuronal+groups.">A synaptic organizing principle for cortical neuronal groups.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 5419-5424 (2011).</li><li>Berger, T. K., Silberberg, G., Perin, R., Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+Bursts+Self-Inhibit+and+Correlate+the+Pyramidal+Network.">Brief Bursts Self-Inhibit and Correlate the Pyramidal Network.</a> PLoS Biol. 8, e1000473 (2010).</li><li>Fino, E., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dense+inhibitory+connectivity+in+neocortex.">Dense inhibitory connectivity in neocortex.</a> Neuron. 69, 1188-1203 (2011).</li><li>Packer, A. M., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dense,+Unspecific+Connectivity+of+Neocortical+Parvalbumin-Positive+Interneurons:+A+Canonical+Microcircuit+for+Inhibition.">Dense, Unspecific Connectivity of Neocortical Parvalbumin-Positive Interneurons: A Canonical Microcircuit for Inhibition.</a> J. Neurosci. 31, 13260-13271 (2011).</li><li>Berger, T. K., Perin, R., Silberberg, G., Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency-dependent+disynaptic+inhibition+in+the+pyramidal+network:+a+ubiquitous+pathway+in+the+developing+rat+neocortex.">Frequency-dependent disynaptic inhibition in the pyramidal network: a ubiquitous pathway in the developing rat neocortex.</a> J. Physiol. 587, 5411-5425 (2009).</li><li>Kodandaramaiah, S. B., Franzesi, G. T., Chow, B. Y., Boyden, E. S., Forest, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+whole-cell+patch-clamp+electrophysiology+of+neurons+in+vivo.">Automated whole-cell patch-clamp electrophysiology of neurons in vivo.</a> Nat. Methods. 9, 585-587 (2012).</li><li>Anastassiou, C. A., Perin, R., Markram, H., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ephaptic+coupling+of+cortical+neurons.">Ephaptic coupling of cortical neurons.</a> Nat. Neurosci. 14, 217-223 (2011).</li><li>Prakash, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+optogenetic+toolbox+for+fast+inhibition,+excitation+and+bistable+modulation.">Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation.</a> Nat. Methods. 9, 1171-1179 (2012).</li><li>Papagiakoumou, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanless+two-photon+excitation+of+channelrhodopsin-2.">Scanless two-photon excitation of channelrhodopsin-2.</a> Nat Methods. 7, 848-854 (2010).</li><li>Ko, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+specificity+of+local+synaptic+connections+in+neocortical+networks.">Functional specificity of local synaptic connections in neocortical networks.</a> Nature. 473, 87-91 (2011).</li><li>Wickersham, I. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+Restriction+of+Transsynaptic+Tracing+from+Single,+Genetically+Targeted+Neurons.">Monosynaptic Restriction of Transsynaptic Tracing from Single, Genetically Targeted Neurons.</a> Neuron. 53, 639-647 (2007).</li><li>Liang, C. W., Mohammadi, M., Santos, M. D., Tang, C. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+Photostimulation+with+Digital+Micromirror+Devices+to+Investigate+Dendritic+Integration+Across+Branch+Points.">Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points.</a> J. Vis. Exp. (49), e2003 (2011).</li><li>Nikolenko, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLM+Microscopy:+Scanless+Two-Photon+Imaging+and+Photostimulation+with+Spatial+Light+Modulators.">SLM Microscopy: Scanless Two-Photon Imaging and Photostimulation with Spatial Light Modulators.</a> Front Neural Circuits. 2, (2008).</li></ol>

החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

שאלה מיידית שעולה בדרך כלל בדבר שיעור הצלחה של ההליך שתארנו מ. לשיעורי הצלחה גבוהים הכנה היא חיונית. טפטפות חייבת להיות פתחי קצה שנחשבים כמספיקים לאדם התאים שנרשם. סינון הפתרון תאיים, כדי למנוע טפטפות הסתומה הוא גם חשוב. טפטפות מאוד נקייה, טרי משכה הן דרישה אחרת. התפלג...

היכולת להקליט ולעורר מספר אתרים עם דיוק מיקרומטר היא מאוד שימושית עבור ניסוי השגת הבנה טובה יותר של מערכות עצביות. טכניקות רבות פותחו למטרה זו אבל אף מאפשרות הרזולוציה submillivolt מושגת על ידי טכניקת תיקון מהדק, חיונית ללימוד פעילות התת ופוטנציאלי postsynaptic בודדים. כאן אנו מכסים את פיתוחה של מערכת התיקון-clamp שנתי עשר האלקטרודה בעזרת מחשב שמטרתה הקלטה בו זמנית ומגרה מספר רב של תאים בודדים בדייקנות מספיק ללימוד קישוריות עצבית. אמנם יכולים להיות יזום יישומים רבים אחרים למערכת כזו, זה משאיל את עצמו במיוחד גם ללימוד קישוריות הסינפטית בהתחשב בעובדה שמספר חיבורים אפשריים בתוך קבוצה של נוירונים גדל באופן פרופורציונלי לריבוע מספר הנוירונים בשאלה. לכן, בזמן שמערכת עם שלוש אלקטרודות מאפשרת בדיקהמופע של עד שישה חיבורים ולרוב הקלטה אחת, הקלטת עשרה נוירונים מאפשר בדיקת ההתרחשות של עד 132 קשרים ולעתים קרובות התבוננות למעלה מתריסר (איור 1). התצפית של עשרות חיבורים בו זמנית מאפשרת לנתח את הארגון של רשתות קטנות ולהסיק מאפיינים סטטיסטיים של מבנה הרשת שלא יכול להיות נחקר אחר 1. יתר על כן, גירוי מדויק של תאים רבים גם מאפשר כימות של גיוס של תאי postsynaptic 2.

<table border="1">
	<tbody>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>40X Immersion Objective</td>
		</tr>
		<tr>
			<td>Manipulators</td>
			<td>Luigs &#38; Neumann</td>
			<td>SM-5</td>
			<td>Serial protocol used</td>
		</tr>
		<tr>
			<td>Amplifiers</td>
			<td>Axon Instruments</td>
			<td>MultiClamp 700B</td>
			<td>SDK used</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Till Photonics</td>
			<td>VS 55</td>
			<td>BNC analog output</td>
		</tr>
		<tr>
			<td>Framegrabber</td>
			<td>Data Translation</td>
			<td>DT3120</td>
			<td>SDK used</td>
		</tr>
		<tr>
			<td>Oscilloscopes</td>
			<td>Tektronix</td>
			<td>TDS 2014</td>
			<td>Serial communication</td>
		</tr>
		<tr>
			<td>Data acquisition</td>
			<td>InstruTECH</td>
			<td>ITC 1600</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition</td>
			<td>National Instruments</td>
			<td>PCI-6221</td>
			<td>Library used (.dll)</td>
		</tr>
		<tr>
			<td>Pressure valve</td>
			<td>SMC</td>
			<td>SMC070C-6BG-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure sensor</td>
			<td>Honeywell</td>
			<td>24PCDFA6G</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane pump</td>
			<td>Schego</td>
			<td>Optimal</td>
			<td></td>
		</tr>
	</tbody>
</table>

a computer-assisted multi-electrode patch-clamp system

טכניקת התיקון-clamp היא כיום השיטה הכי המבוססת היטב להקלטת פעילות חשמלית מנוירונים בודדים או תאי subcellular שלהם. עם זאת, להשגת הקלטות יציבה, גם מהתאים בודדים, נותר הליך מורכב למדי זמן רב. אוטומציה של שלבים רבים בשילוב עם תצוגת מידע יעילה יכולה מאוד לסייע experimentalists בביצוע מספר רב יותר של הקלטות עם אמינות גבוהה יותר ובפחות זמן. על מנת להשיג את הקלטות בקנה מידה גדולה שסכמנו את הגישה היעילה ביותר היא לא automatize באופן מלא בתהליך, אבל כדי לפשט את הצעדים הניסיוניים ולהפחית את הסיכוי לטעויות אנוש תוך שילוב ניסיונו של הנסיין ומשוב חזותי ביעילות. עם מטרות אלה בחשבון שפיתחנו מערכת ממוחשבת המרכזת את כל הבקרות הנדרשות לניסוי רב האלקטרודה תיקון מהדק בממשק יחיד, commercgamepad האלחוטי זמין ially, תוך הצגה ברמזי מידע והדרכה ניסוי קשורות במסך המחשב. כאן אנו מתארים את המרכיבים השונים של המערכת שאפשרה לנו להפחית את הזמן הנדרש להשגת את תצורת ההקלטה ולהגדיל באופן משמעותי את הסיכוי להקלטה בהצלחה מספר גדול של תאי עצב בו זמנית.

בעקבות השיטות שתוארו לעיל הצלחנו בביצוע הקלטת כל התא של עד שנים עשר נוירונים בו זמנית, כמעט ואכפיל את המספר הגדול ביותר של תאי עצב בו זמנית ובכך מרחיק הידק התיקון. דוגמאות לרשתות של קשרים סינפטיים ישירים בין פירמידת נוירונים נרשמו בשכבת V של הקליפה החושית של חולדות מו...

Watch this Scientific Journal Video about A Computer-assisted Multi-electrode Patch-clamp System at JoVE.com

A Computer-assisted Multi-electrode Patch-clamp System

הקלטות תיקון מהדק Multi-אלקטרודה מהוות משימה מורכבת. כאן אנו מראים כיצד, על ידי אוטומציה של רבים מהשלבים הניסיוניים, זה אפשרי כדי להאיץ את התהליך מוביל לשיפור איכותי בביצועים ומספר ההקלטות.

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 ...

a-computer-assisted-multi-electrode-patch-clamp-system

Research

JoVE Journal

Neuroscience

13.7K Views.  Ecole Polytechnique Federale de Lausanne.  הקלטות תיקון מהדק Multi-אלקטרודה מהוות משימה מורכבת. כאן אנו מראים כיצד, על ידי אוטומציה של רבים מהשלבים הניסיוניים, זה אפשרי כדי להאיץ את התהליך מוביל לשיפור איכותי בביצועים ומספר ההקלטות. 

Video: A Computer-assisted Multi-electrode Patch-clamp System

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 ...

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  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<sub&gt; 1</sub&gt; F<sub&gt; O</sub&gt; הכנת שלפוחיות ATPase וטכניקה לביצוע הקלטות מהדק תיקון של קרום שלפוחית ​​Submitochondrial

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

הצמד תיקון נייד לכל חוקר את המנגנונים של גירוי עצבי אינפרא אדום

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

בידוד של נוירונים חושיים של<em&gt; Aplysia californica</em&gt; להקלטות מהדק תיקון של זרמי glutamatergic

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

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 ...

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 ...

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 ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.