Wir danken Dr. Fred Rieke für seine freundliche Erklärung des Agar-embedded Netzhaut Scheibe Vorbereitung, wenn wir mit dem Protokoll in unserem Labor begonnen. Wir danken auch Lori Vaskalis für die Darstellung der schematischen Überblick und Drs. Veeramuthu Balakrishnan und Soyoun Cho für die hilfreichen Kommentare zum Text und Video. Diese Arbeit wurde durch einen NEI-NIH RO1 gewähren unterstützt, und wurde teilweise auch durch einen Korea Research Foundation Grant von der koreanischen Regierung finanziert unterstützt [KRF-2008 bis 357-E00032].

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Wir haben keine konkurrierenden Interessen offen zu legen.

Ein kritischer und schwieriger Schritt in unserem Protokoll ist die Übertragung des Stückes von Netzhaut in den Agar-Lösung (Protokoll 3.4). Es ist notwendig, entfernen Sie vorsichtig den Glaskörper und Rest-slice-Lösung aus der Netzhaut Stück und übertragen sie ohne Verzerrung oder Biegen. Um dies zu erreichen, verwenden wir einen kleinen Spatel (21 bis 401-25B, Fisherbrand) mit einer Spitze gebogen, um einen 90 ° Winkel bilden, zusammen mit gebogener Spitze Pinzette (11251-35, Fine Science Tools), um die Netzhaut Position Stück in den Transfer-Prozess in Scheibe-Lösung. Residual Scheibe Lösung auf der Netzhaut schneiden und Spachtel kann durch vorsichtiges Betupfen der Oberfläche mit der Ecke eines gefaltet oder gerollt Kimwipes (Kimberly-Clark) entfernt werden. Ein weiteres gemeinsames Weg quer Netzhaut Scheiben vorzubereiten, ist das Filterpapier-basiertes Protokoll 23. Kurz gesagt, ist ein umgekehrtes Stück ganze Netzhaut zu einem Rechteck Stück Millipore-Filter disk (Ganglienzellen lagen befestigter gegen das Filterpapier, Photorezeptorschicht nach oben) und Schnitt in 250 mu m dicke Scheiben mit einem manuellen vertikalen &quot;thumb&quot; Allesschneider (zB Narishige ST-20). Wir finden, dass die Vorteile der Filterpapier-basiertes Protokoll gesünder Photorezeptoren und eine Erhöhung Verhältnis von axotomized auf intakte Mb Terminals enthalten. Wir benutzen also diese Filterpapier-Methode ans Licht hervorgerufenen Reaktionen von einzelnen Mb-Terminals in der Netzhaut Scheiben eingebettet entlocken. Die Vorteile unserer Agar-basiertes Protokoll über das Filterpapier-basiertes Protokoll, dass 1) der Netzhaut Scheibe produziert flach ist, auch und relativ unbeschädigt mit klarer Trennung zwischen Netzhautschichten, die das Patchen ermöglicht in jedem inneren Körnerschicht oder plexiformen Schicht der Netzhaut Zelle, die gewünscht wird, ist und 2) Immunhistochemie folgende elektrophysiologische Ableitung ist leichter, weil der intakten und flache Geometrie der Scheibe und die Faktoren in Artikel erwähnt (1) oben durchgeführt. Ein Protokoll für die Aufnahme leichte Anspraches der retinalen Nervenzellen in einem Agar-basierten Präparat wurde bereits in JoVE 2 veröffentlicht. Eine horizontale Netzhaut Scheibe Präparat, das eine Kombination von Agar und Filterpapier verwendeten Techniken war auch zuvor in JoVE 5 veröffentlicht. Wir empfehlen gerade diese Videos in Kombination mit unseren eigenen in die verschiedenen Techniken zu vergleichen. Direkter Zugriff auf Band-Typ präsynaptischen in Wirbeltiere Netzhaut Scheiben ermöglicht es uns, die synaptische Übertragung an Band-Typ aktiven Zonen zu untersuchen. Es erlaubt uns auch direkt Studie der synaptischen Physiologie der Netzhaut Mikroschaltungen. Diese Technik ist besonders nützlich für gepaarte Aufnahmen von Pre-und Post-synaptische Signale, mit postsynaptischen Partnern, darunter hemmende Amakrinzellen und spiking Ganglienzellen 1, 16, 10. Gepaart Aufnahmen auf Band-Typ Synapsen zwischen Ratte Cochlea inneren Haarzellen und afferenten Dendriten sind möglich und wurden um 7 beschrieben worden. Calcium IMAGIng des Mb Bipolarzelle Terminals hat in akut dissoziierten Zellen 8 und in der Netzhaut Scheiben 17, sowie in Goldfisch Amakrinzellen 3 wurde durchgeführt. Jüngste Studien haben große Verbesserungen für die gezeigte in vivo und in vitro optogenetische Ansätze in transgenen Zebrafisch Retina 4. Künftige Ausrichtung wird wahrscheinlich beinhalten diese transgenen Techniken, zusammen mit elektrophysiologischen Methoden, die es uns ermöglichen, die Lücke zwischen In-vitro-slice-Aufnahmen und in-vivo-Bildgebung Brücke, was letztendlich zu Verhaltensstudien wird.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Low Gelier-Temperatur-Agar </td><td> Sigma </td><td> A0701 </td><td> Agarose Typ VII-A </td></tr><tr><td> Patch-Pipette </td><td> World Precision Instruments </td><td> 1B150F-4 </td><td> Dickwandige (1,5 mm Außendurchmesser) Borosilikatglas </td></tr><tr><td> Vertical puller </td><td> Narishige </td><td> PP830 </td><td></td></tr><tr><td> Dental Wachs </td><td> Cavex </td><td></td><td></td></tr><tr><td> Frühling Schere </td><td> Feine Science Tools </td><td> 15003-08 </td><td></td></tr><tr><td> 45 ° abgewinkelt feine Spitze Pinzette </td><td> Feine Science Tools </td><td> 11251-35 </td><td></td></tr><tr><td> Rasierklinge </td><td> Personna </td><td></td><td> Zweischneidig, mit 70% Ethanol und H 2 O gereinigt </td></tr><tr><td> Zylindrischen Rohr </td><td> Fisherbrand </td><td> 03 bis 338-1B </td><td> Polyethylen Probenfläschchen 2,5 ml </td></tr><tr><td> Hyaluronidase </td><td> Sigma </td><td> H6254 </td><td></td></tr><tr><td> Vibratom Allesschneider </td><td> Leica </td><td> VT1000S oder VT1200S </td><td></td></tr><tr><td> Aufrechte Mikroskope </td><td> Olymp </td><td> BX51WI </td><td></td></tr><tr><td> 60x Wasser-Immersions-objektive </td><td> Olymp </td><td> LUMPlanFl </td><td> NA 0,90 </td></tr><tr><td> CCD-Kamera </td><td> Sony </td><td> XC-75 </td><td></td></tr><tr><td> Kamera-Controller </td><td> Hamamatsu </td><td> C2400 </td><td></td></tr><tr><td> Monitor &lt;/ Td&gt; <td> Sony </td><td></td><td> 13 &quot;Schwarz-Weiß-Monitor </td></tr><tr><td> Spritzenfilter </td><td> Nalgene </td><td></td><td> 0,2 um </td></tr><tr><td> Mikromanipulator </td><td> Sutter Instrument </td><td> MPC-200 </td><td></td></tr><tr><td> Lock-in-Verstärker </td><td> HEKA </td><td></td><td> EPC-9/10 Verstärker haben eine Software-Emulation </td></tr><tr><td> Spinning Disk Laser konfokalen Mikroskop </td><td> Yokogawa </td><td> CSU-X1 </td><td> Live Cell Imaging nach Patch-Clamp-Ganzzell Aufnahme </td></tr><tr><td> Slidebook Software </td><td> Intelligent Imaging Instruments (3i) </td><td></td><td> Imaging Datenerfassung und-analyse </td></tr><tr><td> Paraformaldehyd </td><td> Sigma </td><td> P6148 </td><td></td></tr><tr><td> Phosphatpufferlösung </td><td> GIBCO </td><td> 70013 </td><td></td></tr><tr><td> Superfrost slide </td><td> Fisher Scientific </td><td></td><td> Slide Glas </td></tr><tr><td> Anti-Fading-Agenten </td><td> Biomeda corp. </td><td></td><td></td></tr><tr><td> Konfokalen Laser-Scanning-Mikroskop </td><td> Carl Zeiss </td><td> LSM 710 </td><td> Imaging von fixiertem Gewebe </td></tr><tr><td> Spachtel </td><td> Fisherbrand </td><td> 21 bis 401-25B </td><td></td></tr><tr><td> Manuel vertikale Allesschneider </td><td> Narishige </td><td> ST-20 </td><td></td></tr><tr><td> Oregon Green 488 BAPTA-1 </td><td> Invitrogen </td><td> O-6806 </td><td> Ca 2 + Fluoreszenzfarbstoff </td></tr><tr><td> Alexa 555 Hydrazid </td><td> Invitrogen </td><td> A-20501MP </td><td> Fluoreszenzfarbstoff </td></tr></tbody></table>

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

Visuelle Reize werden erkannt und über einen weiten Dynamikbereich von Lichtintensitäten und Frequenzänderungen von spezialisierten Nervenzellen in der Retina vermittelt. Zwei Klassen von retinalen Nervenzellen Photorezeptoren und bipolaren Zellen, dies zu erreichen, indem Sie Lappenkäfig aktiven Zonen, die nachhaltig und mit hohem Durchsatz ermöglicht die Freisetzung von Neurotransmittern über lange Zeiträume. ON-Typ gemischt bipolare Zelle (Mb)-Terminals in der Goldfisch Netzhaut, die auf Lichtreize depolarisiert und erhalten gemischte Stäbchen und Zapfen Photorezeptor-Eingang, sind für das Studium der Band-Typ Synapsen sowohl aufgrund ihrer Größe (~ 10-12 geeignet Mikrometer Durchmesser) und ihre zahlreichen Quer-und wechselseitigen synaptischen Verbindungen mit amakrine Zelle Dendriten. Direkter Zugriff auf Mb Bipolarzelle Terminals in Goldfisch Netzhaut Scheiben mit der Patch-Clamp-Technik ermöglicht die Messung von präsynaptischen Ca 2 + Ströme, Membran-Kapazität ändert, und die gegenseitige synaptischen Feedback-Hemmung durch GABA vermittelte &lt;sub&gt; A und GABA C-Rezeptoren exprimiert auf den Terminals. Präsynaptischen Membran Kapazitätsmessungen der Exozytose erlauben es, die kurzfristige Plastizität der erregenden Neurotransmitter-Freisetzung 14,15 studieren. Darüber hinaus können kurzfristige und langfristige Plastizität inhibitorischer Neurotransmitter-Freisetzung aus Amakrinzellen auch durch Aufnahmen von gegenseitigen Feedback-Hemmung bei der Ankunft am Mb Klemme 21 untersucht werden. Über kurze Zeiträume (zB ~ 10 s), erfährt GABAergen gegenseitige Feedback-Hemmung von Amakrinzellen paired-Puls Depression über GABA Vesikel Pool Erschöpfung 11. Die synaptische Dynamik der retinalen Mikroschaltungen in der inneren plexiformen Schicht der Netzhaut kann somit direkt untersucht werden. Das Gehirn-Slice-Technik wurde vor mehr als 40 Jahren führte jedoch immer noch sehr nützlich für die Untersuchung der elektrischen Eigenschaften von Neuronen, sowohl auf die einzelne Zelle soma, single Dendrit oder Axon, und microcircuit synaptischen Ebene 19. Gewebe, die zu klein, um direkt auf die Schneiden Kammer verklebt werden sollen, werden oft erst in Agar eingebettet (oder platziert auf einem Filterpapier) und dann in Scheiben geschnitten 20, 23, 18, ​​9. In diesem Video, verwenden wir die Pre-embedding-Agar-Technik mit Goldfischen Netzhaut. Einige der riesigen bipolaren Zellen Terminals in unserer Scheiben Goldfisch Netzhaut axotomized (Axon-cut) beim Schneiden Verfahren. Dies ermöglicht uns, einzelne präsynaptischen Nervenendigung Eingänge zu isolieren, da die Aufnahme von axotomized Terminals schließt die Signale von der soma-dendritischen Kompartiment. Alternativ kann man auch aus intakten Mb Bipolarzellen aufzunehmen, durch die Aufnahme von Klemmen befestigt Axone, die nicht beim Schneiden Verfahren geschnitten wurden. Insgesamt wird diesen experimentellen Protokoll in Studien der retinalen synaptischen Physiologie, microcircuit funktionale Analyse und synaptische Übertragung an Synapsen Band zu unterstützen.

Watch this Scientific Journal Video about Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices at JoVE.com

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

Hier beschreiben wir ein Protokoll für die Herstellung von Agar-embedded Netzhaut Scheiben, die sich für Elektrophysiologie und Ca2 +-Imaging sind. Diese Methode erlaubt es, Band-Typ Synapsen der Netzhaut Mikroschaltungen mit direkten Patch-Clamp Messungen von einzelnen präsynaptischen Nervenendigungen zu studieren.

Patch-Clamp Kapazitätsmessungen und Ca 2 + Imaging bei Single Nervenendigungen in Retinal Slices

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

patch-clamp-capacitance-measurements-ca2-imaging-at-single-nerve

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

18.1K Aufrufe.  Oregon Health and Science University. Hier beschreiben wir ein Protokoll für die Herstellung von Agar-embedded Netzhaut Scheiben, die sich für Elektrophysiologie und Ca2 +-Imaging sind. Diese Methode erlaubt es, Band-Typ Synapsen der Netzhaut Mikroschaltungen mit direkten Patch-Clamp Messungen von einzelnen präsynaptischen Nervenendigungen zu studieren.

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

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Patch Clamp Messungen von Mouse Netzhautneuronen in einem Dark-Scheiben angepasst Vorbereitung

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

Forschung

Neurowissenschaften

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

Methoden für die Patch-Clamp Kapazität Recordings aus dem Kelch

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

Biologie

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Immunhistochemischen und Calcium Imaging Methoden in der Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

Patch-Clamp-Aufzeichnung von Starburst-Amakrinzellen in einer Wohnung Montage Herstellung von Deafferentated Maus Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

Laden eines Kalziumfarbstoffs in Froschnervenendungen durch den Nervenstumpf: Calciumübergangsregistrierung im Frosch Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Längerer Inkubation von Akute neuronales Gewebe für Elektrophysiologie und Calcium-Bildgebung

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Zwei-Photonen-Kalzium Imaging in neuronalen Dendriten in Hirnschnitten

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Bewertung der Kalziumaktivität in retinalen Ganglienzellen Schicht

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Autor im Rampenlicht: Ein innovativer Ansatz zur neuronalen Elektrostimulation mit Kalzium-Bildgebung 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

Kalzium-Bildgebung bei elektrisch stimulierten, flach montierten Netzhäuten

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...

Patch-Clamp Kapazitätsmessungen und Ca^{2 +} Imaging bei Single Nervenendigungen in Retinal Slices

Zusammenfassung

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