Wir danken dem späten Rudy Limburg für die Kardan Armumfang Herstellung, Kimberly Jamison, Matthew McHenry, und Raju Metherate uns Ausrüstung für die Kreditvergabe, und Almut Kelber und Kentaro Arikawa, für Ermutigung. Diese Arbeit wurde von einem National Science Foundation (NSF) Graduate Research Fellowship an KJM und NSF Zuschuss IOS-1257627 ADB unterstützt

Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Die intrazelluläre Aufnahme kann eine schwierige Technik, aufgrund der vielen technischen Schritte zu meistern beteiligt. Für eine erfolgreiche Experimente müssen einige wichtige Punkte berücksichtigt werden. Erstens ist es wichtig, eine richtig schwingungsisolierten Tisch zu haben, an dem das Experiment durchgeführt wird. Viele Forscher verwenden Luft-Tabellen, die vollständig die Tischplatte von der Basis trennen, überlegene Schwingungsisolierung zu geben. Unser Setup beinhaltet einen dicken Marmortisch mit ein...

Die elektrischen Eigenschaften von Zellen, wie Neuronen werden durch Messen Ionenfluss durch die Zellmembranen als Änderung der Spannung oder des Stroms beobachtet. Eine Vielzahl von elektrophysiologischen Techniken entwickelt worden bioelektrischen Ereignisse in Zellen zu messen. Neurone in den Augen von Tieren zugänglich sind und ihre Schaltungsanordnung ist oft weniger komplex als im Gehirn, diese Zellen gute Kandidaten für eine elektrophysiologische Untersuchung zu machen. Gängige Anwendungen von Elektro im Auge umfassen Elektroretinogramm (ERG) 1,2 und Mikroelektroden intrazelluläre Aufnahme. ERG beinhaltet in oder auf dem Auge eines Tieres eine Elektrode platziert, ein Lichtreiz Anwendung und Messung der Änderung der Spannung als Summe der Antworten aller Zellen in der Nähe 3-6. Wenn man bei der Charakterisierung von spektralen Empfindlichkeiten einzelner Sehzellen speziell interessiert ist, oft mehrere Zelltypen gleichzeitig an verschiedenen Stärken auf einen bestimmten Reiz reagieren; somitschwierig sein kann, die Empfindlichkeiten von spezifischen Zelltypen aus ERG-Daten zu bestimmen, insbesondere dann, wenn verschiedene Arten von spektral ähnlichen Sehzellen im Auge sind. Eine mögliche Lösung ist transgene Drosophila mit dem Photorezeptor (Opsin) Gen von Interesse in den meisten R1-6 Zellen im Auge ausgedrückt zu schaffen und führen Sie dann ERG 7. Mögliche Nachteile dieser Methode sind nicht zu niedrig Expression des Proteins Photorezeptor 8, und die lange Zeitrahmen für die Erzeugung und das Screening von transgenen Tieren. Für Augen mit weniger Arten von spektral unterschiedlichen Photorezeptoren, die Anpassung des Auges mit farbigen Filter können mit Absenken der Beitrag einiger Zelltypen zu dem ERG, wodurch Schätzung der spektralen Empfindlichkeitsmaxima 9 helfen. Die intrazelluläre Aufnahme ist eine weitere Technik, wo eine feine Elektrode, die eine Zelle und ein Stimulus spießt angewendet wird. Die Elektrodenaufzeichnungen nur, dass indivIdual Zelle Antwort , so dass von der Aufnahme und mehreren einzelnen Zellen 10-14 spezifischen Empfindlichkeiten von physiologisch unterschiedlichen Zelltypen zu analysieren ergeben. Obwohl unser Protokoll auf der Analyse der spektralen Empfindlichkeit konzentriert, sind die grundlegenden Prinzipien der intrazellulären Aufnahme mit scharfen Elektroden für andere Anwendungen modifizierbar. Mit einer anderen Vorbereitung einer Probe, zum Beispiel, und mit scharfen Quarzelektroden kann man aus tiefer in den optischen Loben oder anderen Regionen im Gehirn, die sich an der Frage abhängig gefragt. Zum Beispiel Antwortzeiten einzelner Sehzellen 15, Zell - Aktivität in den optischen Loben 16 (Lamina, Medulla oder Lobula 17), Gehirn 18 oder andere Ganglien 19 kann auch mit ähnlichen Techniken aufgezeichnet werden, oder Farbreize mit Polarisation ersetzt werden könnte 20 -22 oder Bewegungsreize 23,24. Phototransduktion, das Verfahren, mit dem LichtEnergie absorbiert und in ein elektrochemisches Signal, ist ein altes Merkmal gemeinsam fast alle heutigen Tierstämme 25. Die visuelle Pigment gefunden in Sehzellen und verantwortlich für Phototransduktion Einleitung ist Rhodopsin. Rhodopsine in allen Tieren sind aus einem Opsin - Protein, einem Mitglied der 7 transmembranen G - Protein-gekoppelten Rezeptorfamilie und ein zugehöriges Chromophor hergestellt , das aus der Netzhaut oder ein ähnliches Molekül 26,27 ableitet. Opsin Aminosäuresequenz und chromophore Struktur beeinflussen die Absorption von auf verschiedene Wellenlängen von Licht Rhodopsin. Wenn ein Photon durch den Chromophor absorbiert wird das Rhodopsin aktiviert wird, einen G-Protein - Kaskade in der Zelle initiiert, die an der Öffnung der membrangebundenen Ionenkanäle 28 schließlich führt. Anders als die meisten Neuronen unterziehen Sehzellen abgestufter Potentialänderungen, die Lichtreizes als relative Änderung der Reaktionsamplitude mit wechselnden gemessen werden kann. Typischerweise eine gegebenePhotorezeptortyp drückt nur ein Opsin - Gen (obwohl Ausnahmen existieren 8,10,29-31). Anspruchsvolle Farbsehen, wie sie in vielen Wirbeltieren und Gliederfüßern gefunden wird, wird mit einem komplexen Auge von Hunderten oder Tausenden von Sehzellen erreicht jeweils ein oder gelegentlich mehr Rhodopsin Arten ausdrücken. Visuelle Information wird durch den Vergleich Antworten, die über dem Fotoaufnehmer Mosaik über komplexe nachgeschalteten neuronalen Signalisierung im Auge und Gehirn erfasst, was in der Wahrnehmung eines Bildes komplett mit Farbe und Bewegung. die rohen Antworten eines Sehzellen auf unterschiedliche Wellenlängen des Lichts über die intrazelluläre Aufnahme Nach der Messung ist es möglich, seine spektrale Empfindlichkeit zu berechnen. Diese Berechnung basiert auf dem Prinzip der Univariance, die besagt , dass eine Reaktion der Photorezeptorzelle auf der Zahl der Photonen abhängig ist, aber nicht auf die besonderen Eigenschaften der Photonen absorbiert es 32 absorbiert. Jedes Photon, das abso istrbed von Rhodopsin wird die gleiche Art von Reaktion induzieren. In der Praxis bedeutet dies , dass eine rohe Reaktionsamplitude der Zelle entweder aufgrund einer Erhöhung der Lichtintensität (mehr Photonen zu absorbieren) zu erhöhen, oder zu einer Verschiebung der Wellenlänge in Richtung auf seine maximale Empfindlichkeit (höhere Wahrscheinlichkeit von Rhodopsin , dass die Wellenlänge absorbieren). Wir nutzen dieses Prinzip in Bezug zellulären Reaktionen bei bekannter Intensität und der gleichen Wellenlänge zu Reaktionen bei unterschiedlichen Wellenlängen und der gleichen Intensität, aber unbekannten relative Empfindlichkeit. Zelltypen werden oft von der Wellenlänge, bei der ihre Empfindlichkeit Peaks identifiziert. Hier zeigen wir eine Methode für die intrazelluläre Aufnahme und Analyse der spektralen Empfindlichkeit von Photorezeptoren im Auge eines Schmetterlings, mit einem Fokus auf machen diese Methode besser zugänglich zu der weiteren Forschung. Obwohl die intrazelluläre Aufnahme in der Literatur häufig bleibt, vor allem in Bezug auf das Farbsehen bei Insekten, haben wir gefunden, that Beschreibungen der Materialien und Verfahren sind in der Regel zu kurz für die Wiedergabe der Technik zu ermöglichen. Wir präsentieren diese Methode im Video-Format mit dem Ziel, die leichter Replikation ermöglicht. Wir beschreiben auch die Technik leicht erhältlich und preisgünstige Ausrüstung. Wir wenden uns an gemeinsamen Vorbehalte, die oft nicht gemeldet werden, die Forschung verlangsamen, wenn eine neue und komplexe Technik zu optimieren.

determination of photoreceptor cell spectral sensitivity in an insect model from in vivo intracellular recordings

Intrazellulärer Aufnahme ist eine leistungsfähige Technik verwendet, um zu bestimmen, wie eine einzelne Zelle auf einen gegebenen Reiz reagieren. In einer Vision Forschung, hat die intrazelluläre Aufnahme war in der Vergangenheit eine übliche Technik zu studieren Empfindlichkeiten einzelner Sehzellen auf verschiedene Lichtreize verwendet, die noch heute verwendet wird. Es bleibt jedoch ein Mangel an detaillierten Methodik in der Literatur für die Forscher die intrazelluläre Aufnahme Experimente im Auge zu replizieren wollen. Hier präsentieren wir das Insekt als Modell für die Untersuchung Augenphysiologie allgemeiner. Insekten Sehzellen sind in der Nähe der Oberfläche des Auges angeordnet und sind daher leicht zu erreichen, und viele der Mechanismen in der Vision beteiligt sind, über Tierphyla konserviert. Wir beschreiben das grundlegende Verfahren für in vivo intrazelluläre Aufnahme von Sehzellen im Auge eines Schmetterlings, mit dem Ziel, diese Technik für Forscher mit wenig Erfahrung in electrophysiology. Wir stellen die Grundausstattung benötigt, wie ein Live-Schmetterling zur Vorbereitung der Aufnahme, wie ein Glas-Mikroelektrode in eine einzelne Zelle einfügen, und schließlich die Aufnahmeverfahren selbst. Wir erklären auch die grundlegende Analyse der rohen Reaktionsdaten für die spektrale Empfindlichkeit der einzelnen Zelltypen zu bestimmen. Obwohl unser Protokoll über die Bestimmung spektraler Empfindlichkeit konzentriert, andere Reize (zB polarisiertes Licht) und Variationen des Verfahrens sind zu diesem Setup anwendbar.

Für viele Elemente der Aufnahme - Setup, eine schriftliche Beschreibung liefert nicht genügend Detail. 1 ist eine schematische Darstellung der Komponenten in der gesamten Aufnahme - Setup beteiligt. In Figur 2 Spektren werden für weißes Licht und jeden Interferenzfilter ein Gefühl dafür , warum ein Korrekturfaktor benötigt wird, aufgetragen , und was benötigt wird , um diese Korrektur zu berechnen. 3 zeigt Bilder und ein Diagramm des Cardan Arm , die f?...

Watch this Scientific Journal Video about Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings at JoVE.com

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Die elektrophysiologische Technik der intrazellulären Aufnahme wird gezeigt, und verwendet, um spektralen Empfindlichkeiten einzelner Sehzellen in der Verbindung Auge eines Schmetterlings bestimmen.

Bestimmung der Sehzellen Spektrale Empfindlichkeit in einem Insekt Modell aus<em&gt; In Vivo</em&gt; Intrazellulärer Recordings

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

determination-photoreceptor-cell-spectral-sensitivity-an-insect-model

Research

JoVE Journal

Neuroscience

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

11.3K Aufrufe.  University of California, Irvine.  Die elektrophysiologische Technik der intrazellulären Aufnahme wird gezeigt, und verwendet, um spektralen Empfindlichkeiten einzelner Sehzellen in der Verbindung Auge eines Schmetterlings bestimmen. 

Serie: Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissection und Kultur kommissuralen Nervenzellen aus embryonalen Rückenmark

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Forschung

Neurowissenschaften

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Single-Einheit In-vivo- Aufnahmen aus dem Chiasma der Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograde Fluoreszenzmarkierung Ermöglicht Gezielte Extracellular Einzel-Einheit Aufnahme von identifizierten Neuronen<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Funktionelle Bildgebung mit Ultraschall-Blut-Hirnschranke Störung und Mangan-MRT

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

Konstruktion und Montage eines Ultra-light Motorisierte Microdrive für chronische Neural Recordings in Kleintiere

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

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.

Dual-Elektrophysiologische Recordings synaptisch-evozierte astrogliale und neuronale Antworten in Acute Hippocampusschnitte

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

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

Gleichzeitige Ganzzellableitungen von Photorezeptoren und Neuronen zweiter Ordnung in eine Amphibie Retinal Scheibe Vorbereitung

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Eine einfache und kostengünstige Methode zur Bestimmung der Überempfindlichkeit auf Kälte und Anpassung an der Maus

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Gleichzeitig<em&gt; Ex vivo</em&gt; Funktionsprüfung von zwei Netzhäute von<em&gt; In vivo</em&gt; Elektroretinogramm-System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Elektrophysiologische Verfahren zur Aufzeichnung von intrazellulärer Spannung Antworten von<em&gt; Drosophila</em&gt; Fotorezeptoren und Inter auf Lichtreize<em&gt; In Vivo</em

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