Soluzioni <ul><li> Soluzione intracellulare di cellule gangliari (in mm): 100 K-gluconato, 8 KCl, 1 MgCl 2, 1 EGTA, 10 HEPES, 4 ATP, GTP 0,5, 90 mM solforodamina B (per la colorazione), portato a pH = 7,4 con KOH </li><li> Soluzione intracellulare di cellule bipolari (in mm): 90 K-gluconato, 8 KCl, 1 MgCl 2, 10 Bapta, 10 HEPES, 4 ATP, GTP 0,5, 90 mM solforodamina B (per la colorazione), portato a pH = 7,4 con KOH </li><li> Ringer extracellulare della soluzione (in mm): 112 NaCl, 5 Glucosio, 5 HEPES, 2 KCl, 2 CaCl 2, 1 MgCl 2, portato a pH = 7.75 con NaOH. Ossigenazione della soluzione non è necessario. </li></ul> Preparare camere di registrazione La nostra camera di registrazione è costituito da un vetrino da microscopio con un adesivo ben in cui blocchi di silicone piccolo ritagliato da un altro adesivo e sono in grado di dare stabilità al fette di tessuto. Preparazione di fette della retina Per evitare lo sbiancamento dei fotorecettori, la dissezione viene effettuata in IR o luce fioca luce rossa. Se uno è interessato a registrare le risposte dal sentiero asta, solo luce IR deve essere usato (IR dissezione microscopio e nessuna luce ambiente. Aggiuntive occhiali a infrarossi devono essere utilizzati per le procedure non fanno sotto il microscopio). Se uno è principalmente interessato a registrare le risposte cono di luce, fioca luce della stanza rossa o una luce del flash rosso può essere utilizzato. Il microscopio, tuttavia, deve ancora oculari IR. Se l&#39;ingresso dei fotorecettori è irrilevante per l&#39;esperimento, le procedure possono essere effettuate in luce ambiente normale utilizzando un microscopio normale. <ol><li> Preparare due vetrini da microscopio con un adesivo siliconico bene: creare due bande parallele di grasso al silicone (circa 0,5 centimetri di distanza) in ogni diapositiva e luogo un pezzo rettangolare di carta da filtro su un lato del vetrino, attraversando entrambe le bande grasso (spingere il filtro carta con parte di una lama di rasoio in modo che sia ben collegato alla diapositiva) </li><li> Sacrificare una salamandra secondo i protocolli stabiliti: l&#39;animale viene posto in un bagno di ghiaccio per 30 minuti, poi decapitato e doppio pithed. </li><li> Eliminare l&#39;effetto occhi dalla testa salamandra e metterla sotto il microscopio da dissezione su un pezzo di carta velina inumidita con soluzione ghiacciata di Ringer </li><li> Rimuovere il tessuto connettivo dall&#39;occhio </li><li> Usa lama di rasoio per fare un piccolo taglio nella cornea </li><li> Tenere taglio della cornea con una pinza, mentre la rimozione con le forbici lungo il suo attaccamento per l&#39;occhio </li><li> Rimuovere l&#39;obiettivo con una pinza </li><li> Tenere l&#39;iride con una pinza e taglio lungo la serrata per rimuovere l&#39;iride </li><li> Tagliare l&#39;oculare in due pezzi rettangolari </li><li> Prendete uno dei pezzi e mettetelo lato sclera sul filtro di carta (assicuratevi di appiattirsi il pezzo mentre si spinge verso il basso con molta attenzione) </li><li> Sollevare delicatamente la sclera al largo della retina, che ora dovrà essere allegata alla carta filer </li><li> Aggiungere rapidamente una soluzione ghiacciata di Ringer al pozzo </li><li> Ripetere lo stesso per il secondo pezzo </li><li> Utilizzare una lama di rasoio posto in una affettatrice su misura per tagliare 200-300μm fette larghe. </li><li> Usare un paio di pinze per prendere le fette a lato della carta da filtro, li girare così gli strati della retina diventano visibili, e metterli su bande grasso il vuoto </li><li> Scegli una fetta che sembra intatto e mostra fasce orizzontali che indicano gli strati della retina (i livelli sono a volte difficili da vedere al microscopio dissezione come l&#39;ingrandimento non è abbastanza alta, se si può riconoscere qualche stratificazione la fetta di solito è abbastanza buono. La decisione finale sull&#39;opportunità di utilizzare la fetta, comunque, è fatta una volta si è vista al microscopio in posizione verticale). Spostare alla diapositiva camera di registrazione con la costruzione di un ponte Ringer tra i due vetrini </li><li> Assicurarsi che la carta da filtro viene spinto contro i blocchi di gomma nella camera di registrazione in modo che la fetta non sia inclinato (cellule gangliari e fotorecettori dovrebbe essere a fuoco allo stesso tempo) </li></ol> Posizionamento del tessuto nel rig registrazione <ol><li> La slitta di registrazione è posto sotto il microscopio e perfusi con soluzioni di Ringer attraverso un syste perfusione gravità </li><li> La preparazione è visualizzato con un microscopio verticale con un obiettivo 10x e 40x acqua ad immersione. </li><li> Ancora una volta, usiamo solo la luce a infrarossi per prevenire lo sbiancamento dei fotorecettori. Una telecamera a infrarossi è collegato alla porta laterale del microscopio, che è collegato ad uno schermo TV. </li></ol> Stimolazione visiva Stimoli visivi sono programmati in Matlab utilizzando la Psychtoolbox e vengono proiettate sulla retina attraverso la porta superiore del microscopio utilizzando un microscopio immagine iniettore (MBF bioscienze). A Bits + + processore video digitale (Cambridge Research Systems) è usato per ottenere una scala a 14 bit di luminanza e di sincronoonize presentazione dello stimolo con l&#39;acquisizione dei dati. Gli stimoli visivi utilizzati in questo video sono barre chiare o scure (width = 460μm) del 100% di contrasto, che sono stati presentati per due secondi su uno sfondo uniforme costante (luminanza = 8 * 10 4 fotoni / micron / s, dimensioni: 1,84 x 1,38 millimetri). Elettrofisiologia <ul><li> Elettrodi di patch sono tirati da vetro borosilicato (OD:.. 1,5 mm, ID 84mm) con un estrattore micropipetta. Per le cellule gangliari, le resistenze sono tra 2 e 4 MOhm. Per le cellule bipolari, più resistenze tra 4 e 8 MOhm sono utilizzati. </li><li> Registrazioni morsetto di tensione sono eseguiti utilizzando una 700A Multiclamp (Molecular Devices), un amplificatore e software laboratorio interno programmato in Labview. </li><li> I farmaci sono perfusi attraverso un sistema a 8 canali perfusione. L&#39;elettrodo microperfusione si trova vicino alla fetta (il tessuto dovrebbe visibilmente muoversi quando si accende la perfusione e fuori, ma bisogna stare attenti che la pressione non è troppo forte in modo che la fetta potrebbero staccarsi dalla carta da filtro). Durante gli esperimenti il ​​microperfusione deve essere costantemente acceso (soluzione di controllo perfusione se non sono necessari farmaci) in modo che la registrazione non viene disturbato dal passaggio della perfusione e si spegne. </li></ul>

<ol>
	<li>Cook, P. B., Lukasiewicz, P. D., McReynnolds, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA(C)+receptors+control+adaptive+changes+in+a+glycinergic+inhibitory+pathway+in+salamander+retina.">GABA(C) receptors control adaptive changes in a glycinergic inhibitory pathway in salamander retina.</a> Journal of Neuroscience. 20, 806-812 (2000).</li><li>Heflin, S. J., Cook, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narrow+and+wide+field+amacrine+cells+fire+action+potentials+in+response+to+depolarization+and+light+stimulation.">Narrow and wide field amacrine cells fire action potentials in response to depolarization and light stimulation.</a> Visual Neuroscience. 24, 197-206 (2007).</li><li>Ichinose, T., Shields, C. R., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+Channels+in+the+Transient+Retinal+Bipolar+Cells+Enhance+Visual+Responses+in+Ganglion+Cells.">Sodium Channels in the Transient Retinal Bipolar Cells Enhance Visual Responses in Ganglion Cells.</a> J Neurosci.. 25, 1856-1865 (2005).</li></ol>

Vantaggi: <ol><li> Tutti i tipi di cellule sono accessibili </li><li> Facile identificazione dei tipi di cellule, in particolare se un colorante fluorescente viene aggiunto alla soluzione elettrodo </li><li> Agenti farmacologici possono facilmente raggiungere le cellule bersaglio </li><li> La tecnica di patch clamp consente di indagare il ruolo dei canali ionici diversi nei calcoli della retina. Le stesse informazioni non possono essere ottenute attraverso le registrazioni extracellulare picco o registrazioni con elett...

<table><tbody><tr><td>Multiclamp 700A</td><td>Patch Clamp Amplifier</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>SZX9</td><td>Dissection Micropscope</td><td>Olympus Corporation</td><td></td><td></td></tr><tr><td>BX50WI</td><td>Upright Microscope</td><td>Olympus Corporation</td><td></td><td>Equipped with 40x, 10x water immersion objective, fluorescent filters and mercury lamp</td></tr><tr><td>P-97</td><td>Micropipette Puller</td><td>Sutter Instrument Co.</td><td></td><td></td></tr><tr><td>Lucivid</td><td>Microscope image injector</td><td>MBF Bioscience</td><td></td><td></td></tr><tr><td>8-channel perfusion system</td><td>Microperfusion</td><td>Parker Hannifin Corporation</td><td></td><td></td></tr><tr><td>Bits++</td><td>Digital Video Processor</td><td>Cambridge Research Systems</td><td></td><td></td></tr><tr><td>Infrared Oculars</td><td>Other</td><td>ITT Visual Information Solutions</td><td></td><td></td></tr><tr><td>Adhesive silicone wells</td><td>Other</td><td>Molecular Probes, Life Technologies</td><td></td><td>20mm diameter, 1.0mm deep</td></tr><tr><td>Membrane Filters</td><td>Filter</td><td>EMD Millipore</td><td>HAWPO1300</td><td>.45&amp;#x03BC;m HA</td></tr><tr><td>Borosilicate Glass Capillaries</td><td>Electrode glass</td><td>World Precision Instruments, Inc.</td><td>1B150-4</td><td></td></tr><tr><td>Syringe Filters</td><td>Filter</td><td>Whatman, GE Healthcare</td><td>6789-0402</td><td>4mm filters, .2um Nylon Membrane, Polypropylene housing</td></tr><tr><td>IR camera</td><td>Micropscope mounted camera</td><td>Sony Corporation</td><td>SPT-M324</td><td>Extrawave HAD, B7W video camera</td></tr><tr><td>Picrotoxin</td><td>Reagent</td><td>Sigma-Aldrich</td><td>P1675</td><td></td></tr><tr><td>Strychnine</td><td>Reagent</td><td>Sigma-Aldrich</td><td>S0532</td><td></td></tr><tr><td>Imidazole-4-acetic acid sodium salt</td><td>Reagent</td><td>Sigma-Aldrich</td><td>I7013</td><td></td></tr><tr><td>Larval tiger salamanders</td><td>Animal</td><td>Charles E. Sullivan</td><td></td><td></td></tr></tbody></table>

whole-cell recordings of light evoked excitatory synaptic currents in the retinal slice

Usiamo la cellula intera tecnica di patch clamp per lo studio dei circuiti sinaptici che sottende l&#39;elaborazione delle informazioni visive nella retina. In questo video, vi guiderà attraverso il processo di esecuzione a cellula intera registrazioni di luce correnti evocato delle singole cellule nella preparazione fetta della retina. Usiamo il acquatici tigre salamandra come un modello animale. Cominciamo con la descrizione della dissezione degli occhi e mostrare come le fette sono montati per le registrazioni elettrofisiologiche. Una volta che la fetta si trova nella camera di registrazione, dimostriamo come eseguire a cellula intera registrazioni clamp di tensione. Abbiamo poi progetto stimoli visivi sul fotorecettori nella fetta di suscitare luce evocata attuali risposte. Durante la registrazione abbiamo profumato la fetta con agenti farmacologici, per cui un sistema a 8 canali perfusione ci permette di passare velocemente tra diversi agenti. La preparazione fetta della retina è ampiamente usato per le registrazioni di patch clamp nella retina, in particolare per studiare amacrine e cellule bipolari, che non sono accessibili in un tutto-mount preparazione.

Watch this Scientific Journal Video about Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice at JoVE.com

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

Questo video mostra il processo di registrazione di tensione a cellula intera pinza nella fetta della retina della tigre salamandra acquatica. Dimostriamo la preparazione della fetta e come effettuare registrazioni di patch clamp durante la stimolazione visiva della retina.

A cellula intera Recordings of Light Evocati eccitatorio Correnti Synaptic nel Slice Retinal

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

whole-cell-recordings-light-evoked-excitatory-synaptic-currents

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.7K Views.  Boston University. Questo video mostra il processo di registrazione di tensione a cellula intera pinza nella fetta della retina della tigre salamandra acquatica. Dimostriamo la preparazione della fetta e come effettuare registrazioni di patch clamp durante la stimolazione visiva della retina.

Video: Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

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.

Bloccare Registrazioni patch da neuroni della retina del mouse in un Dark-adattato Preparazione Slice

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

Ricerca

Neuroscienze

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals 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 retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

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

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Patch-clamp Misure di capacità e Ca 2 + Imaging terminazioni nervose della retina singola in Fette

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

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.

Registrazioni simultanee a cellule di fotorecettori e dei neuroni di secondo ordine in un anfibio Retinal Fetta Preparazione

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

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Registrazione Light-evocato Risposte postsinaptica nei neuroni in Dark-adattati, i preparativi della retina del mouse Slice Utilizzando tecniche di patch clamp

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imaging Ca<sup&gt; 2+</sup&gt; Dinamica a cono fotorecettore Axon Terminali della retina di topo

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Preparazione di orizzontali Fette di topo adulto Retina per elettrofisiologiche Studi

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

Indagini elettrofisiologiche sulla funzione di sinapla Retinogeniculate e Corticogeniculate

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

Studio delle cellule neuronali bipolari e orizzontali nella retina divisa murina

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.

Autore in primo piano: Utilizzo della tecnica Split Retina per un accesso migliorato e esperimenti accelerati 

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.

A cellula intera Recordings of Light Evocati eccitatorio Correnti Synaptic nel Slice Retinal

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Bloccare Registrazioni patch da neuroni della retina del mouse in un Dark-adattato Preparazione Slice

Patch-clamp Misure di capacità e Ca^{2 +} Imaging terminazioni nervose della retina singola in Fette

Registrazioni simultanee a cellule di fotorecettori e dei neuroni di secondo ordine in un anfibio Retinal Fetta Preparazione

Registrazione Light-evocato Risposte postsinaptica nei neuroni in Dark-adattati, i preparativi della retina del mouse Slice Utilizzando tecniche di patch clamp

Imaging Ca

Preparazione di orizzontali Fette di topo adulto Retina per elettrofisiologiche Studi

Indagini elettrofisiologiche sulla funzione di sinapla Retinogeniculate e Corticogeniculate

Retina divisa come preparazione flatmount migliorata per lo studio dei neuroni dello strato nucleare interno nella retina dei vertebrati

Retina divisa come preparazione flatmount migliorata per lo studio dei neuroni dello strato nucleare interno nella retina dei vertebrati

Retina divisa come preparazione flatmount migliorata per lo studio dei neuroni dello strato nucleare interno nella retina dei vertebrati