Funding for this project was provided by the National Health and Medical Research Council (NHMRC) 1046203 (BVB, AJV) and Melbourne Neuroscience Institute Fellowship (CTN).

<ol>
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The authors have no disclosures relevant to this work.

L&#39;ERG e VEP sono misure oggettive della funzione visiva dalla retina e la corteccia, rispettivamente. Il vantaggio di registrazione simultanea è che una visione più completa di tutto il percorso visivo è prestata. In particolare, le informazioni complementari da loro valutazione concomitante potrebbe fornire una più chiara delimitazione del sito di lesioni in via visiva (per esempio, per i disturbi con sovrapposizione ERG ancora distinto VEP manifestazioni 18, quando neuropatia ottica può coesistere con primaria atrofia cerebrale 19, 20, o quando la perdita di VEP può essere confuso con la manifestazione di lesioni in diverse località nel percorso visivo 21,22). Misurando la ERG e PEV contemporaneamente, un indice di guadagno tra la risposta della retina e corticale può anche essere derivato. Questo può fornire un utile strumento per rilevare i cambiamenti patologici sottili. L&#39;attuale protocollo consente la misurazione ERG e PEV nei ratti di laboratorio comunemente usati, ma può facilmente essere annuncioApted ad altre specie di mammiferi 23-25. ERG e PEV forme d&#39;onda da roditori forniscono un surrogato preclinico ragionevole per risposte osservate in occhi umani 26-28. Progettando uno specifico protocollo di stimolo, sia la risposta ERG e PEV può essere ottenuto durante una singola sessione di registrazione. La tabella 1 mostra una progressione a livelli di luce con un&#39;adeguata considerazione del tempo di recupero tra lampi consecutivi. Questo protocollo fornisce un equilibrio tra la necessità di ottimizzare le caratteristiche di segnale-rumore e per limitare il tempo di registrazione nella finestra anestetico fornito da una singola dose di ketamina: xilazina. Pertanto, questa tecnica può essere utile per una misura quantitativa oggettiva della funzione visiva per la ricerca in fisiologia di base e la malattia. Una valutazione completa del sistema visivo può essere ottenuto valutando contemporaneamente le risposte retiniche bilaterali e le risposte corticali evocati visivamente. Tuttavia, ogni tecnica può anche essere condotta in isolamento e monocularmente anziché binocularmente semplificare la procedura. L&#39;attuale protocollo descrive scotopica segnali ERG e PEV scelti per isolare l&#39;asta-via dato che i ratti hanno una retina asta-dominato. Se le risposte luce adattato sono di maggiore interesse per lo studio, è anche possibile condurre fotopica segnali ERG e PEV pre-adattamento ad uno sfondo chiaro. Una limitazione importante di questa tecnica è la necessità di condurre la procedura in condizioni anestetizzati per consentire il posizionamento degli elettrodi stabile. Tuttavia questo approccio fornisce caratteristiche di resistenza del segnale-rumore che permettono la rilevazione di cambiamenti di trattamento sottili. A causa della piccola ampiezza del STR e la sua sensibilità alla luce adattamento, diversi passaggi devono essere attentamente controllati per garantire una scrittura di questa risposta. In primo luogo, sufficiente adattamento al buio deve essere attuata, che comprendedurante la notte adattamento al buio (≥ 8 ore), il posizionamento degli elettrodi in condizioni di luce rossa fioca (17,4 cd.m -2, λ max = 600 nm), e l&#39;adattamento ri-scuro a seguito di una fioca di test-flash (10 min per - 0,52 log cd. sm -2). Inoltre, le caratteristiche del segnale-rumore del STR possono essere migliorate la media su più segnali (cioè, 20 segnali) raccolti con brevi intervalli inter-stimolo (cioè 2 sec). Uno dei vantaggi di questa valutazione globale di entrambi gli occhi e cortecce è quello di consentire un confronto alla registrazione controlaterale 3. In quanto tale, particolare attenzione dovrebbe essere presa nel processo elettrodo (vale a dire, stessi elettrodi di dimensioni e forma), al fine di garantire il minimo tra gli occhi e la variabilità inter-corticale. Dato l&#39;ampio uso di entrambe le tecniche ERG e PEV per fornire a misure in vivo del percorso visivo e dei suoi processi correlati alla malattia, sarebbe utile per raccogliere altre specifiche del percorso protocols (ad esempio, ON / OFF o cono sottotipo specifico), e di eseguire simultaneamente le registrazioni ERG / VEP con diverse modalità di stimolo (ad esempio, sfarfallio, modello, dente di sega) per estendere l&#39;applicazione di questa tecnica nella diagnosi clinica. Un altro passo logico di questa applicazione in futuro sarebbe anche di registrare l&#39;ERG e PEV contemporaneamente da 29 cosciente, animali liberamente muoversi per evitare influenze anestetici sulla fisiologia neurale 30.

Misura della elettroretinogramma (ERG) e potenziali evocati visivi (PEV) fornire valutazioni quantitative utili dell&#39;integrità della via visiva. L&#39;ERG misura le risposte elettriche della retina alla stimolazione luminosa, mentre il VEP misura corrispondente integrità funzionale delle vie visive dalla retina alla corteccia visiva primaria seguendo lo stesso evento luce. Questo manoscritto descrive un protocollo per la registrazione e analisi delle risposte ERG e PEV in un modello di laboratorio comunemente usato, il ratto. Il ERG fornisce un indice della integrità funzionale di un certo numero di classi principali di cellule retiniche quantificando risposta elettrica lordo del retina un lampo di luce. Una serie coordinata di flussi ionici iniziata da insorgenza luce e offset, producono cambiamenti rilevabili in tensione che può essere misurato con elettrodi di superficie posizionati al di fuori dell&#39;occhio. La forma d&#39;onda risultante rappresenta la combinazione di un séRies di componenti ben definiti, che differiscono in ampiezza, i tempi e la frequenza. Una importante ricerca ha dimostrato che questi componenti sono relativamente ben conservati tra i molti retine vertebrati e che i componenti possono essere separati l&#39;uno dall&#39;altro. Selezionando giudiziosamente lo stimolo (stimolo flash, sfondo, intervallo interstimulus) le condizioni e la scelta di caratteristiche specifiche della forma d&#39;onda composita di analizzare si può essere sicuri di tornare una misura di un gruppo specifico di cellule retiniche 1,2. Queste caratteristiche sono alla base del programma di utilità e quindi le applicazioni diffuse della ERG come una misura non invasiva della funzione della retina. Questo manoscritto si concentra sulla metodologia per misurare l&#39;ERG e analizzando le sue caratteristiche per restituire informazioni su alcune delle principali classi di cellule nella retina, vale a dire fotorecettori (il componente PIII), cellule bipolari (il componente PII) e le cellule gangliari della retina (il positivo risposta soglia scotopica o PSTR). <p class= &quot;Jove_content&quot;&gt; Il VEP offre un saggio della risposta corticale alla luce; prima proveniente dalla retina e successivamente comunicato seriale tramite il nervo ottico, tratto ottico, talamo (genicolato laterale nucleo, LGN) e la radiazione ottica all&#39;area V1 della corteccia 3. Nei roditori, la maggioranza (90 - 95%) di fibre del nervo ottico da ciascun occhio decussate 4 e innervano controlaterale cervello medio. A differenza della ERG, è ancora possibile attribuire diversi componenti del VEP a classi cellulari specifici, 5 cambia così ovunque lungo il percorso visivo potrebbe influenzare la forma d&#39;onda VEP. Tuttavia, il VEP è una misura utile non invasiva di prestazioni visive e dell&#39;integrità via visiva. Il VEP, quando utilizzato in combinazione con l&#39;ERG, può fornire una valutazione più completa del sistema visivo (cioè, retina / via visiva). ERG e PEV registrazioni possono essere condotte isolatamente o in combinazione, a seconda del applicazione. La metodologia descritta in questo documento permette la valutazione contemporanea di elettrofisiologia evocati visivi della retina e corticale da entrambi gli occhi e entrambi gli emisferi nei ratti anestetizzati. Questo è un modo utile per valutare in modo più completo la funzione della retina e gli effetti a monte che i cambiamenti nella funzione retinica possono avere sulla funzione corticale evocati visivi.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alligator clip</td>
			<td>generic brand</td>
			<td>HM3022</td>
			<td>Stainless steel 26 mm clip for connecting VEP screw electrodes to cables</td>
		</tr>
		<tr>
			<td>Bioamplifier</td>
			<td>ADInstruments</td>
			<td>ML 135</td>
			<td>For amplifying ERG and VEP signals</td>
		</tr>
		<tr>
			<td>Carboxymethylcellulose sodium 1.0%</td>
			<td>Allergan</td>
			<td>CAS 0009000-11-7</td>
			<td>Viscous fluid for improving signal quality of the active ERG electrode</td>
		</tr>
		<tr>
			<td>Carprofen 0.5%</td>
			<td>Pfizer Animal Health Group</td>
			<td>CAS 53716-49-7</td>
			<td>Proprietary name: Rimadyl injectable (50 mg/mL). For post-surgery analgesia, diluted to 0.5% (5 mg/mL) in normal saline</td>
		</tr>
		<tr>
			<td>Chlorhexadine 0.5%</td>
			<td>Orion Laboratories</td>
			<td>27411, 80085</td>
			<td>For disinfecting surgical instruments</td>
		</tr>
		<tr>
			<td>Circulating water bath</td>
			<td>Lauda-K&#246;nigshoffen</td>
			<td>MGW Lauda</td>
			<td>For maintaining body temperature of the anesthetized animal during surgery and electrophysiological recordings</td>
		</tr>
		<tr>
			<td>Dental amalgam</td>
			<td>DeguDent GmbH</td>
			<td>64020024</td>
			<td>For encasing the electrode-skull assembly to make it more robust</td>
		</tr>
		<tr>
			<td>Dental burr</td>
			<td>Storz Instruments, Bausch and Lomb</td>
			<td>#E0824A</td>
			<td>A miniature drill head of ~0.7mm diameter for making a small hole in the skull over each hemisphere to implant VEP screws</td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Bosch</td>
			<td>Dremel 300 series</td>
			<td>An automatic drill for trepanning</td>
		</tr>
		<tr>
			<td>Electrode lead</td>
			<td>Grass Telefactor&#160;</td>
			<td>F-E2-30</td>
			<td>Platinum cables for connecting silver wire electrodes to the amplifier</td>
		</tr>
		<tr>
			<td>Faraday Cage</td>
			<td>custom-made</td>
			<td></td>
			<td>Ensures light proof to maintain dark adaptation. Encloses the Ganzfeld setup to improve signal to noise ratio</td>
		</tr>
		<tr>
			<td>Gauze swabs</td>
			<td>Multigate Medical Products Pty Ltd</td>
			<td>57-100B</td>
			<td>For drying the surgical incision and exposed skull surface during surgery</td>
		</tr>
		<tr>
			<td>Ganzfeld integrating sphere</td>
			<td>Photometric Solutions International</td>
			<td></td>
			<td>Custom designed light stimulator: 36 mm diameter, 13 cm aperture size</td>
		</tr>
		<tr>
			<td>Velcro</td>
			<td>VELCRO Australia Pty Ltd</td>
			<td>VELCRO Brand Reusable Wrap</td>
			<td>Hook-and-loop fastener to secure the electrodes and the animal on the recording platform</td>
		</tr>
		<tr>
			<td>Isoflurane 99.9%</td>
			<td>Abbott Australasia Pty Ltd</td>
			<td>CAS 26675-46-7</td>
			<td>Proprietary Name: Isoflo(TM) Inhalation anaaesthetic. Pharmaceutical-grade inhalation anesthetic mixed with oxygen gas for VEP electrode implant surgery</td>
		</tr>
		<tr>
			<td>Ketamine&#160;</td>
			<td>Troy Laboratories</td>
			<td>Ilium Ketamil</td>
			<td>Proprietary name: Ketamil Injection, Brand: Ilium. Pharmaceutical-grade anesthetic for electrophysiological recording</td>
		</tr>
		<tr>
			<td>Luxeon LEDs</td>
			<td>Phillips Lighting Co.</td>
			<td></td>
			<td>For light stimulation twenty 5 watt and one 1 watt LEDs.</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Harvard Apparatus</td>
			<td>BS4 50-2625</td>
			<td>Holds the ERG active electrode during recordings</td>
		</tr>
		<tr>
			<td>Needle electrode</td>
			<td>Grass Telefactor&#160;</td>
			<td>F-E2-30</td>
			<td>Subcutaneously inserted in the tail to serve as the ground electrode for both the ERG and VEP</td>
		</tr>
		<tr>
			<td>Phenylephrine 2.5% minims&#160;</td>
			<td>Bausch and Lomb</td>
			<td>CAS 61-76-7</td>
			<td>Instilled with Tropicamide to achieve maximal dilation for ERG recording</td>
		</tr>
		<tr>
			<td>Povidone iodine 10%</td>
			<td>Sanofi-Aventis</td>
			<td>CAS 25655-41-8</td>
			<td>Proprietory name: Betadine, Antiseptic to prepare the shaved skin for surgery 10%, 500 mL</td>
		</tr>
		<tr>
			<td>Powerlab data acquisition system</td>
			<td>ADInstruments</td>
			<td>ML 785</td>
			<td>Controls the LEDs</td>
		</tr>
		<tr>
			<td>Proxymetacaine 0.5%</td>
			<td>Alcon Laboratories&#160;</td>
			<td>CAS 5875-06-9</td>
			<td>For corneal anaesthesia during ERG recordings</td>
		</tr>
		<tr>
			<td>Saline solution</td>
			<td>Gelflex</td>
			<td></td>
			<td>Non-injectable, for electroplating silver wire electrodes</td>
		</tr>
		<tr>
			<td>Scope Software</td>
			<td>ADInstruments</td>
			<td>version 3.7.6</td>
			<td>Simultaneously triggers the stimulus via the Powerlab system and collects data</td>
		</tr>
		<tr>
			<td>Silver (fine round wire)</td>
			<td>A&#38;E metal</td>
			<td>0.3 mm</td>
			<td>Used to make active and inactive ERG electrodes, and the inactive VEP electrode</td>
		</tr>
		<tr>
			<td>Stainless streel screws&#160;</td>
			<td>MicroFasterners</td>
			<td></td>
			<td>0.7 mm shaft diameter, 3 mm in length to be implanted over the primary visual cortex and serve as the active VEP electrodes</td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>David Kopf</td>
			<td>Model 900</td>
			<td>A small animal stereotaxic instrument for locating the primary visual cortices according to Paxinos &#38; Watson&#39;s 2007 rat brain atlas coordinates</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Swann-Morton Ltd.</td>
			<td>0206</td>
			<td>For incising the area of skin overlaying the primary visual cortex to implant the VEP electrodes</td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co.,Ltd</td>
			<td></td>
			<td>3-0 silk braided suture non-absorbable, for skin retraction during VEP electrode implantation surgery</td>
		</tr>
		<tr>
			<td>Tobramycine eye ointment 0.3%</td>
			<td>Alcon Laboratories&#160;</td>
			<td>CAS 32986-56-4</td>
			<td>Proprietary name: Tobrex. Prophylactic antibiotic ointment applied around the skin wound after surgery</td>
		</tr>
		<tr>
			<td>Tropicamide 0.5%</td>
			<td>Alcon Laboratories&#160;</td>
			<td>CAS 1508-75-4</td>
			<td>Proprietary name: 0.5% Mydriacyl eye drop, Instilled to achieve mydriasis for ERG recording</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Troy Laboratories</td>
			<td>Ilium Xylazil-100</td>
			<td>Pharmaceutical-grade anesthetic for electrophysiological recording</td>
		</tr>
		<tr>
			<td>Pipette tip&#160;</td>
			<td>Eppendorf Pty Ltd</td>
			<td>0030 073.169</td>
			<td>Eppendorf epTIPS 100 - 5000 mL, for custom-made electrodes</td>
		</tr>
		<tr>
			<td>Microsoft Office Excel</td>
			<td>Microsoft</td>
			<td>version 2010</td>
			<td>spreadsheet software for data analysis</td>
		</tr>
		<tr>
			<td>Lethabarb Euthanazia Injection</td>
			<td>Virbac (Australia) Pty Ltd</td>
			<td>LETHA450</td>
			<td>325 mg/mL pentobarbital sodium for rapid euthanazia</td>
		</tr>
	</tbody>
</table>

simultaneous recording of electroretinography and visual evoked potentials in anesthetized rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

L&#39;ERG un onda (&gt; -1.38 log cd.sm -2), b-onde (&gt; - 4,99 log cd.sm -2) STR (&lt;- 4,99 log cd.sm -2) e le PEV (&gt; - 0,52 cd.sm log -2) sono state registrate contemporaneamente (Figura 1 e 3). Al molto dim lampeggia, un STR positivo (PSTR) è visto a circa 110 msec dopo il flash, e STR ​​negativo (nSTR) in circa 220 msec (Figure 1 e 2). Un ERG con un grande b-wave, picchi tra 50 a 100 msec dopo l&#39;inizio di un lampo moderata che può essere analizzato per la sua risposta PII (Figure 1 e 2). A questa energia stimolo, il negativo di una onda prima del picco è trascurabile. Ad energie luminose luminose deflessione negativo a-onda diventa più importante che può essere quantificata con la risposta PIII (Figura 2). La forma d&#39;onda scotopic VEP mostra una risposta negativa (P1N1; 15 - 70 finestra msec) seguito da<html> una deviazione positiva (N1P2; 30 - 100 msec) (figure 3 e 4). <img alt="Figura 1" src="/files/ftp_upload/54158/54158fig1.jpg" /> Figura 1. Gruppo medio ERG forme d&#39;onda. L&#39;ERG altera con crescente intensità dello stimolo. Numeri a sinistra della forma d&#39;onda indicano l&#39;esposizione luminosa utilizzata per suscitare la forma d&#39;onda. Notare le diverse scale di ampiezza e di tempo per ogni pannello. Al dimmer energie luminose le componenti positive e negative della risposta soglia di scotopica possono essere suscitato (PSTR, nSTR). Come le energie di stimolo ottenere più luminoso, la A e B-wave risposta può essere analizzato, e un paradigma accoppiato-flash permette la risposta cono da misurare. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54158/54158fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> PLOAD / 54158 / 54158fig2.jpg &quot;/&gt; Figura 2.. (A) funzione fotorecettore asta analisi ERG può essere saggiata usando un PIII per modellare l&#39;a-onda. A-onde a 1,22 e 1,52 log cd.sm -2 (cerchi vuoti, ○) sono in forma come un insieme con un PIII (linee grigie, l&#39;equazione 1) al 90% del minimo che restituisce Rm PIII (ampiezza saturi, mV) S (sensibilità, m 2 .CD -1 .s -3) e TD (ritardo di temporizzazione, msec) parametri. (B) la funzione delle cellule bipolari Rod (media ± SEM) possono essere analizzati modellando la serie risposta intensità del PII dell&#39;asta (cerchi vuoti ○) con un (linea grigia) funzione di Naka-Rushton. Ciò restituisce V max (ampiezza saturo, mV), k (1 / sensibilità, log cd sm -2) e n (pendenza). (C) la funzione delle cellule gangliari della retina è analizzato ad energie luminose fioche e quantificato da PSTR picco di ampiezza (PSTR amp) e tempi (PSTR esso </sub&gt;). (D) la funzione delle cellule bipolari Cono è suscitato con un paradigma abbinato-flash quantificato dal cono PII picco di ampiezza (cono PII amp) e la tempistica (cono PII esso). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54158/54158fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54158/54158fig3.jpg" /> Figura 3. Gruppo medio VEP forme d&#39;onda. La forma della forma d&#39;onda PEV altera al crescere dell&#39;energia stimolo. I numeri a sinistra della forma d&#39;onda indicano l&#39;esposizione luminosa utilizzata per suscitare la forma d&#39;onda. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54158/54158fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/54158/54158fig4.jpg" /> Figure 4. Analisi VEP e funzione di risposta intensità. (A) Analisi ampiezza del VEP è preso come massimo al minimo (P1N1) e concentrazioni minime e massime ampiezze (N1P2). I tempi impliciti (it) di queste risposte è anche restituito (P1 esso, N1 esso, P2 esso). (B) L&#39;ampiezza VEP P1N1 (media ± SEM) aumenta con l&#39;aumentare l&#39;energia dello stimolo. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54158/54158fig4large.jpg" target="_blank">Cliccate qui per vedere una versione più grande questa figura.</a>

Watch this Scientific Journal Video about Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats at JoVE.com

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

Registrazione simultanea di elettroretinografia e Potenziali evocati visivi nei ratti anestetizzati

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

simultaneous-recording-electroretinography-visual-evoked-potentials

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.2K Views.  University of Melbourne. This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats. 

Video: Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

Registrazione Ensembles neuronali su larga scala con sonde di silicio sotto Rat anestetizzati

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

Ricerca

Neuroscienze

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 ...

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 ...

Extracting Visual Evoked Potentials from EEG Data Recorded During fMRI-guided Transcranial Magnetic Stimulation

Transcranial Magnetic Stimulation (TMS) is an effective method for establishing a causal link between a cortical area and cognitive/neurophysiological effects. Specifically, by creating a transient interference with the normal activity of a target region and measuring changes in an electrophysiological signal, we can establish a causal link between the stimulated brain area or network and the electrophysiological signal that we record. If target brain areas are functionally defined with prior fMRI scan, TMS could be used to link the fMRI activations with evoked potentials recorded. However, conducting such experiments presents significant technical challenges given the high amplitude artifacts introduced into the EEG signal by the magnetic pulse, and the difficulty to successfully target areas that were functionally defined by fMRI. Here we describe a methodology for combining these three common tools: TMS, EEG, and fMRI. We explain how to guide the stimulator&#39;s coil to the desired target area using anatomical or functional MRI data, how to record EEG during concurrent TMS, how to design an ERP study suitable for EEG-TMS combination and how to extract reliable ERP from the recorded data. We will provide representative results from a previously published study, in which fMRI-guided TMS was used concurrently with EEG to show that the face-selective N1 and the body-selective N1 component of the ERP are associated with distinct neural networks in extrastriate cortex. This method allows us to combine the high spatial resolution of fMRI with the high temporal resolution of TMS and EEG and therefore obtain a comprehensive understanding of the neural basis of various cognitive processes.

This paper describes a method for collecting and analyzing electroencephalography (EEG) data during concurrent transcranial magnetic stimulation (TMS) guided by activations revealed with functional magnetic resonance imaging (fMRI). A method for TMS artifact removal and extraction of event related potentials is described as well as considerations in paradigm design and experimental setup.

Estrazione Potenziali visivi evocati da EEG dati registrati durante fMRI-guidata Stimolazione Magnetica Transcranica

Transcranial Magnetic Stimulation (TMS) is an effective method for establishing a causal link between a cortical area and cognitive/neurophysiological ...

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.

Simultaneo<em&gt; Ex vivo</em&gt; Test funzionale di due retine per<em&gt; In vivo</em&gt; Sistema Elettroretinogramma

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

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

Registrazione potenziale evocato visivo in un modello murino di sperimentale del nervo ottico demielinizzazione

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

acuto<em&gt; In Vivo</em&gt; Registrazioni elettrofisiologiche dei potenziali del campo locale e dell&#39;attività multi-unità dal percorso iperdirezionale nei ratti anestetizzati

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Stimolazione elettrica transcranica cronica e registrazione intracorticale in ratti

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

Registrazioni di potenziali evocate visive nei topi utilizzando un multicanale Non invasiva a secco del cuoio capelluto EEG sensore

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

Modelli morfologici potenziali evocati di tempo evocati da corticali specifici dello stimolo

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...