Supported by grants from CIHR (MOP231122) and NSERC (RGPIN-2014-06503). We would like to thank Genevi&#232;ve Cyr for her help preparing experiments and supervising laboratory work. MAL received a scholarship from The Natural Sciences and Engineering Research Council of Canada (NSERC).

NOTA: Tutte le procedure sono state eseguite in conformità alle direttive del Consiglio canadese per la protezione degli animali e la revisione consiglio Etico della Université de Montréal. 1. Assemblea della pipetta registrazione iniezione <ol><li> Estrarre un capillare di vetro di circa 7 cm di lunghezza (diametro esterno 1 mm) con un estrattore pipetta. </li><li> Rompere la punta del capillare e controllare l&#39;apertura al microscopio ottico. Verificare che il diametro interno è compreso tra 30 micron a 40 micron. </li><li> Inserire un lungo filo Silver 7 cm nel capillare di vetro con circa 1 cm sporgenti dall&#39;estremità non rastremata della pipetta di vetro. </li><li> Piegare il filamento eccesso ortogonalmente al capillare di vetro. </li><li> Applicare una goccia di adesivo plastica flessibile sull&#39;albero di un ago ipodermico 30 G. </li><li> Inserire un ago ipodermico 30 G nella pipetta di vetro secondo gli schemi presentati nella figura 1 </strong&gt;. </li><li> Aggiungere un secondo strato di colla per garantire un&#39;adeguata tenuta dalla giunzione tra la pipetta di vetro e l&#39;ago ipodermico. </li><li> Lasciare la pipetta asciugare con la punta rivolta verso l&#39;alto per circa 12 ore per garantire il corretto indurimento della colla. Il risultato finale è mostrato in Figura 2. </li></ol> NOTA: Questa procedura viene eseguita su esperimenti acute e la sterilizzazione del puntale non è necessaria. 2. Preparazione degli animali <ol><li> Posizionare il topo in una scatola di anestesia. </li><li> Indurre l&#39;anestesia con il 4% isoflurano per 5 a 10 min. </li><li> Posto l&#39;animale su un tavolo stereotassico con il rilievo di riscaldamento e sonda rettale per mantenere una temperatura corporea di 37 ° C. Utilizzare un cono per mantenere l&#39;anestesia con il 2% isoflurano. Fissare la testa del topo con le orecchie bar e titolare denti. </li><li> Applicare unguento oftalmico o collirio, che includono 1% Atropina scende a facilitare la dilatazione della pupilla. Per prevenire la secchezza, applicare goccia lubrificantes circa ogni 30 min. </li><li> Shave testa e pulirlo con 10% iodopovidone. </li><li> Per anestesia locale, iniettare 0,5 ml di 2% lidocaina sotto il cuoio capelluto in 2-3 posizioni sollevando la pelle ed inserendo la punta dell&#39;ago. </li><li> Conferma adeguato livello di anestesia eseguendo un pizzico piedi e osservando la mancanza di movimento. Inoltre, monitorare la frequenza cardiaca per garantire che sia entro valori normali (da 300 a 400 battiti / min). </li><li> Incidere il cuoio capelluto in una linea retta lungo la mediana con una lama di 10 # bisturi per esporre entrambe le suture coronale e sagittale. </li><li> Rivela i punti di Lambda e Bregma spingendo da parte del tessuto che copre il cranio con una spatola chirurgico. </li><li> Livellare il cranio in modo che le posizioni bregma e lambda sono sullo stesso piano. </li><li> Per impostare il punto di riferimento, utilizzare un dispositivo stereotassico con un tubo di vetro montato impostarla proprio sopra Bregma. Questo sarà il &quot;zero&quot; antero-posteriore e medcoordina le misure ial-laterali. </li><li> Impostare il punto di interesse spostando il stereotassico di montaggio alle coordinate richieste, annotare le coordinate stereotassica e disegnare un quadrato intorno alla zona di destinazione che segna dove verrà eseguita la craniotomia. </li><li> Utilizzare un trapano chirurgico con un trapano sterilizzato lungo la piazza marcata lentamente senza pressione per rimuovere lentamente il materiale osseo. Fare attenzione a non forare troppo a lungo nella stessa zona, in quanto la produzione di calore e causare lesioni della corteccia. </li><li> Quando l&#39;osso che delimita la craniotomia è diventata sufficientemente sottile, rimuovere con attenzione la sezione del cranio con le pinzette per esporre la corteccia. </li><li> Irrigare Spesso la corteccia esposta con liquido cerebro-spinale artificiale per evitare l&#39;essiccamento dei tessuti. NOTA: la rimozione mater Dura è inutile per il ratto come la punta di un injectrode è abbastanza robusto da penetrare. </li></ol> 3. Riempimento e montaggio del sistema di iniezione <ol><li> Riempire il5-10 microlitri microsiringa da aspirazione con olio minerale. </li><li> Riempire il ago ipodermico con la pipetta di vetro fissa con una soluzione di 0,5% Chicago Sky Blu (CSB) e acido 300 pM γ-amminobutirrico (GABA), o una soluzione di 2% lidocaina con 0,5% CSB 9. Diluire tutte le soluzioni con soluzione salina. <ol><li> Nel caso di una sostanza abbondante, riempire con una siringa regolare e utilizzare le usuali tecniche precauzionali per evitare la formazione di bolle d&#39;aria. </li><li> Nel caso di sostanze più costose, utilizzare olio minerale per riempire il injectrode e l&#39;agente chimico può quindi essere introdotto tramite aspirazione. Poiché la differenza di densità tra l&#39;olio minerale e l&#39;acqua è relativamente elevata, questa sostanza è un buon candidato per l&#39;iniezione di soluzioni acquose. NOTA: Un colorante può essere aggiunto per confermare la separazione tra i due liquidi. </li></ol></li><li> Per riempire la pipetta di iniezione, riempire una siringa da 1 ml con la soluzione per aspirazione e poi iniettare lentamente la soluzionenella pipetta di iniezione. </li><li> Prestare attenzione per perdite in regioni indicate in figura 1 con tampone queste aree pulite e osservando perdite iniettando ulteriormente la soluzione lentamente con la siringa. </li><li> Rimuovere la siringa da 1 ml. Nel fare così, essere sicuri di mantenere una leggera pressione sullo stantuffo in modo che il vuoto non rimuove la soluzione dalla pipetta iniezione. </li><li> Riempire la microsiringa con olio minerale tramite aspirazione e fissarlo saldamente alla pipetta di iniezione riempita, poi pulire attentamente tutta la soluzione in eccesso dalla injectrode assemblato con una garza. </li><li> Verificare che la punta non è bloccato iniettando un volume molto piccolo, abbastanza per vedere una piccola goccia formando sulla punta della pipetta di vetro. </li><li> Montare il injectrode sul sistema micropompa e assicurarsi che sia ben fissata. </li><li> Posizionare accuratamente la punta injectrode alle coordinate di destinazione e abbassare la punta alla superficie della corteccia. </li><li> Abbassare lentamente il injectrODE usando l&#39;apparato stereotassico alla struttura di destinazione (collicolo superiore in questo caso) utilizzando gli appositi coordinate anatomiche. </li><li> Coprire la corteccia esposta con agar calda per prevenire l&#39;essiccamento dei tessuti. </li></ol> 4. Iniezione e inattivazione reversibile <ol><li> Impostare la pompa microiniezione di iniettare 400-800 nl a 40 nl / min e premere Esegui per avviare l&#39;iniezione. Si noti che il tasso di picco mostrerà riduzione durante l&#39;iniezione. NOTA: Nel setup sperimentale, l&#39;attività neurale recuperato nel giro di un&#39;ora dopo la fine della consegna GABA. Qualsiasi dispositivo meccanico tarato può essere usato per applicare pressione sulla siringa microiniezione per condurre l&#39;iniezione. </li><li> Dopo l&#39;acquisizione dei dati elettrofisiologici, eutanasia con un metodo approvato dalla locale Comunità etico degli animali. </li></ol>

<ol>
	<li>Martin, J. H., Ghez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+inactivation+in+the+analysis+of+the+central+control+of+movement.">Pharmacological inactivation in the analysis of the central control of movement.</a> Journal Of Neuroscience Methods. 86 (2), 145-159 (1999).</li><li>Ponce, C. R., Hunter, J. N., Pack, C. C., Lomber, S. G., Born, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+indirect+pathways+to+visual+response+properties+in+macaque+middle+temporal+area+MT.">Contributions of indirect pathways to visual response properties in macaque middle temporal area MT.</a> The Journal Of Neuroscience The Official Journal Of The Society For Neuroscience. 31 (10), 3894-3903 (2011).</li><li>Lomber, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advantages+and+limitations+of+permanent+or+reversible+deactivation+techniques+in+the+assessment+of+neural+function.">The advantages and limitations of permanent or reversible deactivation techniques in the assessment of neural function.</a> Journal Of Neuroscience Methods. 86 (2), 109-117 (1999).</li><li>Malpeli, J., Schiller, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+reversible+inactivation+of+small+regions+of+brain+tissue.">A method of reversible inactivation of small regions of brain tissue.</a> Journal Of Neuroscience Methods. 1 (2), 143-151 (1979).</li><li>Lomber, S. G., Payne, B. R., Horel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cryoloop:+an+adaptable+reversible+cooling+deactivation+method+for+behavioral+or+electrophysiological+assessment+of+neural+function.">The cryoloop: an adaptable reversible cooling deactivation method for behavioral or electrophysiological assessment of neural function.</a> Journal Of Neuroscience Methods. 86 (2), 179-194 (1999).</li><li>Gonzalez-Perez, O., Guerrero-Cazares, H., Qui&#241;ones-Hinojosa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+deep+brain+structures+with+microinjections+for+delivery+of+drugs,+viral+vectors,+or+cell+transplants.">Targeting of deep brain structures with microinjections for delivery of drugs, viral vectors, or cell transplants.</a> Journal Of Visualized Experiments. (46), (2010).</li><li>Hup&#233;, J., Chouvet, G., Bullier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+parameters+of+cortical+inactivation+by+GABA.">Spatial and temporal parameters of cortical inactivation by GABA.</a> Journal Of Neuroscience Methods. 86 (2), 129-143 (1999).</li><li>Casanova, C., McKinley, P., Molotchnikofff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsiveness+of+Reorganized+Primary+Somatosensory+(SI)+Cortex+after+Local+Inactivation+of+Normal+SI+Cortex+in+Chronic+Spinal+Cats.">Responsiveness of Reorganized Primary Somatosensory (SI) Cortex after Local Inactivation of Normal SI Cortex in Chronic Spinal Cats.</a> Somatosensory &amp; Motor Research. 8 (1), 65-76 (1991).</li><li>Malpeli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+inactivation+of+subcortical+sites+by+drug+injection.">Reversible inactivation of subcortical sites by drug injection.</a> Journal Of Neuroscience Methods. 86 (2), 119-128 (1999).</li><li>Minville, K., Casanova, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+frequency+processing+in+posteromedial+lateral+suprasylvian+cortex+does+not+depend+on+the+projections+from+the+striate-recipient+zone+of+the+cat&#8217;s+lateral+posterior-pulvinar+complex.">Spatial frequency processing in posteromedial lateral suprasylvian cortex does not depend on the projections from the striate-recipient zone of the cat&#8217;s lateral posterior-pulvinar complex.</a> Neuroscience. 84 (3), 699-711 (1998).</li><li>Diao, Y., Wang, Y., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Representation+of+the+binocular+visual+field+in+the+superior+colliculus+of+the+albino+rat.">Representation of the binocular visual field in the superior colliculus of the albino rat.</a> Experimental Brain Research. 52 (1), 67-72 (1983).</li></ol>

We have nothing to disclose.

Il protocollo proposto è stato progettato per risolvere le sfide poste dagli attuali metodi di inattivazione reversibile. In particolare, questo progetto mira a perfezionare i metodi utilizzati per microiniezioni chimiche delle sostanze che modulano l&#39;attività neurale, in particolare nelle strutture cerebrali profonde. Una sfida tecnica emergente da questo tipo di configurazione è la necessità per entrambe le sonde da colocalized nello stesso spazio ristretto in vivo al fine di derivare registrazioni pr...

L&#39;inattivazione di aree corticali e nuclei sottocorticali è importante nello studio delle relazioni funzionali tra varie strutture cerebrali 2-4. La letteratura recente ha impiegato tecniche criogeniche chimica perdita-di-funzione o per studiare il ruolo delle strutture cerebrali 2,5. Per quanto riguarda microiniezioni farmacologiche, piccoli volumi di farmaci possono essere somministrati in una regione del cervello ad una velocità controllata, riducendo al minimo i danni collaterali al 6,7 tessuto circostante. Questa tecnica può essere usata per fornire agonisti specifici, agonisti inversi o antagonisti per studiare l&#39;effetto di differenti bersagli farmacologici sulla attività neuronale. Tali effetti possono essere studiati da cambiamenti nelle risposte neuronali provenienti da località lontane misura, consentendo ai ricercatori di studiare i rapporti tra diverse strutture corticali e subcorticali. Qui, dimostriamo l&#39;assemblaggio di un dispositivo, il injectrode, capace di both registrazione di segnali elettrofisiologici e consegna di piccole quantità di droga nel luogo di destinazione. Dimostriamo le capacità di questo sistema iniettando GABA, un inibitore comune di attività neuronale, nel ratto collicolo superiore. Questa regione è sensibile alla stimolazione visiva, che ci ha permesso di utilizzare l&#39;attività multiunit evocata visivamente per confermare la localizzazione injectrode. La reversibilità della inattivazione è stata valutata dalla ripresa della normale attività neuronale dopo la fine dell&#39;iniezione GABA. La possibilità di monitorare l&#39;attività multi-unit dal sito di iniezione consente la messa a punto dei tassi di iniezione e volumi necessari per ottenere la risposta farmacodinamica desiderato. Pertanto, un vantaggio di questa tecnica è il potenziale limitazione di danni ai tessuti causati da microperfusione, poiché i volumi efficaci piccoli vengono iniettati. Il protocollo proposto fornisce un metodo efficiente di costo per la generazione del necessar hardware monousoy per esperimenti che conducono in cui si desidera la consegna della droga e la registrazione dell&#39;attività neuronale locale.

<table border="0" cellpadding="0" cellspacing="0" width="981">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Name of the reagent</td>
			<td style="width:209px;">Company</td>
			<td style="width:184px;">Catalogue number</td>
			<td style="width:308px;">Comments (optional)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Injection pump (UltraMicroPump III)</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#UMP3</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Injection console (Micro4 Controller)</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#SYS-MICRO4</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Hamilton syringe</td>
			<td style="width:209px;">Hamliton</td>
			<td style="width:184px;">(80301) 701LT 10 &#181;L SYR</td>
			<td style="width:308px;">Syringes between 5 and 10 &#956;L used</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Gel cyanoacrylate adhesive</td>
			<td style="width:209px;">Krazy Glue</td>
			<td style="width:184px;">KG86648R</td>
			<td style="width:308px;">The gel form is easier to apply on the shaft of the 30G hypodermic needle</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Glass pipettes</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#TW100F-4</td>
			<td style="width:308px;">Thin wall, 1mm OD, 0.75mm ID with filament pipettes used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">720 Needle Pipette Puller</td>
			<td style="width:209px;">Kopf</td>
			<td style="width:184px;">720</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Silver wire</td>
			<td style="width:209px;">A-M Systems, Inc.</td>
			<td style="width:184px;">782500</td>
			<td style="width:308px;">Bare 0.010&#8221;</td>
		</tr>
	</tbody>
</table>

simultaneous electrophysiological recording and micro-injections of inhibitory agents in the rodent brain

Qui si descrive un metodo per la costruzione di un mono-uso &quot;injectrode&quot; utilizzando parti commercialmente accessibili e convenienti. Un sistema di ispezione è stato sviluppato che consente l&#39;iniezione di un farmaco durante la registrazione di segnali elettrofisiologici dalla popolazione neuronale colpita. Questo metodo fornisce una semplice ed economica alternativa alle soluzioni commerciali. Una pipetta di vetro è stato modificato combinandolo con un ago ipodermico e un filamento d&#39;argento. Il injectrode è collegato a pompa Microsiringa commerciale per la somministrazione di farmaci. Ciò si traduce in una tecnica che fornisce feedback farmacodinamica tempo reale attraverso segnali extracellulari più unità provenienti dal sito di somministrazione di farmaci. Come un proof of concept, abbiamo registrato l&#39;attività neuronale del collicolo superiore suscitata da lampi di luce nei ratti, in concomitanza con la consegna di farmaci attraverso il injectrode. La capacità di registrazione injectrode permette la caratterizzazione funzionale del injsito ezione favorendo un controllo preciso sulla localizzazione del drug delivery. L&#39;applicazione di questo metodo si estende anche ben oltre ciò che è dimostrato qui, come la scelta di sostanza chimica caricato nel injectrode è vasta, tra cui tracciare i marcatori per esperimenti anatomici.

La costruzione del injectrode è illustrato in Figura 1. Un filo d&#39;argento (C) viene alimentato in una pipetta di vetro (D) con la porzione di filo e piegata sporge fuori dall&#39;apertura. Un ago 30 G (B) viene fissato e sigillato all&#39;apertura della pipetta di vetro con colla. Dopo la pipetta è stata riempita con la sostanza di iniezione, una siringa di vetro micro (A) è collegata all&#39;ago. È importante che vi sia una buona tenuta in cui il micro siringa collega con l&#39;ago (E) e dove i...

Watch this Scientific Journal Video about Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain at JoVE.com

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Registrazione simultanea elettrofisiologici e micro-iniezioni di agenti inibitori nel cervello del roditore

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

simultaneous-electrophysiological-recording-micro-injections

Research

JoVE Journal

Neuroscience

8.0K Views.  Université de Montréal. Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Video: Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

FMRI simultanea ed Elettrofisiologia nel cervello roditori

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Ricerca

Neuroscienze

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

Progettazione e Costruzione di un Headstage conveniente per simultanea Stimolazione Neurale e registrazione nel Labirinto d&#39;acqua

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

Proencefalo registrazione elettrofisiologica in larvale Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

Simultanea di pre-e post-sinaptica registrazione elettrofisiologica da<em&gt; Xenopus</em&gt; Nervo-muscolo Co-colture

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

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 ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Registrazione elettrofisiologica nel cervello di intatto Zebrafish adulti

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Registrazione di plasticità sinaptica in fettine ippocampali Acute mantenuti in un piccolo volume riciclaggio - aspersione- e sistema di immersione-tipo camera

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most neuroimaging or neurophysiological investigations in humans have been confined to the study of &#947;-Amino-Butyric-Acid-(GABA)-receptor-agonist-based anesthetics, while the effects of dissociative N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthetics ketamine, nitrous oxide (N2O) and xenon (Xe) are largely unknown. This paper describes the methods underlying the simultaneous recording of magnetoencephalography (MEG) and electroencephalography (EEG) from healthy males during inhalation of the gaseous anesthetic agents N2O and Xe. Combining MEG and EEG data enables the assessment of electromagnetic brain activity during anesthesia at high temporal, and moderate spatial, resolution. Here we describe a detailed protocol, refined over multiple recording sessions, that includes subject recruitment, anesthesia equipment setup in the MEG scanner room, data collection and basic data analysis. In this protocol each participant is exposed to varying levels of Xe and N2O in a repeated measures cross-over design. Following relevant baseline recordings participants are exposed to step-wise increasing inspired concentrations of Xe and N2O of 8, 16, 24 and 42%, and 16, 32 and 47% respectively, during which their level of responsiveness is tracked with an auditory continuous performance task (aCPT). Results are presented for a number of recordings to highlight the sensor-level properties of the raw data, the spectral topography, the minimization of head movements, and the unequivocal level dependent effects on the auditory evoked responses. This paradigm describes a general approach to the recording of electromagnetic signals associated with the action of different kinds of gaseous anesthetics, which can be readily adapted to be used with volatile and intravenous anesthetic agents. It is expected that the method outlined can contribute to the understanding of the macro-scale mechanisms of anesthesia by enabling methodological extensions involving source space imaging and functional network analysis.

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

Registrazione attività elettromagnetica cerebrale durante l'amministrazione del xeno di agenti anestetici gassosi e protossido di azoto in volontari sani

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Registrazione e modulazione di attività Epileptiform in fettine di cervello del roditore accoppiate a matrici di microelettrodi

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Cryosectioning simultanea di più cervelli roditore

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...