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: Todos los procedimientos se realizaron de acuerdo con las directrices del Consejo Canadiense para la Protección de los Animales y de la junta de revisión de Ética de la Universidad de Montreal. 1. Asamblea de la pipeta de grabación por inyección <ol><li> Tire de un capilar de vidrio de aproximadamente 7 cm de largo (1 mm de diámetro exterior) con un extractor pipeta. </li><li> Romper la punta del capilar y compruebe la abertura con un microscopio óptico. Confirmar que el diámetro interior es de entre 30 micras a 40 micras. </li><li> Insertar un alambre de plata 7 cm de largo en el capilar de vidrio con aproximadamente 1 cm que sobresale del extremo no ahusada de la pipeta de vidrio. </li><li> Doble el exceso de filamento ortogonal al capilar de vidrio. </li><li> Aplicar una gota de adhesivo de plástico flexible en el eje de una aguja hipodérmica 30 G. </li><li> Inserte una aguja hipodérmica 30 G en la pipeta de vidrio de acuerdo con los esquemas presentados en la Figura 1 </strong&gt;. </li><li> Añadir un segundo recubrimiento de pegamento para asegurar un sellado adecuado de la unión entre la pipeta de vidrio y la aguja hipodérmica. </li><li> Deja la pipeta para secar con la punta hacia arriba durante aproximadamente 12 horas para asegurar un curado adecuado de la cola. El resultado final se muestra en la Figura 2. </li></ol> NOTA: Este procedimiento se realiza en experimentos agudos y esterilización de la punta de la pipeta no es necesario. 2. Preparación de los animales <ol><li> Coloque la rata en una caja de la anestesia. </li><li> Inducir la anestesia utilizando 4% isoflurano por 5 a 10 minutos. </li><li> Colocar el animal sobre una mesa estereotáxica con el cojín de calefacción y sonda rectal para mantener una temperatura corporal de 37 ° C. Use un cono de la nariz para mantener la anestesia con isoflurano 2%. Asegure la cabeza de la rata utilizando barras de oído y titular de los dientes. </li><li> Aplicar pomadas o gotas oftálmicas oculares, que incluyen 1% atropina gotas para ayudar en la dilatación de la pupila. Para evitar la sequedad, aplicar lubricante gotas aproximadamente cada 30 min. </li><li> Afeitarse la cabeza y límpielo con un 10% de povidona-yodo. </li><li> Para anestesia local, inyectar 0,5 ml de lidocaína al 2% bajo el cuero cabelludo en 2-3 ubicaciones levantando la piel y la inserción de la punta de la aguja. </li><li> Confirmar nivel adecuado de anestesia mediante la realización de una pizca dedo del pie y la observación de la falta de movimiento. Además, controlar la frecuencia cardíaca para asegurar que está dentro de los valores normales (300 a 400 latidos / min). </li><li> Incisión en el cuero cabelludo en una línea recta a lo largo de la mediana con una cuchilla de bisturí # 10 para exponer tanto las suturas coronal y sagital. </li><li> Revela los puntos Lambda y bregma por empujar a un lado el tejido que cubre el cráneo con una espátula quirúrgica. </li><li> Nivelar el cráneo de modo que las posiciones de bregma y lambda están en el mismo plano. </li><li> Para establecer el punto de referencia, utilice un dispositivo estereotáxico con un tubo de vidrio montada para configurarlo justo encima Bregma. Este será el &quot;cero&quot; para el antero-posterior y medmediciones ial-laterales coordenadas. </li><li> Establecer el lugar de interés moviendo el estereotáxica montaje a las coordenadas necesarias, tenga en cuenta las coordenadas estereotáxicas y dibujar un cuadrado alrededor de la zona objetivo que marca donde se realizará la craneotomía. </li><li> Use un taladro con una broca quirúrgica esterilizada a lo largo del marcado plaza lentamente y sin presión para eliminar lentamente el material óseo. Tenga cuidado de no perforar demasiado tiempo en la misma zona, ya que producirá calor y causar lesiones en la corteza. </li><li> Cuando el hueso que delimita la craneotomía se ha convertido en lo suficientemente delgada, retire con cuidado la sección craneal con pinzas para exponer la corteza. </li><li> Irrigar frecuentes la corteza expuesta con líquido cefalorraquídeo artificial para evitar la desecación de los tejidos. NOTA: la eliminación mater Dura es innecesario en la rata como la punta de la injectrode es lo suficientemente resistente como para penetrar. </li></ol> 3. Llenar y Montaje del Sistema de Inyección <ol><li> Llenar el5-10 l microjeringa por aspiración con aceite mineral. </li><li> Llene la aguja hipodérmica con la pipeta de vidrio fijo con una solución de 0,5% Chicago Sky Azul (CSB) y 300 M-γ aminobutírico (GABA) o una solución de lidocaína al 2% con 0,5% CSB 9. Diluir todas las soluciones con solución salina. <ol><li> En el caso de una sustancia abundante, llenar utilizando una jeringa regular y utilizar las técnicas habituales de precaución para evitar la formación de burbujas de aire. </li><li> En el caso de las sustancias más caras, utilice aceite mineral para llenar el injectrode y el agente químico puede ser introducido por aspiración. Como la diferencia de densidad entre el aceite mineral y el agua es relativamente alta, esta sustancia es un buen candidato para la inyección de soluciones acuosas. NOTA: Un colorante puede ser añadido a confirmar la separación entre ambos líquidos. </li></ol></li><li> Para llenar la pipeta de inyección, llenar una jeringa de 1 ml con la solución por aspiración y luego inyectar lentamente la soluciónen la pipeta de inyección. </li><li> Prestar especial atención para detectar fugas en las regiones indicadas en la Figura 1 en muestras de secreciones estas áreas limpias y la observación de fugas mediante la inyección aún más la solución lentamente con la jeringa. </li><li> Retire la jeringa de 1 ml. Al hacerlo, asegúrese de mantener una ligera presión sobre el émbolo para que el vacío no elimina la solución de la pipeta de inyección. </li><li> Llene el microjeringa con aceite mineral por aspiración y adjuntarlo firmemente a la pipeta de inyección lleno, a continuación, limpie cuidadosamente cualquier exceso de solución de la injectrode montado con una gasa. </li><li> Verifique que la punta no está bloqueada mediante la inyección de un volumen muy pequeño, lo suficiente para ver una pequeña formación en la punta de la pipeta de vidrio gota. </li><li> Monte el injectrode en el sistema microbomba y asegurarse de que está bien fijada. </li><li> Coloque con cuidado la punta injectrode en las coordenadas de destino y de bajar la punta a la superficie de la corteza. </li><li> Baje lentamente la injectrOda usando el aparato estereotáxico a la estructura objetivo (colículo superior en este caso) utilizando las coordenadas anatómicas apropiadas. </li><li> Cubra la corteza expuesta con agar caliente para evitar la desecación de los tejidos. </li></ol> 4. Inyección y inactivación reversible <ol><li> Ajuste la bomba de microinyección para inyectar 400 a 800 nl a 40 nl / min y pulse Ejecutar para iniciar la inyección. Tenga en cuenta que la tasa pico mostrará reducción durante la inyección. NOTA: En la configuración experimental, la actividad neuronal se recuperó dentro de una hora después del final de la entrega de GABA. Cualquier dispositivo mecánico calibrado se puede utilizar para aplicar presión en la jeringa la microinyección con el fin de llevar a cabo la inyección. </li><li> Después de la adquisición de los datos electrofisiológicos, la eutanasia utilizando un método aprobado por la Comunidad de Ética Animal local. </li></ol>

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	<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.

El protocolo propuesto fue diseñado para resolver los retos derivados de los actuales métodos de inactivación reversible. En concreto, este proyecto destinada a perfeccionar los métodos utilizados para microinyecciones químicas de las sustancias que modulan la actividad neuronal, particularmente en las estructuras profundas del cerebro. Un reto técnico que emerge de este tipo de configuración es la necesidad de ambas sondas a ser colocalized en el mismo espacio restringido in vivo a fin de obtener grabac...

La inactivación de áreas corticales y núcleos subcorticales es importante en el estudio de las relaciones funcionales entre varias estructuras cerebrales 2-4. La literatura reciente ha empleado química de pérdida de función o técnicas criogénicas para estudiar el papel de las estructuras cerebrales 2,5. En lo que se refiere a las microinyecciones farmacológicas, pequeños volúmenes de medicamentos pueden ser administrados en una región del cerebro a una velocidad controlada y reducir al mínimo el daño colateral al tejido circundante 6,7. Esta técnica se puede utilizar para entregar agonistas específicos, agonistas inversos o antagonistas para estudiar el efecto de diferentes dianas farmacológicas sobre la actividad neuronal. Estos efectos también se pueden estudiar mediante la medición de cambios en las respuestas neuronales desde lugares distantes, lo que permite a los investigadores estudiar las relaciones entre las diferentes estructuras corticales y subcorticales. Aquí, nos demuestran el montaje de un dispositivo, el injectrode, capaz de boº grabación de señales electrofisiológicas y entregar pequeñas cantidades de drogas en el lugar de destino. Se demuestra las capacidades de este sistema mediante la inyección de GABA, un inhibidor de la actividad neuronal común, en la rata colículo superior. Esta región es sensible a la estimulación visual, que nos permitió utilizar la actividad multiunit evocado visual para confirmar la localización injectrode. La reversibilidad de la inactivación se evaluó por la recuperación de la actividad neuronal normal tras el final de la inyección GABA. La capacidad de controlar la actividad de varias unidades de la zona de la inyección permite la puesta a punto de las tasas de inyección y los volúmenes necesarios para lograr la respuesta farmacodinámica deseada. Por lo tanto, una ventaja de esta técnica es el potencial de limitar el daño tisular causado por microperfusión, ya que se inyectan los más pequeños volúmenes efectivos. El protocolo propuesto proporciona un método rentable para generar la necessar hardware desechabley para la realización de experimentos donde se desea la administración de fármacos y la grabación de la actividad neuronal local.

<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

Aquí se describe un método para la construcción de un solo uso &quot;injectrode&quot; utilizando piezas comercialmente accesibles y asequibles. Un sistema de sondeo fue desarrollado que permite la inyección de un medicamento durante la grabación de señales electrofisiológicas de la población neuronal afectada. Este método proporciona una alternativa sencilla y económica a las soluciones comerciales. Una pipeta de vidrio se modificó mediante la combinación con una aguja hipodérmica y un filamento de plata. El injectrode está unido a la bomba microjeringa comercial para la administración de fármacos. Esto resulta en una técnica que proporciona retroalimentación farmacodinámica en tiempo real a través de señales extracelulares de unidades múltiples originarios desde el sitio de administración de fármacos. Como una prueba de concepto, se registró la actividad neuronal del colículo superior provocada por destellos de luz en ratas, concomitantemente con la entrega de medicamentos a través de la injectrode. La capacidad de grabación injectrode permite la caracterización funcional de la injEl sitio de reflexión favoreciendo un control preciso sobre la localización del suministro del fármaco. La aplicación de este método también se extiende mucho más allá de lo que se ha demostrado aquí, como la elección de la sustancia química cargado en el injectrode es vasto, incluyendo rastreo de marcadores para experimentos anatómicos.

La construcción de la injectrode se ilustra en la Figura 1. Un alambre de plata (C) se alimenta a una pipeta de vidrio (D) con una porción de la alambre doblado y que sobresale hacia fuera de la abertura. Una aguja de 30 G (B) se une y se sella a la abertura de la pipeta de vidrio con pegamento. Después de la pipeta se ha llenado con la sustancia de la inyección, una jeringa micro de vidrio (A) está unido a la aguja. Es importante que haya un buen cierre hermético, donde la jeringa micro conecta c...

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.

Grabación simultánea electrofisiológico y micro-inyecciones de agentes inhibitorios en el cerebro de roedores

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 vistas.  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 simultánea y electrofisiología en el cerebro de roedores

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

Investigación

Neurociencias

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.

Diseño y construcción de un cabezal rentable para la estimulación simultánea neural y de grabación en el laberinto de agua

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.

Prosencéfalo electrofisiológico de grabación en larvas de pez cebra

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.

Simultánea pre-y post-sináptica de registro electrofisiológico<em&gt; Xenopus</em&gt; Músculo Nervio-Co-culturas

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.

Registro electrofisiológico en el cerebro del pez cebra adultos Intacto

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.

Grabación de la plasticidad sináptica en rebanadas Hippocampal agudas mantenidas en un pequeño volumen reciclado - perfusión- y sistema de cámara de tipo inmersión

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.

Registro actividad electromagnética cerebral durante la administración del xenón gaseoso agentes anestésicos y óxido nitroso en voluntarios sanos

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.

Grabación y modulación de la actividad epileptiforme en rebanadas de cerebro de roedor juntada a arreglos de microelectrodos

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 simultánea de varios cerebros de roedores

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