Los fondos de la Fundación Nacional para la Ciencia (IOS-1050701 a BAC), los Institutos Nacionales de Salud (NS54174 a SM y F30DC0111907 de AML-W.), La Fundación Uehara Memorial de la Sociedad Japonesa para la Promoción de la Ciencia (G2205 a los conocimientos tradicionales ). Damos las gracias a Julian Meeks por su apoyo y orientación sobre el tema de las grabaciones extracelulares axonal. Damos las gracias a Carl Hopkins para proporcionar una cámara de registro prototipo.

<ol>
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No hay conflictos de interés declarado.

Una vez dominado, esta técnica permitirá una para apuntar a las neuronas identificadas, incluyendo los axones individuales en grabaciones in vivo en muchos sistemas modelo. Además, esta técnica permite grabar fiable de salida de pico de las neuronas con características anatómicas únicas que lo hacen tradicional en métodos de grabación que desafían in vivo. Hemos utilizado esta técnica para grabar desde ELA &quot;pequeñas células&quot; en mormyrid peces débilmente eléctrica. Los intentos anteriores para estudiar las propiedades de ajuste de &quot;celdas pequeñas&quot; no tuvieron éxito debido a las condiciones difíciles de grabación 11, 14. Características anatómicas similares crean barreras para obtener grabaciones de una sola unidad de diversos vertebrados y neuronas auditivas electrosensoriales 8-10. Para superar estos retos de nuestro sistema, tomamos ventaja del hecho de que &quot;células pequeñas&quot; son las únicas células en ELA que proyectan a ELP. Por lo tanto, el transporte retrógrado de colorante colocado en ELP limita etiquetado en ELA para &quot;pequeña&quot;somas celulares y axones. etiquetado fluorescente de los axones permite la colocación precisa del electrodo al lado de los axones marcados, por lo que las grabaciones de una sola unidad de células identificadas posibles a pesar de somas inaccesibles. Se intentó grabaciones somáticas, pero no tuvieron éxito, probablemente debido a las sinapsis envolventes alrededor de 11 , 17. Sin embargo, somática etiquetado era claramente visible lo que sugiere que esta técnica podría ser utilizada para orientar grabaciones somáticas en otros tipos de células y otros circuitos. etiquetado fluorescente de las neuronas a través de transporte retrógrado in vivo se ha usado para guiar grabaciones específicas in vitro 19-21 . Una técnica similar se utiliza para dirigido en grabaciones in vivo de las neuronas motoras en la médula espinal pez cebra 22. Nuestro trabajo representa una nueva expansión de este enfoque, en el que tanto el etiquetado y la grabación se realiza in vivo en el cerebro. Nuestro método demuestra que en vivo etiquetado de las áreas del sistema nervioso central con una retrógrada tcorredor se puede ampliar para el estudio de otros circuitos intactos con las neuronas de proyección similar selectivos. Por ejemplo, en el procesamiento auditivo mamífero, el colículo inferior (CI) sirve como un centro de retransmisión importante para las entradas de múltiples estructuras rhombencephalic 23. Inyección de un contraste en el IC se etiquete selectivamente las células de proyección de cada uno de estos núcleos. El colículo superior (SC) cumple una función similar para la visión 24. Preparaciones de médula espinal son particularmente adecuados para esta técnica, como la médula espinal es de fácil acceso, la inyección de medio de contraste puede ocurrir lejos del sitio de registro, y que se pueden combinar con el registro intracelular y el llenado de las neuronas seleccione para adquirir información más detallada anatómica 25. Finalmente, el trazado de las vías es una técnica bien establecida usada en todo el sistema nervioso central para asignar circuitos complejo 26. Nuestro método se puede utilizar para añadir información funcional a estos estudios como se ha hecho windicadores sensibles al calcio ith de gato corteza visual 27. La cirugía, que está bien establecida, fiable, y se usa regularmente para ciego in vivo grabaciones 16, debe ser completada con un sangrado mínimo y ningún daño a la superficie del cerebro para permitir que el animal y el tejido para sobrevivir. Con la práctica, la aplicación de la cirugía y el tinte se puede completar en 30-45 minutos. Hemos marcado con éxito &quot;pequeñas células&quot; axones en el 67% de las preparaciones. La mayoría de las preparaciones de tener sólo 1 o 2 axones marcados visibles, pero algunos tienen el mayor número 8. De las 119 unidades etiquetadas intentos, se obtuvo grabaciones de una sola unidad de 26 unidades distribuidas en 12 preparaciones (Tabla 2). Por lo tanto, los datos se obtuvieron de 41% de las preparaciones con axones etiquetados para una tasa de éxito del 28%. El aspecto crítico de la aplicación de tinte es el etiquetado profundidad. Poca inserción del alambre de tungsteno dará lugar a que el colorante de ser lavada away. Sin embargo, si la penetración es demasiado profunda, los axones marcados no serán visibles para la orientación. Además, algunos daños mecánicos en la celda debe ocurrir para que el medio de contraste para ser tomada adecuadamente hasta 25, 28. Sin embargo, demasiado daño matará a las células. Hemos intentado marcar con otros tintes y otros métodos (Tabla 2), incluido el etiquetado anterógrada a través de inyección de tinta en ELA junto con la grabación de los axones marcados en ELP (Tabla 3). Nuestra hipótesis es que el etiquetado anterógrada no tuvo éxito porque el etiquetado se limita a las células con daños somáticos, por lo que no responde. Además, los terminales presinápticos pueden haber sido perturbado. Por el contrario, el etiquetado retrógrada minimiza tanto de estas preocupaciones. La cantidad y la ubicación de la aplicación del colorante pueden ser modificados de acuerdo con el circuito particular que está siendo estudiado. Etiquetado máxima se produce con la mayor concentración de colorante, lo que hemos logrado con alambres de tungsteno recubiertas. Sin embargo, para la rotulación de los axones conproyecciones ep, colorante podrían salir de una aguja de tungsteno, ya que es avanzado. En estos casos, la inyección de presión sería más apropiado. La absorción de colorante y el transporte son rápidos, con los axones marcados en nuestra preparación son visibles tan temprano como 2 horas después de la inyección y los axones marcados adicionales que aparecen tan tarde como 6 horas después de la inyección. Por lo tanto, el etiquetado y registro pueden llevarse a cabo en un solo día, la eliminación de dificultades técnicas asociadas con la cirugía de la supervivencia. El tiempo variará para cada aplicación en función de la distancia necesaria para el transporte de tinte. Otro aspecto crítico es la colocación de electrodos. Es importante para entrar en el tejido cerca del sitio del axón marcado para evitar la obstrucción de la punta. Para tejido denso como en ELA, un largo, pierna delgada en el electrodo de registro minimiza el movimiento excesivo de los tejidos circundantes. Si una grabación correcta no se consigue en el primer intento, repita con electrodos frescos hasta que se obtiene una grabación o el TISSue se interrumpe hasta el punto en el que el axón es ya no es visible. Sin embargo, también es importante para colocar rápidamente el electrodo al lado del axón para reducir al mínimo la cantidad de exposición fluorescente, que puede causar fototoxicidad, blanqueo y puede afectar a las propiedades fisiológicas de la célula 29-31. Una vez que un segmento de axón se filtra con succión con éxito en el electrodo de registro, las grabaciones se puede conseguir por varias horas. Si las unidades se pierden constantemente en menos de 1 hora, considerar la posibilidad de puntas de los electrodos más pequeños para prevenir el axón se deslice fuera. Por otra parte, demasiado pequeño de una punta puede dar lugar a la obstrucción, baja relación señal-ruido, o daños en el axón. Una disminución constante de la amplitud de la espiga y el &quot;retorno&quot; de una unidad con succión adicional es una indicación de que la punta es demasiado grande. El exceso de aspiración puede causar daños irreparables en el axón. Una solución es permitir que una pequeña fuga en la línea de aire para que la presión lentamente vuelve a cero. Rápidamente igualar ªpresión e dará lugar a un &quot;empuje&quot; relativa-transitoria de salida que puede expulsar al axón. Aunque esta técnica representa una gran ventaja para la obtención de grabaciones específicas de las neuronas de proyección identificados, no será útil para distinguir interneuronas locales, como colorante estaría ocupado por todos los tipos de células en el sitio de la inyección. Teóricamente, el uso de múltiples fluoróforos con distintos lugares de inyección puede permitir que este método se expanda. Por ejemplo, la comparación de un solo frente de doble etiquetado podría ser utilizado para distinguir interneuronas de las neuronas de proyección después de las inyecciones de doble en dos puntos en el circuito 32. Del mismo modo, los trazadores retrógrados se pueden combinar con otras técnicas de imagen avanzadas, tales como la imagen de dos fotones, como se hizo recientemente en el centro de Vocal zebra finch High (HVc) 32. Además, las regiones de grabación se limitan a los que están cerca de la superficie cuando se utiliza microscopios de epifluorescencia, ya que sólo hemos sido capaces de resolver 1 &amp;mu; estructuras m dentro de los primeros 30 micras de tejido. Sin embargo, esta profundidad se podría extender a través de la utilización de otras técnicas de microscopía, como la microscopía de dos fotones 33 u objetivo-junto microscopía de iluminación plana 34. En general, esta técnica representa un avance importante en el estudio de los circuitos neuronales in vivo, ya que puede ser utilizado para grabar desde las neuronas individuales en muchos circuitos diferentes en una variedad de sistemas de modelos - incluyendo aquellos que son relativamente inaccesibles.

<table border="1" cellspacing="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tricaine Methanesulfonate</td>
 <td>Sigma-Aldrich</td>
 <td>E10521</td>
 <td>MS-222</td>
 </tr>
 <tr>
 <td>Gallamine Triethiodide </td>
 <td>Sigma-Aldrich</td>
 <td>G8134</td>
 <td>Flaxedil</td>
 </tr>
 <tr>
 <td>Lidocaine Hydrochloride</td>
 <td>Sigma-Aldrich</td>
 <td>L5647</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hickman's Ringer Solution </td>
 <td>NA</td>
 <td>NA</td>
 <td>NaCl (6.48 g/l), KCl (0.15 g/l), CaCl2&bull;2H20 (0.29 g/l), MgSO4 (0.12 g/l), NaHCO3 (0.084 g/l), NaH2PO4 (0.06 g/l)</td>
 </tr>
 <tr>
 <td>Peristaltic Pump</td>
 <td>Gilson or Rainin</td>
 <td>Minipulse 3 Model 312; RP-1</td>
 <td>10.0 &plusmn; 1.0 rpm, Alternatively, a gravity-fed line with a flow-meter</td>
 </tr>
 <tr>
 <td>Dissection Microscope </td>
 <td>Nikon</td>
 <td>SM2645</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Super Glue</td>
 <td>Super Glue Corporation</td>
 <td>SGH2</td>
 <td>2g tube</td>
 </tr>
 <tr>
 <td>Omni Drill 35</td>
 <td>World Precision Instruments</td>
 <td>503-598</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ball Mill Carbide Drill Bit #1/4</td>
 <td>World Precision Instruments</td>
 <td>501860</td>
 <td>0.19 DIA (bit diameter = 0.48 mm) </td>
 </tr>
 <tr>
 <td>Low Temp Cautery Kit</td>
 <td>World Precision Instruments</td>
 <td>500391</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Manual Micromanipulator</td>
 <td>World Precision Instruments</td>
 <td>M3301R</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dextran Conjugated Alexa Fluor 568</td>
 <td>Invitrogen</td>
 <td>D-22912</td>
 <td>2mM concentration; 10,000MW The choice of dye wavelength should be selected to match the microscope filter settings</td>
 </tr>
 <tr>
 <td>Epifluorescent Microscope</td>
 <td>Nikon </td>
 <td>E600 FN</td>
 <td>A remote focus accessory is helpful for fine focus TRITC filter - or appropriate filter to match focal planes for different dye wavelengths</td>
 </tr>
 <tr>
 <td>Low Power Objective</td>
 <td>Nikon</td>
 <td>93182</td>
 <td>CFI Plan Achromat Series, 4X N.A. 0.1, W.D. 30mm</td>
 </tr>
 <tr>
 <td>High Power Objective</td>
 <td>Nikon</td>
 <td>93148</td>
 <td>CFI Fluor Series, 40X WI N.A. 0.8, W.D. 2.0 mm</td>
 </tr>
 <tr>
 <td>White Light Source</td>
 <td>Dolan-Jenner</td>
 <td>Model 190</td>
 <td>Fiber Optic Illuminator</td>
 </tr>
 <tr>
 <td>Fluorescent Light Source </td>
 <td>Lumen Dynamics</td>
 <td>X-Cite 120Q</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Low-light Level Camera</td>
 <td>Photometrics</td>
 <td>CoolSnap ES</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digital Manometer</td>
 <td>Omega Engineering</td>
 <td>HHP-201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Motorized Micromanipulator</td>
 <td>Sutter Instruments</td>
 <td>MP-285</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Headstage</td>
 <td>Molecular Devices</td>
 <td>CV-7B</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Molecular Devices</td>
 <td>MultiClamp 700B</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Data Acquisition System</td>
 <td>Molecular Devices</td>
 <td>Digidata 1322A</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Isolated Pulse Stimulator</td>
 <td>A-M Systems</td>
 <td>2100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Flaming/Brown Micropipette Puller</td>
 <td>Sutter Instruments</td>
 <td>P-97</td>
 <td>Program settings for our application: Heat: ramp + 1, Pull: '0', Velocity: 60, Time: 90 Box filament</td>
 </tr>
 <tr>
 <td>Pipette Glass</td>
 <td>World Precision Instruments</td>
 <td>1B100F-4</td>
 <td>Borosilicate capillary glass with filament (1 mm OD, 0.58 mm ID) </td>
 </tr>
 </tbody>
</table>

retrograde fluorescent labeling allows for targeted extracellular single-unit recording from identified neurons in vivo

El objetivo general de este método consiste en registrar las respuestas de una sola unidad de una población seleccionada de las neuronas. En los registros electrofisiológicos in vivo de neuronas individuales son fundamentales para la comprensión de cómo funcionan los circuitos neuronales en condiciones naturales. Tradicionalmente, estas grabaciones se han realizado &quot;a ciegas&quot;, es decir, la identidad de la célula registrada es desconocido al inicio de la grabación. Identidad celular puede determinarse posteriormente mediante intracelular 1, juxtacellular 2 o sueltas-patch 3 iontoforesis de tinte, pero estas grabaciones no se puede pre-dirigido a neuronas específicas en regiones con tipos de células funcionalmente heterogéneas. Las proteínas fluorescentes se pueden expresar de una manera específica del tipo de célula que permite visualmente guiada por electrofisiología de una sola célula 4-6. Sin embargo, hay muchos sistemas modelo para el que estas herramientas genéticas no están disponibles. Incluso en sistemas de modelos genéticamente accesibles, el programa deseadopromotor puede ser desconocida o neuronas genéticamente homogéneos puede haber diferentes patrones de proyección. Del mismo modo, los vectores virales se han utilizado para etiquetar los subgrupos específicos de las neuronas de proyección 7, pero el uso de este método está limitada por la toxicidad y la falta de especificidad trans-sináptica. Por lo tanto, se necesitan técnicas adicionales que ofrecen pre-visualización específica para grabar desde neuronas individuales identificados in vivo. Pre-visualización de la neurona diana es particularmente útil para condiciones de grabación desafiantes, para los que las grabaciones de una sola célula clásicos a menudo son prohibitivamente difícil 8-11. La novedosa técnica descrita en este documento se utiliza el transporte retrógrado de un colorante fluorescente aplicada, utilizando agujas de tungsteno para etiquetar rápidamente y selectivamente un subconjunto específico de células dentro de una región particular del cerebro sobre la base de sus proyecciones axonales únicas, proporcionando de ese modo una señal visual para obtener registros electrofisiológicos dirigidos de las neuronas identificadas en un circuito intacto withina vertebrado SNC. La novela avance más significativo de nuestro método es el uso de etiquetado fluorescente para apuntar a tipos de células específicos en un sistema modelo accesible no genéticamente. Débilmente peces eléctricos son un excelente sistema modelo para el estudio de los circuitos neuronales en despierto, comportándose los animales 12. Hemos utilizado esta técnica para estudiar el procesamiento sensorial de &quot;células pequeñas&quot; en el núcleo exterolateral anterior (ELA) de peces mormyrid débilmente eléctrica. &quot;Células pequeñas&quot; son la hipótesis de que el tiempo de comparación neuronas importantes para detectar diferencias submillisecond en los tiempos de llegada de los picos presináptica 13. Sin embargo, las características anatómicas tales como la mielina densa, que envuelve las sinapsis, y cuerpos celulares pequeños han hecho extremadamente difícil para grabar a partir de estas células utilizando métodos tradicionales 11, 14. Aquí nos demuestran que nuestro método novedoso etiqueta selectivamente estas células en el 28% de los preparativos, lo que permite, grabaciones robustos y fiables caracrización de las respuestas a la estimulación electrosensory.

Watch this Scientific Journal Video about Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo at JoVE.com

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

Transporte retrógrado de tinte fluorescente etiqueta a una subpoblación de neuronas en base a la proyección anatómica. Axones etiquetadas pueden orientarse visualmente<em&gt; In vivo</em&gt;, Que permite la grabación extracelular de los axones identificados. Esta técnica facilita la grabación cuando las neuronas no se pueden etiquetar a través de la manipulación genética o son difíciles de aislar el uso de &quot;ciego&quot;<em&gt; In vivo</em&gt; Enfoques.

Marcaje fluorescente retrógrada Permite dirigida extracelular sola unidad de grabación de neuronas identificadas<em&gt; En vivo</em

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

retrograde-fluorescent-labeling-allows-for-targeted-extracellular

Research

JoVE Journal

Neuroscience

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

19.8K vistas.  Washington University in St. Louis. Transporte retrógrado de tinte fluorescente etiqueta a una subpoblación de neuronas en base a la proyección anatómica. Axones etiquetadas pueden orientarse visualmente In vivo, Que permite la grabación extracelular de los axones identificados. Esta técnica facilita la grabación cuando las neuronas no se pueden etiquetar a través de la manipulación genética o son difíciles de aislar el uso de "ciego" In vivo Enfoques.

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

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3).
Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons.
The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons.

Dual Somatic Recordings from Gonadotropin-Releasing Hormone GnRH Neurons Identified by Green Fluorescent Protein GFP in Hypothalamic Slices

Activity in neuronal systems often requires synchronous action potential discharges from neurons within a specific population. For example, pulses of gonadotropin-releasing hormone (GnRH) likely require coordinated activity between GnRH neurons. We present our methodological approach for reliably obtaining simultaneous electrophysiological recordings from the diffusely distributed GnRH neurons.

Las grabaciones de doble somáticas de la hormona liberadora de gonadotropina (GnRH) identificados por la proteína verde fluorescente (GFP) en cortes de hipotálamo

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating ...

Investigación

Neurociencias

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

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

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

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

Una sola unidad En vivo Grabaciones de la quiasma óptico de la rata

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

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

La implantación quirúrgica de electrodos neuronales crónicas para registrar la actividad y señales de una sola unidad electrocorticográfica

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

La grabación simultánea de señales de calcio en neuronas identificadas y comportamiento de alimentación de Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

Cargando retrógrada de los nervios, panfletos, y las raíces espinales con tintes fluorescentes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

Registro de la actividad eléctrica de las neuronas identificadas en el cerebro intacto de peces transgénicos

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Intracelular de grabación, Sensorial Asignación de campos, y el cultivo de neuronas identificadas en el Leech, Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

Etiquetado retrógrada de células ganglionares retinianas en adultos de pez cebra con tintes fluorescentes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

Una guía para<em&gt; En vivo</em&gt; Single-unidad de grabación de Optogenetically Identificado cortical inhibitorio interneuronas

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforado grabación de patch-clamp de ratón neuronas sensoriales olfativas en neuroepitelio Intacto: Análisis funcional de las neuronas que expresan un odorante del receptor Identificado

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...