Name: optogenetics identification of a neuronal type with a glass optrode in awake mice
Uploaded: 2018-06-28T14:30:00.000+00:00
Duration: 7 min 51 s
Description: Watch this Scientific Journal Video about Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice at JoVE.com

Los autores fueron apoyados por la sociedad japonesa para la promoción de la ciencia KAKENHI Grant JP16K11200 y 17H 02223 y la subvención para la investigación de Kanazawa médica Universidad S2016-8 y C2017-3. Damos las gracias por su apoyo en la toma de las fotos Yuhichi Kuda.

Todos los procedimientos se realizan con arreglo a los principios rectores de la sociedad fisiológica de Japón y con la aprobación de la Animal cuidado Comité de Kanazawa Universidad médica.
1. construcción del vidrio Optrode titular
Nota: Para construir un soporte de optrode de vidrio, se utiliza un soporte de electrodo comercial (figura 1A).
<ol><li>Extraiga con cuidado el tubo de acero para control de la presión del barril del titular.</li>
	<li>Por perforación, ampliar el agujero del lado del asiento de pin en el cañón del titular. Hacer el agujero 3 mm de diámetro y 12 mm de profundidad.</li>
	<li>Poner un tubo de acero inoxidable (3 mm de diámetro, 0,5 mm de espesor y 8 mm de longitud; Figura 1A 2) en el agujero.</li>
	<li>Inserte el partido manga de acoplamiento para virolas de ⌀2.5 mm (figura 1A1) en el orificio de cerámica.</li>
	<li>Fijar el cilindro de la pinza portaelectrodos para una barbilla en forma de L con adhesivo epoxi.</li>
	<li>Hacer los agujeros (3 mm de diámetro) en la barbilla y adjuntarlo a un manipulador con tornillos.</li>
</ol>2. cabeza Post instalación
<ol><li>Preparar un post de cabeza a la medida de un separador de circuitos. Hacer el espaciador cilíndrico de circuitos en forma cuboidal con una fresa y cortar en 2 puestos de cabeza con una sierra (figura 1B). Aplanar la parte inferior del post principal con una fresa.</li>
	<li>Preparar el animal transgénico en el que un opsin de sensibles a la luz se expresa en un tipo de neurona específica para el estudio. En este trabajo, se utiliza el ratón transgénico (ChR2 VGAT ratones) en el cual las neuronas inhibitorias expresan channelrhodopsin-2 (ChR2)11 .</li>
	<li>Anestesiar al animal con una mezcla de 0,3 mg/kg de medetomidina, 4,0 mg/kg de midazolam y 5,0 mg/kg de butorfanol por administración intraperitoneal. Para confirmar que el animal está completamente anestesiado, prueba sus reacciones para pellizcar la cola.</li>
	<li>Coloque el ratón en un marco estereotáctica en un cojín de calefacción. Limpiar el marco estereotáctica y la calefacción almohadilla con alcohol al 70% por adelantado. Colocar los dientes en el agujero de la barra de bocado y apriete ligeramente la abrazadera de la nariz. Fijar ambos lados de la cabeza por medio de barras de oído. Cuando la cabeza se fija correctamente por las barras de oído, apriete la abrazadera de la nariz. Aplicar una pomada oftálmica en los ojos.</li>
	<li>Afeitarse la piel cabeza con pequeñas tijeras y limpiar el cuero cabelludo con un algodón de gluconato de clorhexidina. Antes de la cirugía, esterilizar todos que los instrumentos quirúrgicos y una cabeza de correos con un autoclave (121 ° C, 15 min).</li>
	<li>Corte el cuero cabelludo a lo largo de la línea media con unas tijeras y empuje a un lado. Hacer la incisión de la parte posterior de la cabeza a los ojos (aproximadamente 20 milímetros). Retirar el periostio con un hisopo de algodón humedecido en alcohol al 70% y secar el cráneo.</li>
	<li>Coloque el poste principal en el manipulador. Coloque el post cabeza plana sobre el cráneo en el vértice con el manipulador.</li>
	<li>Mezcla el monómero (4 gotas), catalizador (1 gota) y polímero (1 cuchara pequeña) de cemento dental sobre un pequeño plato que se enfría en la nevera con antelación. Poner el cemento dental (por debajo de 1 g) en el frontal y el hueso parietal del cráneo para fijar el perno de cabeza en el cráneo.</li>
	<li>Después de que el cemento dental se convierte en duro, quitar el ratón en la estructura estereotáxicas. Pongo un ungüento antibiótico en la piel expuesta. Inyecte el antagonista de la medetomidina (0,3 mg/kg) y los antibióticos (oxitetraciclina; 20 mg/kg). Y controlar el mouse sobre el cojín del calor en una jaula hasta que despierta y volver a una jaula cubierta.</li>
	<li>Casa el animal en una jaula de vivienda individual con algún dispositivo de enriquecimiento. Cambiar el papel animal ropa de cama todos los días para mantener la jaula limpia y prevenir infecciones. Controle cuidadosamente si el animal muestra signos de malestar. Se aplica lidocaína a la herida si se sospecha de cualquier dolor. Administrar meloxicam o el carprofeno una vez cada 24 h durante 48 h después de la cabeza de tanto post cirugías instalación y craneotomía. La lidocaína local se puede dar además si el AINE no es adecuada.
</li>
</ol>3. aclimatación
<ol><li>(Mínimo) dos días después de la recuperación de la anestesia, aclimatar a los animales para el estado de cabeza fija en la cámara de grabación. Realizar las sesiones de aclimatación durante al menos 5 días.</li>
	<li>Durante las sesiones de aclimatación, coloque el animal en la sala de grabación con el post principal fijado a una mordaza a la medida. Durante la sesión, con el fin de limitar el movimiento, tapa el cuerpo con un tubo de medio de resina por encargo (figura 1), hecho con una impresora 3D. Nota: El día 1, la sesión de aclimatación debe ser 5 minutos de largo. Es entonces aumentado diariamente a 10, 20, 40 y 60 min para los días 2, 3, 4 y 5, respectivamente.</li>
	<li>Al final de cada sesión, dar al animal una recompensa de comida. Si el animal se esfuerza continuamente durante la aclimatación, detener la sesión.</li>
</ol>4. craneotomía
Nota: Después de la aclimatación, se realiza una craneotomía sobre la región del cerebro para la grabación. La craneotomía se realiza en el marco estereotáctica bajo anestesia, como con la cabeza post instalación. El procedimiento posterior a la operación es el mismo como la cabeza post instalación.
<ol><li>Antes de la craneotomía, limpiar el cráneo con un hisopo de algodón con clorhexidina gluconato y el 70% de alcohol.</li>
	<li>Raspar cuidadosamente el cráneo en el sitio de grabación con un taladro dental. Cuando el hueso se convierte en lo suficientemente delgado, cortar con la punta del bisturí y retírela.</li>
	<li>Poner cemento dental alrededor de la región expuesta a fin de fortalecer el cráneo.</li>
	<li>Cubra el área expuesta del cerebro con adhesivo de silicona después de que el cemento dental se convierte en duro. El procedimiento después de la cirugía es como se describe en el paso 2.8.</li>
</ol>5. grabación electrofisiológico
<ol><li>Permitir que el animal recuperar al menos 1 día después de la craneotomía antes de realizar la grabación electrofisiológica. Colocar el animal en la cámara de grabación de la misma manera que se ha hecho durante las sesiones de aclimatación cuando se inicie la grabación.</li>
	<li>Hacer pipetas de vidrio de Capillares de vidrio de borosilicato (OD = 1,5 mm, ID = 0,9 mm, largo 90,0 mm) tirado en un tirador de electrodo. El diámetro de su punta es 2 – 3 μm, y su resistencia es de 4 a 6 MΩ cuando están llenas de 10 mM PBS. La longitud del electrodo es de 40-50 mm. En algunas grabaciones, neurobiotin 2% se agrega a lo 10 mM PBS.</li>
	<li>Coloque la pipeta de cristal llenado al titular de la optrode por encargo.</li>
	<li>Poner el alambre de plata revestido de Ag-Cl (0,2 mm de diámetro) con la pipeta de cristal. Conecte el alambre de plata a un pin a través de un alambre eléctrico revestido fino. Conectar la clavija en el headstage del amplificador de grabación.</li>
	<li>Conecte el cable de fibra óptica parche (núcleo = 960 μm, revestimiento = 1.000 μm, NA = 0,63) con una virola de zirconia (OD = 2,5 mm) a la división de manga en el soporte de optrode de acoplamiento. Introduzca el cable de parche en la manga hasta la parte posterior de la pipeta de vidrio en contacto con la punta de la virola. Entrega de la cuerda de remiendo de la luz (longitud de onda = 465 nm) del LED. La luz LED es controlada por un controlador de LED de canal único.</li>
	<li>Remover el pegamento de silicona sobre la craneotomía. Poner caliente solución salina (38 ° C) en la superficie del cerebro periódicamente para evitar que la superficie del cerebro se seque durante la sesión de grabación. Si es necesario, retire la duramadre con pinzas afiladas.</li>
	<li>Insertar el electrodo de vidrio en el tejido cerebral con un manipulador. Monitorear la resistencia del electrodo cuando se inserta. Vuelva a colocar el electrodo cuando la resistencia es menor de lo esperado porque la punta del electrodo puede dañarse. En la profundidad de interés, buscar y aislar las puntas de la unidad de ajuste de la profundidad en pasos de 2 a 5 μm.</li>
	<li>Después de que la unidad está aislada, entregar la luz y observar la respuesta a los estímulos de luz.</li>
	<li>Registrar las actividades neuronales después de la identificación de los tipos de células. Realizar las grabaciones para 2-3 h. Si la grabación se realiza en días consecutivos (máxima de 2 días), cubrir la craneotomía con adhesivo de silicona. Cuando la grabación está terminada, y el animal es sacrificado, recuperar el post principal y ponerlo en acetona para lavar y reutilícelo.</li>
</ol>

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Los autores no tienen nada que revelar.

Optogenetics se ha convertido en una herramienta poderosa en la neurociencia. Se ha utilizado para identificar tipos específicos neuronas en vivo así como manipular las actividades de vías neuronales específicas. La clarificación de las actividades neuronales de diferentes tipos neuronales promueve la comprensión del mecanismo de los circuitos neuronales. Aquí, hemos demostrado un método para proporcionar la luz en el sitio de grabación a través de un electrodo de vidrio en el IC de despiertas ratones ...

El sistema nervioso central consta de varios tipos de neuronas, que tienen diferentes funciones. Cómo funcionan estos tipos de neuronas dentro del circuito neural es una de las preocupaciones principales de la neurociencia. Sin embargo, en muchas regiones del cerebro, ha sido imposible distinguir los tipos neuronales en grabaciones en vivo de actividades eléctricas porque no hay clara diferencia en la señal de punto eléctrico, con algunas excepciones. Los avances recientes en optogenetics han hecho un gran avance1,2. Uso de animales transgénicos que sensibles a la luz opsina (p. ej., channelrhodopsin-2) se expresa en tipos neuronales específicos, llegó a ser posible distinguir los tipos neuronales eficientemente en vivo grabaciones3, 4,5,6. En estos animales se excitan las neuronas con sensibles a la luz opsina dando estímulos de luz durante las grabaciones eléctricas, pero otras neuronas no son. Las neuronas de la opsina-positivo, por lo tanto, se distinguen fácilmente de otros tipos de neurona por sus respuestas a la luz.
En experimentos de optogenetics, es fundamental para entregar la luz en el sitio de grabación. Como un método no invasivo, la luz es dirigida a menudo de la superficie del cerebro. Sin embargo, porque la fuerza de la luz reduce ya que pasa a través de tejido fino del cerebro, es difícil estimular las regiones profundas del cerebro de la superficie del cerebro. Sobre todo, es difícil para la luz de estimulación llegar a las regiones profundas del cerebro cuando la transparencia óptica de la superficie del cerebro es baja, como sucede a menudo con grabaciones de animales despiertos. Experimentos electrofisiológicos a menudo se han realizado en animales anestesiados ya que el movimiento del cuerpo hace ruido en las grabaciones. Como está bien documentado, sin embargo, la anestesia es conocida por cambiar las respuestas neuronales7,8,9,10. Por lo tanto, es necesario utilizar animales despiertos para estudiar las respuestas neuronales sin el efecto artificial de la anestesia. A diferencia de los experimentos con animales anestesiados, las grabaciones electrofisiológicas se realizan después de la recuperación de la cirugía en los experimentos con animales despiertos. Durante el intervalo entre la cirugía y las grabaciones, el exudado del tejido a menudo se acumula en la superficie del cerebro y hace poco la transparencia óptica de la superficie del cerebro.
Aquí, describimos un método para registrar grabaciones de una sola unidad de un ratón despierto utilizando un optrode de cristal a medida. En este método, la luz se proporciona mediante el electrodo de cristal de la grabación para que sea posible estimular confiablemente la neurona grabada con luz en regiones profundas del cerebro. Este sistema de optrode por encargo consiste en materiales accesibles y de bajo costo y es fácil de montar.

<table><tbody><tr><td>Electrode holder</td><td>Molecular Device</td><td>1-HL-U</td><td>pipette holder for microelectrode amplifier</td></tr><tr><td>Ceramic split mating sleeve</td><td>Thorlabs</td><td>ADAF1</td><td>f2.5 mm ferrule</td></tr><tr><td>Circuit board spacer</td><td>Teishin Denki</td><td>SPA-320</td><td>f8.0 mm, 20.0 mm long</td></tr><tr><td>Stereotaxic frame for mice</td><td>Narishige</td><td>SR-6M-HT</td><td>Stereotaxic instruments for mice</td></tr><tr><td>Manipulator</td><td>Narishige</td><td>NA</td><td>Manual manipulator</td></tr><tr><td>Superbond</td><td>Sun Medical</td><td>M: 204610557</td><td>Dental adhesive resin cement</td></tr><tr><td>Form2</td><td>Formlabs</td><td>NA</td><td>3D printer</td></tr><tr><td>Kwik-Sil</td><td>WPI</td><td>KWIK-SIL</td><td>Low toxicity silicone adhesive</td></tr><tr><td>Borosilicate glass capillaries</td><td>Narishige</td><td>GD-1.5</td><td>OD 1.5 mm, ID 0.9 mm, 90.0 mm long</td></tr><tr><td>Fiber-optic patch cord</td><td>Doric Lenses</td><td>MFP_960/1000/2200-0.63_1m_FCM-ZF2.5</td><td>Monofiberoptic patchcord, OD, 2.5 mm, core = 960 mm, cladding = 1000 mm, NA = 0.63</td></tr><tr><td>Connectrized LED</td><td>Doric Lenses</td><td>LEDC-1B_FC</td><td>Central wave length = 465 nm, output power = 45 mW (Core 960 mm 0.63 NA )</td></tr><tr><td>LED driver</td><td>Doric Lenses</td><td>LEDRV_1CH_1000</td><td>1 ch LED driver, maximum output = 1000 mA</td></tr><tr><td>Electrode puller</td><td>Narishige</td><td>PB-7</td><td>Dual-stage glass micropipette puller</td></tr><tr><td>Borosilicate glass capillary</td><td>Narishige</td><td>GD-1.5</td><td>Bolosilicate glass capillary, OD, 1.5mm, ID, 0.9 mm, 90.0 mm long</td></tr><tr><td>GENTACIN</td><td>MSD CO., Ltd</td><td>185711173</td><td>Antibiotic ointment</td></tr><tr><td>Terramycin&amp;reg;-LA</td><td>Zoetis</td><td>G 333</td><td>Oxytetracycline</td></tr><tr><td>Tg(Slc32a1- COP4*H134R/EYFP)8Gfng/J</td><td>Jackson Labs</td><td>#14548</td><td>VGAT-ChR2 mice</td></tr><tr><td>Multiclamp 700B</td><td>Molecular Devices</td><td>2500-0157</td><td>Microelectrode amplifier</td></tr></tbody></table>

optogenetics identification of a neuronal type with a glass optrode in awake mice

Es una preocupación importante en Neurociencia cómo diferente tipos de neuronas funcionan en circuitos neurales. Los avances recientes en optogenetics han permitido la identificación del tipo neuronal en vivo experimentos electrofisiológicos en regiones amplias del cerebro. En experimentos de optogenetics, es fundamental para entregar la luz en el sitio de grabación. Sin embargo, a menudo es difícil entregar la luz del estímulo a las regiones profundas del cerebro de la superficie del cerebro. Sobre todo, es difícil para la luz de estimulación llegar a las regiones profundas del cerebro cuando la transparencia óptica de la superficie del cerebro es baja, como sucede a menudo con grabaciones de animales despiertos. Aquí, describimos un método para registrar respuestas de espiga a la luz de un ratón despierto utilizando un optrode de cristal a medida. En este método, la luz se proporciona mediante el electrodo de cristal de la grabación para que sea posible estimular confiablemente la neurona grabada con luz en las regiones profundas del cerebro. Este sistema de optrode por encargo consiste en materiales accesibles y de bajo costo y es fácil de montar.

En la figura 2, se analizaron los efectos del tamaño de la punta y la longitud de las pipetas de vidrio en la potencia de luz en la punta de las pipetas (figura 2A-B). La potencia de luz fue medida por un medidor de potencia óptica a 1 mm de la punta. La longitud total se estableció en 50 ± 2 mm cuando el tamaño de la punta varía, mientras que el tamaño de la punta fue a 2.5 μm cuando la longitud total va...

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Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

Optogenetics identificación de un tipo Neuronal con un vidrio Optrode en ratones despiertos

Este trabajo presenta un método para realizar un optogenetic de una sola unidad grabación fiable desde un ratón despierto utilizando un optrode de cristal a medida.

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

6.7K vistas.  Kanazawa Medical University. Este trabajo presenta un método para realizar un optogenetic de una sola unidad grabación fiable desde un ratón despierto utilizando un optrode de cristal a medida.

Video: Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Caracterización electrofisiológica de las buenas prácticas agrarias que expresan las poblaciones de células en la retina intacta

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Investigación

Neurociencias

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

Un método para la alta fidelidad Optogenética control de un individuo neuronas piramidales<em&gt; En vivo</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

Manipulación optogenética de la actividad neuronal para modular el comportamiento en ratones que se mueven libremente

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

Comportamiento

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo optogenético la estimulación del sistema nervioso central de roedores

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

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 ...

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser.&#160;Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible.&#160;The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

La visualización directa de la murino Dorsal coclear Núcleo de optogenética La estimulación de la vía auditiva

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

Manipulación optogenética de circuitos neuronales durante la supervisión de los estados de sueño/vigilia en ratones

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective neural modulation or monitoring. As specific types of neuronal cells can be genetically modified to express opsin proteins, optogenetics enables optical stimulation or inhibition of the selected neurons. There have been several technological advances in the optical system for optogenetics. Recently, it was proposed to combine the optical waveguide for light delivery with electrophysiological recording to simultaneously monitor the neural responses to optogenetic stimulation or inhibition. In this study, an implantable optrode array (2x2 optical fibers) was developed with embedded multichannel electrodes. 
A light-emitting diode (LED) was employed as a light source, and a microfabricated microlens array was integrated to provide sufficient light power at the tip of the optical fibers. The optrode array system comprises the disposable part and the reusable part. The disposable part has optical fibers and electrodes, while the reusable part has the LED and electronic circuitry for light control and neural signal processing. The novel design of the implantable optrode array system is introduced in the accompanying video in addition to the procedure of the optrode implantation surgery, optogenetic light stimulation, and the electrophysiological neural recording. The results of in vivo experiments successfully showed time-locked neural spikes evoked by the light stimuli from hippocampal excitatory neurons of mice.

Here, we present the fabrication method of an optrode system with optical fibers for light delivery and an electrode array for neural recording. In vivo experiments with transgenic mice expressing channelrhodopsin-2 show the feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording.

Optrode Array para modulación optogenética simultánea y grabación neuronal eléctrica

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective ...

Installation of Optical Windows in the Brain of Transgenic Mice to Image Neural Activities

Source: Lin, X., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56297">Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice.</a> J. Vis. Exp. (2019).
This video demonstrates the brain&#39;s neural activity recording through an optical window in transgenic mice. The mice express a fluorescent protein that enables the detection of neural activities. A small portion of the skull is carefully removed to install an optical window, and the skull is then covered with black cement to block light before imaging.

Instalación de ventanas ópticas en el cerebro de ratones transgénicos para visualizar actividades neuronales

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Optogenetics identificación de un tipo Neuronal con un vidrio Optrode en ratones despiertos

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