Os autores foram apoiados pela sociedade de Japão para a promoção da ciência KAKENHI Grant JP16K11200 e 17H 02223 e o subsídio para a pesquisa da universidade médica S2016 de Kanazawa-8 e C2017-3. Yuhichi Kuda Agradecemos seu apoio em tirar as fotos.

Todos os procedimentos são feitos em conformidade com os princípios orientadores da sociedade Physiological do Japão e com a aprovação do Animal conta Comissão de Kanazawa médica Universidade.
1. construção do titular do Optrode de vidro
Nota: Para construir um suporte de optrode de vidro, um porta-eletrodo comercial é utilizado (figura 1A).
<ol><li>Suavemente retire o tubo de aço para controle de pressão do cilindro do titular.</li>
	<li>Por perfuração, alargar o buraco do lado do assento pin no cano do titular. Faça o furo de 3 mm de diâmetro e 12 mm em profundidade.</li>
	<li>Colocar um tubo de aço inoxidável (3 mm de diâmetro, de espessura 0,5 mm e 8 mm de comprimento; Figura 1A 2) no buraco.</li>
	<li>Inserir uma cerâmica de dividir o acasalamento manga para virolas de ⌀2.5 mm (figura 1A1) no orifício.</li>
	<li>Consertar o cano do porta eletrodo para uma ranhura em forma de L com adesivo epóxi.</li>
	<li>Faça os furos (3 mm de diâmetro) na ranhura e anexá-lo para um manipulador com parafusos.</li>
</ol>2. cabeça pós instalação
<ol><li>Prepare um Custom-Built post cabeça feito de um espaçador de placa de circuito. Fazer o espaçador de placa de circuito colunar em forma cuboidal com uma fresa e corte em 2 lugares de cabeça com uma serra (figura 1B). Achate a parte inferior do post principal com uma fresa.</li>
	<li>Prepare o animal geneticamente modificado em que um opsin sensível à luz é expresso em um tipo de neurônio específico para o estudo. Neste trabalho, é usado o rato transgénico (ratos VGAT-ChR2) em que os neurônios inibitórios expressam channelrhodopsin-2 (ChR2)11 .</li>
	<li>Anestesia o animal com uma mistura de 0,3 mg/kg de medetomidina, 4,0 mg/kg de midazolam e 5,0 mg/kg de butorfanol pela administração intraperitoneal. Para confirmar que o animal é totalmente anestesiado, teste suas reações para beliscar da cauda.</li>
	<li>Coloque o mouse em uma armação estereotáxica em uma almofada de aquecimento. Limpo, o aquecimento e a armação estereotáxica almofada antecipadamente com álcool a 70%. Coloque os dentes no buraco da barra dentada e aperte levemente o grampo do nariz. Corrigi a ambos os lados da cabeça usando barras de orelha. Quando a cabeça é fixada corretamente pelas barras de orelha, aperte o grampo do nariz. Aplica uma pomada oftálmica nos olhos.</li>
	<li>Raspar a pele da cabeça com uma tesoura pequena e limpar o couro cabeludo com um cotonete de gluconato de clorexidina. Antes da cirurgia, esterilize todos que os instrumentais cirúrgicos e uma cabeça de postar com uma autoclave (121 ° C, 15 min).</li>
	<li>Corte o couro cabeludo ao longo da linha mediana com uma tesoura e empurrá-lo de lado. Fazer a incisão na parte de trás da cabeça para os olhos (cerca de 20 mm). Remover o periósteo com um cotonete embebido em álcool a 70% e secar o crânio.</li>
	<li>Anexe o posto de cabeça para o manipulador. Posicione o post cabeça plana no crânio em torno do bregma usando o manipulador.</li>
	<li>Misture o monômero (4 gotas), catalisador (1 gota) e polímero (1 colher pequena) de cimento dental em um pequeno prato que é tranquilo na geladeira com antecedência. Ponha o cimento dental (abaixo de 1 g) frontal e parietal osso do crânio para fixar a coifa cabeça no crânio.</li>
	<li>Depois que o cimento dental torna-se difícil, retire o mouse da armação estereotáxica. Coloque uma pomada antibiótica na pele exposta. Injete o antagonista de medetomidina (0,3 mg/kg) e os antibióticos (oxitetraciclina, 20 mg/kg). Coloque e monitorar o mouse sobre a almofada do calor em uma gaiola até que ele acorda e devolvê-lo para uma gaiola de habitação.</li>
	<li>Casa do animal em uma gaiola de habitação individual com algum dispositivo de enriquecimento. Mude o papel de animal fundamento diariamente para manter a gaiola limpa e prevenir a infecção. Monitore cuidadosamente se o animal mostra qualquer sinal de desconforto. Aplicam-se lidocaína a ferida se houver suspeita de qualquer dor. Administrar meloxicam ou carprofeno uma vez a cada 24h por 48h após ambos a cabeça pós instalação e craniotomia cirurgias. A lidocaína local pode ser dado além disso se o AINE não é adequada.
</li>
</ol>3. a aclimatação
<ol><li>(No mínimo) dois dias após a recuperação da anestesia, adapte os animais ao estado cabeça-fixo na câmara de gravação. Realize as sessões de aclimatação para pelo menos 5 dias.</li>
	<li>Durante as sessões de aclimatação, coloca o animal na câmara de gravação com o post cabeça fixado para uma pinça custom-built. Durante a sessão, a fim de limitar o movimento, a tampa do corpo com um tubo de resina feito por metade (Figura 1), feita usando uma impressora 3D. Nota: No dia 1, a sessão de aclimatação deve ser longo 5 min. Ele é então aumentado diariamente para 10, 20, 40 e 60 min para dias 2, 3, 4 e 5, respectivamente.</li>
	<li>No final de cada sessão, o animal de dar uma recompensa de comida. Se o animal se esforça continuamente durante a aclimatação, interromper a sessão.</li>
</ol>4. craniotomia
Nota: Após a aclimatação, uma craniotomia é feita sobre a região do cérebro para a gravação. A craniotomia é realizada no quadro estereotáxicos sob anestesia, como com a cabeça pós instalação. O procedimento de pós operação é o mesmo como a cabeça pós instalação.
<ol><li>Antes a craniotomia, limpe o crânio com um cotonete de algodão com clorexidina gluconato e 70% de álcool.</li>
	<li>Raspe cuidadosamente o crânio sobre o local de gravação com uma broca de dentista. Quando o osso torna-se bastante fino, cortar com a ponta do bisturi e removê-lo.</li>
	<li>Colocar cimento dental em torno da região exposta a fim de reforçar o crânio.</li>
	<li>Cobrir a área do cérebro exposto com adesivo de silicone após o cimento dental torna-se difícil. O procedimento pós-operatório é conforme descrito na etapa 2.8.</li>
</ol>5. eletrofisiológica gravação
<ol><li>Permitir que o animal se recuperar pelo menos 1 dia após a craniotomia antes de executar a gravação eletrofisiológica. Coloque o animal na câmara de gravação da mesma forma que foi feito durante as sessões de aclimatação, quando inicia a gravação.</li>
	<li>Fazer vidro pipetas de capilares de vidro de borosilicato (OD = 1,5 mm, ID = 0,9 mm, 90,0 mm de comprimento) apontou um extrator de eletrodo. Seu diâmetro de ponta é de 2 – 3 µm, e sua resistência é 4-6 MΩ quando eles estão cheios de 10mm PBS. O comprimento do eletrodo é de 40 – 50 mm. Em algumas gravações, 2% neurobiotin é adicionado para o PBS de 10 mM.</li>
	<li>Anexe a pipeta de vidro preenchida ao titular do optrode sob medida.</li>
	<li>Coloque o fio de prata Ag-Cl revestido (0,2 mm de diâmetro) dentro da pipeta de vidro. Ligue o fio de prata a um pino através de um fio elétrico revestido fino. Conecte o pino para o headstage do amplificador de gravação.</li>
	<li>Conecte o cabo de remendo de fibra óptica (núcleo = 960 µm, revestimento = 1.000 µm, at = 0,63) com uma virola de zircônia (OD = 2,5 mm) para a divisão de acasalamento manga no porta-optrode. Empurre o cabo de remendo na manga até a ponta da virola entra em contato com as costas da pipeta vidro. O cabo de remendo oferece a luz (comprimento de onda = 465 nm) de LED. O diodo emissor de luz é controlada por um controlador de LED de canal único.</li>
	<li>Remova o adesivo de silicone sobre a craniotomia. Colocar soro aquecido (38 ° C) sobre a superfície do cérebro periodicamente para evitar que a superfície do cérebro de secagem durante a sessão de gravação. Se necessário, remova a dura-máter com Pinças afiadas.</li>
	<li>Introduza o eléctrodo de vidro o tecido do cérebro com um manipulador. Monitore a resistência do eletrodo, quando ele é inserido. Substitua o eletrodo quando a resistência é menor do que o esperado porque a ponta do eletrodo pode ser danificada. Na profundidade de interesse, Pesquisar e isolar os picos da única unidade ajustando a profundidade em passos de 2 a 5 µm.</li>
	<li>Depois que a unidade está isolada, entregar o diodo emissor de luz e observar a resposta aos estímulos leves.</li>
	<li>Grave as atividades neurais após a identificação dos tipos de célula. Realize as gravações de 2 – 3 h. Se a gravação é feita em dias consecutivos (máxima de 2 dias), cubra a craniotomia com adesivo de silicone. Quando a gravação estiver concluída, e o animal é sacrificado, recuperar o post cabeça e colocá-lo em acetona para lavar e reutilizá-lo.</li>
</ol>

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Optogenetics tornou-se uma ferramenta poderosa em neurociência. Ele tem sido utilizado para identificar neurônio específico tipos na vivo , bem como manipular as atividades das vias neuronais específicas. O esclarecimento da actividade neural de diferentes tipos neuronais promove a compreensão do mecanismo dos circuitos neurais. Aqui, temos demonstrado um método para fornecer a luz para o local de gravação através de um eléctrodo de vidro na IC de ratos de VGAT-ChR2 acordados.
<p class="jove_conten...

O sistema nervoso central consiste de vários tipos de neurônios, que têm funções diferentes. Como esses diferentes tipos de neurônios funcionam dentro do circuito neural é uma das principais preocupações da neurociência. No entanto, em muitas regiões do cérebro, tem sido impossível distinguir os tipos neuronais em gravações na vivo de actividades eléctricas, porque não há nenhuma diferença clara no sinal elétrico spike em si, com algumas exceções. Avanços recentes em optogenetics fizeram uma descoberta1,2. Usando animais transgénicos na qual opsin sensíveis à luz (por exemplo, channelrhodopsin-2) é expressa em tipos específicos de neurônios, tornou-se possível distinguir os tipos neuronais eficientemente na vivo gravações3, 4,5,6. Nestes animais, os neurônios com opsin sensíveis à luz estão animados, dando estímulos de luz durante as gravações elétricas, mas outros neurônios não são. Os neurônios opsin-positivo, portanto, facilmente são distintos dos outros tipos de neurônio por suas respostas à luz.
Em experimentos de optogenetics, é essencial para fornecer a luz para o local de gravação. Como um método não-invasivo, a luz é muitas vezes dirigida da superfície do cérebro. No entanto, porque a força da luz reduz conforme passa pelo tecido cerebral, é difícil estimular as regiões cerebrais profundas da superfície do cérebro. Especialmente, é difícil para a luz de estimulação atingir as regiões profundas do cérebro quando a transparência óptica da superfície do cérebro é baixa, como é frequentemente o caso com gravações de animais acordados. Eletrofisiológicos experimentos frequentemente foram realizados em animais anestesiados porque o movimento do corpo provoca ruído nas gravações. Como está bem documentado, no entanto, anestesia é conhecida para alterar as respostas neurais7,8,9,10. Assim, é necessário utilizar animais acordados para estudar as respostas neurais, sem os efeitos artificiais da anestesia. Ao contrário das experiências com animais anestesiados, as gravações eletrofisiológicas são executadas após a recuperação da cirurgia nos experimentos com animais acordados. Durante o intervalo entre a cirurgia e as gravações, o exsudato tecido frequentemente se acumula na superfície do cérebro e faz com que a transparência óptica da superfície do cérebro baixa.
Aqui, descrevemos um método para gravar gravações unitárias de um rato acordado usando um optrode de vidro sob medida. Neste método, a luz é fornecida através do eléctrodo de vidro de gravação é possível estimular confiantemente o neurônio gravado com luz em regiões profundas do cérebro. Este sistema de optrode sob medida consiste de materiais acessíveis e baratos e é 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

É uma grande preocupação em neurociência como diferente tipos de neurônios funcionam em circuitos neurais. Avanços recentes em optogenetics permitiram a identificação do tipo neuronal na vivo eletrofisiológicos experiências em regiões do cérebro amplo. Em experimentos de optogenetics, é essencial para fornecer a luz para o local de gravação. No entanto, muitas vezes é difícil entregar-se a luz de estimulação às regiões profundas do cérebro da superfície do cérebro. Especialmente, é difícil para a luz de estimulação atingir as regiões profundas do cérebro quando a transparência óptica da superfície do cérebro é baixa, como é frequentemente o caso com gravações de animais acordados. Aqui, descrevemos um método para gravar respostas de pico à luz de um rato acordado usando um optrode de vidro sob medida. Neste método, a luz é fornecida através do eléctrodo de vidro de gravação é possível estimular confiantemente o neurônio gravado com luz nas regiões profundas do cérebro. Este sistema de optrode sob medida consiste de materiais acessíveis e baratos e é fácil de montar.

Na Figura 2, examinamos os efeitos do tamanho da ponta e do comprimento das pipetas de vidro sobre o poder de luz na ponta das pipetas (Fig. 2A-B). O poder da luz foi medido por um medidor de potência óptica colocado 1 mm da ponta. O comprimento total foi definido como 50 ± 2 mm quando o tamanho da ponta variado, enquanto o tamanho da ponta foi definido como 2,5 µm, quando todo o comprimento variado. A haste ...

Watch this Scientific Journal Video about Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice at JoVE.com

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

Identificação de Optogenetics de um tipo Neuronal com um Optrode de vidro em ratos acordados

Este trabalho apresenta um método para executar uma optogenetic unidade única gravação confiável de um rato acordado usando um optrode de vidro sob 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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Research

JoVE Journal

Neuroscience

6.7K visualizações.  Kanazawa Medical University. Este trabalho apresenta um método para executar uma optogenetic unidade única gravação confiável de um rato acordado usando um optrode de vidro sob medida.

Vídeo: 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.

Caracterização eletrofisiológica de GFP-Expressando populações de células na retina intacta

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

Neurociência

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.

Um Método para High Fidelity Optogenetic Controle de Individual Piramidal neurônios<em&gt; In 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.

Manipulação optogenética da atividade neuronal para modular o comportamento em camundongos em movimento livre

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

Comportamento

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 Optogenetic estimulação do sistema nervoso 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.

Um Guia para<em&gt; In vivo</em&gt; Gravação única unidade de Optogenetically Identificado Cortical Inibitória Interneurônios

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.

Visualização direta do Murino Dorsal Coclear Nucleus para Optogenetic Estimulação da via 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. ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

Neuronal Tracing em explantes cérebro guiadas a laser

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

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.

Manipulação optogenetic de circuitos neural durante a monitoração sono/Estados do wakefulness nos ratos

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.

Oprode Array para modulação optogenética simultânea e gravação neural elétrica

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

Identificação de Optogenetics de um tipo Neuronal com um Optrode de vidro em ratos acordados

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