We thank Tran Huynh, Andrew San Antonio, Jerry Lin for their technical assistance. This work was funded by the National Institutes of Health grants DA023700 and DA023700-04S1 to X.X.

1. Preparação fatia do cérebro <ol><li> Camundongos transgênicos estão profundamente anestesiados com pentobarbital sódico (&gt; 100 mg / kg, ip) e rapidamente decapitados, e seus cérebros extraídos para uma solução de corte congelados e oxigenado. </li><li> Óculos de GFP são usados ​​para visualmente tela se o cérebro de camundongo na verdade expressa GFP. </li><li> Cortical somatossensorial primário seções de 400 mm de espessura são cortados com uma vibratome em sacarose contendo líquido cefalorraquidiano artificial (ACSF). Fatias são os primeiros incubados em sacarose contendo ACSF por 30 min a 1 h em 32 ° C, e após o período de incubação inicial, transferido para ACSF gravação em temperatura ambiente. Todo o período de incubação e de gravação, as fatias são continuamente aeradas com 95% O 2 -5% CO 2. </li></ol> 2. Equipamentos de inicialização A implementação total do sistema é ilustrada na Figura 1. <ol><li> O sistema de resfriamento a laser e fornecedor de energia é ligada, recebendo o laser pronto. </li><li> Todo o hardware relacionados ao controle fotoestimulação (incluindo o sistema de varredura do espelho, um modulador óptico, um obturador eletrônico e um amplificador de entrada fotodiodo) está ligado. </li><li> O equipamento eletrofisiológicos (incluindo um amplificador Multiclamp 700B e micromanipuladores) é ligado. Gravações eletrofisiológicas, fotoestimulação e imagens dos preparativos fatia é feito em uma câmara de perfusão fatia montada no palco motorizado do microscópio. </li><li> A câmera de imagem (Retiga 2000, Q-imagem, Austin, TX) é ligado. Fatias são visualizados com o microscópio vertical (BW51X, Olympus) com contraste de infravermelho interferência diferencial (DIC) e epi-fluorescente óptica através do sistema de imagem. </li><li> O Matlab baseado em software EPHUS e Q captura de software da câmera são iniciado. A versão personalizada modificada da Ephus software (Ephus, disponível em <a href="https://openwiki.janelia.org/">https://openwiki.janelia.org/</a> ) é usado para controlar protocolos fotoestimulação e adquirir dados fotoestimulação. </li><li> Depois que o sistema está ligado, deve-se verificar para ver se o sistema de fotoestimulação laser é em um estado de funcionamento. Como a objetiva do microscópio 4x é utilizado para enviar flashes UV para uncaging glutamato, um pedaço de papel branco pode ser colocado sob a observar o padrão de varredura a laser, enquanto o sistema é executado. </li></ol> 3. Criação de perfusão fatia <ol><li> Um sistema de perfusão pressurizado é ligada para alimentar a ACSF gravação na câmara de fatia. Cuidado é tomado para garantir um nível de fluido constante de 2,0 mm acima da fatia na câmara. </li><li> Uma alíquota da solução estoque de MNI-enjaulado-glutamato é adicionado a 25 ml de circulação ACSF para uma concentração de 0,2 mM glutamato enjaulado. Por favor note que após 5-6 h de experimentação, a solução de banho e MNI-glutamato será atualizada. </li><li> A fatia do cérebro é movido para dentro da câmara de gravação. Pode-se executar o sistema de imagem para verificar a qualidade fatia e anatomicamente localizar a área somatossensorial primário. Em seguida, a fatia está ancorado com um anel de platina custom-made de cordas. Tenha cuidado para não colocar a âncora sobre a região do cérebro destinada para as gravações. </li></ol> 4. Patch-fixação de células inteiras de gravação <ol><li> Eletrodos de vidro (4-6 mohms resistência) são puxados e preenchido com uma solução interna contendo biocytin 0,1% para a rotulagem e identificação de células morfológica. </li><li> Para executar a gravação patch, as células são visualizadas no objetivo 60x. Tipos de células inibitórias são os primeiros identificados e seleccionados com base na visualização de expressão GFP sob um microscópio Olympus DIC / fluorescentes; gravações são posteriormente executados sob controle visual, auxiliada pela monitorização de vídeo infravermelho DIC. Por favor note que algumas vezes de alternar entre os modos de DIC e fluorescentes é necessário para confirmar a localização de células GFP quando se aproxima o eletrodo para a célula-alvo. </li><li> Técnicas convencionais de patch-fixação são aplicadas. O eletrodo é preenchido com pressão positiva, mudou-se perto da superfície da célula para estabelecer uma ondulação perceptível sobre a membrana da célula alvo, e então a pressão negativa é aplicada rapidamente para formar um gigaseal e quebrar em monitorar as respostas ao mesmo tempo de injeção de corrente no monitor de vídeo. </li><li> Ao romper dentro, as imagens da célula em 60x sob os modos de DIC e fluorescentes são levados para a verificação on-line. Antes da coleta de dados fotoestimulação, hiperpolarizante e despolarizante pulsos de corrente são injetados para examinar cada célula básica propriedades eletrofisiológicas. </li><li> Uma vez estável gravações de células inteiras são alcançados com a resistência de bons acessos (geralmente &lt;20 mohms), a objetiva do microscópio é alternado de 60x para 4x para o laser fotoestimulação digitalização. A imagem fatia em 4x é adquirido e será utilizado para orientar e registrar os sites fotoestimulação. </li></ol> 5. Laser scanning fotoestimulação <ol><li> Os parâmetros de aquisição fotoestimulação e os dados são definidos pela ativação do módulo de switch de configuração de Ephus. Normalmente, 1 ms laser (20 mW) com um intervalo de estímulo de um segundo é utilizado para o mapeamento fotoestimulação. Dados os traços de um segundo amostrados a 10 kHz são adquiridos. </li><li> A imagem fatia 4x é carregado no módulo de mapeador de Ephus. A localização da soma das células é definido e sites fotoestimulação de um padrão 16x16 (80 mM mM espaçamento x 80) são criados em torno da localização da célula, abrangendo todas as camadas corticais. </li><li> O perfil de excitação do neurônio gravado é mapeado através do exame dos locais spiking em resposta a fotoestimulação no modo de fixação atual. Nosso software user-friendly com os recursos on-line facilita a visualização dos experimentos de mapeamento. </li><li> Ligações locais circuito excitatório são mapeados pela detecção de correntes excitatórias pós-sinápticas (EPSC) a partir da célula gravado durante a varredura a laser em diferentes locais. A célula é mantida a -70 mV no modo braçadeira de tensão para detectar dentro correntes excitatórias. Os mapas são repetidas EPSC 2-3 vezes com o padrão de fotoestimulação girado ou invertido. </li><li> Opcional: Local conexões do circuito inibitório também pode mapeado pela detecção de correntes pós-sináptico inibitório (IPSC) a partir da célula registrado realizada no próximo a 0 mV no modo braçadeira de tensão para detectar as correntes exteriores inibitória. Note-se que o melhor é usar a solução interna do eletrodo com o potássio substituído com césio para o mapeamento de IPSC. </li><li> Depois de todos os ensaios fisiológicos são concluídas, o eletrodo é delicadamente retirado da célula gravada. A fatia do cérebro é retirado e fixado em paraformaldeído a 4% durante a noite. As células gravado é manchada contra biocytin. Morfologia das células é examinada com confocal ou epi-fluorescente de microscopia, o que também confirma que cada célula registrada é de fato o interneurônio expressando GFP-originalmente-alvo. </li></ol> 6. Fotoestimulação análise de dados Nossa metodologia recentemente desenvolvida e implementação de software 11 é aplicada à detecção e medição de fotoestimulação evocadas eventos sinápticos nos mapas de dados brutos. Como exemplificado na Figura 2, codificados por cores mapas são construídos para ilustrar o padrão de entrada sináptica para o neurônio gravado. 7. Resultados representativos: Exemplo de resultados de gravação eletrofisiológico e mapeamento fotoestimulação são mostrados na Figura 2. Gravações alvo de tipos específicos de células inibitórias são possíveis usando camundongos transgênicos expressando GFP em conhecidos tipos de células inibitórias. Dada a diversidade de interneurônios inibitórios, análises de expressão GFP, eletrofisiologia intrínseca e características morfológicas (Figura 2A) são combinados para chegar à classificação final célula tipo. Figura 2B-C ilustrar que fotoestimulação de varredura a laser permite mapeamento extensivo de ligações locais circuito cortical a única neurônios inibitórios. O mapa de dados brutos é mostrada na Figura 2C. As respostas uncaging directos são excluídos da análise de dados (Figura 2C). Os mapas de entrada quantitativas são codificados por cores, mostrado na Figura 2D-F. O exemplo fast-spiking interneurônios inibitórios recebeu forte entrada excitatórios sinápticos (EPSCs) de camada 4 e camadas mais profundas. Ao relacionar a organização da célula entradas sinápticas definido vias corticais, somos capazes de inferir o seu papel possível (por exemplo, feedback e inibição feedforward) no processamento da informação cortical. <img alt="Figura 1" src="/files/ftp_upload/3109/3109fig1.jpg" /> Figura 1. Instrumentação sistema geral de fotoestimulação laser de varredura. Nosso sistema completo é composto de controle fotoestimulação, imagem, vídeo e sistemas de gravação eletrofisiológico. Adotamos o projeto do sistema de LSPs descrito anteriormente 7,17. A unidade de laser é usado para gerar 355 nm laser UV para uncaging glutamato. O feixe de laser é direcionado através do caminho óptico do nosso sistema. Curta duração das ondas de laser (por exemplo, 1-3 ms) são controlados por meio de um modulador eletro-óptico (isto é, células Pockels) e um obturador mecânico. Laser potência do feixe é modulado por uma roda de densidade neutra gradiente e monitorado por desviar uma pequena fração do feixe de laser com uma lamela de vidro para um fotodiodo. O sistema de varredura a laser inclui um par XY de espelhos scan, a lente de digitalização, a lente do tubo, ea lente objetiva. Os espelhos entregar o feixe de laser através de uma lente de digitalização, em seguida, o feixe entra no microscópio por meio de um espelho dicróico e é focalizado por uma lente do tubo custom-made UV-transmissão. O feixe underfills a abertura parte de trás da objetiva do microscópio para fornecer um colunar mais (em oposição a cônicas) feixe de iluminação, mantendo o mapeamento bidimensional quanto possível, reduzindo a resolução axial. No âmbito do objectivo de 4x, o feixe de laser formas spots uncaging, cada approximating um perfil Gaussiano com uma largura de 153 M lateralmente no plano focal (ver a inserção). Posições diversas estimulação laser pode ser alcançado através de galvanômetros-driven espelhos de varredura XY, como os espelhos ea abertura de trás do objetivo são em planos conjugados, traduzindo posições espelho em diferentes localizações de varredura no plano focal da lente objetiva. Durante uncaging, um número variável de sites padronizados que cobre todo o campo são estimulados em seqüência em um nonraster, seqüência não aleatória para evitar revisitar as proximidades de locais recentemente estimulada. Por favor, consulte Xu et al. (2010) para obter informações mais detalhadas. <img alt="Figura 2" src="/files/ftp_upload/3109/3109fig2.jpg" /> Figura 2. Mapeamento local conexões do circuito de excitação a uma expressando GFP-interneurônio fast-spiking na camada 4 do córtex sensorial primário do mouse. A1 e A2 mostram as imagens DIC e GFP fluorescente da célula-alvo no âmbito do objectivo 60x no fatia cérebro vivo, enquanto mostra A3 a morfologia celular revelado pela coloração biocytin no pós-gravação, fatia fixa. A4 mostra os padrões de disparo das células GFP gravado, característica de um fast-interneurônio spiking inibitória, em resposta a intrassomáticas injeções de corrente de forças diferentes. B mostra a imagem sobreposta com a fatia de 16 sites fotoestimulação x16 (*). A localização da célula é indicada por um pequeno círculo vermelho. C mostra uma série de fotoestimulação evocadas traços de resposta dos locais de estimulação mostrado na B, enquanto a célula foi realizada a -70 mV para detectar dentro correntes excitatórias. Diferentes formas de respostas fotoestimulação são ilustrados pelos traços em locais de 1 e 2, que são expandidos e de forma separada na inserção. O traço 1 é um exemplo das respostas diretas (indicado por setas a vermelho) para glutamato uncaging no corpo celular e dendritos promximal. Outros vestígios em 2 são exemplos típicos de respostas excitatórias entrada sináptica (azul). Respostas diretas e entradas sinápticas podem ser distinguidos por suas amplitudes e latências de resposta. D, E e F são os mapas com código de cores (16x16 sites) da média EPSC amplitude, os números EPSC, ea latência detectados primeiro EPSC por site, respectivamente, para os dados brutos mapa mostrado na C. A amplitude média de entrada de cada local de estimulação é a amplitude média de EPSCs na janela de análise, com a resposta inicial espontânea subtraída da resposta fotoestimulação do mesmo site. O número de EPSCs ea hora de chegada ou latência do EPSC detectado pela primeira vez por site também são medidos e plotados. Por favor, consulte (Shi et al., 2010) para obter mais detalhes.

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No conflicts of interest declared.

Photostimulation-based mapping techniques have been effectively applied for analyzing cortical circuits. Laser scanning photostimulation combined with whole cell recording allows high resolution mapping of laminar distributions of presynaptic input sources to single neurons, because the simultaneous recording from a postsynaptic neuron with photostimulation of clusters of presynaptic neurons at many different locations provides quantitative measures of spatial distribution of excitatory or inhibitory inputs. This tech...

<table border="1"> <tbody> <tr> <td>Name of the reagent</td> <td>Company</td> <td>Catalogue number</td> <td>Comments</td> </tr> <tr> <td>transgenic mouse lines</td> <td>Jackson lab or other sources</td> <td></td> <td>Please refer to Xu and Callaway (2009)</td> </tr> <tr> <td>GFP goggles</td> <td>BLS Ltd., Hungary</td> <td></td> <td></td> </tr> <tr> <td>vibratome</td> <td>Leica Systems</td> <td>VT1200S</td> <td></td> </tr> <tr> <td>MNI caged glutamate (4-methoxy-7-nitroindolinyl-caged l-glutamate)</td> <td>Tocris Bioscience, Ellisville, MO</td> <td>Cat No. 1490</td> <td></td> </tr> <tr> <td>biocytin</td> <td></td> <td>B4261</td> <td></td> </tr> <tr> <td>electrode puller</td> <td>Sutter Instrument, Novato, CA</td> <td>P-97</td> <td></td> </tr> <tr> <td>glass tubes for making electrodes</td> <td></td> <td>BF150-86-10</td> <td></td> </tr> <tr> <td>Multiclamp 700B amplifier</td> <td>Molecular Devices, Sunnyvale, CA</td> <td>Multiclamp 700B</td> <td></td> </tr> <tr> <td>digital CCD camera</td> <td>Q-imaging, Austin, TX</td> <td>Retiga 2000</td> <td></td> </tr> <tr> <td>Research microscope</td> <td>Olympus, Tokyo, Japan</td> <td>BW51X</td> <td></td> </tr> <tr> <td>UV laser unit</td> <td>DPSS Lasers, Santa Clara, CA</td> <td>model 3501</td> <td></td> </tr> <tr> <td>Other equipment for Laser scanning phostimulation</td> <td></td> <td></td> <td>Please refer to Xu et al. (2010)</td> </tr> </tbody> </table> Solutions: <ul> <li>Sucrose-containing artificial cerebrospinal fluid (ACSF) for slice cutting (in mM: 85 NaCl, 75 sucrose, 2.5 KCl, 25 glucose, 1.25 NaH2PO4, 4 MgCl2, 0.5 CaCl2, and 24 NaHCO3).</li> <li>Recording ACSF (in mM: 126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, and 10 glucose)</li> <li>Electrode internal solution (in mM: 126 K-gluconate, 4 KCl, 10 HEPES, 4 ATP-Mg, 0.3 GTP-Na, and 10 phosphocreatine; pH 7.2, 300 mOsm).</li> </ul>

mapping inhibitory neuronal circuits by laser scanning photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.

Watch this Scientific Journal Video about Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation at JoVE.com

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

Mapeamento Inibitória Circuitos Neuronal por Laser Scanning fotoestimulação

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

mapping-inhibitory-neuronal-circuits-laser-scanning

Research

JoVE Journal

Neuroscience

17.2K visualizações.  University of California, Irvine. This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

Vídeo: Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Rápido Micro-iontoforese de glutamato e GABA: uma ferramenta útil para investigar Integração Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Neurociência

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 ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Induzir Plasticidade de astrocítica Receptores de Manipulação de Neuronal queima de moeda

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.
A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

Desenho, Tratamento de Superfícies, Cellular Galvanização e Cultura de modulares Neuronal de redes formadas por Circuitos Funcionalmente Inter-conectados

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

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 ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Rewiring Neuronal Circuits: um novo método para Fast Neurite Extension e Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Estimulação elétrica transcraniana crônica e gravação Intracortical em ratos

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

Combinação de Optogenetics com microRNAs Artificial para caracterizar os efeitos do Gene Knockdown na função pré-sináptica dentro circuitos neuronais intacta

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic de arrastamento de Theta Hippocampal oscilações em comportar-se ratos

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

Preparação de fatia de cunha in vitro para imitar conectividade do circuito neuronal vivo

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...