Agradecemos ao Dr. Chris Rogers (Tufts University) para sugestões de programação e manuscrito. Agradecemos Alex Giuliano e Deborah Lee para apoio à produção de vídeo. Financiamento concedido por um NSF Graduate Research Fellowship e um MURI ONR em Biologia Sintética.

1. Construir o modelo Robot <ol><li> Escolha um organismo modelo para o estudo que está bem representado na literatura neuroetológica. Invertebrados geralmente fazem bons candidatos, porque seu sistema nervoso relativamente simples foram bem estudados e são, basicamente, composta de reflexos inatos. Vamos demonstrar esta abordagem utilizando o Lobster americana, Homarus americanus. </li><li> Escolha bem estudado comportamentos reflexivos para fins de modelagem. Nós escolhemos as respostas da lagosta a curva da antena e detecção de colisão garra como o animal reflexivamente responde à flexão de antenas para obter reotaxia (orientação para o fluxo de água), enquanto contatos garra mediar desvio de obstáculos. Reflexos simples contando com decussating conexões neurais a partir de sensores bilateralmente simétricos são adequados para este tipo de estudo 1. </li><li> Construir ou escolher uma plataforma robótica com sensores apropriados para os comportamentos selecionados, a fim de executar o neuralsimulação da rede. Enquanto um robô dedicado pode ser construído como RoboLobster 12 ou o Sprawl (barata) família robot 13, aqui nós usamos um kit disponível comercialmente para agilizar o processo experimental. A LEGO Mindstorms 2.0 kit NXT, proporcionando moldes, componentes modulares e sensores estabelecidos, permite a construção física rápida do robô. </li><li> Selecione ou construir sensores para combinar as modalidades dos comportamentos selecionados na etapa 1.2. Premade sensores do kit LEGO Mindstorms pode ser usado homebrew ou sensores podem ser projetados 14,15. Nós selecionamos o sensor de toque LEGO incluído e um sensor de curvatura da antena costume que consiste em um sensor de curva FlexPoint emendado a um conector NXT. Para construir um conector resistiva costume, cortou um fio conector NXT e soldar os cabos preto e branco para as ligações do sensor. </li><li> Use neuroetológica estudos publicados anteriormente para encontrar ou desenvolver uma rede neural hipotética para a modelagem purposes. A literatura tem proposto várias redes neurais para explicar o comportamento de vários organismos modelo, a partir de lagostas 16 a lampreia 17. Unidades neurais funcionais devem ser identificados e suas conexões sinápticas teorizou. Experimentos neuroetológica romance também pode ser desenvolvido e implementado se as circunstâncias de laboratório permitem. </li></ol> 2. Programação do Sistema Nervoso <ol><li> Use o software LabVIEW, juntamente com o Módulo LabVIEW para LEGO Mindstorms NXT para criar instrumentos virtuais (VIs), que funcionam equações para um neurônio matemático e modelo sinapse. Embora qualquer modelo publicado pode ser usado, recomendamos o tempo discreto baseado em mapa (DTM), modelo 18. Isto permite uma operação eficiente computacionalmente tempo real, mantendo uma variedade de regimes de saída de disparo neuronal. Dois parâmetros de controle, alfa e sigma, determinar a dinâmica do neurônio simulado para produzir uma saída neural variado, incluindo tonic spiking, disparo de caótico, o silêncio de ruptura e intrínseco. VIs do LabVIEW para LEGO Mindstorms para este modelo neurônio pode ser encontrado online aqui: <a href="http://myfiles.neu.edu/blustein.d/neurobots" target="_blank">http://myfiles.neu.edu/blustein.d/neurobots</a> </li><li> Lugar VIs em um loop while para que a rede irá atualizar de forma iterativa. </li><li> Ligue as saídas dos neurônios pré-sinápticos pico através das sinapses dos neurônios pós-sinápticos. </li><li> Passe iteratively atualizar os parâmetros através de registradores de deslocamento para o próximo ciclo de cálculo. O DTM modelo atualizações rápido (x) e lentas (y) variáveis ​​dinâmicas que são passados ​​para a próxima iteração de cálculos. </li><li> Defina os parâmetros de sinapse para definir as características de rede. Para cada sinapse, a força sináptica (gsyn), a taxa de relaxamento (gama), e o potencial de inversão (XRP) são coeficientes nas equações do modelo DTM que determinam o comportamento sináptica. O valor de gsyn é um multialicate que determina a magnitude do efeito da corrente sináptica de um evento de pico, variando entre 0,0 (nenhum efeito) a 1,0 (100% de efeito). O valor da gama é um multiplicador que determina a velocidade de decaimento da corrente sináptica, independentemente dos eventos de pico, variando entre 0,0 (decomposição completa) e 1,0 (nenhuma deterioração). O valor de xrp determina o nível de excitação ou inibição da sinapse, valores geralmente gama de -3,0 a 3,0, com valores positivos representam excitação e os valores negativos representam a inibição. Os valores podem ser ajustados para as suas características específicas da rede. Aqui usamos as seguintes configurações: Gamma = 0,95, gsyn = 0,60 e xrp = 2,2 (sinapse excitatória) ou -1.8 (sinapse inibitória). </li><li> Ajuste alfa e parâmetros de controle sigma para modificar a atividade dos neurônios de base. A dinâmica do modelo de parâmetros de controle variados são descritos em detalhes em outra parte 18, mas normalmente, alpha </em&gt; &lt;4 produz intrinsecamente atividade neural em silêncio por valores sigma baixos ou tônicas spiking para altos valores de sigma. A transição entre &quot;baixo&quot; e &quot;alto&quot; valores sigma ocorre geralmente entre 1,0 e 1,0, dependendo do valor alfa. Estourando a atividade dos neurônios é produzido para alpha&gt; 4, quando os valores sigma estão perto de zero. Aqui usamos alfa = 4,05 e sigma = -3,10. </li><li> Use LabVIEW VIs a informação do sensor de entrada em sua rede. Para sensores fornecidos, o VIS já estão disponíveis no módulo LabVIEW Mindstorms. Para sensores resistivos personalizados, use o Sensor VI Luz como um quadro de programação. Informações sobre a criação de sensores homebrew pode ser encontrada em outro lugar 14. </li><li> Inserir gráficos gráficas no painel frontal do programa LabVIEW para visualizar a actividade neuronal. </li><li> Temporariamente substituir entradas de sensores com caixas de controle do painel frontal, a fim de manipular manualmente o sensorinformação vai da rede neural. </li><li> Funcionamento da rede neuronal e ajustar manualmente os controles do painel frontal para apresentar estímulos sensoriais variadas. Confirmar que a rede funciona como qualitativamente previsto para as condições conhecidas. </li><li> Ajuste a rede, conforme necessário, alterando os parâmetros de neurônios e sinapses. Primeiras tentativas de ajuste da rede deve ser feito alterando a força sináptica (gsyn) das várias sinapses. Outros parâmetros podem necessitar de ser ajustado. </li><li> Uma vez que as funções de rede com precisão qualitativa, substituir as caixas de controle do painel frontal com a entrada do sensor VIs. </li></ol> 3. Testes de Simulação do Sistema Nervoso <ol><li> Defina-se num ambiente controlado para observar o animal e o robô sob condições semelhantes. Usamos um tanque rectangular contendo obstáculos naturais (Figura 1). O tanque pode ser esvaziado para acomodar o robô LEGO no mesmo espaço. </li><li> Monte avideo câmera aérea para gravar o animal / comportamento do robô. Certifique-se o contraste entre o motivo eo fundo é elevada para permitir o rastreamento automático. Nós pintada no fundo do tanque de ensaio branco. </li><li> Colocar o animal no ambiente controlado e de gravação de vídeo do comportamento. Certifique-se de limitar as capacidades sensoriais da lagosta para coincidir com as do robô. Aqui vamos restringir as capacidades sensoriais do lagosta, cobrindo os olhos com uma máscara de folha de alumínio e deafferenting chemoreceptors com água doce 19. </li><li> Ajuste o ambiente para atender um robô LEGO (remoção de água, se necessário) e executar o robô no mesmo ambiente controlado. </li><li> Use um script MATLAB para rastrear automaticamente os marcadores nas amostras. Usamos MouseLabTracker 20. </li><li> Criar e comparar parcelas vetor do animal e os movimentos do robô. </li><li> Modificar o sistema nervoso e repita os passos de 3,3-3,6 para observar como os diferentes aspectos da eletrônicasistema nervoso afeta o comportamento do robô. Aqui ajustar a força sináptico entre as garras da colisão neurónios sensor e o sistema de desempenho do motor e do monitor do robô comparado àquele de uma lagosta. Alternativamente, as condições sensoriais na arena de teste pode ser modificado e os efeitos observados no robô e animal. Por exemplo, a arena descrita aqui pode ser sujeita a diferentes taxas de fluxo de água. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Ao iniciar experimentos de simulação do sistema nervoso biorobotic, existem algumas orientações importantes a seguir. Escolhendo o organismo modelo certo é fundamental: escolher um organismo que é fácil de obter e manter. Invertebrados são ideais porque eles geralmente não necessitam de aprovação institucional para a experimentação e as suas necessidades de criação são menos exigentes do que aqueles dos vertebrados. De uma perspectiva científica, é benéfico para escolher um animal que tem um registro ...

Investigações neurofisiológicas ao longo dos últimos 100 anos, têm ampliado enormemente nosso conhecimento da estrutura e função do sistema nervoso. No entanto, a maioria das pesquisas sistema nervoso até à data tem contado com o uso de preparações isoladas ou assuntos contidos. Embora tenha havido muitos esforços bem-sucedidos para registrar a atividade neural de se comportar livremente animais 2-5, a abordagem biorobotic fornece uma ferramenta valiosa para permitir a manipulação do sistema nervoso, a fim de testar os sistemas de nível neurociência hipóteses 6. Sistemas nervosos simulados operacionais em robôs podem ser experimentalmente manipulada e permitir a extensão de modelagem de software para o mundo físico. Esta abordagem tem sido bem implementado no mundo acadêmico 7,8, mas o processo de construção de um robô biomimético para o teste de hipóteses pode ser caro e demorado. Apresenta-se um método para executar a abordagem biorobotic disponível comercialmente utilizando um k robóticaele (LEGO Mindstorms NXT 2.0). O objetivo deste método é fornecer uma maneira rápida e eficiente para testar sistemas de nível neurociência hipóteses sobre robótica 9 ou 10 incorporados simulações de rede neural bio-híbridos. Acelerar o processo de hipótese de experimentar melhora a produtividade de pesquisa. O simples plataforma LEGO Mindstorms fornece uma plataforma de teste para sensores biomiméticos e redes neurais que demonstrar usando a lagosta americana (Homarus americanus) como um organismo modelo. O método também fornece uma poderosa hands-on ferramenta pedagógica em sala de aula como os alunos podem projetar e manipular sistemas nervosos de seus próprios robôs 11.

designing and implementing nervous system simulations on lego robots

Nós apresentamos um método para usar o Mindstorms plataforma disponível comercialmente LEGO NXT robótica para testar sistemas de nível neurociência hipóteses. O primeiro passo do método é o de desenvolver um sistema de simulação de comportamentos nervoso reflexivas especiais de um organismo modelo adequado; aqui usamos a lagosta americana. Reflexos exteroceptivos mediadas por decussating (passagem) conexões neurais podem explicar táxis de um animal ou para longe de um estímulo, tal como descrito por Braitenberg e são particularmente bem adequado para investigação, utilizando a plataforma NXT. 1 A simulação do sistema nervoso está programado utilizando software LabVIEW no LEGO plataforma Mindstorms. Uma vez que o sistema nervoso está sintonizado corretamente, experimentos comportamentais são executados no robô e sobre o animal sob condições ambientais idênticas. Ao controlar o meio sensorial experimentada pelas amostras, as diferenças nos resultados comportamentais podem ser observadas. Estas diferenças podem apontar para deficienc específicos no modelo de sistema nervoso e servem para informar a iteração do modelo para o comportamento particular em estudo. Este método permite a manipulação experimental do sistema nervoso electrónicos e serve como um meio para explorar neuroscience hipóteses especificamente em relação à base de neurofisiológicos simples comportamentos reflexivos inatas. O LEGO Mindstorms NXT kit fornece uma plataforma acessível e eficiente em que para testar esquemas de controle de robôs biomiméticos preliminares. A abordagem também é adequado para a sala de aula do ensino médio para servir de base para um hands-on baseada na investigação currículo Biorobotics.

Entradas de garras de um lagosta no seu sistema nervoso mediar negociação obstáculo num ambiente novo. Figura 1 mostra uma imagem do vídeo usadas para analisar o comportamento de um robô LEGO (Figura 1A) e uma lagosta (Figura 1B) na arena de teste. A arena de teste foi inalterado entre os testes de robô e animal, exceto que a água foi esvaziado do tanque para os ensaios do robô. Resultados de rastreamento de vídeo são apresentados ...

Watch this Scientific Journal Video about Designing and Implementing Nervous System Simulations on LEGO Robots at JoVE.com

Designing and Implementing Nervous System Simulations on LEGO Robots

É apresentada uma abordagem para a modelagem de rede neural na plataforma robótica LEGO Mindstorms. O método fornece uma ferramenta de simulação para invertebrados pesquisa em neurociência, tanto no laboratório de pesquisa e sala de aula. Esta técnica permite a investigação de princípios de controle de robôs biomiméticos.

Projetando e implementando Simulações do Sistema Nervoso em LEGO Robots

We present a method to use the commercially available LEGO Mindstorms NXT robotics platform to test systems level neuroscience hypotheses. The first step ...

designing-and-implementing-nervous-system-simulations-on-lego-robots

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Neuroscience

14.9K visualizações.  Northeastern University. É apresentada uma abordagem para a modelagem de rede neural na plataforma robótica LEGO Mindstorms. O método fornece uma ferramenta de simulação para invertebrados pesquisa em neurociência, tanto no laboratório de pesquisa e sala de aula. Esta técnica permite a investigação de princípios de controle de robôs biomiméticos.

Vídeo: Designing and Implementing Nervous System Simulations on LEGO Robots

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Visualização do Sistema Nervoso embrionárias no Whole-mount Drosophila Os embriões

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Neurociência

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Produção de lentivirais para Células Transdução do Sistema Nervoso Central

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Marcação de células individuais no Sistema Nervoso Central de<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

Neural Circuit gravação de um Sistema Nervoso Barata Intacta

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

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 ...

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

Usando insetos eletroantenogramas Sensores em robôs autônomos para Pesquisas olfativas

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

In Situ Ca 2+ Imagem do Sistema Nervoso Entérico

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

Geração de hidrogéis com base em colágeno 3-D para analisar o crescimento e o comportamento axonal durante o desenvolvimento do sistema nervoso

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Transplantação dos progenitors corticais Quimiogenetically projetados do interneuron em cérebros Postnatal adiantados do rato

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often rodents. This approach limits the interrogation of blood-brain barrier (BBB) properties to the cortex and does not allow for individual comparison. This project focuses on the development of an isolation method that allows for the comparison of the neurovascular unit (NVU) from multiple CNS regions: cortex, cerebellum, optic lobe, hypothalamus, pituitary, brainstem, and spinal cord. Moreover, this protocol, originally developed for murine samples, was successfully adapted for use on CNS tissues from small and large vertebrate species from which we are also able to isolate microvessels from brain hemisphere white matter. This method, when paired with immunolabeling, allows for quantitation of protein expression and statistical comparison between individuals, tissue type, or treatment. We proved this applicability by evaluating changes in protein expression during experimental autoimmune encephalomyelitis (EAE), a murine model of a neuroinflammatory disease, multiple sclerosis. Additionally, microvessels isolated by this method could be used for downstream applications like qPCR, RNA-seq, and Western blot, among others. Even though this is not the first attempt to isolate CNS microvessels without the use of ultracentrifugation or enzymatic dissociation, it is unique in its adeptness for the comparison of single individuals and multiple CNS regions. Therefore, it allows for investigation of a range of differences that may otherwise remain obscure: CNS portions (cortex, cerebellum, optic lobe, brainstem, hypothalamus, pituitary, and spinal cord), CNS tissue type (gray or white matter), individuals, experimental treatment groups, and species.

The goal of this protocol is to isolate microvessels from multiple regions of the central nervous system of lissencephalic and gyrencephalic vertebrates.

Isolamento confiável de micronavios do sistema nervoso central em cinco grupos de vertebrados

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often ...