Agradecemos al Dr. Chris Rogers (Universidad de Tufts) para sugerencias de programación y el manuscrito. Damos las gracias a Alex Giuliano y Deborah Lee de apoyo a la producción de vídeo. Los fondos proporcionados por la NSF Graduate Research Fellowship y una MURI ONR en Biología Sintética.

1. Construyendo el Modelo Robot <ol><li> Elige un organismo modelo para el estudio que está bien representada en la literatura neuroethological. Los invertebrados en general son buenos candidatos debido a que sus sistemas nerviosos relativamente simples han sido bien estudiado y se componen principalmente de los reflejos innatos. Vamos a demostrar este enfoque con la langosta americana, Homarus americanus. </li><li> Seleccione bien estudiado comportamientos reflexivos con fines de modelización. Hemos elegido las respuestas de la langosta de curva antenal y para la detección de golpe de garra como el animal responde instintivamente a la flexión de la antena para obtener rheotaxis (orientación al flujo de agua), mientras que los contactos garra median evitar obstáculos. Reflejos simples que dependen de decussating conexiones neuronales de los sensores de simetría bilateral son muy adecuadas para este tipo de estudio 1. </li><li> Crear o elegir una plataforma robótica con sensores adecuados para los comportamientos seleccionados con el fin de ejecutar el neuralsimulación de la red. Mientras que un robot dedicado puede ser construido como RoboLobster 12 o la dispersión (cucaracha) familia de robots 13, aquí se utiliza un kit disponible en el mercado para agilizar el proceso experimental. El kit de LEGO MINDSTORMS NXT 2.0, proporcionando plantillas, componentes modulares y sensores establecidos, permite la rápida construcción física del robot. </li><li> Seleccione o construir sensores para que coincida con las modalidades de las conductas seleccionadas en el paso 1.2. Premade sensores del kit LEGO Mindstorms pueden ser utilizados o sensores caseros pueden ser diseñados 14,15. Hemos seleccionado el sensor táctil LEGO incluido y un sensor de flexión antenal costumbre que consiste en un sensor de flexión FlexPoint empalmada a un conector de NXT. Para crear un conector resistente costumbre, cortar un cable conector de NXT y soldar los cables blanco y negro a los cables del sensor. </li><li> Utilice estudios neuroethological publicados anteriormente para encontrar o desarrollar una red neuronal hipotética para el modelado purposes. La literatura ha propuesto varias redes neuronales para explicar el comportamiento en varios organismos modelo, de langostas 16 a 17 lamprea. Unidades neuronales funcionales deben ser identificados y sus conexiones sinápticas teorizaron. Experimentos neuroethological novela también se pueden desarrollar y aplicar, si las circunstancias de laboratorio permiten. </li></ol> 2. Programación del Sistema Nervioso <ol><li> Utilice el software LabVIEW con el Módulo LabVIEW para LEGO MINDSTORMS NXT para crear instrumentos virtuales (VIs) que se ejecutan las ecuaciones para una neurona matemática y el modelo de sinapsis. Mientras que cualquier modelo que se publica se puede utilizar, se recomienda el tiempo discreto basado en mapas (DTM) modelo 18. Esto permite la operación en tiempo real computacionalmente eficiente manteniendo al mismo tiempo una variedad de regímenes de salida de disparo neuronal. Dos parámetros de control, alfa y sigma, determinan la dinámica de la neurona simulada para producir variada de salida neuronal incluyendo Tonic Rematar, caótica despido, el silencio que estalla e intrínseca. VIs de LabVIEW para LEGO Mindstorms para este modelo neuronal se puede encontrar en línea en: <a href="http://myfiles.neu.edu/blustein.d/neurobots" target="_blank">http://myfiles.neu.edu/blustein.d/neurobots</a> </li><li> Lugar VIs en un bucle While para que la red se actualizará iterativa. </li><li> Conecte la neurona presináptica salidas pico través de las sinapsis en las neuronas postsinápticas. </li><li> Pass iterativamente la actualización de parámetros a través de registros de desplazamiento para el siguiente ciclo de cálculo. El DTM modelo actualizaciones rápido (x) y variables dinámicas lentas (y) que se pasan a la siguiente iteración de los cálculos. </li><li> Establezca los parámetros de sinapsis para definir las características de la red. Para cada sinapsis, la fuerza sináptica (gsyn), la velocidad de relajación (gamma), y el potencial de inversión (XRP) son coeficientes de las ecuaciones del modelo DTM que determinan el comportamiento sináptica. El valor de gsyn es un multialicates que determina la magnitud de la corriente sináptica efecto de un evento de pico, que van desde 0.0 (sin efecto) a 1,0 (100% de efecto). El valor de gamma es un multiplicador que determina la tasa de decaimiento de la corriente sináptica independientemente de los eventos pico, que van desde 0,0 (descomposición completa) a 1.0 (sin descomposición). El valor de XRP determina el nivel de la excitación o la inhibición de la sinapsis, valora típicamente rango -3,0 a 3,0 con valores positivos representan excitación y los valores negativos representan la inhibición. Los valores pueden ajustarse a sus características específicas de la red. Aquí se utiliza la siguiente configuración: gamma = 0,95, gsyn = 0,60 y XRP = 2,2 (sinapsis excitatoria) o -1,8 (sinapsis inhibitoria). </li><li> Ajuste alfa y parámetros de control de sigma para modificar la actividad neuronal basal. La dinámica del modelo de variados parámetros de control se describen en detalle en otra parte 18 pero por lo general, alfa </em&gt; &lt;4 produce intrínsecamente la actividad neuronal en silencio por valores o tónicas adición de altos valores de sigma sigma bajos. La transición entre &quot;baja&quot; y &quot;alta&quot; valores sigma generalmente ocurre entre -1,0 y 1,0 en función del valor alfa. Repartir la actividad neuronal se produce para alpha&gt; 4, cuando los valores de sigma están cerca de cero. Aquí se utiliza alfa = 4,05 y sigma = -3,10. </li><li> Utilice LabVIEW VIs a la información del sensor de entrada a la red. Para los sensores previstos, los VIs ya están disponibles en el módulo LabVIEW Mindstorms. Para los sensores resistivos personalizados, utilice el sensor VI Luz como un marco de programación. Información sobre la creación de sensores caseros se puede encontrar en otro lugar 14. </li><li> Insertar diagramas gráficos en el panel frontal del programa LabVIEW para visualizar la actividad neuronal. </li><li> Reemplace temporalmente las entradas del sensor con cajas de control del panel frontal para manipular manualmente el sensorinformación va a la red neuronal. </li><li> Ejecutar la red neuronal y ajustar manualmente los controles del panel frontal para presentar variados estímulos sensoriales. Asegúrese de que la red funciona cualitativamente como se predijo por condiciones conocidas. </li><li> Ajuste de la red según sea necesario mediante el cambio de los parámetros de neuronas y sinapsis. Primeros intentos de ajuste de la red se debe hacer mediante el cambio de la fuerza sináptica (gsyn) de las distintas sinapsis. Pueden necesitar ser ajustado así como otros parámetros. </li><li> Una vez que las funciones de la red con una precisión cualitativa, reemplace las cajas de control del panel frontal, con la entrada del sensor VIs. </li></ol> 3. Pruebas de Simulación del Sistema Nervioso <ol><li> Establecer un ambiente controlado en el que observar el animal y el robot en condiciones similares. Utilizamos un tanque rectangular que contiene obstáculos roca (Figura 1). El tanque puede ser vaciado para alojar el robot LEGO en el mismo espacio. </li><li> Mount avideo sobrecarga cámara para grabar el animal / comportamiento del robot. Asegúrese de que el contraste entre el motivo y el fondo es alta para permitir el seguimiento automático. Pintamos la parte inferior de la prueba del tanque blanca. </li><li> Colocar el animal en el ambiente controlado y el comportamiento de grabación de vídeo. Asegúrese de limitar las capacidades sensoriales de la langosta para que coincida con los del robot. Aquí nos limitamos capacidades sensoriales de la langosta, cubriendo los ojos con una máscara de papel de aluminio y deafferenting quimiorreceptores con agua dulce 19. </li><li> Ajuste el entorno para adaptarse a un robot LEGO (eliminación de agua si es necesario) y ejecutar el robot en el mismo entorno controlado. </li><li> Utilice una secuencia de comandos de MATLAB para seguir automáticamente los marcadores en las muestras. Utilizamos MouseLabTracker 20. </li><li> Crear y comparar representaciones gráficas de vectores del animal y los movimientos del robot. </li><li> Modificar el sistema nervioso y repita los pasos 3.3 a 3.6 para observar cómo los diferentes aspectos de la electrónicasistema nervioso afecta el comportamiento del robot. Aquí nos ajustamos la fuerza sináptica entre las neuronas garra bump sensor y el sistema del motor y el rendimiento del robot de monitor en comparación con la de una langosta. Alternativamente, las condiciones sensoriales en la zona de ensayo pueden ser modificados y los efectos observados en el robot y animal. Por ejemplo, la arena se describe aquí podría ser sometido a diferentes tasas de flujo de agua. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Al iniciar experimentos de simulación del sistema nervioso biorobotic, hay algunas pautas importantes a seguir. Elegir el organismo modelo adecuado es fundamental: elegir un organismo que es fácil de obtener y de mantener. Los invertebrados son ideales, ya que por lo general no requieren la aprobación institucional para la experimentación y sus necesidades de cría suelen ser menos exigentes que los de los vertebrados. Desde una perspectiva científica, es beneficioso para elegir un animal que tiene un registro lite...

Investigaciones neurofisiológicas en los últimos 100 años se han ampliado enormemente nuestro conocimiento de la estructura del sistema nervioso y la función. Sin embargo, la mayor parte de la investigación del sistema nervioso hasta la fecha se ha basado en el uso de preparaciones aisladas o materias restringidas. Aunque ha habido muchos esfuerzos exitosos para registrar la actividad neuronal de comportarse libremente animales 2-5, el enfoque biorobotic proporciona una valiosa herramienta para permitir la manipulación del sistema nervioso con el fin de probar los sistemas de nivel de hipótesis neurociencia 6. Sistemas nerviosos simulados que operan en los robots pueden ser experimentalmente manipulados y permiten la extensión de modelado de software con el mundo físico. Este enfoque ha sido bien implementada en el mundo académico de 7,8, pero el proceso de construcción de un robot biomimético para la prueba de hipótesis puede ser costoso y consume tiempo. Se presenta un método para ejecutar el enfoque biorobotic utilizando un comercialmente disponible robótica kél (LEGO Mindstorms NXT 2.0). El objetivo de este método es proporcionar una manera rápida y eficaz para poner a prueba los sistemas de nivel de neurociencia hipótesis sobre robótica 9 o bio-híbridos 10 incorporados simulaciones de redes neuronales. Acelerar el proceso de hipótesis y experimentación mejora la productividad de la investigación. La plataforma sencilla LEGO Mindstorms proporciona un banco de pruebas para sensores biomiméticos y redes neuronales que muestran el uso de la langosta americana (Homarus americanus) como organismo modelo. El método también proporciona una potente herramienta práctica educativa en el aula, los estudiantes pueden diseñar y manipular sistemas nerviosos por sus propios robots 11.

designing and implementing nervous system simulations on lego robots

Se presenta un método para utilizar la plataforma disponible en el mercado LEGO Mindstorms NXT robótica para poner a prueba los sistemas de nivel de la neurociencia hipótesis. La primera etapa del método es el desarrollo de una simulación del sistema nervioso de los comportamientos reflexivos específicos de un organismo modelo apropiado; aquí usamos la langosta americana. Reflejos exteroceptivos mediadas por decussating (cruce) conexiones neuronales pueden explicar los taxis de un animal hacia o lejos de un estímulo tal como se describe por Braitenberg y se adaptan particularmente bien para la investigación usando la plataforma de NXT. 1 La simulación del sistema nervioso se programa usando el software LabVIEW en el LEGO plataforma Mindstorms. Una vez que el sistema nervioso está sintonizado correctamente, los experimentos de comportamiento se ejecutan en el robot y en el animal en las condiciones ambientales idénticas. Al controlar el ambiente sensorial experimentado por las muestras, se pueden observar diferencias en resultados conductuales. Estas diferencias pueden indicar deficienc específicas en el modelo del sistema nervioso y sirve para informar a la versión del modelo para el comportamiento particular de estudio. Este método permite la manipulación experimental de los sistemas nerviosos electrónicos y sirve como una forma de explorar hipótesis neurociencia específicamente con respecto a la base neurofisiológica de comportamientos reflexivos innatas simples. El kit de LEGO Mindstorms NXT ofrece una plataforma asequible y eficiente en el que poner a prueba sistemas de control de robots biomiméticos preliminares. El enfoque también es muy adecuado para el aula de secundaria para servir como la base para una práctica en el plan de estudios basado en la investigación biorrobótica.

Las aportaciones de las garras de una langosta en su sistema nervioso median en la negociación de obstáculos en un ambiente nuevo. Figura 1 se muestra una captura de pantalla del video utilizado para analizar el comportamiento de un robot LEGO (Figura 1A) y una langosta (Figura 1B) en el campo de prueba. La zona de ensayo no se modificó entre las pruebas robot animales y excepto que el agua se vació desde el tanque para los ensayos robot. <p class="jove_content"...

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

Se presenta un enfoque para el modelado de redes neuronales en la plataforma de robótica LEGO Mindstorms. El método proporciona una herramienta de simulación para invertebrados investigación en neurociencias, tanto en el laboratorio de investigación y en el aula. Esta técnica permite la investigación de los principios de control de robots biomiméticos.

Diseño e implementación de simulaciones del sistema nervioso en 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 ...

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14.9K vistas.  Northeastern University. Se presenta un enfoque para el modelado de redes neuronales en la plataforma de robótica LEGO Mindstorms. El método proporciona una herramienta de simulación para invertebrados investigación en neurociencias, tanto en el laboratorio de investigación y en el aula. Esta técnica permite la investigación de los principios de control de robots biomiméticos.

Video: Designing and Implementing Nervous System Simulations on LEGO Robots

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.

La producción de vectores lentivirales de células transductoras del Sistema Nervioso Central

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

Investigación

Neurociencias

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.

El marcaje de células individuales en el Sistema Nervioso 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 ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

Un paradigma de Lesiones de investigar reparación del sistema nervioso central en<em&gt; Drosophila</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

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 Circuito de grabación de una cucaracha sistema nervioso intacto

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 optogenético la estimulación del sistema nervioso central de roedores

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

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.

El uso de insectos electroantenograma Sensores de Robots Autónoma de Búsquedas 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+ Imaging del sistema nervioso 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 ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Clearing óptico del sistema nervioso central del ratón Utilizando CLARITY pasiva

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

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.

Generación de hidrogeles a base de colágeno 3D para analizar el crecimiento y el comportamiento axonal durante el desarrollo del sistema nervioso

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.

Trasplante de progenitores interneuronas corticales de ingeniería química en cerebros de ratón postnatal temprano

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