Ringraziamo il Dott. Chris Rogers (Tufts University) per la programmazione e il manoscritto suggerimenti. Ringraziamo Alex Giuliano e Deborah Lee per il sostegno alla produzione video. Finanziamenti erogati da un NSF Graduate Research Fellowship e un MURI ONR in biologia sintetica.

1. Costruire il modello di robot <ol><li> Scegli un organismo modello per lo studio che è ben rappresentata nella letteratura neuroethological. Invertebrati generalmente fanno buoni candidati perché i loro sistemi nervosi relativamente semplici sono stati ben studiati e sono in prevalenza costituiti di riflessi innati. Dimostreremo questo approccio con l&#39;aragosta americana, Homarus americanus. </li><li> Selezionare ben studiato i comportamenti riflessivi per scopi di modellazione. Abbiamo scelto le risposte del aragosta alla curva antenne e di rilevazione urto artiglio come l&#39;animale risponde riflessivamente alla piegatura di antenne per suscitare rheotaxis (orientamento al flusso d&#39;acqua), mentre i contatti artiglio mediano l&#39;evitamento di ostacoli. Riflessi semplici basandosi su decussating connessioni neurali da sensori bilateralmente simmetriche sono adatti per questo tipo di studio 1. </li><li> Costruire o scegliere una piattaforma robotica con opportuni sensori per i comportamenti selezionati al fine di eseguire il neuralesimulazione della rete. Mentre un robot dedicato può essere costruito come RoboLobster 12 o il Sprawl (scarafaggio) famiglia di robot 13, qui usiamo un kit disponibile in commercio per accelerare il processo di sperimentazione. Il LEGO Mindstorms NXT kit 2.0, fornendo modelli, componenti modulari e sensori stabilite, consente la rapida costruzione fisica del robot. </li><li> Selezionare o costruire sensori per abbinare le modalità di comportamenti selezionati al punto 1.2. Premade sensori del kit Mindstorms LEGO possono essere usati o sensori homebrew possono essere progettate 14,15. Abbiamo selezionato il sensore di contatto LEGO inclusa e un sensore di curva antenne personalizzato costituito da un sensore di curva FlexPoint impiombato a un connettore NXT. Per costruire un connettore resistivo personalizzato, tagliato un filo connettore NXT e saldare i cavi bianco e nero per i cavi del sensore. </li><li> Utilizzare studi neuroethological precedentemente pubblicati da trovare o sviluppare una rete neurale ipotetico per la modellazione purposes. La letteratura ha proposto varie reti neurali di spiegare il comportamento in vari organismi modello, dalle aragoste da 16 a 17 lampreda. Unità neurali funzionali dovrebbero essere identificati e le loro connessioni sinaptiche teorizzato. Esperimenti neuroethological romanzo può anche essere sviluppate e attuate se le circostanze consentono di laboratorio. </li></ol> 2. Programmazione del Sistema Nervoso <ol><li> Utilizzare il software LabVIEW con il modulo LabVIEW per LEGO MINDSTORMS NXT per creare strumenti virtuali (VI) che corrono equazioni per un neurone matematico e modello di sinapsi. Mentre qualsiasi modello pubblicato può essere utilizzato, si consiglia il tempo discreto map-based (DTM) modello 18. Questo permette computazionalmente efficiente funzionamento in tempo reale mantenendo una varietà di regimi di uscita neuronale. Due parametri di controllo, alfa e sigma, determinano la dinamica del neurone simulato per produrre vario uscita neurale compreso tonic Schiacciata, cottura caotico, il silenzio di rottura e intrinseco. VI di LabVIEW per LEGO Mindstorms per questo modello di neurone può essere trovato on-line qui: <a href="http://myfiles.neu.edu/blustein.d/neurobots" target="_blank">http://myfiles.neu.edu/blustein.d/neurobots</a> </li><li> Luogo VI in un ciclo while in modo che la rete si aggiornerà iterativo. </li><li> Collegare presinaptiche dei neuroni uscite picco attraverso le sinapsi ai neuroni post-sinaptici. </li><li> Passano iterativamente l&#39;aggiornamento dei parametri tramite registri a scorrimento per il ciclo di calcolo successivo. Il modello DTM aggiornamenti veloce (x) e lenta (y) variabili dinamiche che vengono passate alla prossima iterazione di calcoli. </li><li> Impostare i parametri di sinapsi per definire le caratteristiche della rete. Per ogni sinapsi, la forza sinaptica (gsyn), il tasso di rilassamento (gamma), e il potenziale di inversione (XRP) sono coefficienti nelle equazioni del modello DTM che determinano il comportamento sinaptica. Il valore di gsyn è un multipinza che determina l&#39;entità dell&#39;effetto corrente sinaptica da un evento spike, che vanno da 0.0 (nessun effetto) a 1.0 (100% di effetto). Il valore di gamma è un moltiplicatore che determina il tasso di decadimento della corrente sinaptica indipendentemente eventi spike, che vanno da 0,0 (decadimento completo) a 1.0 (nessun decadimento). Il valore del XRP determina il livello di eccitazione o inibizione della sinapsi, valori normalmente va da -3.0 a 3.0 con valori positivi rappresentano eccitazione e valori negativi rappresentano inibizione. I valori possono essere sintonizzati per le caratteristiche specifiche della rete. Qui usiamo le seguenti impostazioni: gamma = 0.95, gsyn = 0.60 e XRP = 2.2 (sinapsi eccitatoria) o -1.8 (sinapsi inibitoria). </li><li> Regolare i parametri di controllo sigma alfa e di modificare l&#39;attività dei neuroni basale. La dinamica del modello per vari parametri di controllo sono descritti in dettaglio in altra 18 ma tipicamente, alfa </em&gt; &lt;4 produce intrinsecamente silenziosa attività neurale per bassi valori di sigma o tonico chiodare per alti valori di sigma. La transizione tra il &#39;basso&#39; e &#39;alta&#39; valori di sigma si verifica in genere tra -1.0 e 1.0 a seconda del valore alfa. Scoppio attività neuronale è prodotta per alfa&gt; 4, quando i valori di sigma sono quasi a zero. Qui usiamo alpha = 4.05 e sigma = -3.10. </li><li> Utilizzare LabVIEW per informazioni sensore di ingresso nella rete. Per i sensori forniti, il VIS sono già disponibili all&#39;interno del modulo Mindstorms LabVIEW. Per i sensori resistivi personalizzati, utilizzare il sensore di luce VI come un quadro di programmazione. Informazioni sulla creazione di sensori homebrew può essere trovato altrove 14. </li><li> Inserire di grafici sul pannello frontale del programma LabVIEW per visualizzare l&#39;attività neuronale. </li><li> Sostituire temporaneamente ingressi sensori con centraline di controllo del pannello frontale, al fine di modificare manualmente il sensoreInformazioni di andare a rete neurale. </li><li> Eseguire la rete neuronale e regolare manualmente i comandi del pannello frontale per presentare vario input sensoriale. Verificare che la rete funzioni qualitativamente come previsto per le condizioni note. </li><li> Regolare la rete in base alle esigenze, modificando i parametri di neuroni e sinapsi. Primi tentativi di messa a punto della rete dovrebbe essere fatto modificando la forza sinaptica (gsyn) dei vari sinapsi. Altri parametri possono essere necessario un aggiustamento così. </li><li> Una volta che le funzioni di rete con una precisione qualitativa, sostituire le caselle di controllo del pannello frontale con l&#39;ingresso del sensore VI. </li></ol> 3. Sistema nervoso Test Simulation <ol><li> Impostare un ambiente controllato in cui osservare l&#39;animale e il robot in condizioni simili. Usiamo una vasca rettangolare che contiene gli ostacoli rocciosi (Figura 1). Il serbatoio può essere svuotato per accogliere il robot LEGO nello stesso spazio. </li><li> Monte avideo telecamera in testa per registrare l&#39;animale / comportamento del robot. Assicurarsi che il contrasto tra il soggetto e lo sfondo è elevato per consentire il monitoraggio automatico. Abbiamo dipinto il fondo del serbatoio di prova bianco. </li><li> Posto l&#39;animale in ambiente controllato e registrare video il comportamento. Assicurati di limitare le capacità sensoriali del aragosta per adeguarli a quelli del robot. Qui ci limitiamo sensoriali del aragosta coprendo gli occhi con un foglio di alluminio e maschera deafferenting chemiorecettori con acqua dolce 19. </li><li> Regolare l&#39;ambiente per soddisfare un robot LEGO (rimuovere l&#39;acqua se necessario) ed eseguire il robot nello stesso ambiente controllato. </li><li> Utilizzare uno script MATLAB per tracciare automaticamente i marcatori sui provini. Usiamo MouseLabTracker 20. </li><li> Creare e confrontare vettoriali trame dei movimenti del robot e animali. </li><li> Modificare il sistema nervoso e ripetere i passaggi 3,3-3,6 per osservare come diversi aspetti della elettronicasistema nervoso influenza il comportamento del robot. Qui registriamo il forza sinaptica tra gli artigli di bump neuroni sensori e il sistema del motore e le prestazioni del robot del monitor rispetto a quella di un astice. Alternativamente, le condizioni sensoriali in campo prova possono essere cambiate e gli effetti osservati in robot e animale. Per esempio, l&#39;arena qui descritta potrebbe essere sottoposto a diverse portate d&#39;acqua. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

Quando si inizia esperimenti di simulazione del sistema nervoso biorobotic, ci sono alcune importanti linee guida da seguire. La scelta del giusto organismo modello è critica: scegliere un organismo che è facile da ottenere e mantenere. Gli invertebrati sono l&#39;ideale, perché di solito non richiedono l&#39;approvazione istituzionale per la sperimentazione e le loro esigenze di allevamento sono spesso meno esigenti di quelli dei vertebrati. Dal punto di vista scientifico, è utile scegliere un animale che ha un rec...

Indagini neurofisiologiche negli ultimi 100 anni hanno ampliato enormemente la nostra conoscenza della struttura del sistema nervoso e la funzione. Tuttavia, la maggior parte della ricerca del sistema nervoso fino ad oggi ha invocato l&#39;uso di preparati isolati o soggetti sobri. Mentre ci sono stati molti sforzi riusciti a registrare l&#39;attività neurale di comportarsi liberamente animali 2-5, l&#39;approccio biorobotic fornisce un valido strumento per consentire la manipolazione del sistema nervoso al fine di testare i sistemi di livello neuroscienze ipotesi 6. Sistemi nervosi simulati operanti sul robot possono essere sperimentalmente manipolati e permettono l&#39;estensione del software di modellazione per il mondo fisico. Questo approccio è stato ben implementato nel mondo accademico 7,8, ma il processo di costruzione di un robot biomimetico per la verifica di ipotesi può essere costoso e richiede tempo. Vi presentiamo un metodo per eseguire l&#39;approccio biorobotic utilizzando un robot disponibile in commercio kesso (LEGO Mindstorms NXT 2.0). L&#39;obiettivo di questo metodo è quello di fornire un modo rapido ed efficiente per testare sistemi livello neuroscienze ipotesi sul robotizzato 9 o bio-ibridi 10 incarnata simulazioni di reti neurali. Accelerare il processo di ipotesi di sperimentare migliora la produttività della ricerca. Il semplice piattaforma LEGO Mindstorms fornisce un banco di prova per sensori biomimetici e le reti neurali che dimostriamo con l&#39;aragosta americano (Homarus americanus) come organismo modello. Il metodo fornisce anche un potente hands-on strumento educativo in classe gli studenti in grado di progettare e gestire sistemi nervosi per i propri robot 11.

designing and implementing nervous system simulations on lego robots

Vi presentiamo un metodo per utilizzare il LEGO Mindstorms NXT piattaforma robotica disponibile in commercio per testare i sistemi di livello di neuroscienze ipotesi. Il primo passo del metodo è quello di sviluppare una simulazione del sistema nervoso di specifici comportamenti riflessivi di un adeguato organismo modello, qui usiamo l&#39;aragosta americana. Riflessi esterocettivi mediate da decussating (incrocio) connessioni neurali in grado di spiegare i taxi di un animale verso o lontano da uno stimolo come descritto da Braitenberg e sono particolarmente adatti per le indagini utilizzando la piattaforma NXT. Viene programmato 1 La simulazione del sistema nervoso utilizzando il software LabVIEW sul LEGO piattaforma Mindstorms. Una volta che il sistema nervoso è adeguatamente regolati, esperimenti comportamentali passino sul robot e sull&#39;animale sotto identiche condizioni ambientali. Controllando l&#39;ambiente sensoriale vissuta dai campioni, si possono osservare differenze di uscita comportamentali. Queste differenze possono puntare a specifiche deficiencrie del modello del sistema nervoso e servono per informare l&#39;iterazione del modello per il particolare comportamento in fase di studio. Questo metodo consente la manipolazione sperimentale dei sistemi nervosi elettronici e serve come un modo per esplorare neuroscienze ipotesi specificamente per quanto riguarda la base neurofisiologica di comportamenti riflessivi innati semplici. Il kit Mindstorms NXT LEGO costituisce una piattaforma accessibile ed efficiente su cui testare schemi di controllo di robot biomimetici preliminari. L&#39;approccio è adatto anche per l&#39;aula di scuola per servire come base per un hands-on basato sulla ricerca curriculum biorobotica.

Ingressi da artigli di un astice nel suo sistema nervoso mediano trattativa ostacolo in un ambiente nuovo. Figura 1 mostra uno screenshot del video utilizzato per analizzare il comportamento di un LEGO robot (Figura 1A) e un&#39;aragosta (Figura 1B) in ambito di test. Dell&#39;arena test è stato non modificata tra i test robot animale e tranne che l&#39;acqua è stata svuotata dal serbatoio per le prove robot. Risultati di monitoraggio vide...

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Designing and Implementing Nervous System Simulations on LEGO Robots

Viene presentato un approccio alla modellazione rete neurale sulla piattaforma robotica LEGO Mindstorms. Il metodo fornisce uno strumento di simulazione per invertebrati ricerca neuroscientifica sia nel laboratorio di ricerca e l&#39;aula. Questa tecnica permette la ricerca di principi di controllo di robot biomimetici.

Progettazione e implementazione di simulazioni del sistema nervoso su LEGO Robot

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 Views.  Northeastern University. Viene presentato un approccio alla modellazione rete neurale sulla piattaforma robotica LEGO Mindstorms. Il metodo fornisce uno strumento di simulazione per invertebrati ricerca neuroscientifica sia nel laboratorio di ricerca e l'aula. Questa tecnica permette la ricerca di principi di controllo di robot biomimetici.

Video: 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.

Visualizzazione del sistema nervoso embrionale in Whole-mount Drosophila Embrioni

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

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Neuroscienze

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.

Produzione di vettori lentivirali per Celle di trasduzione del Sistema Nervoso Centrale

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.

Etichettatura di singole cellule nel sistema nervoso centrale di<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 Registrazione da un intatto scarafaggio Sistema Nervoso

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 stimolazione del sistema nervoso centrale Roditore

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.

Utilizzando Insetto Electroantennogram sensori sul robot autonomi per le ricerche olfattive

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 nervoso enterico

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.

Generazione di idrogel 3D basati su collagene per analizzare la crescita e il comportamento assonali durante lo sviluppo del 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.

Trapianto di progenitori interneuroni corticali chemiogeneticamente ingegnerizzati nei primi cervelli dei topi postnatali

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 affidabile dei micronavi del sistema nervoso centrale in cinque gruppi di vertebrati

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