Name: designing and implementing nervous system simulations on lego robots
Uploaded: 2013-05-25T14:00:00
Description: Watch this Scientific Journal Video about Designing and Implementing Nervous System Simulations on LEGO Robots at JoVE.com

Vi takker Dr. Chris Rogers (Tufts University) til programmering og manuskript forslag. Vi takker Alex Giuliano og Deborah Lee til videoproduktion support. Finansiering leveres af en NSF Graduate Research Fellowship og en ONR MURI i syntetisk biologi.

1.. Opbygning af Robot Model <ol><li> Vælg en model organisme for at studere, som er godt repræsenteret i neuroethological litteratur. Hvirvelløse generelt gøre gode kandidater, fordi deres relativt simple nervesystem er blevet godt undersøgt og er primært sammensat af medfødte reflekser. Vi vil demonstrere denne tilgang med den amerikanske hummer, Homarus americanus. </li><li> Vælg velundersøgte refleksive adfærd til modellering formål. Vi har valgt den hummer svar til antennal bøjning og klo bum...

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Fluid Mech. 2 (1), 95-114 (2002).</li><li>Westphal, A., Rulkov, N. F., Ayers, J., Brady, D., Hunt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+a+lamprey-based+robot+with+an+electronic+nervous+system.">Controlling a lamprey-based robot with an electronic nervous system.</a> Smart Struct. and Systems. 8 (1), 39-52 (2011).</li><li>Chao, Z. C., Bakkum, D. J., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+embodied+neural+networks+for+adaptive+goal-directed+behavior.">Shaping embodied neural networks for adaptive goal-directed behavior.</a> PLoS Computational Biology. 4 (3), e1000042 (2008).</li><li>Blustein, D., Schultheis, K., Ayers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+nervous+systems+for+robots:+an+interactive+and+collaborative+neuroscience+curriculum.">Building nervous systems for robots: an interactive and collaborative neuroscience curriculum.</a> , (2011).</li><li>Ayers, J., Blustein, D., Westphal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Conserved+Biomimetic+Control+Architecture+for+Walking,+Swimming+and+Flying+Robots.">A Conserved Biomimetic Control Architecture for Walking, Swimming and Flying Robots.</a> Lect. 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Intelli. 7375, 1-12 (2012).</li><li>Cowan, N., Ma, E., Cutkosky, M., Full, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Biologically+Inspired+Passive+Antenna+for+Steering+Control+of+a+Running+Robot.">A Biologically Inspired Passive Antenna for Steering Control of a Running Robot.</a> , 541-550 (2003).</li><li>Gasperi, M., Hurbain, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Extreme NXT: Extending the LEGO MINDSTORMS NXT to the Next Level. , (2007).</li><li>Gasperi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> LabVIEW for LEGO MINDSTORMS NXT. , (2008).</li><li>Kennedy, D., Davis, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+27+Organization+of+invertebrate+motor+systems.">Chapter 27 Organization of invertebrate motor systems.</a> Handbook of Physiology. 1, 1023-1088 (1977).</li><li>Buchanan, J., Grillner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+identified+"glutamate+interneurons"+and+their+role+in+locomotion+in+the+lamprey+spinal+cord.">Newly identified "glutamate interneurons" and their role in locomotion in the lamprey spinal cord.</a> Science. 236 (4799), 312-314 (1987).</li><li>Rulkov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+spiking-bursting+neural+behavior+using+two-dimensional+map.">Modeling of spiking-bursting neural behavior using two-dimensional map.</a> Physical Review E. 65, (2002).</li><li>Derby, C. D., Atema, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narrow-spectrum+chemoreceptor+cells+in+the+walking+legs+of+the+lobster,+Homarus+americanus:+Taste+specialists.">Narrow-spectrum chemoreceptor cells in the walking legs of the lobster, Homarus americanus: Taste specialists.</a> Journal of Comparative Physiology A. 146 (2), 181-189 (1982).</li><li>Tort, A. B. L., Neto, W. P., Amaral, O. B., Kazlauckas, V., Souza, D. O., Lara, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+webcam-based+approach+for+the+measurement+of+rodent+locomotion+and+other+behavioural+parameters.">A simple webcam-based approach for the measurement of rodent locomotion and other behavioural parameters.</a> Journal of Neuroscience Methods. 157 (1), 91-97 (2006).</li><li>Huxley, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Crayfish, An Introduction to the Study of Zoology. , (1880).</li><li>Blustein, D., Ayers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+network+for+control+of+arthropod+exteroceptive+optical+flow+reflexes+during+locomotion.">A conserved network for control of arthropod exteroceptive optical flow reflexes during locomotion.</a> Lect. Notes on Artif. Intelli. 6226, 72-81 (2010).</li><li>Webb, B., Scutt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+latency-dependent+spiking-neuron+model+of+cricket+phonotaxis.">A simple latency-dependent spiking-neuron model of cricket phonotaxis.</a> Biological Cybernetics. 82 (3), 247-269 (2000).</li><li>Demarse, T. B., Wagenaar, D. A., Blau, A. W., Potter, S. 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Ved indledning biorobotic nervesystem simulation eksperimenter, er der et par vigtige retningslinjer at følge. At vælge den rigtige model organisme er kritisk: vælge en organisme, der er let at opnå og vedligeholde. Hvirvelløse er ideelle, fordi de ikke kræver normalt institutionel godkendelse til eksperimenter og deres opdræt behov er ofte mindre krævende end hvirveldyr. Fra et videnskabeligt perspektiv, er det gavnligt at vælge et dyr, der har en etableret neuroethological litteratur rekord hvorfra man kan tr...

Neurofysiologiske undersøgelser i løbet af de seneste 100 år har udvidet enormt vores viden om nervesystemet struktur og funktion. Imidlertid har de fleste af nervesystemet hidtidige forskning påberåbt anvendelsen af ​​isolerede præparater eller tilbageholdende emner. Mens der har været mange vellykkede bestræbelser på at optage neurale aktivitet i frit opfører dyr 2-5, den biorobotic tilgang giver et værdifuldt redskab til at give mulighed for nervesystemet manipulation for at teste systemer niveau neurovidenskab hypoteser 6.. Simuleret nervesystem opererer på robotter kan eksperimentelt manipuleres og give mulighed for udvidelse af software modellering til den fysiske verden. Denne tilgang er blevet godt gennemført i den akademiske verden 7,8, men processen med at opbygge et biomimetisk robot til hypotesetest kan være dyrt og tidskrævende. Vi præsenterer en metode til at udføre biorobotic tilgang med en kommercielt tilgængelig robotteknologi kdet (LEGO Mindstorms NXT 2.0). Målet med denne metode er at give en hurtig og effektiv måde at teste systemerne niveau neurovidenskab hypoteser om robot 9 eller bio-hybrid 10 legemliggjort neurale netværk simuleringer. Fremskynde processen fra hypotesen at eksperimentere forbedrer forskningen produktivitet. Den simple LEGO Mindstorms platform giver et prøveanlæg for biomimetiske sensorer og neurale netværk, som vi vise ved hjælp af amerikanske hummer (Homarus americanus) som en model organisme. Metoden giver også en stærk hands-on pædagogisk redskab i klasseværelset, da eleverne kan designe og manipulere nervesystemer for deres egne robotter 11..

designing and implementing nervous system simulations on lego robots

Vi præsenterer en metode til at bruge kommercielt tilgængelige LEGO Mindstorms NXT robotter platform til at teste systemerne niveau neurovidenskab hypoteser. Det første trin i metoden er at udvikle et nervesystem simulering af specifikke refleksive adfærd af en passende model organisme, her bruger vi den amerikanske hummer. Eksteroceptiv reflekser medieret af decussating (krydsning) neurale forbindelser kan forklare et dyrs taxaer mod eller væk fra en stimulus som beskrevet af Braitenberg og er især velegnet til undersøgelse ved hjælp af NXT platformen. 1. nervesystemet simulation er programmeret ved hjælp af LabVIEW software på LEGO Mindstorms-platform. Når nervesystemet er indstillet korrekt, bliver adfærdsmæssige eksperimenter køre på robotten og på dyret under identiske miljøforhold. Ved at styre den sensoriske miljø opleves af prøverne, kan forskellene i adfærdsmæssige udgange overholdes. Disse forskelle kan pege på specifikke deficiencerne i nervesystemet model og tjener til at informere iteration af model for en bestemt adfærd under studiet. Denne metode giver mulighed for den eksperimentelle manipulering af elektroniske nervesystemer og tjener som en måde at udforske neuroscience hypoteser specifikt vedrørende neurofysiologiske grundlag af simple medfødte refleksive adfærd. LEGO Mindstorms NXT kit giver en billig og effektiv platform til at teste de indledende biomimetiske robotstyring ordninger. Den fremgangsmåde er også velegnet til high school klasseværelset til at tjene som grundlag for en hands-on inquiry-baserede biorobotics pensum.

Input fra en hummer kløer i sit nervesystem mægle hindring forhandling i en roman miljø. Figur 1 viser et skærmbillede af den video bruges til at analysere opførslen af en LEGO robot (figur 1A) og en hummer (figur 1B) i testen arena. Testområdet blev umodificeret mellem dyr og robot test bortset fra, at vandet blev tømt fra tanken til robotten forsøg. Videosporingssystemet resultaterne vises i figur 2.. Dyre-og robot...

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

En tilgang til neurale netværk modellering på LEGO Mindstorms robot platform præsenteres. Metoden giver et simuleringsværktøj for hvirvelløse neurovidenskab forskning i både forskningslaboratorium og klasseværelset. Denne teknik gør det muligt at undersøge biomimetiske robotstyring principper.

Design og implementering af Nervesystemet Simuleringer på LEGO robotter

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

Research

JoVE Journal

Neuroscience

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.

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

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.

Produktion af lentivirusvektorer til transduktion af celler fra det centrale nervesystem

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.

Mærkning af enkeltceller i centralnervesystemet hos<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 Optagelse fra en intakt Kakerlak Nervous System

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 Stimulering af Rodent centralnervesystemet

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.

Brug Insekt Electroantennogram Sensorer på selvstyrende robotter til Olfaktoriske søgninger

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+ billeddannelse af enteriske nervesystem

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.

Generation af 3-D kollagen-baserede Hydrogels til at analysere Axonal vækst og adfærd under nervesystemet udvikling

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.

Transplantation af kemo genetisk manipulerede kortikale interneuron-Progenitorer til tidlige postnatale musehjerner

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.

Pålidelig isolering af Mikrofartøjer i central nervesystemet på tværs af fem hvirveldyr grupper

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