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) for programmering og manuskript forslag. Vi takker Alex Giuliano og Deborah Lee for video produksjonsstøtte. Finansiering gitt av en NSF Graduate Research Fellowship og en ONR MURI i syntetisk biologi.

En. Bygging av Robot Model <ol><li> Velg en modell organisme for å studere som er godt representert i neuroethological litteratur. Virvelløse dyr generelt gjør gode kandidater fordi deres relativt enkle nervesystem har blitt godt undersøkt og består primært av medfødte reflekser. Vi vil demonstrere denne tilnærmingen ved hjelp av den amerikanske hummer, Homarus americanus. </li><li> Velg godt studert refleksive atferd for modellering formål. Vi har valgt hummer reaksjoner på antennal bøy og å klor...

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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. 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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 initiering biorobotic nervesystemet simulering eksperimenter, er det noen viktige retningslinjer å følge. Å velge riktig modell organisme er kritisk: plukke en organisme som er lett å oppnå og opprettholde. Virvelløse dyr er ideelle fordi de ikke krever vanligvis institusjonelle godkjenning for eksperimentering og deres dyrehold behov er ofte mindre krevende enn de av virveldyr. Fra et vitenskapelig perspektiv er det gunstig å velge et dyr som har en etablert neuroethological litteratur posten for å trekke h...

Nevrofysiologiske undersøkelser i løpet av de siste 100 årene har utvidet enormt vår kunnskap om nervesystemet struktur og funksjon. Imidlertid har de fleste av nervesystemet forskning hittil støttet seg på bruk av isolerte preparater eller behersket fag. Mens det har vært mange vellykkede innsats for å ta opp nevral aktivitet fra fritt oppfører dyr 2-5, gir biorobotic tilnærming et verdifullt verktøy for å tillate nervesystemet manipulasjon for å teste systemer nivå nevrovitenskap hypoteser seks. Simulerte nervesystem som opererer på roboter kan eksperimentelt manipulert og gi rom for utvidelse av programvare modellering til den fysiske verden. Denne tilnærmingen har blitt godt implementert i den akademiske verden 7,8, men prosessen med å bygge en biomimetic robot for hypotesetesting kan være dyrt og tidkrevende. Vi presenterer en metode for å utføre biorobotic tilnærming ved hjelp av en kommersielt tilgjengelig robotikk kdet (LEGO Mindstorms NXT 2.0). Målet med denne metoden er å gi en rask og effektiv måte å teste systemene nivå nevrovitenskap hypoteser på robot 9 eller bio-hybrid 10 nedfelt nevrale nettverk simuleringer. Akselererer prosessen fra hypotesen å eksperimentere forbedrer forskning produktivitet. Den enkle LEGO Mindstorms plattformen gir en test seng for biomimetic sensorer og nevrale nettverk som vi demonstrere ved hjelp av amerikansk hummer (Homarus americanus) som modell organisme. Metoden gir også et kraftig hands-on pedagogisk verktøy i klasserommet så elevene kan designe og manipulere nervesystem for sine egne roboter 11.

designing and implementing nervous system simulations on lego robots

Vi presenterer en metode for å bruke den kommersielt tilgjengelige LEGO Mindstorms NXT robotikk plattform for å teste systemer nivå nevrovitenskap hypoteser. Det første trinn i metoden er å utvikle et nervesystem simulering av bestemte refleksive oppførsel av en passende modell organisme, her bruker vi den amerikanske hummer. Exteroceptive reflekser formidlet av decussating (krysset) nerveforbindelser kan forklare et dyrs drosjer mot eller bort fra en stimulus som beskrevet av Braitenberg og er spesielt godt egnet for etterforskning ved hjelp av NXT-plattformen. En Nervesystemet simulering programmeres via LabVIEW programvare på LEGO Mindstorms plattform. Når nervesystemet er innstilt på riktig måte, blir atferdsmessige eksperimenter kjørt på roboten, og på dyret under identiske forhold i omgivelsene. Ved å kontrollere den sensoriske miljøet oppleves av prøvene, kan forskjeller i atferdsmessige utganger følges. Disse forskjellene kan peke på spesifikke deficienckaper i nervesystemet modell og tjene til å informere gjentakelse av modellen for den spesielle oppførselen under studien. Denne metoden gjør det mulig for den eksperimentelle manipulasjon av elektroniske nervesystem og fungerer som en måte å utforske nevrovitenskap hypoteser spesifikt om nevrofysiologiske grunnlaget for enkle medfødte refleksive atferd. LEGO Mindstorms NXT kit gir en rimelig og effektiv plattform for å teste foreløpige biomimetic robot kontroll ordninger. Tilnærmingen er også godt egnet for den videregående skolen klasserommet for å tjene som grunnlag for en hands-on forespørsel-basert biorobotics pensum.

Innspill fra en hummer klør i sin nervesystemet megle hinder forhandling i en roman miljø. Figur 1 viser et skjermbilde av videoen som brukes til å analysere atferden til en LEGO robot (figur 1A) og en hummer (figur 1B) i testen arena. Testen arena ble umodifisert mellom dyr og robot tester bortsett fra at vannet ble tømt fra tanken for roboten prøvelser. Video sporing resultatene vises i figur 2. Dyre-og robot reise st...

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 tilnærming til nevrale nettverk modellering på LEGO Mindstorms robotikkplattformen er presentert. Metoden gir et simuleringsverktøy for virvelløse nevrovitenskapelig forskning i både forskningslaboratorium og klasserommet. Denne teknikken gjør at etterforskningen av biomimetic robot kontroll prinsipper.

Designe og implementere Nevrologiske Simuleringer på LEGO Robots

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

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

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.

Produksjon av Lentiviral vektorer for Transducing Celler fra Central Nervous System

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.

Merking av enkeltceller i Central Nervous System av<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 Opptak fra et intakt Cockroach 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 av gnagere sentralnervesystemet

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.

Bruke insekt Electroantennogram Sensorer på autonome roboter for Olfactory søk

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 av enteric nervesystemet

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.

Generering av 3-D kollagen-baserte Hydrogeler å analysere axonal vekst og atferd under nervesystemet utvikling

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.

Transplantasjon av Chemogenetically utviklet kortikale Interneuron forfedre inn tidlig postnatal Mouse hjerner

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.

Pålitelig isolering av sentrale nervesystemet Microårene over fem virveldyr grupper

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