Diese Arbeit wurde vom neuen Ermittler Anschubfinanzierung von Albert Einstein College of Medicine der Yeshiva University RVS unterstützt.

<ol>
	<li>Apps, R., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+cortical+organization:+a+one-map+hypothesis.">Cerebellar cortical organization: a one-map hypothesis.</a> Nat. Rev. Neurosci. 10, 670-681 (2009).</li><li>Grishkat, H. L., Eisenman, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+spinocerebellar+projection+in+the+prenatal+mouse.">Development of the spinocerebellar projection in the prenatal mouse.</a> J. Comp. Neurol. 363, 93-108 (1995).</li><li>Mesulam, 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> Tracing neural connections with horseradish peroxidase. , (1982).</li><li>Reeber, S. L., Sillitoe, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+expression+of+a+cocaine-+and+amphetamine+regulated+transcript+(CART)+peptide+reveals+complex+circuit+topography+in+the+rodent+cerebellar+cortex.">Patterned expression of a cocaine- and amphetamine regulated transcript (CART) peptide reveals complex circuit topography in the rodent cerebellar cortex.</a> Journal of Comparative Neurology. , (2011).</li><li>Reeber, S. L., Gebre, S. A., Sillitoe, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+mapping+of+afferent+topography+in+three+dimensions.">Fluorescence mapping of afferent topography in three dimensions.</a> Brain. Struct. Funct. , (2011).</li><li>Sillitoe, R. V., Stephen, D., Lao, Z., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engrailed+homeobox+genes+determine+the+organization+of+Purkinje+cell+sagittal+stripe+gene+expression+in+the+adult+cerebellum.">Engrailed homeobox genes determine the organization of Purkinje cell sagittal stripe gene expression in the adult cerebellum.</a> J. Neurosci. 28, 12150-12162 (2008).</li><li>Sillitoe, R. V., Vogel, M. W., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engrailed+homeobox+genes+regulate+establishment+of+the+cerebellar+afferent+circuit+map.">Engrailed homeobox genes regulate establishment of the cerebellar afferent circuit map.</a> J. Neurosci. 30, 10015-10024 (2010).</li><li>Vig, J., Goldowitz, D., Steindler, D. A., Eisenman, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentation+of+the+reeler+cerebellum:+segregation+and+overlap+of+spinocerebellar+and+secondary+vestibulocerebellar+fibers+and+their+target+cells.">Compartmentation of the reeler cerebellum: segregation and overlap of spinocerebellar and secondary vestibulocerebellar fibers and their target cells.</a> Neuroscience. 130, 735-744 (2005).</li><li>Vogel, M. W., Prittie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographic+spinocerebellar+mossy+fiber+projections+are+maintained+in+the+lurcher+mutant.">Topographic spinocerebellar mossy fiber projections are maintained in the lurcher mutant.</a> J. Comp. Neurol. 343, 341-351 (1994).</li><li>Voogd, J., Broere, G., van Rossum, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+medio-lateral+distribution+of+the+spinocerebellar+projection+in+the+anterior+lobe+and+the+simple+lobule+in+the+cat+and+a+comparison+with+some+other+afferent+fibre+systems.">The medio-lateral distribution of the spinocerebellar projection in the anterior lobe and the simple lobule in the cat and a comparison with some other afferent fibre systems.</a> Psychiatr. Neurol. Neurochir. 72, 137-151 (1969).</li><li>Wu, H. S., Sugihara, I., Shinoda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projection+patterns+of+single+mossy+fibers+originating+from+the+lateral+reticular+nucleus+in+the+rat+cerebellar+cortex+and+nuclei.">Projection patterns of single mossy fibers originating from the lateral reticular nucleus in the rat cerebellar cortex and nuclei.</a> J. Comp. Neurol. 411, 97-118 (1999).</li></ol>

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Wir haben die operative und technische Details für eine erfolgreiche axonalen und dendritischen Tracing unter Verwendung eines neuartigen Fluoreszenz-basierten Ansatz für die schnelle Kennzeichnung neuronalen Projektionen in der Entwicklung und erwachsenen Mäusen erforderlich beschrieben. Mit WGA-Alexa zeigen wir, wie Tracer und Marker für die Analyse von strukturierten Topografie elektrischer Schaltungen mit hoher Auflösung und in drei Dimensionen verwendet werden kann. Viele Indikator...

<table border="1"><tbody><tr><td> Equipment / Reagenzien </td><td> Model / Katalog-Nummer </td><td> Firma </td></tr><tr><td> Bead-Sterilisator </td><td> Modell Steri 250 Cat. # 18000-45 </td><td> Feine Science Tools </td></tr><tr><td> Cauterizer </td><td> Cat. # 18000-00 </td><td> Feine Science Tools </td></tr><tr><td> Borosilikatglas Kapillaren </td><td> Cat. # 300056 </td><td> Harvard Apparatus </td></tr><tr><td> Zweistufige Glasmikropipette Puller </td><td> Modell 001-PC-10 </td><td> Narishige </td></tr><tr><td> Mikrometer Spritze </td><td> Cat. # GS-1100 </td><td> Gilmont Instruments </td></tr><tr><td> Small Animal stereotaktische Instrument </td><td> Modell 940-A </td><td> Kopf Instrumentation </td></tr><tr><td> Electrode Manipulator </td><td> Modell 960 </td><td> Kopf Instrumentation </td></tr><tr><td> Vetcare Kammer </td><td> Cat. #340508 </td><td> Harvard Apparatus </td></tr><tr><td> Heizkissen </td><td> Cat. # 341241 </td><td> Harvard Apparatus </td></tr><tr><td> Leica DFC360 FX Kamera </td><td> DFC360 FX </td><td> Leica </td></tr><tr><td> Leica DFC490 Kamera </td><td> DFC490 </td><td> Leica </td></tr><tr><td> Leica DM5500 Mikroskop </td><td> DM5500 </td><td> Leica </td></tr><tr><td> Leica DFC3000 FX-Kamera </td><td> DFC3000 FX </td><td> Leica </td></tr><tr><td> Leica MZ16 FA-Mikroskop </td><td> MZ16 FA </td><td> Leica </td></tr><tr><td> CY3 Filter </td><td> Model # 11600231 </td><td> Leica </td></tr><tr><td> FITC Filter </td><td> Model # 11513880 </td><td> Leica </td></tr><tr><td> A4 DAPI / UV-Filter </td><td> Model # 11504162 </td><td> Leica </td></tr><tr><td> Weizenkeimagglutinin, Alexa Fluor 488-Konjugat </td><td> Cat. # W11261 </td><td> Invitrogen </td></tr><tr><td> Weizen germ Agglutinin, Alexa 555-Konjugat </td><td> Cat. # W32464 </td><td> Invitrogen </td></tr></tbody></table>

revealing neural circuit topography in multi-color

Neuronale Schaltkreise sind in funktionale topografische Karten organisiert. Um komplexe Schaltung Architektur visualisieren wir entwickelten einen Ansatz, um zuverlässig Etikett der globalen Strukturierung von mehreren topographischen Projektionen. Das Kleinhirn ist ein ideales Modell, um die geordnete Anordnung der neuronalen Schaltkreise zu untersuchen. Zum Beispiel hat die räumliche Organisation der spinozerebellären Moosfasern sich als unverzichtbare System für das Studium Moosfaser Strukturierung werden. Wir konnten kürzlich zeigen, dass Weizenkeimagglutinin (WGA) konjugiert Alexa 555 und 488 können für die Rückverfolgung spinozerebellären Moosfaser Projektionen in der Entwicklung und erwachsenen Mäusen verwendet werden (Reeber et al. 2011). Wir fanden drei großen Eigenschaften, die die WGA-Alexa Tracer wünschenswert Tools machen für die Kennzeichnung neuronalen Projektionen. Erstens sind Alexa Fluorophore intensive und ihre Helligkeit lässt wholemount Bildgebung direkt nach Tracing. Zweitens Label WGA-Alexa Tracer die gesamte Flugbahn des sich entwickelnden und adulten neuralen projections. Drittens WGA-Alexa Tracer sind schnell in beide retrograde und anterograde Richtungen transportiert. Hier beschreiben wir detailliert, wie die Tracer und andere erforderliche Tools, wie die Operation für spinozerebellären Tracing durchführen und wie man am besten zum Bild zurück Projektionen in drei Dimensionen vorzubereiten. Zusammenfassend stellen wir eine Schritt-für-Schritt-Tracing-Protokoll, das von Nutzen sein wird für die Entschlüsselung der Organisation und Vernetzung der funktionellen Karten nicht nur im Kleinhirn, sondern auch in der Hirnrinde, Hirnstamm und Rückenmark.

Watch this Scientific Journal Video about Revealing Neural Circuit Topography in Multi-Color at JoVE.com

Revealing Neural Circuit Topography in Multi-Color

Wir bieten eine praktische Anleitung für die Bereitstellung von Tracer<em&gt; In vivo</em&gt; Und die spinozerebellären Weg als Modellsystem, um wesentliche Schritte für eine erfolgreiche Analyse neuronaler Schaltkreise in Mäusen zu demonstrieren. Wir beschreiben im Detail unsere vielseitigen Tracing-Protokoll, das Weizenkeimagglutinin (WGA) konjugiert Alexa Fluorophore Exploits.

Revealing Neural Circuits Topographie in Multi-Color

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably ...

revealing-neural-circuit-topography-in-multi-color

Research

JoVE Journal

Neuroscience

14.9K Aufrufe.  Yeshiva University. Wir bieten eine praktische Anleitung für die Bereitstellung von Tracer In vivo Und die spinozerebellären Weg als Modellsystem, um wesentliche Schritte für eine erfolgreiche Analyse neuronaler Schaltkreise in Mäusen zu demonstrieren. Wir beschreiben im Detail unsere vielseitigen Tracing-Protokoll, das Weizenkeimagglutinin (WGA) konjugiert Alexa Fluorophore Exploits.

Serie: Revealing Neural Circuit Topography in Multi-Color

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissection und Kultur kommissuralen Nervenzellen aus embryonalen Rückenmark

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Forschung

Neurowissenschaften

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Die Kombination Behavioral Endokrinologie und Experimentelle Wirtschaftsforschung: Testosteron und Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

Physiologische, morphologische und neurochemische Charakterisierung von Neuronen Modulated durch Bewegung

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

Erkennung von mikroregionale Hypoxie in Maus-Großhirnrinde Zwei-Photonen-Imaging von endogenen NADH Fluoreszenz

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

Wholemount Immunhistochemie für die Aufdeckung von komplexen Gehirn Topographie

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Multi-unit Recording Methoden zur neuronalen Aktivität in der Locust Charakterisieren (<em&gt; Schistocerca Americana</em&gt;) Olfaktorische Circuits

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

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.

Neuronalen Schaltkreis-Aufnahme von einem intakten Nervensystem Schabe

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

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Ganze Berge Immunmarkierung der olfaktorischen Rezeptorneuronen in der<em&gt; Drosophila</em&gt; Antennen

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

Neuronale Aktivitätsausbreitung in einem ungefalteten Hippocampus Vorbereitung mit einem durchdringenden Mikroelektroden-Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Zeitabhängige Erhöhung der Netzwerkreaktion auf die Stimulation von neuronalen Zellkulturen auf Mikroelektroden-Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...