Nous remercions le Dr M. Yuriy Usachev pour son aide dans la normalisation initiale de la microscopie confocale / imagerie de la souris immunomarquage plantaires biopsie, et le Dr Donna L. Hammond pour son aide continue et la critique constructive à ce travail. Ce travail a été financé par des subventions du NINDS / NIH (NS069898), et une attribution de subvention Idée de développement du ministère de la Défense de la prostate Cancer Research Programme (DoD-PCRP-101096) pour DPM

<ol>
	<li>Hoffman, E. M., Schechter, R., Miller, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixative+Composition+Alters+Distributions+of+Immunoreactivity+for+Glutaminase+and+Two+Markers+of+Nociceptive+Neurons,+Nav+1.8+and+TRPV1,+in+the+Rat+Dorsal+Root+Ganglion.">Fixative Composition Alters Distributions of Immunoreactivity for Glutaminase and Two Markers of Nociceptive Neurons, Nav 1.8 and TRPV1, in the Rat Dorsal Root Ganglion.</a> Journal of Histochemistry &#38; Cytochemistry. 58, 329-344 (2010).</li><li>Neves, J. D. S., Omar, N. F., Narvaes, E. A. O., Gomes, J. R., Novaes, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+different+decalcifying+agents+on+EGF+and+EGFR+immunostaining.">Influence of different decalcifying agents on EGF and EGFR immunostaining.</a> Acta Histochemica. 113, 484-488 (2011).</li><li>Watson, R. E., Wiegand, S. J., Clough, R. W., Hoffman, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+cryoprotectant+to+maintain+long-term+peptide+immunoreactivity+and+tissue+morphology.">Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology.</a> Peptides. 7, 155-159 (1986).</li><li>Yen, L. D., Bennett, G. J., Ribeiro-da-Silva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympathetic+sprouting+and+changes+in+nociceptive+sensory+innervation+in+the+glabrous+skin+of+the+rat+hind+paw+following+partial+peripheral+nerve+injury.">Sympathetic sprouting and changes in nociceptive sensory innervation in the glabrous skin of the rat hind paw following partial peripheral nerve injury.</a> The Journal of Comparative Neurology. 495, 679-690 (2006).</li><li>Boyette-Davis, J., Xin, W., Zhang, H., Dougherty, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraepidermal+nerve+fiber+loss+corresponds+to+the+development+of+Taxol-induced+hyperalgesia+and+can+be+prevented+by+treatment+with+minocycline.">Intraepidermal nerve fiber loss corresponds to the development of Taxol-induced hyperalgesia and can be prevented by treatment with minocycline.</a> Pain. 152, 308-313 (2011).</li><li>Bloom, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+Cancer-Induced+Bone+Remodeling,+Skeletal+Pain,+and+Sprouting+of+Sensory+Nerve+Fibers.">Breast Cancer-Induced Bone Remodeling, Skeletal Pain, and Sprouting of Sensory Nerve Fibers.</a> The Journal of Pain. 12, 698-711 (2011).</li><li>Constantin, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+Tumor+Necrosis+Factor+&#945;+(TNF&#945;)+Requires+TNF+Receptor+Type+2+to+Generate+Heat+Hyperalgesia+in+a+Mouse+Cancer+Model.">Endogenous Tumor Necrosis Factor &#945; (TNF&#945;) Requires TNF Receptor Type 2 to Generate Heat Hyperalgesia in a Mouse Cancer Model.</a> J. Neurosci. 28, 5072-5081 (2008).</li><li>Jankowski, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitization+of+Cutaneous+Nociceptors+after+Nerve+Transection+and+Regeneration:+Possible+Role+of+Target-Derived+Neurotrophic+Factor+Signaling.">Sensitization of Cutaneous Nociceptors after Nerve Transection and Regeneration: Possible Role of Target-Derived Neurotrophic Factor Signaling.</a> The Journal of Neuroscience. 29, 1636-1647 (2009).</li><li>Jimenez-Andrade, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathological+Sprouting+of+Adult+Nociceptors+in+Chronic+Prostate+Cancer-Induced+Bone+Pain.">Pathological Sprouting of Adult Nociceptors in Chronic Prostate Cancer-Induced Bone Pain.</a> J. Neurosci. 30, 14649-14656 (2010).</li><li>Persson, A. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium-calcium+exchanger+and+multiple+sodium+channel+isoforms+in+intra-epidermal+nerve+terminals.">Sodium-calcium exchanger and multiple sodium channel isoforms in intra-epidermal nerve terminals.</a> Molecular Pain. 6, 84-84 (2010).</li><li>Ohshima, M., Miyake, M., Takeda, M., Kamijima, M., Sakamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+Enterotoxin+B+Causes+Proliferation+of+Sensory+C-Fibers+and+Subsequent+Enhancement+of+Neurogenic+Inflammation+in+Rat+Skin.">Staphylococcal Enterotoxin B Causes Proliferation of Sensory C-Fibers and Subsequent Enhancement of Neurogenic Inflammation in Rat Skin.</a> Journal of Infectious Diseases. 203, 862-869 (2011).</li><li>Johnson, M. S., Ryals, J. M., Wright, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+loss+of+peptidergic+intraepidermal+nerve+fibers+in+an+STZ-induced+mouse+model+of+insensate+diabetic+neuropathy.">Early loss of peptidergic intraepidermal nerve fibers in an STZ-induced mouse model of insensate diabetic neuropathy.</a> Pain. 140, 35-47 (2008).</li></ol>

Pas de conflits d&#39;intérêts déclarés.

Ici, nous avons détaillé les méthodes de préparation de la peau des souris et des coupes de tissus osseux pour immunomarquage et la détection des périphériques fibres nerveuses sensitives. Les sections produite à partir de biopsies plantaires contiennent à la fois la peau glabre et poilue, ce qui signifie que le protocole peut être utilisé sur tout type de peau. Ces techniques peuvent également être employées pour colorer les autres types de cellules dans ces tissus (leucocytes, vasculaire endothélium, mu...

<table border="1"><tbody><tr><td> Nom du produit </td><td> Tapez </td><td> Société </td><td> No de catalogue </td><td> Commentaire </td></tr><tr><td> 3mm Harris Micro-Punch </td><td> Matériel </td><td> Ted Pella </td><td> 15094 </td><td></td></tr><tr><td> Pompe à perfusion </td><td> Matériel </td><td> VWR International </td><td> 23609-170 </td><td></td></tr><tr><td> Paraformaldéhyde </td><td> Réactif </td><td> Fisher Scientific </td><td> T353 </td><td></td></tr><tr><td> L&#39;acide picrique </td><td> Réactif </td><td> Sigma-Aldrich </td><td> 239801 </td><td></td></tr><tr><td> Composé Embedding octobre </td><td> Réactif </td><td> Tissue-Tek </td><td> 4583 </td><td></td></tr><tr><td> Cyto-Freeze aérosol cryogénique </td><td>Matériel </td><td> Société de contrôle </td><td> 3118 </td><td></td></tr><tr><td> Sérum de chèvre </td><td> Réactif </td><td> Sigma-Aldrich </td><td> G9023 </td><td></td></tr><tr><td> Bac Incubation et couvercle pour Immunocoloration (Large) </td><td> Matériel </td><td> RPI Corp </td><td> 248270 (bac) 248 270-A (couvercle) </td><td></td></tr><tr><td> Stylo ImmEdge barrière hydrophobe </td><td> Matériel </td><td> Vector Laboratories </td><td> H-4000 </td><td></td></tr><tr><td> Brosses à cheveux de Camel (# 1 épaisseur) </td><td> Matériel </td><td> Ted Pella </td><td> 11859 </td><td></td></tr><tr><td> Pro-Long Or Milieu de montage </td><td> Réactif </td><td> Invitrogen </td><td> P36930 </td><td></td></tr></tbody></table>

tissue preparation and immunostaining of mouse sensory nerve fibers innervating skin and limb bones

Détection et traitement primaire des propriétés physiques, chimiques et thermiques par des stimuli sensoriels périphériques fibres nerveuses sensorielles est la clé de la perception sensorielle chez les animaux et les humains. Ces fibres nerveuses sensitives périphériques expriment une multitude de récepteurs et de protéines des canaux ioniques qui détectent et initier des stimuli sensoriels spécifiques. Les méthodes sont disponibles pour caractériser les propriétés électriques des fibres nerveuses sensorielles périphériques innervant la peau, qui peut également être utilisé pour identifier l&#39;expression fonctionnelle des protéines des canaux ioniques spécifiques dans ces fibres. Toutefois, les méthodes électrophysiologiques similaires ne sont pas disponibles (et sont également difficiles à développer) pour la détection de l&#39;expression fonctionnelle des récepteurs et des protéines des canaux ioniques dans les fibres nerveuses sensorielles périphériques innervant les autres viscères, y compris les tissus les plus difficiles tels que les os. Par ailleurs, ces méthodes électrophysiologiques ne peut pas être utilisé pour déterminer l&#39;expression de la protéine non excitabless dans les zones périphériques des fibres nerveuses sensitives. Par conséquent, immunomarquage d&#39;échantillons de tissus périphériques / viscérale pour fivers nerveuses sensorielles fournit la meilleure façon possible afin de déterminer l&#39;expression de protéines spécifiques de l&#39;intérêt pour ces fibres nerveuses. Jusqu&#39;ici, la plupart des études d&#39;expression de protéines dans les neurones sensoriels ont utilisé immunomarquage procédures dans les ganglions sensitifs, où l&#39;information est limitée à l&#39;expression de protéines spécifiques dans le corps cellulaire de certains types ou sous-ensembles de neurones sensoriels. Nous rapportons ici les méthodes détaillées / protocoles pour la préparation d&#39;échantillons de tissus périphériques / viscérale pour l&#39;immunocoloration des périphériques fibres nerveuses sensitives. Nous détail les méthodes spécifiquement pour la préparation de la peau ou plantaire biopsie et d&#39;os (fémur) les articles de la souris pour l&#39;immunocoloration des périphériques fibres nerveuses sensitives. Ces méthodes ne sont pas seulement essentiels pour la détermination qualitative de l&#39;expression des protéines dans les neurones sensoriels périphériques, mais aussi fournir une méthode de dosage quantitatif pourdéterminer les changements dans les niveaux d&#39;expression de protéines dans des types spécifiques ou des sous-ensembles de fibres sensorielles, ainsi que pour déterminer les modifications morphologiques et / ou anatomiques dans le nombre et la densité des fibres sensorielles au cours de divers états pathologiques. De plus, ces méthodes ne sont pas confinés à la coloration des fibres nerveuses sensorielles que, mais peut aussi être utilisé pour la coloration des types de fibres nerveuses dans la peau, les os et autres tissus viscéraux.

Watch this Scientific Journal Video about Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones at JoVE.com

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

L&#39;identification immunocytochimique de sensitive périphérique sous-types de fibres nerveuses (et de détection de l&#39;expression des protéines qui y sont) sont essentiels à la compréhension des mécanismes moléculaires sous-jacents sensations périphériques. Nous décrivons ici les méthodes de préparation d&#39;échantillons de tissus périphériques / viscérale, tels que les os de la peau et des membres, par immunomarquage spécifique des fibres nerveuses périphériques sensorielles.

Préparation des tissus et des fibres Immunocoloration souris nerveuses sensitives innervant la peau et les os du membre

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

tissue-preparation-immunostaining-mouse-sensory-nerve-fibers

Research

JoVE Journal

Neuroscience

23.0K vues.  The University of Iowa. L'identification immunocytochimique de sensitive périphérique sous-types de fibres nerveuses (et de détection de l'expression des protéines qui y sont) sont essentiels à la compréhension des mécanismes moléculaires sous-jacents sensations périphériques. Nous décrivons ici les méthodes de préparation d'échantillons de tissus périphériques / viscérale, tels que les os de la peau et des membres, par immunomarquage spécifique des fibres nerveuses...

Vidéo: Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Préparation du tissu cérébral de souris pour microscopie électronique

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

Recherche

Neurosciences

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Imagerie phéromones détection dans une souris Vomeronasal tissus Préparation Trancher aiguë

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

Sélective traçage des fibres auditives dans le nerf vestibulo-cochléaire aviaire embryonnaire

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Une préparation semi-intact d&#39;isolement de la souris vestibulaire épithélium sensoriel pour électrophysiologie et haute résolution microscopie à deux photons

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Appartement Mont imagerie de la peau de souris et son application à l&#39;analyse des follicules pileux Modélisation et sensorielle Axon Morphologie

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Whole-monter imagerie de l&#39;embryon de souris projections sensorielles Axon

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

Imagerie en trois dimensions et l’analyse des mitochondries dans les fibres nerveuses humaines intra-épidermiques

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Une méthode pour cibler et Isoler Airway-innervant neurones sensoriels chez la souris

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

Préparation physiologique des cellules ciliées de l&#39;sacculus du ouaouaron (<em&gt; Rana catesbeiana</em&gt;)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

Immunomarquage quadruple de l&#39;ampoule olfactive pour la visualisation des codes d&#39;identité moléculaire des axes sensoriels olfactifs

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...