Les auteurs tiennent à remercier le Dr Candace Hsieh des suggestions et de l&#39;aide des techniques d&#39;imagerie et Dr Doris Wu d&#39;expertise sur l&#39;anatomie poussin oreille interne au cours de l&#39;embryogenèse précoce. Ce travail a été soutenu par la NSF IOS-0642346, NIH T32-DC010775, NIH T32-GM008620, NIH R01-DC010796, et le DOE GAANN P200A120165.

1. Préparer les outils de dissection et réactifs suivants <ul><li> Liquide céphalo-rachidien artificiel (ACSF, 130 mM NaCl, 3 mM de KCl, 1,2 mM KH 2 PO 4, 20 mM NaHCO 3, 3 mM HEPES, 10 mM de glucose, 2 mM de CaCl2, 1,3 mM MgSO 4) perfusion continue avec 95% 2/5% de CO 2 à la température ambiante. Pour la perfusion, remplir au 2/3 de 500 ml à large ouverture Nalgene pot avec un trou percé dans le couvercle. Réservoir sera fixé par une tubulure à une tige de verre barboteur, qui pénètre dans la aCSF à travers le trou dans le couvercle du bocal. Régler la pression afin d&#39;obtenir un flux constant de bulles qui atteignent environ 1/3 du liquide, mais ne sont pas suffisamment puissants pour provoquer des éclaboussures au-dessus de la jante. Submerger 5 ml flacons de verre (jusqu&#39;à 6) dans le pot Nalgene pour séparer les échantillons individuels les uns des autres et de la turbulence de l&#39;écoulement de gaz. ACSF doit être oxygéné pendant au moins 20 min avant l&#39;une incubation de tissus. </li><li> Petit plat dissection de silicone (35 x 10 mmPetri mm revêtu en utilisant Sylgard Elastomere Kit) avec deux broches de dissection placés en polystyrène hexagonale petite (47 mm en bas) pèsent bateau. </li><li> Deux pinces à pointe fine, une pointe recourbée-pinces, un bécher de 50 ml, et un récipient pour les déchets de tissu. </li><li> Tiré de verre micropipette (1,2 OD / 0,9 ID) rompu à environ 20-50 um d&#39;ouverture à l&#39;extrémité et rempli avec de la rhodamine dextrane aminé (RDA, MW = 3.000, Invitrogen) dans une solution de 6,25% avec 0,4% de Triton X-100 dans du PBS . Fixer par un tube fin pour picospritzer et stabiliser à micromanipulateur. </li></ul> 2. Micro-dissection pour révéler Papille basilaire à 4,5 E8 <ol><li> Avec des pinces courbes, couper E8 embryon juste en dessous du tronc cérébral, en plaçant directement sur la tête de dissection plat enduit de silicone à l&#39;intérieur de peser bateau. </li><li> Utilisant des broches de dissection, positionner la tête avec une vue ventrale afin de l&#39;oreille sur le côté de l&#39;intérêt est légèrement visible, la tête légèrement inclinée sur le dos et pointant 10-30 degrés loin de CEnter (figure 2A). Immédiatement fixer la tête avec des épingles. Si bilatérale traçage est désiré, un centre de la tête à 0 degrés, ou une vue plein ventrale afin que les deux côtés peuvent être consultées. De même, pour une injection du côté gauche, fixer la tête de -10 à -30 degrés à partir du centre de sorte que le côté gauche est accessible. </li><li> En utilisant le bécher de 50 ml, 30 ml de transférer aCSF oxygéné dans le plat, entièrement immerger le tissu. </li><li> Saisissez doucement vers le bas et le zeste de la mâchoire inférieure en utilisant une pince fine pointe, puis retirez la paupière inférieure sur le côté de l&#39;intérêt. </li><li> Retirez le tissu vasculaire sus-jacente, y compris tous les palais restant et l&#39;œsophage. Ces tissus mous devrait décoller révélant la surface lisse de développer chondrocrâne avec les artères vertébrales distinctifs à sa surface. À ce stade, la caractéristique masse blanche otoconial doit être visible. </li><li> Disséquer soigneusement la peau autour du conduit auditif externe, le cartilage de l&#39;articulation de la mâchoire et l&#39;oreille moyenne, tout en laissant l&#39;enner l&#39;oreille intacte. Utilisez la pince pour couper délicatement ces structures cartilagineuses. </li><li> Retirer artères vertébrales et l&#39;accumulation de sang à l&#39;aide d&#39;un léger mouvement de balayage avec les extrémités de la pince fine. Le sang peut obscurcir vue et la coagulation peut boucher l&#39;ouverture pipette d&#39;injection. </li><li> Le canal cochléaire avec épithélium sensoriel adjacente (papille basilaire) et le ganglion acoustique se trouve directement sous le chondrocrâne 6 et peut être visualisée comme une forme allongée en forme de doigt saillie s&#39;étendant à partir de l&#39;oreille interne ventralement, avec la pointe la plus distale (apex) près de la partie antérieure médiane 5. Il peut y avoir une petite accumulation de sang (rouge) de la dissection et / ou des calcifications otolithiques (blanc) à l&#39;apex de la cochlée conduit (figure 2B) qui peuvent aider à visualiser. Au sommet se trouve la macula lagenar, un patch sensorielle pensé pour être de la fonction vestibulaire 7,8. </li></ol> 3. Étiquetage sélective des fibres NC VIII auditif <ol><li&gt; Placez la pipette d&#39;injection près de la surface embryon à la région papille basilaire et conduit cochléaire. </li><li> Abaissez lentement la micropipette à première ponction du crâne. Cette région crânienne est plus mince que les zones environnantes et nécessite moins de force pour pénétrer. La micropipette devrait maintenant être à l&#39;intérieur du canal cochléaire. En option: une petite injection de colorant de traçage peut être faite ici pour aider à la visualisation de la papille basilaire. </li><li> Sans avoir à repositionner la micropipette, continuer à baisser jusqu&#39;à ce qu&#39;il seulement à l&#39;atteinte de la frontière profondeur du canal cochléaire (Figure 1). Effectuer une injection ici et répéter dans de multiples régions le long de la longueur de la papille basilaire (figures 2C, 2D), à l&#39;exclusion de la pointe la plus proximale où le lagena vestibulaire est situé. Avec le picospritzer mis entre 10-30 psi, plusieurs injections sont faites. Le volume de fluide injecté de colorant varie, et dépend du degré de marquage souhaitée. Chaque injection doit se traduire par un colorant fluorescent-laBeled place d&#39;environ 200 à 400 um de diamètre (figure 2D). </li><li> Une fois l&#39;injection terminée, utilisez une pince pour sectionner l&#39;échantillon au-dessus du tronc cérébral et d&#39;utiliser une pipette de transfert pour placer la partie caudale dans un petit pot dans le conteneur de aCSF continuellement perfusés avec 95% O 2/5% de CO 2. </li><li> Pour chaque embryon, 20-30 min pour permettre le transfert de colorant, selon la distance du centre de traçage souhaitée. </li><li> Éliminer les tissus et préparer pour la coupe histologique. Dans l&#39;exemple présenté ici, le tissu est immergé dans du paraformaldéhyde à 4% (dans du PBS 1X) pendant une nuit, le saccharose 30% (dans du PBS 1X) pendant une nuit. </li></ol> 4. La contre et analyse <ol><li> La contre est utile pour aider à orienter l&#39;observateur de la localisation anatomique ainsi que d&#39;identifier les régions d&#39;intérêt particulier. Une contre-coloration efficace dans cette préparation est immunomarquage neurofilaments (figures 1, 3b, 3e), où L vivementAbel axones et permet de délimitation des voies axonales dans le SNC. </li><li> L&#39;analyse doit être effectuée dans toute la région étiqueté, comme les saillies peuvent s&#39;étendre sur plusieurs centaines de microns ou plus long de l&#39;axe rostro-caudal. Quelques exemples d&#39;analyses de base sont les suivants: taux d&#39;erreur de ciblage, le calendrier et / ou des changements dans la divergence des modèles de projections. Axones RDA-étiquetés sont visualisées dans le système nerveux périphérique et central à haute résolution (figures 3A, 3D). </li></ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brn3a+is+a+transcriptional+regulator+of+soma+size,+target+field+innervation+and+axon+pathfinding+of+inner+ear+sensory+neurons.">Brn3a is a transcriptional regulator of soma size, target field innervation and axon pathfinding of inner ear sensory neurons.</a> Development. 128, 2421-2432 (2001).</li><li>Jones, J. M., Warchol, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+Gata3+transcription+factor+in+the+acoustic+ganglion+of+the+developing+avian+inner+ear.">Expression of the Gata3 transcription factor in the acoustic ganglion of the developing avian inner ear.</a> The Journal of Comparative Neurology. 516, 507-518 (2009).</li><li>Lu, C. C., Appler, J. M., Houseman, E. A., Goodrich, L. 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Pas de conflit d&#39;intérêts sont déclarés.

Les études sur le développement précoce du nerf VIII ont été limitées, en partie à cause de la difficulté d&#39;identifier les axones embryonnaires provenant de ganglions distincts multiples. Plusieurs études ont exploré les signaux moléculaires directeurs auditif et vestibulaire cellule sensorielle et destins des cellules ganglionnaires au cours du développement précoce, 5,11,12, mais les processus de régulation innervation centrale n&#39;ont pas encore été déterminés. Rapports des acoustiq...

<table border="1"><tbody><tr><td> Nom de réactif / Matériel </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Peser vaisselle en polystyrène </td><td> Fisher Scientific </td><td> 02-202-101 </td><td></td></tr><tr><td> Boîte de Pétri, 35 X 10 mm </td><td> Fisher Scientific </td><td> 50820644 </td><td> Utilisez-le pour faire la dissection plat en silicone </td></tr><tr><td> Kit Élastomère silicone Sylgard </td><td> World Precision Instruments </td><td> SYLG184 </td><td> Manteau de Petri plat pour faire la dissection </td></tr><tr><td> Dissection Pins </td><td> Divers </td><td></td><td> Dit embryon en place lors de la dissection </td></tr><tr><td> NaCl </td><td> Divers </td><td></td><td> une partie de la recette aCSF </td></tr><tr><td> KCl </td><td> Divers </td><td></td><td> une partie de la recette aCSF </td&gt; </tr><tr><td> KH 2 PO 4 </td><td> Divers </td><td></td><td> une partie de la recette aCSF </td></tr><tr><td> NaHCO 3 </td><td> Divers </td><td></td><td> une partie de la recette aCSF </td></tr><tr><td> Glucose </td><td> Divers </td><td></td><td> une partie de la recette aCSF </td></tr><tr><td> CaCl 2 </td><td> Divers </td><td></td><td> une partie de la recette aCSF </td></tr><tr><td> MgSO 4 </td><td> Divers </td><td></td><td> une partie de la recette aCSF </td></tr><tr><td> Récipient pour aCSF. Proposer translucide à large ouverture Nalgene pot de 500 ml (16 oz) avec couvercle. </td><td> CPLabSafety </td><td> QP-PLC-03717 </td><td> Trou de perçage en haut du couvercle pour tige de verre bubling de pénétrer de liquide </td></tr><tr><td> Vider flacon de 5 ml en verre transparent ou comparables flacon </td><td> American Pharmaceutical Partners, Inc </td><td> 6332300105 </td><td&gt; Utilisation pendant l&#39;incubation aCSF de conserver des échantillons séparés l&#39;un de l&#39;autre et du courant de bouillonnement </td></tr><tr><td> Réservoir de carbogène (95% O 2/5% CO 2) relié par un tube à barbotage </td><td> Divers </td><td></td><td> Fixer par un tube de barbotage tige de verre pour perfusion en aCSF </td></tr><tr><td> Verre à pied barboteur </td><td> Divers </td><td></td><td> Pour infuser dans carbogène aCSF </td></tr><tr><td> Courbé en bout d&#39;une pince </td><td> World Precision Instruments </td><td> 501008 </td><td> Pour retirer la tête de l&#39;embryon de l&#39;oeuf </td></tr><tr><td> Deux fine pointe de pince </td><td> World Precision Instruments </td><td> 501985 </td><td> Pour les micro-dissection </td></tr><tr><td> 50 ml Bécher </td><td> divers </td><td></td><td></td></tr><tr><td> Rhodamine dextrane Amine (RDA) </td><td> Invitrogen </td><td> divers </td><td> Fluorescent axone traceur </td></tr><tr><td> Triton X-100 </td><td> ICN Biomedicals </td><td></td><td></td></tr><tr><td> Phosphate Buffered Saline (PBS 1X) </td><td> Divers </td><td></td><td> Réactif de laboratoire standard </td></tr><tr><td> Thin Wall capillaires en verre, 1,2 OD, .9 ID 4 &quot;(100 mm) de longueur </td><td> World Precision Instruments </td><td> TW120F-4 </td><td> Chargez-RDA. Chaque capillaire fait deux micropipettes de verre </td></tr><tr><td> Aiguille / extracteur pipette </td><td> David Kopf Instruments </td><td> Modèle 720 </td><td> Paramètres utilisés: Chaleur 16,4, 2,2 solénoïde </td></tr><tr><td> Picospritzer </td><td> Parker Instrumentation </td><td> divers </td><td> Fixer par un tube de verre fin pour micropipette </td></tr><tr><td> Micromanipulateur </td><td> Narishige </td><td> divers </td><td></td></tr><tr><td> Microscope à dissection avec la fluorescence </td><td> Divers </td><td></td><td></td&gt; </tr><tr><td> Paraformaldéhyde 4% </td><td> Divers </td><td></td><td> Réactif de laboratoire standard </td></tr><tr><td> anti-neurofilaments anticorps, en option </td><td> Millipore </td><td> AB1991 </td><td> Suivre le protocole histologique recommandé par le fabricant </td></tr><tr><td> Cryostat et matériaux associés pour la coupe </td><td> Leica </td><td> divers </td><td></td></tr><tr><td> Microscope à épifluorescence de formation d&#39;image </td><td> Zeiss, divers </td><td></td><td></td></tr></tbody></table>

selective tracing of auditory fibers in the avian embryonic vestibulocochlear nerve

Le poussin embryonnaire est un modèle largement utilisé pour l&#39;étude des périphériques et centraux projections des cellules ganglionnaires. Dans le système auditif, marquage sélectif des axones du nerf auditif dans VIIIe crânienne améliorerait l&#39;étude du développement central circuit auditif. Cette approche est difficile en raison de multiples organes sensoriels de l&#39;oreille interne contribuer à la VIIIe nerf 1. De plus, des marqueurs qui distinguent de manière fiable contre les groupes auditif vestibulaires des axones dans le nerf VIII aviaire doivent encore être identifiés. Voies auditives et vestibulaires peuvent pas être distinguées fonctionnellement dans les embryons précoces, comme sensorielles-réponses évoquées ne sont pas présents avant que les circuits sont formés. Idéalement saillie axones VIIIe ont été tracées dans certaines études, mais auditives axone étiquetage a été accompagnée par l&#39;étiquetage des autres composants nerveuses VIIIe 2,3. Ici, nous décrivons une méthode de traçage antérograde du ganglion acoustique de façon sélective labeaxones auditives l sein du VIIIe nerf développement. Tout d&#39;abord, après dissection partielle de la région céphalique antérieure d&#39;un embryon de poulet 8-jour immergé dans le liquide céphalorachidien artificiel oxygéné, le canal cochléaire est identifié par des repères anatomiques. Ensuite, une micropipette de verre fine tiré est positionnée pour injecter une petite quantité de rhodamine dextrane aminé dans la région de canal adjacent et profonde, où les cellules ganglionnaires acoustiques sont situés. Dans les trente minutes qui suivent l&#39;injection, les axones auditives sont tracées de manière centrale dans le cerveau postérieur et peuvent ensuite être visualisées après une préparation histologique. Cette méthode fournit un outil utile pour les études sur le développement de la formation auditive périphérique circuit central.

Les composants du nerf VIII et l&#39;anatomie du nerf lui-même sont complexes et alambiquées (figures 1, 3). Par sélectivement le traçage des fibres provenant de cellules ganglionnaires acoustiques, les segments du nerf VIII ainsi que les afférences auditives primaires dans le tronc cérébral peuvent être tracées et proprement distinguent de leurs homologues vestibulaires (Figures 2, 3). De même, cette technique pourrait être utilisée pour étudier les projections périphéri...

Watch this Scientific Journal Video about Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve at JoVE.com

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Nous décrivons ici une technique de microdissection suivie par l&#39;injection de colorant fluorescent dans le ganglion acoustique des embryons de poulet au début de traçage sélective des fibres axonales auditives dans le nerf et du cerveau postérieur.

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 ...

selective-tracing-auditory-fibers-avian-embryonic-vestibulocochlear

Research

JoVE Journal

Neuroscience

9.1K vues.  University of California, Irvine. Nous décrivons ici une technique de microdissection suivie par l'injection de colorant fluorescent dans le ganglion acoustique des embryons de poulet au début de traçage sélective des fibres axonales auditives dans le nerf et du cerveau postérieur.

Vidéo: Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

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

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

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 ...

Recherche

Neurosciences

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

Un léger Casque-based System pour manipuler Rétroaction sonore chez les oiseaux chanteurs

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Stimulation optogenetic du nerf auditif

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Transsynaptique génétique conditionnelle traçage dans le cerveau embryonnaire de souris

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

Neuronal Tracing dans des explants de cerveau à guidage laser

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

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 ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Imagerie fibres sérotoninergiques dans la moelle épinière de souris Utilisation du CLARITY / Technique CUBIC

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

In Vitro Caractérisation des propriétés électrophysiologiques des fibres afférentes du côlon chez le rat

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

Traçage neuroanatomique rétrograde des motoneurones phréniques chez la souris

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

Cibler les agrégats alpha synuclein dans les fibres nerveuses périphériques cutanées par l'évaluation de l'immunofluorescence flottante

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...