Ce travail a été soutenu par la Muscular Dystrophy Association, le NIH (NS062320).

1. Administration sous-cutanée du récepteur de l&#39;acétylcholine muscariniques (mAChR) antagonistes <ol><li> Préparer dans des conditions aseptiques la dose appropriée de l&#39;antagoniste mAChR, (cf. tableau) en dissolvant le médicament dans du sérum physiologique stérile dans le tube de réaction 1.5ml. Les antagonistes suivants ont été utilisés: l&#39;atropine, méthoctramine, 4-DAMP, AFDX-116, AFDX-384, MT 7. </li><li> Tirage 50 pl de la solution dans la seringue d&#39;insuline et de a1cc utiliser une seringue pour chaque souris. Également préparer les seringues contenant du sérum physiologique seulement pour chaque souris du groupe contrôle. Tenez les seringues sur la glace jusqu&#39;à ce que prêt à injecter. </li><li> Anesthetize souris avec de la kétamine-xylazine cocktails (120 mg / kg de kétamine, de 8 mg / kg de xylazine) Vérifier profondeur de l&#39;anesthésie appropriée en observant l&#39;absence de réponse à l&#39;orteil de pincement. </li><li> Après une sédation adéquate, raser les poils couvrant la région rostrale de la tête à la région caudale du cou, essuyez restantes cheveux loin avec de l&#39;éthanol à 70%. </li><li> Placez la souris sous stéréomicroscope, et d&#39;insérer l&#39;aiguille parallèlement au muscle LAL tout en tirant légèrement sur ​​l&#39;aiguille de la seringue (Fig. 1A, B). Cela garantit que l&#39;aiguille est insérée dans le tissu conjonctif recouvrant et ne pas directement endommager le muscle du labo lui-même. Insérez l&#39;aiguille pour que la pointe couvre la partie caudale du muscle LAL. </li><li> Très lentement commencer à injecter la solution sur le muscle droit LAL tout en retirant la seringue, le temps d&#39;injection totale devrait être d&#39;environ 1 minute. Si elle est injectée correctement, la solution doit former un «dôme» qui est d&#39;environ 1 cm de diamètre, dans la peau recouvrant qui restera pour au moins une heure. Une solution uniforme injecté couvrira les régions entières rostrale et caudale du muscle droit LAL. </li><li> Répétez la procédure toutes les 12 heures pour 1 à 7 jours. Pour d&#39;autres études1, nous avons utilisé le même protocole d&#39;injecter des facteurs de croissance toutes les 12 heures pour un maximum de 21 jours. Aucune des réponses anormales ont été observées chez des souris administré une anesthésie avec 2x par jour pour un maximum de 21 jours. </li></ol> 2. Dissection des muscles du labo <ol><li> Euthanasier les souris avec une surdose de pentobarbital de sodium (390 mg / kg). La mort est assurée en évaluant le manque de rythme cardiaque, après thoracotomie est réalisée. </li><li> Pin bas de la souris dans un plat de dissection, face dorsale avec une épingle dans chaque patte et une aiguille à travers le nez. </li><li> Faire la première incision dans la peau uniquement, en utilisant des ciseaux pour couper petit ressort le long de la LAL à gauche de la région juste en amont de l&#39;oreille à la région autour de l&#39;omoplate gauche. </li><li> Retirer délicatement la peau de cette région, mais évitez de couper trop près de l&#39;oreille droite comme cela est l&#39;un des points de fixation pour le muscle droit LAL. </li><li> Repérez la bande adipeux en position centrale tissu de lipides le long de la ligne médiane, sur la surface superficielle de la tête, où les muscles droit et gauche du labo rencontrer. Avec des ciseaux petit ressort, couper 1 cm sur la droite du tissu adipeux (vers l&#39;oreille gauche) à partir du bord proximal du muscle gauche LAL jusqu&#39;à atteindre l&#39;épaule (Fig. 1C). </li><li> Une fois l&#39;incision est faite, peler le muscle droit LAL avec petits forceps pour exposer la face ventrale du muscle. Coupez le tissu conjonctif et fascia tandis tirant doucement sur ​​le muscle droit LAL avec une pince (Fig. 1C). </li><li> Couper autour l&#39;extrémité caudale du LAL à droite à la base de l&#39;oreille. Continuer la coupe, le déplacement vers la fin rostrale de l&#39;oreille droite, en gardant le droit de LAL remis (Fig. 1C).. Il est préférable d&#39;inclure plus de tissu à ce stade que de risquer d&#39;endommager le labo lui-même. Si le muscle commence à sécher, une pipette PBS 1X sur le muscle. </li><li> Placez disséqué droit LAL dans la culture plat 30mm Sylgard contenant du PBS 1X, face dorsale en place. Pin bas des quatre coins de muscles avec des épingles petit insecte. </li><li> En utilisant des pinces et ciseaux petits au printemps, nettoyez le tissu conjonctif des surfaces dorsale et ventrale du muscle droit LAL. Tournez musculaire plus et re-broches en vue de nettoyer la surface ventrale. 2-3 muscles peuvent être placés dans le même plat de 30mm. </li></ol> 3. Immunomarquage Triple NMJs LAL: Jour 1 <ol><li> Avant immunomarquage début, préparer tous les réactifs nécessaires. PBS 1X, 2% d&#39;albumine sérique bovine BSA) / PBS, glycine 0,1 M à 2% de BSA / PBS, Triton X100 à 0,2% dans 2% BSA / PBS, et 4% de paraformaldéhyde (PFA) dans du PBS 1X seront nécessaires. Pré-refroidir dans le méthanol à -20 ° C au congélateur. </li><li> Laisser les muscles LAL épinglé dans la face dorsale vaisselle pendant ce processus. Jeter PBS 1X solution et ajouter assez de 4% (PFA) pour couvrir le muscle. Placer sur agitateur plat à la vitesse de 2 ou 3 pendant 20 minutes. Sauf mention contraire, le plat doit être placé sur un agitateur pour les étapes suivantes. </li><li> Jeter 4% PFA et laver le muscle avec du PBS 1X 3 fois, 10 &#39;chacune. </li><li> Jeter dernière PBS 1X laver et ajoutez 0,1 M glycine dans 2% BSA / PBS solution pendant 30 &#39;. </li><li> Jeter une solution de glycine 0,1 MD Ajouter du PBS 1X contenant rhodamine conjugué α-bungarotoxine à une concentration de 1:200 pendant 15 &#39;. </li><li> Jeter α-bungarotoxine solution et laver les muscles dans du PBS 1X, 3 fois 10 &#39;chacune. </li><li> Perméabiliser les tissus en y ajoutant de pré-refroidissement du méthanol et de placer le plat au congélateur à -20 ° C pendant exactement 5 minutes, sans secousses nécessaires. </li><li> Retirez le méthanol et laver 3 fois 10 &#39;chacun avec du PBS 1X. Jeter dernier lavage et les tissus bloc avec 0,2% de Triton X-100 dans 2% BSA / PBS pendant 1 heure. Les étapes de lavage et de blocage sont réalisées à température ambiante. </li><li> Incuber les muscles durant la nuit, en secouant, à 4 ° C dans un cocktail d&#39;anticorps primaire dilué dans la solution de blocage (cf. tableau). </li></ol> 4. Immunomarquage Triple NMJs LAL: Jour 2 <ol><li> Lavez les muscles dans du PBS 1X 3 fois, 10 &#39;chacun à température ambiante. </li><li> Ajouter cocktail d&#39;anticorps secondaires dans une solution de blocage pendant 1 heure à température ambiante (cf. tableau). Les muscles doivent être protégés de la lumière à partir de ce pas pour éviter la photo-blanchiment d&#39;Alexa Fluor 647-fluorescence. </li><li> Lavez les muscles dans du PBS 1X 3 fois, 10 &#39;chacune. </li><li> Nettoyez tout tissu conjonctif reste dans du PBS 1X, couper les bords latéraux des muscles du labo et monter sur des lames côté dorsal, en utilisant des lamelles de verre et de milieux de montage. Utilisez vernis à ongles transparent pour sécuriser lamelle en place. </li><li> Protéger les diapositives de la lumière et conserver à -20 ° C jusqu&#39;au moment de l&#39;analyse. </li></ol> 5. Imagerie confocale des NMJs LAL <ol><li> Des images haute résolution confocales sont obtenus avec un objectif Leica Apo plan d&#39;huile 63X (1.4NA) sur un microscope Leica TCS 4D confocale. </li><li> Fluorescéine et Alexa 647 signaux ont été simultanément numérisées avec des lignes laser à 488 nm (argon, 488 nm, 20 mW) et 633 nm (HeNe, 633 nm, 10 mW), respectivement. En séquentiel, le signal de la rhodamine est ensuite scanné avec la ligne laser 561 nm (DPSS, 561, 20 mW). Sections optiques ont été recueillis à intervalles de 0,3 um, et le nombre d&#39;images dans le plan z varie de 60-80. Le sténopé est ajusté à 1 Airy (107,97 UM). Le format d&#39;image 1024 x 1024 (X, Y), avec un facteur de zoom de 1, à la vitesse des résultats 400Hz dans une taille d&#39;image de 234,32 x 234,32 um um et la taille de pixel de 229,05 nm x 229,05 nm. </li><li> Logiciel Leica TCS-NT d&#39;acquisition est alors utilisé pour reconstruire des images de la série Z dans les projections d&#39;intensité maximale. </li><li> Multipanel images sont ajuster la luminosité et contraste à l&#39;aide d&#39;Adobe Photoshop. </li><li> Pour un muscle typiques du labo, on devrait être capable d&#39;analyser environ 75 à 100 fr NMJs visage. La stabilité Synaptic est déterminé par des caractéristiques telles que l&#39;alignement spatial de pré-, péri-et post-synaptiques éléments (par exemple, nerveuses, des cellules de Schwann et les muscles), et les mesures de la zone synaptique (ie, la taille des synapses). </li></ol> Les résultats représentatifs: Lors de la JNM mammifères adultes, un seul axone moteur développe des branches fines qui forme hautement différenciés tonnelles d&#39;une terminaison nerveuse (fig. 2; vert) sur une fibre musculaire, précisément revêtue de grappes postsynaptique de AChR nicotinique (Fig. 2; Rouge) . Perisynaptically, le terminal des cellules de Schwann étroitement couvrent toutes les branches de terminaisons nerveuses présynaptiques (Fig. 2; Bleu). L&#39;intégrité structurale et fonctionnelle de cette organisation tripartite est gravement perturbée par l&#39;application quotidienne d&#39;inhibiteurs mAChR sous-type spécifique. Dans l&#39;exemple présenté ici (fig. 3), 4-humide, un antagoniste du mAChR avec une haute affinité pour les autoroutes M1, M3, M4 et M5 sous-types mAChR, évoque l&#39;élimination sélective des terminaisons nerveuses du NMJs nombreuses sur toute la surface du muscle (figure 3B, C). En outre, le terminal des cellules de Schwann sont anormalement tranquilles 8 comme en témoigne l&#39;étiquetage S100 lumineux sans l&#39;extension du processus (Fig. 3B », 3C&quot;). Postsynaptique, les fibres musculaires sont normales et il n&#39;ya aucune perte de nAChR (figure 3B, 3C). <img src="/files/ftp_upload/3124/3124fig1.jpg" alt="Figure 1"/> Figure 1. Localisation anatomique et l&#39;organisation du muscle LAL. Lieu d&#39;rostrale (rLAL), caudale (CLAL), et le nerf LAL (LALn) et des bandes des plateaux vertébraux correspondants sont présentés (A, en médaillon). L&#39;emplacement et l&#39;orientation de l&#39;aiguille en ce qui concerne le muscle LAL est montré pour la procédure d&#39;injection (B). Incision des points sont présentés pour la procédure de dissection (C). <img src="/files/ftp_upload/3124/3124fig2.jpg" alt="Figure 2"/> Figure 2. Organisation tripartite de la JNM. Fort grossissement vues confocale d&#39;un JNM souris. Un seul axone élabore fines branches terminales (A), bien couvert par la borne cellule de Schwann et de leurs procédés (A &#39;, point astérisques à des organes terminaux des cellules de Schwann). Postsynaptique dans une fibre musculaire, une grappe de AChR nicotinique est précisément apposed aux branches du nerf termiNALS et terminale des cellules de Schwann. <img src="/files/ftp_upload/3124/3124fig3.jpg" alt="Figure 3"/> Figure 3. Muscles LAL traités avec 4-humide, un antagoniste du mAChR. Faible et fort grossissement vues confocale des muscles LAL traités avec le véhicule ou 4-DAMP. Contrairement aux muscles traités avec le véhicule (A-A NMJs&#39;&#39;&#39;), nombreuses dans les terminaux des muscles 4-DAMP traités nerveuses absence (B-B&#39;&#39;&#39;, C-C&#39;&#39;&#39;). Une zone encadrée dans la figure 2B est zoomée à la figure 2C.

<ol>
	<li>Wright, M. C., Son, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+neurotrophic+factor+is+not+required+for+terminal+sprouting+and+compensatory+reinnervation+of+neuromuscular+synapses:+re-evaluation+of+CNTF+null+mice.">Ciliary neurotrophic factor is not required for terminal sprouting and compensatory reinnervation of neuromuscular synapses: re-evaluation of CNTF null mice.</a> Exp Neurol. 205, 437-448 (2007).</li><li>Gurney, M. E., Yamamoto, H., Kwon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+motor+neuron+sprouting+in+vivo+by+ciliary+neurotrophic+factor+and+basic+fibroblast+growth+factor.">Induction of motor neuron sprouting in vivo by ciliary neurotrophic factor and basic fibroblast growth factor.</a> J Neurosci. 12, 3241-3247 (1992).</li><li>Caroni, P., Aigner, L., Schneider, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+neuronal+determinants+locally+regulate+extrasynaptic+and+synaptic+growth+at+the+adult+neuromuscular+junction.">Intrinsic neuronal determinants locally regulate extrasynaptic and synaptic growth at the adult neuromuscular junction.</a> J Cell Biol. 136, 679-692 (1997).</li><li>Witzemann, V., Brenner, H. R., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+factors+regulate+AChR+subunit+mRNAs+at+rat+neuromuscular+synapses.">Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses.</a> J Cell Biol. 114, 125-141 (1991).</li><li>Angaut-Petit, D., Molgo, J., Connold, A. L., Faille, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+levator+auris+longus+muscle+of+the+mouse:+a+convenient+preparation+for+studies+of+short-+and+long-term+presynaptic+effects+of+drugs+or+toxins.">The levator auris longus muscle of the mouse: a convenient preparation for studies of short- and long-term presynaptic effects of drugs or toxins.</a> Neurosci Lett. 82, 83-88 (1987).</li><li>Lanuza, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-+and+postsynaptic+maturation+of+the+neuromuscular+junction+during+neonatal+synapse+elimination+depends+on+protein+kinase.">Pre- and postsynaptic maturation of the neuromuscular junction during neonatal synapse elimination depends on protein kinase.</a> C. J Neurosci Res. 67, 607-617 (2002).</li><li>Garcia, N., Santafe, M. M., Tomas, M., Lanuza, M. A., Tomas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+effects+of+beta-amyloid25-35+peptide+aggregates+on+transmitter+release+in+neuromuscular+synapses.">Short-term effects of beta-amyloid25-35 peptide aggregates on transmitter release in neuromuscular synapses.</a> J Neuropathol Exp Neurol. 67, 250-259 (2008).</li><li>Wright, M. C., Cho, W. J., Son, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+patterns+of+motor+nerve+terminal+sprouting+induced+by+ciliary+neurotrophic+factor+vs.+botulinum+toxin.">Distinct patterns of motor nerve terminal sprouting induced by ciliary neurotrophic factor vs. botulinum toxin.</a> J Comp Neurol. 504, 1-16 (2007).</li><li>Wright, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+muscarinic+acetylcholine+receptor+subtypes+contribute+to+stability+and+growth,+but+not+compensatory+plasticity,+of+neuromuscular+synapses.">Distinct muscarinic acetylcholine receptor subtypes contribute to stability and growth, but not compensatory plasticity, of neuromuscular synapses.</a> J Neurosci. 29, 14942-14955 (2009).</li><li>Voss, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ATP+inhibits+chloride+channels+in+mature+mammalian+skeletal+muscle+by+activating+P2Y1+receptors.">Extracellular ATP inhibits chloride channels in mature mammalian skeletal muscle by activating P2Y1 receptors.</a> J Physiol. 587, 5739-5752 (2009).</li><li>Murray, L. M., Gillingwater, T. H., Parson, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+mouse+cranial+muscles+to+investigate+neuromuscular+pathology+in+vivo.">Using mouse cranial muscles to investigate neuromuscular pathology in vivo.</a> Neuromuscul Disord. 20, 740-743 (2009).</li><li>Dorje, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonist+binding+profiles+of+five+cloned+human+muscarinic+receptor+subtypes.">Antagonist binding profiles of five cloned human muscarinic receptor subtypes.</a> J Pharmacol Exp Ther. 256, 727-733 (1991).</li><li>Caulfield, M. P., Birdsall, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Union+of+Pharmacology.+XVII.+Classification+of+muscarinic+acetylcholine+receptors.">International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors.</a> Pharmacol Rev. 50, 279-290 (1998).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

La méthode présentée ici permet d&#39;établir le rôle précédemment non reconnu de sous-type de signalisation mAChR dans la stabilité et le maintien de NMJs mammifères. Cette méthode sera également utile pour tester les effets de facteurs neurotrophiques et des agents pharmacologiques. Par exemple, notre laboratoire a révélé que le facteur neurotrophique ciliaire (CNTF), a suscité la germination de presque tous les terminaisons nerveuses LAL chez la souris adulte <a>1</a> . Ce résultat contraste avec les ...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> kétamine </td><td> Hospira </td><td> NDC0409-2051-05 </td><td> Dose: 120mg/kg </td></tr><tr><td> xylazine </td><td> Laboratoires Lloyd </td><td> LA33806 </td><td> Dose: 8mg/kg </td></tr><tr><td> atropine </td><td> Sigma-Aldrich </td><td> A0132 </td><td> (Pureté&gt; 98%); Dose: 0.2mg/kg - 20mg/kg </td></tr><tr><td> atropine </td><td> Voigt Global Distribution </td><td> AT105 </td><td> Qualité pharmaceutique </td></tr><tr><td> Méthoctramine </td><td> Sigma-Aldrich </td><td> M105 </td><td> Dose: 100 - 400m </td></tr><tr><td> 4-DAMP </td><td> Sigma-Aldrich </td><td> D142 </td><td> Dose: 2,5 mg </td></tr><tr><td> AFDX-116 </td><td> Bioscience Tocris </td><td> 1105 </td><td> 250 M </td></tr><tr><td> AFDX-384 </td><td> Bioscience Tocris </td><td> 1345 </td><td> 50M - 500M </td></tr><tr><td> MT 7 </td><td> Peptides international </td><td> PMT-4340-S </td><td> 0,1 - 1M </td></tr><tr><td> Phosphate 1X saline tamponnée, pH 7,4 </td><td> Invitrogen </td><td> 10010049 </td><td></td></tr><tr><td> Paraformaldéhyde </td><td> Fisher </td><td> T353-500 </td><td> Faire solution à 10% d&#39;abord par dissolution 10g/100mL eau désionisée distillée; faire 4% avec du PBS 1X, ajuster le pH à 7,4 </td></tr><tr><td> Sodium pentobarbital </td><td> Santé Animale Virbac </td><td> NDC-051311-050-01 </td><td> Dose: 390mg/kg </td></tr><tr><td> Sylgard </td><td> Dow Corning </td><td> Partie # 184 </td><td> Suivez les instructions qui viennent avec un kit, peut utiliser plusieurs boîte de culture de taille (30mm, 60mm, 100mm) en fonction des besoins </td></tr><tr><td> Glycine 0,1 M </td><td> Sigma-Aldrich </td><td> G-7126 </td><td> Ajouter 0.185g à 25ml de 2% de BSA / PBS </td></tr><tr><td> 2% d&#39;albumine de sérum bovin (BSA 2%) </td><td> Sigma-Aldrich </td><td> A3059-100g </td><td> Dissoudre 2g BSA dans 100mL d&#39;PBS 1X </td></tr><tr><td> Triton X100 à 0,2% dans 2% BSA / PBS (tampon de blocage) </td><td> Sigma-Aldrich </td><td> T9284-100mL </td><td> Dissoudre 0.2ml/100mL 2% BSA / PBS </td></tr><tr><td> α-bungarotoxine </td><td> Invitrogen </td><td> T1175 </td><td> Utiliser à une concentration de 1:200 </td></tr><tr><td> SMI-312 </td><td> Monoclonaux Sternberger </td><td> SMI312 </td><td> Utiliser à une concentration de 1:1000 </td></tr><tr><td> SV2 </td><td> Des études de développement de la Banque d&#39;hybridomes </td><td> SV2-Surnageant </td><td> Utiliser à une concentration de 1:10 </td></tr><tr><td> S100 </td><td> Dako </td><td> Z0311 </td><td> Utiliser à une concentration de 1:400 </td></tr><tr><td> FITC de chèvre anti-souris IgG1 </td><td> Roche </td><td> 03117731001 </td><td> Utiliser à une concentration de 1:200, mais si le fond est élevé, essayez 1:400 </td></tr><tr><td> Alexa Fluor 647-chèvre anti-lapin conjugué </td><td> Invitrogen </td><td> A21244 </td><td> Utiliser à une concentration de 1:200 </td></tr><tr><td> Vectashield fluorescentes montage de médias </td><td> Laboratoires Vecteur </td><td> H-1000 </td><td> Ce n&#39;est pas un média dur est défini, vous aurez besoin pour assurer la lamelle avec du vernis à ongles transparent. </td></tr><tr><td> Ciseaux petit ressort </td><td> Outils Fine Science </td><td> 15002-08 </td><td></td></tr><tr><td> Pinces à dissection </td><td> Outils Fine Science </td><td> 11295-51 </td><td></td></tr></tbody></table>

subcutaneous administration of muscarinic antagonists and triple-immunostaining of the levator auris longus muscle in mice

Muscles des membres postérieurs des rongeurs, comme les gastrocnémiens et du jambier antérieur, sont fréquemment utilisés pour les études pharmacologiques in vivo des signaux essentiels pour la formation et la maintenance de NMJs mammifères. Cependant, la pénétration du médicament dans ces muscles sous-cutanée ou intramusculaire est souvent incomplète ou inégale et de nombreuses NMJs peut rester inchangée. Bien que l&#39;administration systémique de dispositifs tels que des mini-pompes peuvent améliorer les effets spatio-temporelle, le caractère invasif de cette approche peut entraîner de confusion réponses inflammatoires et / ou des dommages musculaires directes. Par ailleurs, l&#39;analyse complète de la NMJs dans un muscle du membre postérieur est difficile car elle nécessite beaucoup de temps coupes sériées et immunomarquage vaste. Le labo de la souris est une fine feuille plate de muscle situé superficiellement sur le dos de la nuque. C&#39;est un tic musculaire rapide que les fonctions pour déplacer le pavillon de l&#39;oreille. Il contient des portions rostrales et caudales qui proviennent de la ligne médiane du crâne et s&#39;étendent latéralement à la partie cartilagineuse de chaque penne. Le muscle est innervé par une branche du nerf facial que les projets caudalement à sa sortie du trou stylo-mastoïdien. Nous et d&#39;autres ont trouvé du labo pour être une préparation pratique qui offre des avantages pour l&#39;enquête des deux courts et à long terme les effets in vivo de médicaments sur NMJs et les muscles. Tout d&#39;abord, sa localisation superficielle facilite multiples applications locale de médicaments sous anesthésie légère. Deuxièmement, sa minceur (2-3 couches de fibres musculaires) permet la visualisation et l&#39;analyse de presque tous les NMJs dans le muscle. Troisièmement, la facilité de le disséquer avec ses nerfs intacts avec le modèle de son innervation permet une analyse supplémentaire électrophysiologiques in vitro de 9,5. Enfin, et surtout, un petit volume appliqué (~ 50 pl) couvre facilement la surface du muscle entier, offre une exposition uniforme et prolongée de tous ses NMJs à la drogue et élimine la nécessité d&#39;une approche systémique 1,8.

Watch this Scientific Journal Video about Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice at JoVE.com

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Nous décrivons les procédures de l&#39;administration répétée d&#39;inhibiteurs de la signalisation muscariniques à l&#39;longus releveur Auris (LAL) de muscle de souris adultes jeunes et d&#39;immunomarquage ultérieure de ses jonctions neuromusculaires (NMJs) dans des échantillons entiers. Le muscle a des avantages uniques du labo pour révéler<em&gt; In vivo</em&gt; Effets pharmacologiques sur NMJs.

Administration sous-cutanée des antagonistes muscariniques et Triple-Immunocoloration du muscle releveur Longus Auris chez la souris

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

subcutaneous-administration-muscarinic-antagonists-triple

Research

JoVE Journal

Neuroscience

13.6K vues.  Arcadia University. Nous décrivons les procédures de l'administration répétée d'inhibiteurs de la signalisation muscariniques à l'longus releveur Auris (LAL) de muscle de souris adultes jeunes et d'immunomarquage ultérieure de ses jonctions neuromusculaires (NMJs) dans des échantillons entiers. Le muscle a des avantages uniques du labo pour révéler In vivo Effets pharmacologiques sur NMJs.

Vidéo: Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Évaluation de vocalisations ultrasoniques Pendant médicaments auto-administration chez le rat

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Recherche

Neurosciences

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

Administration orale de la roténone en utilisant un Gavage et analyse d&#39;images d&#39;alpha-synucléine inclusions dans le système nerveux entérique

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Spectral imagerie confocale de fluorescence tels taggés récepteurs nicotiniques dans souris knock-in avec l&#39;administration chronique de nicotine

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Microdialyse d&#39;éthanol au cours de l&#39;éthanol détermination opérant auto-administration et d&#39;éthanol par chromatographie en phase gazeuse

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

Promotion de la survie et de la différenciation des cellules souches neurales de fibrine et du facteur de croissance Cocktails après Spinal Cord Injury sévère

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

Proprioception et tension récepteurs en crabe Membres: Exercices de laboratoire étudiant

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Un protocole pour l’Administration de la Real-Time IRMf formation Neurofeedback

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most neuroimaging or neurophysiological investigations in humans have been confined to the study of &#947;-Amino-Butyric-Acid-(GABA)-receptor-agonist-based anesthetics, while the effects of dissociative N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthetics ketamine, nitrous oxide (N2O) and xenon (Xe) are largely unknown. This paper describes the methods underlying the simultaneous recording of magnetoencephalography (MEG) and electroencephalography (EEG) from healthy males during inhalation of the gaseous anesthetic agents N2O and Xe. Combining MEG and EEG data enables the assessment of electromagnetic brain activity during anesthesia at high temporal, and moderate spatial, resolution. Here we describe a detailed protocol, refined over multiple recording sessions, that includes subject recruitment, anesthesia equipment setup in the MEG scanner room, data collection and basic data analysis. In this protocol each participant is exposed to varying levels of Xe and N2O in a repeated measures cross-over design. Following relevant baseline recordings participants are exposed to step-wise increasing inspired concentrations of Xe and N2O of 8, 16, 24 and 42%, and 16, 32 and 47% respectively, during which their level of responsiveness is tracked with an auditory continuous performance task (aCPT). Results are presented for a number of recordings to highlight the sensor-level properties of the raw data, the spectral topography, the minimization of head movements, and the unequivocal level dependent effects on the auditory evoked responses. This paradigm describes a general approach to the recording of electromagnetic signals associated with the action of different kinds of gaseous anesthetics, which can be readily adapted to be used with volatile and intravenous anesthetic agents. It is expected that the method outlined can contribute to the understanding of the macro-scale mechanisms of anesthesia by enabling methodological extensions involving source space imaging and functional network analysis.

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

Enregistrement activité électromagnétique du cerveau au cours de l’Administration des Agents anesthésiques gazeux Xenon et l’oxyde nitreux chez des volontaires sains

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

Levator Auris Longus Préparation pour l’examen de la Transmission neuromusculaire mammifères dans des Conditions de tension de serrage

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

La détection de 5-Hydroxymethylcytosine dans les cellules souches neurales et le cerveau des souris

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...