Imaging Neuronal Svar på Slice Varer af Vomeronasal Organ udtryk for en genetisk kodet Calcium Sensor
Summary
The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.
Abstract
Den vomeronasal organ (VNO) registrerer chemosensory signaler, der bærer information om de sociale, seksuelle og reproduktive status for individer inden for samme art 1,2. Disse intraspecies signaler, feromoner, samt signaler fra nogle rovdyr 3, aktivere vomeronasal sensoriske neuroner (VSNs) med høje niveauer af specificitet og sensitivitet 4. Mindst tre forskellige familier af G-protein koblede receptorer, V1R, V2R og FPR 5-14, er udtrykt i VNO neuroner at mægle påvisning af chemosensory tidskoder. For at forstå hvordan feromon oplysninger er kodet af VNO, er det kritisk at analysere respons profiler af de enkelte VSNs til forskellige stimuli og identificere de specifikke receptorer, der formidler disse svar.
Den neuroepithelia af VNO er indesluttet i et par vomer knogler. Den semi-blind rørformet struktur VNO har en åben ende (vomeronasal kanal) tilslutning tilnæsehulen. VSNs udvide deres dendritter til lumen del af VNO, hvor feromon signaler er i kontakt med de receptorer udtrykt på dendritiske drejeknapper. Cellen organer VSNs formularen pseudo-stratificerede lag med V1R og V2R til udtryk i den apikale og basale lag henholdsvis 6-8. Adskillige teknikker er blevet brugt til at overvåge svar VSNs på sensoriske stimuli 4,12,15-19. Blandt disse teknikker tilbyder akut skive forberedelse flere fordele. Først, i forhold til dissocieret VSNs 3,17, slice præparater opretholde neuroner i deres eget morfologi og dendritter af cellerne forbliver relativt intakt. For det andet, cellen organer VSNs er let tilgængelige i koronale bid af VNO at tillade elektrofysiologi undersøgelser og billedbehandling eksperimenter i forhold til hele epitel og hel-mount præparater 12,20. For det tredje kan denne metode kombineres med molekylær kloning teknikker, der giver receptor identifikation.
Sensorisk stimulation udløser stærke Ca 2 + tilstrømning i VSNs, der er betegnende for receptor-aktivering 4,21. Vi dermed udvikle transgene mus, der udtrykker G-CAMP2 i olfaktoriske sensoriske neuroner, herunder VSNs 15,22. Følsomheden og den genetiske karakteren af sonden i høj grad lette Ca 2 + billeddannelse eksperimenter. Denne metode har fjernet farvestoffet loading proces, der anvendes i tidligere undersøgelser 4,21. Vi har også ansat en ligand delivery system, der muliggør anvendelsen af forskellige stimuli til VNO skiver. Kombinationen af de to teknikker giver os mulighed for at overvåge flere neuroner på samme tid som reaktion på et stort antal stimuli. Endelig har vi etableret et semi-automatiseret analyse rørledning til at hjælpe billedbehandling.
Protocol
Discussion
The majority of the vomeronasal receptors (VRs) remain as orphan receptors since their discovery by Dulac and Axel 5. The pheromone ligands for these chemosensory receptors and their roles in mediating animal behaviors are not well understood. Until now, only one pair of ligand/receptor, the ESP1 peptide and its cognate receptor, Vmn2r116 (V2Rp5), has been identified and shown to convey specific social information 19,23. Another receptor, V1rb2, has been shown to respond to 2-heptanone, which presum…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We thank Andrea Moran together with members of Lab Animal Service Facility (LASF) at Stowers Institute for their excellent support on animal husbandry and technical services. This work is supported by funding from Stowers Institute and the NIH (NIDCD 008003) to CRY. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Deafness and Other Communication Disorders or the National Institutes of Health. U.S. patent pending for the tetO-G-CaMP2 mice for Stowers Institute, CRY and LM.
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