Live-Imaging af Nikotin induceret Calcium Signaling og neurotransmitterfrigivelse Langs ventrale Hippocampale Axoner
Summary
We developed a gene-chimeric preparation of ventral hippocampal – accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.
Abstract
Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)–accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.
Introduction
Cholinerg modulering af kredsløb excitabilitet bidrager til grundlæggende aspekter af kognition og ændret cholinerg modulation er en funktion af neurodegenerative og neuropsykiatriske lidelser, herunder Alzheimers sygdom, Parkinsons sygdom, skizofreni og afhængighed 1-4. En etableret mekanisme af cholinerge lettelse af synaptisk transmission i CNS er via direkte aktivering af nAChR'er lokaliseret ved præsynaptiske steder. Aktivering af disse præ-synaptiske receptorer fører til øget intracellulær Ca2 + ([Ca2 +] i) i præ-synaptiske terminaler – både direkte, på grund af den relativt høje calcium konduktans af visse nAChR undertyper, og indirekte via intracellulære signalering kaskader 5, hvilket vil styrke neurotransmitterfrigivelse. Faktisk har aktiveringen af præsynaptiske nAChRs blevet forbundet med ændringer i frigivelse af en lang række forskellige neurotransmittere, herunder glutamat, GABA, ACh, ennd dopamin 6-10. Selv om denne proces er blevet undersøgt indirekte ved hjælp elektrofysiologiske metoder på forskellige synapser, optiske reportere i [Ca2 +] i og synaptisk vesikel genanvendelse tillade mere direkte og tidsligt præcis måling af præsynaptiske fænomener.
Præsynaptiske lokalisering af nAChR'er er blevet demonstreret overbevisende med direkte immuno-guld mærkning af nAChR'er ved elektronmikroskopiske (EM) niveau 11,12. Adskillige andre teknikker har også været anvendt til at behandle nAChR lokalisering indirekte, herunder påvisning placeringer af nAChRs subunit- fluorescerende proteinkimærer i dyrkede neuroner 13,14, elektrofysiologiske registrering af nAChR strømme i synaptiske terminaler 15,16, overvågning nikotin inducerede ændringer i [Ca 2 +] i i synaptiske nerveender ved levende celler 17, og indirekte overvågning af neurotransmitter frigørelse ved den synaptiske terminal vedlevende celle billeddannelsesteknikker med fluorescerende indikatorer, herunder exocytose af synaptiske vesikler ses af styryl amfipatiske FM farvestoffer (FM1-43 og FM4-64) og / eller synapto-pHluorin og ved specifikke fluorescerende neurotransmitter reportere, såsom CNiFERs for ACh og iGluSnFr for glutamat 18-20. Samlet set har disse nuværende tilgange til at identificere præ-synaptisk lokalisering af nAChRs er komplicerede, og kræver særlige systemer og teknikker til at tillade pålidelig identifikation og fysiologiske overvågning af præ-synaptisk aktivitet.
Her beskriver vi protokoller og udstyr til en in vitro co-kultur system i en ventral hippocampalt (vHipp) – nucleus accumbens (NACC) kredsløb, der giver direkte adgang til at identificere og analysere både præ- og postsynaptiske komponenter af synaptisk transmission. Vi viser eksempler på præ-synaptisk lokalisering af nAChR'er og levende celler billeddannelse af nAChR medieret Ca2 + signalering og neurotransmitter release along vHipp axoner. En naturlig (og ligetil) forlængelse af protokollen præsenteres her er forberedelsen af præ- og post-synaptiske kontakter består af neuroner fra forskellige genotyper. På denne måde kan vurderes af bidraget fra en bestemt genprodukt til præ- og / eller post-synaptiske mekanismer af gradueringen direkte.
Protocol
Representative Results
Discussion
Forberedelse co-kultur beskrevet re-kapitulerer ventrale hippocampus-accumbens kredsløb i vitro. Denne forberedelse tillader forholdsvis enkel og pålidelig undersøgelse af de rumlige og tidsmæssige profiler, hvorved aktivering af præsynaptiske nAChRs fremkalder forbedrede glutamaterg transmission 5, 21.
Co-kulturer er defineret som væksten af forskellige specifikke celletyper i en skål, som kan give fysiologiske betingelser in vitro til at påvise in v…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank Yehui Qin and Mallory Myers for technical support. We also thank Dr. Sigismund Huck for providing us the anti-α4-ECD antibody. This work is supported by National Institutes of Health grant NS22061 to L. W. R.
Materials
| 1, Culture Media (50 ml) | |||
| Neurobasal | GIBCO | 10888022 | 48 ml |
| B-27 Supplements | GIBCO | 0080085-SA | 1 ml |
| Penicillin-Streptomycin | GIBCO | 10908-010 | 0.5 ml |
| GlutaMAX Supplement | GIBCO | 35050-061 | 0.5 ml |
| Brain-derived neurotrophic factor (BDNF) | GIBCO | 15140-122 | 20 ng/ml |
| 2, washing media (HBSS, 100 ml) | |||
| HBSS, no calcium, no magnesium, no phenol red | GIBCO | 14175-095 | 99 ml |
| HEPES ( 1M) | GIBCO | 15630-130 | 1 ml |
| 3, HEPES buffered saline (HBS) pH=7.3 | |||
| NaCl | Sigma | S9888 | 135 mM |
| KCl | Sigma | P9333 | 5 mM |
| MgCl2 | Sigma | M8266 | 1 mM |
| CaCl2, | Sigma | C1016 | 2 mM |
| HEPES | Sigma | H3375 | 10 mM |
| Glucose | Sigma | G0350500 | 10 mM |
| 4, HBS Cocktail for live imaging pH=7.3 | |||
| NaCl | Sigma | S9888 | 135 mM |
| KCl | Sigma | P9333 | 5 mM |
| MgCl2 | Sigma | M8266 | 1 mM |
| CaCl2, | Sigma | C1016 | 2 mM |
| HEPES | Sigma | H3375 | 10 mM |
| Glucose | Sigma | G0350500 | 10 mM |
| tetrodotoxin | Tocris | 1078 | 2 µM |
| bicuculline | Tocris | 131 | 10 µM |
| D-AP-5 | Tocris | 105 | 50 µM |
| CNQX | Tocris | 1045 | 20 µM |
| LY341495 | Tocris | 1209 | 10 µM |
| 5, Calcium-free HBS pH=7.3 | |||
| NaCl | Sigma | S9888 | 135 mM |
| KCl | Sigma | P9333 | 5 mM |
| MgCl2 | Sigma | M8266 | 1 mM |
| HEPES | Sigma | H3375 | 10 mM |
| Glucose | Sigma | G0350500 | 10 mM |
| 6, 56 mM Potassium ACSF pH=7.4 | |||
| NaCl | Sigma | S9888 | 119 mM |
| KCl | Sigma | P9333 | 56 mM |
| MgSO4.7H | Sigma | M1880 | 1.3 mM |
| CaCl2 | Sigma | C1016 | 2.5 mM |
| NaH2PO4 | Sigma | S8282 | 1 mM |
| NaHCO3 | Sigma | S5761 | 26.2 mM |
| Glucose | Sigma | G0350500 | 10 mM |
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