ニコチン誘導性カルシウムシグナリングおよび腹側海馬軸索に沿って神経伝達物質放出のライブイメージング
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
回路の興奮性のコリン作動性調節は、認知機能の基本的な側面に寄与し、変更されたコリン作動性の調節は、アルツハイマー病、パーキンソン病、統合失調症及び中毒1-4を含む神経変性及び精神神経疾患の特徴です。 CNSにおけるシナプス伝達のコリン作動性促進の確立メカニズムは、シナプス前部位に局在するのnAChRの直接の活性化を介してです。 、間接的に特定のnAChRサブタイプ、および比較的高いカルシウムコンダクタンスの両方に直接起因を介した細胞内シグナル伝達カスケード-これらのシナプス前受容体の活性化は細胞内Ca 2+（[Ca 2+] i） のシナプス前末端での増加につながります図5は 、それによって神経伝達物質の放出を増強します。実際には、シナプス前のnAChRの活性化は、グルタミン酸、GABA、アセチルコリンなどの神経伝達物質の多種多様の放出の変化と関連している、ANDドーパミン6-10。このプロセスは、様々なシナプスにおける電気生理学的方法を用いて、間接的に研究されてきたが、の光学記者の[Ca 2+] iとシナプス小胞のリサイクルは、前シナプス現象のより直接的かつ時間的に正確な測定を可能にします。
nAChR類の前シナプス局在は、電子顕微鏡（EM）レベル11,12でのnAChRの直接の免疫金標識で説得力が実証されています。いくつかの他の技術もまた、培養ニューロン13,14でのnAChR subunit-蛍光タンパク質キメラの位置を検出するなど、間接的のnAChR局在に対処するために使用されている、シナプス端子15,16でのnAChR電流の電気生理学的記録、[内のCaニコチン誘発変化を監視しますによるシナプスターミナルでのライブセルイメージング17、及び神経伝達物質の放出を間接的に監視することにより、シナプス神経終末における2+] i のスチリル両親媒性FM色素（FM1-43とFM4-64）および/またはsynapto-pHluorinによって、そのようなグルタミン酸のためのAChとiGluSnFrためCNiFERsなどの特定の神経伝達物質の蛍光の記者、によって見シナプス小胞のエキソサイトーシスを含む蛍光指示薬と生細胞イメージング技術、 18-20。全体的に、nAChR類の前シナプス局在を同定するため、これらの現在の手法は複雑であり、信頼性の高い同定とプレシナプス活性の生理学的モニタリングを可能にするために、特別なシステム及び技術を必要とします。
識別し、シナプス伝達のシナプス前および後の両方の成分を分析するための直接アクセスを提供し、側坐核（NACC）回路-ここでは、腹側海馬（vHipp） の de vitro共培養システムのためのプロトコルおよび装置が記載されています。我々は、シナプス前のnAChRの局在およびCa 2+シグナル伝達と神経伝達物質の放出ALO媒介のnAChRのライブセルイメージングの例を示しますNG vHipp軸索。ここで紹介するプロトコルの自然（と簡単な）拡張は、異なる遺伝子型からの神経細胞からなる前と後シナプスの接点の製造です。このように、変調の前および/または後シナプスメカニズムの特定の遺伝子産物の寄与を直接評価することができます。
Protocol
Representative Results
Discussion
共培養の準備は、in vitroで前シナプスのnAChRの活性化が強化されたグルタミン酸作動性伝達5、21を誘発することにより、空間的および時間的プロファイルのこの準備を許可比較的簡単で信頼性の高い検査を再降伏し、腹側海馬側坐核回路を説明しました。
共培養は、インビボ様の機能を実証するために、インビトロでの生理学的条件を提供す?…
Declarações
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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