尼古丁诱导的钙信号和神经递质的释放了沿着腹侧海马轴突实时成像
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
电路的兴奋性胆碱能调制有助于认识的基本方面，并改变胆碱能调制的神经变性和神经精神障碍的特征，包括阿耳茨海默氏病，帕金森氏病，精神分裂症和成瘾1-4。建立突触传递在中枢神经系统的胆碱能便利化机制是通过直接激活烟碱受体定位在突触前部位。这些突触前受体的激活导致增加的细胞内Ca 2+（内[Ca 2+] i）的突触前端子-包括直接，由于某些胆碱受体亚型，并间接的相对高的钙传导，通过细胞内的信号级联5，由此增强神经递质释放。事实上，突触前烟碱受体的激活已与变化的各种各样的神经递质包括谷氨酸，GABA，乙酰胆碱，释放一个第二多巴胺6-10。尽管间接使用电生理学方法在各种突触这一过程进行了研究，光学记者的[Ca 2+] i和突触小泡循环允许更直接的和时间上精确测量的突触前的现象。
烟碱受体的突触前本地化已被证明具有令人信服的烟碱受体在电子显微镜（EM）水平11,12直接免疫金标记。几个其它技术也被用于间接寻址nAChR的定位，包括：检测在培养的神经元13,14的nAChRs的亚单位的荧光蛋白嵌合体的位置，电生理记录的nAChR电流在突触末端15,16，监测尼古丁引起的变化的[Ca 2+] 我由活细胞成像17，和神经递质释放的间接监控在由突触终端突触神经末梢用荧光指示剂活细胞成像的技术，包括由苯乙烯两亲调频染料（FM1-43和FM4-64）和/或突触-pHluorin与由特定的荧光神经递质记者，如CNiFERs为乙酰胆碱和iGluSnFr谷氨酸观察突触小泡的胞吐18-20。总体而言，目前的这些方法鉴定烟碱受体的突触前本地化是复杂的，并且需要特殊的系统和技术，让可靠的鉴定和生理监测突触前活动。
在这里，我们描述的协议和设备进行了腹海马（vHipp）的体外共培养体系-伏隔核（NACC）电路，可直接访问识别和分析前和突触后突触传递的组成部分。我们发现烟碱受体的突触前本地化和胆碱受体的活细胞成像介导的钙信号和神经递质的释放ALO的例子NG vHipp轴突。这里介绍的协议的一个自然（和直接的）扩展是由来自不同基因型的神经元前和突触后接触的制备方法。以这种方式，特定的基因产物，以调制的前和/或突触后的机制的贡献可以直接评估。
Protocol
Representative Results
Discussion
共培养制备所述重新投降体外腹侧海马伏电路。该制剂允许相对简单和可靠的空间和时间曲线通过该激活突触前烟碱受体引起增强谷氨酸传输5,21检查。
共培养物被定义为不同的特定细胞类型的在一个菜的生长，可以提供在生理条件的体外证明体内样功能。常规神经元的神经元共培养相继出台神经科学的研究为研究不同类型的细胞之间突触?…
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 |
Referências
- Changeux, J. P., et al. Brain nicotinic receptors: structure and regulation, role in learning and reinforcement. Brain Res Brain Res Rev. 26 (2-3), 198-216 (1998).
- Levin, E. D. Nicotinic receptor subtypes and cognitive function. J Neurobiol. 53 (4), 633-640 (2002).
- Dani, J. A., Bertrand, D. Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system. Annu Rev Pharmacol Toxicol. 47, 699-729 (2007).
- Mineur, Y. S., Picciotto, M. R. Nicotine receptors and depression: revisiting and revising the cholinergic hypothesis. Trends Pharmacol Sci. 31 (12), 580-586 (2010).
- Zhong, C., Talmage, D. A., Role, L. W. Nicotine elicits prolonged calcium signaling along ventral hippocampal axons. PloS one. 8 (12), e82719 (2013).
- McGehee, D. S., Heath, M. J., Gelber, S., Devay, P., Role, L. W. Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors. Science. 269 (5231), 1692-1696 (1995).
- Gray, R., Rajan, A. S., Radcliffe, K. A., Yakehiro, M., Dani, J. A. Hippocampal synaptic transmission enhanced by low concentrations of nicotine. Nature. 383 (6602), 713-716 (1996).
- Dickinson, J. A., Kew, J. N., Wonnacott, S. Presynaptic alpha 7- and beta 2-containing nicotinic acetylcholine receptors modulate excitatory amino acid release from rat prefrontal cortex nerve terminals via distinct cellular mechanisms. Mol Pharmacol. 74 (2), 348-359 (2008).
- Zappettini, S., Grilli, M., Salamone, A., Fedele, E., Marchi, M. Pre-synaptic nicotinic receptors evoke endogenous glutamate and aspartate release from hippocampal synaptosomes by way of distinct coupling mechanisms. Br J Pharmacol. 161 (5), 1161-1171 (2010).
- Zappettini, S., et al. Presynaptic nicotinic alpha7 and non-alpha7 receptors stimulate endogenous GABA release from rat hippocampal synaptosomes through two mechanisms of action. PloS one. 6 (2), e16911 (2011).
- Fabian-Fine, R., et al. Ultrastructural distribution of the alpha7 nicotinic acetylcholine receptor subunit in rat hippocampus. J Neurosci. 21 (20), 7993-8003 (2001).
- Jones, I. W., Barik, J., O’Neill, M. J., Wonnacott, S. Alpha bungarotoxin-1.4 nm gold: a novel conjugate for visualising the precise subcellular distribution of alpha 7* nicotinic acetylcholine receptors. J Neurosci Methods. 134 (1), 65-74 (2004).
- Nashmi, R., et al. Assembly of alpha4beta2 nicotinic acetylcholine receptors assessed with functional fluorescently labeled subunits: effects of localization, trafficking, and nicotine-induced upregulation in clonal mammalian cells and in cultured midbrain neurons. J Neurosci. 23 (37), 11554-11567 (2003).
- Drenan, R. M., et al. Subcellular trafficking, pentameric assembly, and subunit stoichiometry of neuronal nicotinic acetylcholine receptors containing fluorescently labeled alpha6 and beta3 subunits. Mol Pharmacol. 73 (1), 27-41 (2008).
- Wu, J., et al. Electrophysiological, pharmacological, and molecular evidence for alpha7-nicotinic acetylcholine receptors in rat midbrain dopamine neurons. J Pharmacol Exp Ther. 311 (1), 80-91 (2004).
- Parikh, V., Ji, J., Decker, M. W., Sarter, M. Prefrontal beta2 subunit-containing and alpha7 nicotinic acetylcholine receptors differentially control glutamatergic and cholinergic signaling. J Neurosci. 30 (9), 3518-3530 (2010).
- Nayak, S. V., Dougherty, J. J., McIntosh, J. M., Nichols, R. A. Ca(2+) changes induced by different presynaptic nicotinic receptors in separate populations of individual striatal nerve terminals. J Neurochem. 76 (6), 1860-1870 (2001).
- Richards, C. I., et al. Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine. J Biol Chem. 286 (36), 31241-31249 (2011).
- Colombo, S. F., Mazzo, F., Pistillo, F., Gotti, C. Biogenesis, trafficking and up-regulation of nicotinic ACh receptors. Biochem Pharmacol. 86 (8), 1063-1073 (2013).
- St John, P. A. Cellular trafficking of nicotinic acetylcholine receptors. Acta Pharmacol Sin. 30 (6), 656-662 (2009).
- Zhong, C., et al. Presynaptic type III neuregulin 1 is required for sustained enhancement of hippocampal transmission by nicotine and for axonal targeting of alpha7 nicotinic acetylcholine receptors. J Neurosci. 28 (37), 9111-9116 (2008).
- Jacobowitz, D. M., Abbott, L. C. . Chemoarchitectonic Atlas of the Developing Mouse Brain. , (1998).
- Betz, W. J., Mao, F., Bewick, G. S. Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. J Neurosci. 12 (2), 363-375 (1992).
- Amaral, E., Guatimosim, S., Guatimosim, C. Using the fluorescent styryl dye FM1-43 to visualize synaptic vesicles exocytosis and endocytosis in motor nerve terminals. Methods Mol Biol. 689, 137-148 (2011).
- Garduno, J., et al. Presynaptic alpha4beta2 nicotinic acetylcholine receptors increase glutamate release and serotonin neuron excitability in the dorsal raphe nucleus. J Neurosci. 32 (43), 15148-15157 (2012).
- Guo, J. Z., Liu, Y., Sorenson, E. M., Chiappinelli, V. A. Synaptically released and exogenous ACh activates different nicotinic receptors to enhance evoked glutamatergic transmission in the lateral geniculate nucleus. J Neurophysiol. 94 (4), 2549-2560 (2005).
- Szabo, S. I., Zelles, T., Vizi, E. S., Lendvai, B. The effect of nicotine on spiking activity and Ca2+ dynamics of dendritic spines in rat CA1 pyramidal neurons. Hippocampus. 18 (4), 376-385 (2008).
