Brown Laura E, Fuchs Celine, Nicholson Martin W, Stephenson F Anne, Thomson Alex M, Jovanovic Jasmina N
UCL School of Pharmacy, University College London.
UCL School of Pharmacy, University College London;
J Vis Exp. 2014 Nov 14(93):e52115. doi: 10.3791/52115.
Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.
抑制性神经元在中枢神经系统中发挥作用，以调节神经网络的动态和时空协调。γ-氨基丁酸（GABA）是大脑中主要的抑制性神经递质。它从抑制性神经元的突触前末端释放，这些突触前末端位于高度特化的细胞间连接即突触内，在突触处它与存在于突触后神经元质膜上的GABAA受体（GABAARs）结合。这些GABA门控离子通道的激活导致氯离子内流，从而引起突触后电位变化，降低这些神经元产生动作电位的可能性。在发育过程中，具有不同形态、电生理和神经化学特征的多种抑制性神经元能够识别其靶神经元并形成包含特定GABAAR亚型的突触。这种对神经元靶标的选择性支配原则提出了一个问题，即合适的突触伙伴如何相互识别。为了阐明潜在的分子机制，建立了一种新型的体外共培养模型系统，其中将从胚胎纹状体分离的高度同质的中等棘状GABA能神经元群体与稳定转染的表达不同GABAAR亚型的HEK293细胞系一起培养。在这个系统中，突触快速、高效且选择性地形成，并且易于进行定量分析。我们的结果表明，各种GABAAR亚型在促进突触形成的能力上存在差异，这表明这种简化的体外模型系统至少可以部分地重现体内识别合适的突触伙伴并形成特定突触所需的条件。这里首先描述培养中等棘状神经元和生成表达GABAARs的HEK293细胞系的方案，随后详细说明如何将这两种细胞类型进行共培养并分析突触接触的形成。
