Swanwick Catherine Croft, Murthy Namita R, Mtchedlishvili Zakaria, Sieghart Werner, Kapur Jaideep
Neuroscience Graduate Program, University of Virginia, Charlottesville, Virginia 22908, USA.
J Comp Neurol. 2006 Apr 10;495(5):497-510. doi: 10.1002/cne.20897.
The formation and maturation of gamma-aminobutyric acid (GABA)-ergic synapses was studied in cultured hippocampal pyramidal neurons by both performing immunocytochemistry for GABAergic markers and recording miniature inhibitory postsynaptic currents (mIPSCs). Nascent GABAergic synapses appeared between 3 and 8 days in vitro (DIV), with GABAA receptor subunit clusters appearing first, followed by GAD-65 puncta, then functional synapses. The number of GABAergic synapses increased from 7 to 14 DIV, with a corresponding increase in frequency of mIPSCs. Moreover, these new GABAergic synapses formed on neuronal processes farther from the soma, contributing to decreased mIPSC amplitude and slowed mIPSC 19-90% rise time. The mIPSC decay quickened from 7 to 14 DIV, with a parallel change in the distribution of the alpha5 subunit from diffuse expression at 7 DIV to clustered expression at 14 DIV. These alpha5 clusters were mostly extrasynaptic. The alpha1 subunit was expressed as clusters in none of the neurons at 7 DIV, in 20% at 14 DIV, and in 80% at 21 DIV. Most of these alpha1 clusters were expressed at GABAergic synapses. In addition, puncta of GABA transporter 1 (GAT-1) were localized to GABAergic synapses at 14 DIV but were not expressed at 7 DIV. These studies demonstrate that mIPSCs appear after pre- and postsynaptic elements are in place. Furthermore, the process of maturation of GABAergic synapses involves increased synapse formation at distal processes, expression of new GABAA receptor subunits, and GAT-1 expression at synapses; these changes are reflected in altered frequency, kinetics, and drug sensitivity of mIPSCs.
通过对γ-氨基丁酸（GABA）能标志物进行免疫细胞化学检测和记录微小抑制性突触后电流（mIPSCs），研究了培养的海马锥体神经元中GABA能突触的形成和成熟过程。新生的GABA能突触在体外培养3至8天（DIV）时出现，首先出现GABAA受体亚基簇，随后是GAD-65斑点，然后是功能性突触。GABA能突触的数量从7 DIV增加到14 DIV，同时mIPSCs的频率相应增加。此外，这些新的GABA能突触形成于离胞体较远的神经元突起上，导致mIPSC幅度降低和mIPSC 19-90%上升时间减慢。mIPSC衰减从7 DIV加快到14 DIV，同时α5亚基的分布也发生了平行变化，从7 DIV时的弥散表达变为14 DIV时的簇状表达。这些α5簇大多位于突触外。α1亚基在7 DIV时在所有神经元中均未以簇状形式表达，在14 DIV时为20%，在21 DIV时为80%。这些α1簇大多表达于GABA能突触处。此外，GABA转运体1（GAT-1）的斑点在14 DIV时定位于GABA能突触，但在7 DIV时未表达。这些研究表明，mIPSCs在突触前和突触后元件就位后出现。此外，GABA能突触的成熟过程涉及远端突起处突触形成增加、新的GABAA受体亚基表达以及突触处GAT-1表达；这些变化反映在mIPSCs的频率、动力学和药物敏感性改变上。
