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本实验旨在运用64通道(8 × 8)平面微电极阵列记录技术(MED-64系统)探讨5-HT2C受体在调节内嗅区-海马突触传递和突触联系中的作用。将急性分离的大鼠海马脑片置于MED-64系统的电极平皿中,持续灌流通入95% O2和5% CO2混合气的人工脑脊液,孵育2 h后进行多电极阵列同步记录。选一个电极进行电刺激(刺激强度30～199 μA,正负双波脉冲,单波宽0.1 ms,频率0.1 Hz),其余63个电极作为记录电极。最佳有效刺激部位恰好位于前穿质通路(perforant path, PP)上,电刺激结果可以分别在海马CA1区和齿状回(dentate gyrus, DG)记录到兴奋性突触后场电位(field excitatory post-synaptic potential,fEPSP)。我们以往研究结果已显示内嗅区-CA1和内嗅区-DG直接突触联系是由谷氨酸非NMDA受体所介导的兴奋通路。在稳定地诱导出网络fEPSP后,分别给予5-HT2C受体激动剂DOI和选择性拮抗剂SB242084,观察与计算fEPSP的反应幅值与斜率变化。同时利用双线性内插法计算出64个点的电流源(current source)与电流井(current sink),并将其转换为二维电流源密度(two-dimensional current source density, 2D-CSD)分布图。结果显示,结合fEPSP波的极相与2D-CSD成像,可见电刺激PP纤维在CA1区(腔隙分子层、锥体细胞层)和上部DG的分子层均发生突触兴奋引起的去极化,fEPSP为负向波,在空间上形成2D-CSD成像的电流井(蓝色区域)。同时,可在DG的颗粒细胞层和门部检测出正向波,形成2D-CSD成像的电流源(黄色区域),这反映了颗粒细胞在树突部位发生去极化后很快沿门向CA3传播电信号。在此基础上,给予5-HT2C受体激动剂DOI后,海马内有效突触联系(>基线20%的fEPSP)的空间网络范围显著扩大,突触传递效能显著增强。而相对应的是,给予5-HT2C受体的选择性拮抗剂SB242084,则海马内有效突触联系空间网络范围显著缩小,突触反应强度减弱。以上结果提示,内源性5-HT作用于5-HT2C受体可易化内嗅区-海马突触传递效能,扩大有效突触联系,引起突触反应的兴奋作用。
The purpose of this study was to investigate the role of 5-HT2C receptor in the regulation of synaptic transmission and synaptic connection in the entorhinal-hippocampal regions using a 64-channel (8 × 8) planar microelectrode array recording technique (MED-64 system) The acutely isolated rat hippocampal slices were placed in the electrode plate of MED-64 system and perfused continuously with artificial cerebrospinal fluid with a mixture of 95% O2 and 5% CO2 for 2 h. The multi-electrode arrays were synchronously recorded. Select one electrode for electrical stimulation (stimulation intensity 30 ~ 199 μA, positive and negative double-wave pulse width 0.1 ms, 0.1 Hz frequency), and the remaining 63 electrodes as the recording electrode. The most effective site of stimulation is located just on the perforant path (PP), and electrical stimulation results can be recorded in the field excitatory excitatory postsynaptic membrane potential in the hippocampus CA1 and dentate gyrus (DG) post-synaptic potential, fEPSP). Our previous findings have shown that the direct synaptic connection between the entorhinal-CA1 and the entorhinal-DG is an excitatory pathway mediated by non-NMDA receptors of glutamate. After stable induction of network fEPSP, DOI and selective antagonist SB242084, a 5-HT2C receptor agonist, were respectively administered to observe and calculate the response amplitude and slope change of fEPSP. At the same time, the current source and current sink of 64 points are calculated by bilinear interpolation and converted to two-dimensional current source density (2D-CSD) Distribution. The results showed that in combination with the polar phase of fEPSP wave and 2D-CSD imaging, PP-induced electrical shock induced depolarization induced by synaptic excitations in the CA1 (lacunar and pyramidal) and upper DG layers , fEPSP is a negative wave that spatially forms a current well (blue area) for 2D-CSD imaging. At the same time, a positive wave can be detected at the granulosa cell layer and the gate of the DG to form a current source for 2D-CSD imaging (yellow area), reflecting the rapid migration of granular cells along the gate toward the dendrites CA3 propagates electrical signals. On this basis, the spatial network of effective hippocampal synaptic connections (> 20% of baseline fEPSP) in the hippocampus was significantly expanded and the synaptic transmission efficiency was significantly enhanced after DOI, a 5-HT2C receptor agonist, was administered. Correspondingly, given the selective antagonist SB242084 at the 5-HT2C receptor, the spatial network of effective synaptic connections within the hippocampus was significantly reduced and the intensity of the synaptic response weakened. The above results suggest that endogenous 5-HT can enhance the synaptic transmission in the entorhinal hippocampus via the 5-HT2C receptor, enlarge the effective synaptic connections and trigger the excitatory effects of synaptic responses.