电突触在颞叶癫痫海马抑制性神经元死亡中的作用及其分子机制研究

Temporal lobe epilepsy is one common refractory disease which is characterized with hyper excitability and hyper synchrony among brain neurons, accompanied with abundant cell death of HIPP interneurons in hippocampal dentate gyrus. Although there are various studies reporting the regulation of excitability and synchronization between neurons by electrical synapses in epilepsy, whether electrical synapses are involved in HIPP cell specific death during epilepsy is not known. Recently it was found that electrical synapses played a key role in glutamate-dependent neural death, and our preliminary experiments demonstrated that (1) dentate gyrus interneurons have electrical synapses expression, and (2) the probability of electrical synaptic connection among surviving interneurons after epilepsy is significantly decreased. Therefore, we made the hypothesis: electrical synapses between HIPP interneurons are up-regulated after epilepsy, further causing HIPP neuronal death. This project will use paired whole-cell recordings to study the functional changes of electrical synapses and single cell RT-PCR for their gene expression in dentate gyrus interneurons after status epilepticus. We will also use in vitro hippocampal slice organotypic culture and in vivo model to study mechanisms of electrical synapses mediating HIPP specific death after epilepsy. This study will benefit the characterization of dentate electrical synapses network in epilepsy, thus enriching our knowledge of epilepsy pathogenesis and providing novel targets and strategies in clinical treatment.

颞叶癫痫是一种高发的难治性脑病，主要特征是神经元超级兴奋及超级同步发放，伴有海马齿状回HIPP神经元大量死亡。关于神经元间电突触如何调控癫痫神经网络的兴奋性和同步性已有大量研究，但电突触是否以及如何调控癫痫过程中HIPP神经元特异性死亡仍是未知。最近体外实验发现电突触在谷氨酸诱导神经元死亡中起关键作用,我们前期研究证明海马齿状回中各种同类中间神经元间均有电突触，同时癫痫发作后大量存活的中间神经元类型间电突触联系明显降低。因此我们认为：首次癫痫发生后，电突触特异在HIPP神经元上大量表达，从而促进了HIPP神经元大量死亡。本项目将运用膜片钳配对记录和单细胞RT-PCR研究癫痫发生过程中海马齿状回中各类中间神经元电突触及其调控因子变化，同时利用体外脑片培养和在体实验探索电突触介导HIPP神经元特异性死亡的具体机制。本项目将有助于我们进一步了解癫痫的发生机制，从而为癫痫的治疗提供新的思路和靶标。

颞叶癫痫发作常伴有海马齿状回中间神经元大量死亡，电突触主要存在中间神经元之间。关于电突触是否以及如何调控癫痫过程中中间神经元特异性死亡仍是未知。本课题中，我们研究了癫痫发作对于海马齿状回中各类中间神经元间电突触的影响，以及电突触阻断剂对癫痫发作后海马区的影响。我们实验结果显示，在正常大鼠海马中，各类中间神经元（PV和CCK）间具有电突触的存在，但是PV阳性中间神经元间的电突触明显比CCK阳性中间神经元多。利用pilocarpine诱导癫痫发作后，并不会影响规则发放（推测为CCK阳性）中间神经元间的电突触，只特异性减少海马快速发放的PV阳性中间神经元间的电突触。同时癫痫发作后，PV阳性中间神经元并不会明显死亡，而SST以及CCK阳性中间神经元会大量死亡。因此我们在癫痫发作后，给与电突触阻断剂干预，发现它可以逆转癫痫发作后的SST以及CCK阳性中间神经元死亡，并且由此逆转抑制性突触传递的降低，并进一步减少自发癫痫发作的产生。本课题的研究结果提示电突触在癫痫导致的中间神经元死亡过程中发挥重要作用，电突触阻断剂可以有效保护中间神经元，并抑制癫痫发作。本项目成果将为癫痫药物研发提供新的靶标。