小鼠谷氨酸能神经元mGluR5受体调节皮层兴奋性的机制研究

Signaling by the group I metabotropic glutamate receptor 5 (mGluR5) in cortical glutamatergic neurons is required for the proper functional development of GABAergic circuits projected onto glutamatergic neurons (Ballester-Rosado, et al, J Neurosci., 2010). We hypothesis that mGluR5 signaling in cortical glutamatergic neurons is required to regulate cortical excitability. Cortex-specific mGluR5 conditional knockout mice (NEX-mGluR5 cKO mice) were generated by crossing mGluR5 floxed mice with NEX-Cre mice. At the cellular level, we found an increase in the frequency in spontaneous events in cortical layer IV glutamatergic neurons with NEX-mGluR5 cKO mice compared with their littermate control mice. Consistent with the increased excitability at the neuronal level, the threshold to induce status epilepticus by kainic acid was reduced in cKO mice. mGluR5 deletion has been found in ADHD patients. At the behavioral level, NEX-mGluR5 cKO mice also exhibit enhanced novelty-induced locomotion in open field testing. This may make it has a potential to be a novel ADHD model. To further evaluate the alteration of E/I balance in thalamocortical circuitry. we will combine molecular biological methods, electrophysiological experiments, two-photon calcium imaging and a series of behavior assays in our future experiments. It should help us to elucidate the critical role of mGluR5 in regulating the activity of cortex.

从丘脑投射到皮层谷氨酸能神经元的抑制性环路的发育需要功能正常的代谢型谷氨酸受体mGluR5。我们因此推测皮层谷氨酸能神经元mGluR5受体的正常表达对于调节皮层兴奋性是必须的。电生理学结果发现皮层兴奋性神经元mGluR5基因敲除鼠（NEX-cre-mGluR5 knockout mice）的皮层IV（layer IV）谷氨酸能神经元自发兴奋性突触后电流（sEPSCs）的频率相比对照鼠显著增加。海人藻酸诱导癫痫实验也显示条件性基因敲除鼠诱导阈值明显降低。此外，条件性基因敲除鼠在旷场实验中也呈现多动（hyperactivity）的行为学变化，这使得该小鼠有可能成为一个新颖的ADHD模型。我们拟综合分子生物学手段、电生理技术、双光子钙成像技术和一系列行为学方法的测试，进一步探究基因敲除鼠从丘脑到皮层神经通路兴奋/抑制（E/I）平衡的改变，从而揭示mGluR5调节皮层兴奋性的内在机制。

代谢型谷氨酸受体mGluR5（group I metabotropic glutamate receptor 5）通过第二信使发挥生物学效应。它广泛分布于大脑各区域，参与神经兴奋性网络调节、神经发生以及与学习记忆相关的突触可塑性的形成。近来，mGluR5已经被证实与多种神经系统的疾病发生相关，比如脆性X染色体综合症，自闭症和癫痫等疾病。为了检测mGluR5在前脑区域兴奋性神经元中所扮演的角色，我们使用mGLuR5 flox/flox mice 和CamkII alpha-Cre mice杂交，获得了在前脑区域兴奋性神经元敲除mGluR5的条件性敲除小鼠。在此基础上，我们全面开展了电生理和行为学研究，其中电生理学结果发现，海马CA1区谷氨酸能神经元自发兴奋性突触后电流（sEPSCs）和微小兴奋性突触后电流（mEPSCs）的频率相比对照鼠都有显著增加，而振幅变化不大，这显示了突触前的机制参与其中，PPR（Pair Pulse Ratio）的结果同样证实了这一点。动作电位的实验结果显示皮层锥体神经元有更高的兴奋性，证实了前脑部位兴奋/抑制（E/I）平衡的改变。行为学结果显示，条件性基因敲除鼠在旷场实验（Open Field）中也呈现多动（hyperactivity）的行为学变化，但并无焦虑伴随。Morris水迷宫实验基因敲除鼠需要更长的时间去发现水下平台，暗示海马依赖的空间学习和记忆的受损。为进一步证实这种行为学的变化，我们在海马区域 Schaffer collateral pathway进行LTP的记录，以评估mGluR5条件性敲除对突触可塑性产生的影响。实验证实基因敲除鼠NMDA-dependent的LTP受损，和我们的行为学结果相一致。综上所述，本实验使用mGluR5基因条件性敲除小鼠，揭示了mGluR5调节前脑尤其是海马区域E/I平衡的内在机制，mGluR5的敲除改变了锥体神经元的兴奋性和海马的突触可塑性，从而造成学习和记忆的行为学变化。