VGLUT2 Transmission in Prefrontal Cortex and Working Memory

描述（由申请人提供）：精神分裂症是一种发育障碍，假设包括脑内神经元功能改变、异常N-甲基-D-天冬氨酸受体（NMDAR）信号传导和内侧前额叶皮质（mPFC）兴奋/抑制（E/I）平衡改变。在改善用于治疗和预防精神分裂症的策略方面取得进展的关键障碍是缺乏对突触前谷氨酸释放到GABA能中间神经元上的作用的理解，所述作用是通过内在皮层囊泡谷氨酸转运蛋白-2（VGLUT 2）突触在调节E/I平衡中以及这可能如何影响含PV的中间神经元的成熟。我们的目标是通过开发和提供对VGLUT 2介导的谷氨酸释放对PV抑制系统功能障碍的作用和影响的理解来改善治疗和预防精神分裂症的策略。我们的中心假设是，VGLUT 2提供谷氨酸的mPFC的新皮层PV+抑制性中间神经元，这是重要的兴奋/抑制（E/I）平衡的成熟和工作记忆。我们的目标是（1）在体内和体外确定功能性VGLUT 2突触靶向mPFC中的PV+神经元间系统，（2）证明VGLUT 2突触的缺失减少了E-I突触处的谷氨酸释放并抑制了抑制性传递，以及（3）证明条件性VGLUT 2敲除（KO）小鼠显示工作记忆缺陷。我们的预期结果将是（1）理解内在皮层VGLUT 2突触与E/I平衡相关，因为它们靶向复发性抑制性反馈神经元并表现出活动依赖性谷氨酸释放，（2）在条件性VGLUT 2 KO小鼠中内在皮质VGLUT 2表达的丧失导致PV+小鼠中GAD 67减少的知识，（3）证实VGLUT 2编码的传递在皮质边缘回路中对工作记忆至关重要。这些结果对治疗和预防精神分裂症的策略的影响，我们的结果将提供的是一个重要的一步，治疗洞察与精神分裂症相关的认知障碍和理解的作用和影响VGLUT 2介导的释放PV-神经元间系统和E/I平衡的成熟。目的1将检验这样的假设，即内源性皮质VGLUT 2突触与E/I平衡相关，因为a）它们靶向PV+ GABA能中间神经元，B）它们表现出谷氨酸的活性依赖性释放，以及c）它们的丧失抑制了mPFC中的抑制性突触传递。我们将利用GAD 67 gfp+转基因小鼠，选择性地表达绿色荧光蛋白的PV+中间神经元的免疫荧光和电生理研究。目的2将测试的假设，即VGLUT 2编码的兴奋性传输皮质边缘电路在发展过程中是至关重要的工作记忆在成年人中，通过评估工作记忆功能，使用连续延迟交替任务在T-迷宫中的两行小鼠，其中VGLUT 2失活发生在发展早期和青春期小鼠。

DESCRIPTION (provided by applicant): Schizophrenia is a developmental disorder that is hypothesized to include alterations in glutamatergic function, aberrant N-methyl-D-aspartic acid receptor (NMDAR) signaling and altered excitatory/inhibitory (E/I) balance in the medial prefrontal cortex (mPFC). A critical barrier to progress in improving strategies for the treatment and prevention of schizophrenia is a lack of understanding of the role of presynaptic glutamate release onto GABAergic interneurons by intrinsic cortical vesicular glutamate transporter-2 (VGLUT2) synapses in regulating E/I balance and how this may impact the maturation of PV- containing interneurons. Our goal is to improve strategies for the treatment and prevention of schizophrenia by developing and providing an understanding of the role and influence of VGLUT2-mediated glutamate release on the dysfunction of the PV-inhibitory system. Our central hypothesis is that VGLUT2 provides glutamate to neocortical PV+ inhibitory interneurons in the mPFC that is important for the maturation of excitatory/inhibitory (E/I) balance and to working memory. Our objectives are to (1) establish that functional VGLUT2 synapses target the PV+ interneuronal system in mPFC in vivo and in vitro, (2) demonstrate that the loss of VGLUT2 synapses decreases glutamate release at E-I synapses and suppresses inhibitory transmission, and (3) show that conditional VGLUT2 knockout (KO) mice display deficits in working memory. Our expected outcomes will be (1) an understanding that intrinsic cortical VGLUT2 synapses are relevant to E/I balance because they target recurrent inhibitory feedback neurons and exhibit activity-dependent release of glutamate, (2) the knowledge that loss of intrinsic cortical VGLUT2 expression in conditional VGLUT2 KO mice leads to a reduction in GAD67 in PV+ interneurons and suppresses inhibitory synaptic transmission onto pyramidal neurons, and (3) to affirm that VGLUT2-encoded transmission in corticolimbic circuits is critical to working memory. The impact of these results on strategies for treating and preventing schizophrenia that our outcomes will provide is an important step towards therapeutic insight into the cognitive impairment associated with schizophrenia and an understanding of the role and influence of VGLUT2-mediated release on the maturation of the PV-interneuronal system and E/I balance. Aim 1 will test the hypothesis that intrinsic cortical VGLUT2 synapses are relevant to E/I balance because a) they target PV+ GABAergic interneurons, b) they exhibit activity-dependent release of glutamate, and c) their loss suppresses inhibitory synaptic transmission in the mPFC. We will utilize GAD67gfp+ transgenic mice that selectively express EGFP in PV+ interneurons for immunofluorescence and electrophysiologic studies. Aim 2 will test the hypothesis that VGLUT2-encoded excitatory transmission in corticolimbic circuits during development is critical to working memory in adults, by assessing working memory function using the continuous delayed alternation task in a T-maze in two lines of mice where VGLUT2 inactivation occurs early in development and in adolescent mice.