VGLUT2 Transmission in Prefrontal Cortex and Working Memory

描述(申请人提供)：精神分裂症是一种发育障碍，假设包括谷氨酸能功能改变，N-甲基-D-天冬氨酸受体(NMDAR)信号异常以及内侧前额叶皮质(MPFC)兴奋/抑制(E/I)平衡改变。在改善治疗和预防精神分裂症的策略方面取得进展的一个关键障碍是缺乏对内源性皮质囊泡谷氨酸转运体2(VGLUT2)突触向GABA能中间神经元释放突触前谷氨酸在调节E/I平衡中的作用以及这可能如何影响含PV的中间神经元成熟的了解。我们的目标是通过发展和了解VGLUT2介导的谷氨酸释放对PV抑制系统功能障碍的作用和影响来改进精神分裂症的治疗和预防策略。我们的中心假设是，VGLUT2向mPFC中的新皮质PV+抑制中间神经元提供谷氨酸，这对兴奋/抑制(E/I)平衡的成熟和工作记忆至关重要。我们的目标是(1)在体内和体外建立针对mPFC中PV+神经元系统的功能性VGLUT2突触，(2)证明VGLUT2突触的丢失减少了E-I突触的谷氨酸释放并抑制了抑制传递，(3)表明条件VGLUT2基因敲除(KO)小鼠表现出工作记忆缺陷。我们的预期结果将是：(1)了解固有的皮质VGLUT2突触与E/I平衡有关，因为它们针对的是经常性的抑制性反馈神经元，并且表现出活动依赖性的谷氨酸释放；(2)了解条件VGLUT2 KO小鼠固有的皮质VGLUT2表达缺失会导致PV+中间神经元中的GAD67减少，并抑制向锥体神经元的抑制性突触传递；(3)确认皮质边缘回路中的VGLUT2编码的传递对工作记忆至关重要。这些结果对我们的结果将提供的治疗和预防精神分裂症的策略的影响是重要的一步，有助于治疗性地洞察与精神分裂症相关的认知障碍，并理解VGLUT2介导的释放在PV-神经元间系统和E/I平衡成熟中的作用和影响。目的1验证皮质内VGLUT2突触与E/I平衡有关的假设，因为a)它们以PV+GABA能中间神经元为靶标，b)它们表现出活性依赖性的谷氨酸释放，c)它们的丢失抑制了mPFC中的抑制性突触传递。我们将利用GAD67gfp+转基因小鼠在PV+中间神经元选择性表达EGFP，用于免疫荧光和电生理研究。目的2通过在发育早期VGLUT2失活的两个品系和青春期小鼠中使用T迷宫中的连续延迟交替任务来评估工作记忆功能，以检验VGLUT2编码的兴奋性传递在发育过程中的皮质边缘回路对成人工作记忆至关重要的假说。

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.