Neuronal regulation of glutamate homeostasis

 描述(申请人提供)：精神分裂症是一种慢性、破坏性的精神障碍，其特征是多巴胺和谷氨酸信号异常。兴奋回路控制着多巴胺神经元的活动，人们认为这些回路的异常会产生精神分裂症的积极、消极和认知特征。谷氨酸稳态是指控制兴奋性突触内和周围的谷氨酸水平，我们现在有证据表明，谷氨酸稳态在控制精神分裂症涉及的回路中可能是重要的。谷氨酸转运体控制大脑谷氨酸的动态平衡，而大脑中主要的谷氨酸转运体是GLT-1，主要表达于星形胶质细胞。我们发现GLT-1在兴奋性突触前终末也有表达。为了了解GLT-1在神经元中表达的功能，我们建立了一个条件性GLT-1基因敲除小鼠，在其中我们使用突触素-cre来实现神经元中GLT-1的选择性失活。我们对这只小鼠进行了广泛的行为表型分析，包括测试对苯丙胺的反应，苯丙胺受到兴奋性信号的高度调节，因此我们认为很可能受到谷氨酸代谢紊乱的影响。许多研究小组以前的工作已经证明了苯丙胺的敏化现象，在这种现象中，行为或神经(即多巴胺释放)效应随着重复给药而增加。苯丙胺敏化被认为是精神分裂症阳性症状背后的细胞过程的模型。值得注意的是，我们发现神经元中GLT-1的失活显著降低了对苯丙胺的急性和敏化运动反应。此外，我们还发现nGLT-1 KO在新对象识别和明暗显现方面的性能有所改善。NOR和LDE上的缺陷可能反映了工作记忆受损和焦虑增加，这是精神分裂症症状的认知和负性领域的组成部分。这些观察结果使我们假设，nGLT-1 KO可能对与精神分裂症相关的生化和电路障碍表现出韧性。我们进一步假设，我们在nGLT-1 KO中观察到的表型部分源于大脑中依赖神经元GLT-1实现谷氨酸稳态的区域内环境谷氨酸的变化。我们建议进一步表征这些表型，以确定GLT-1是否可能成为治疗药物发现的有效靶点。为此，我们将：1)鉴定亚慢性给予PCP的nGLT-1 KO小鼠的行为表型以建立精神分裂症症状领域的模型；2)鉴定nGLT-1 KO小鼠的生化表型；3)确定神经元敲除GLT-1对网状核切片制备中谷氨酸动态平衡的影响。

 DESCRIPTION (provided by applicant): Schizophrenia is a chronic, devastating, psychiatric disorder characterized attributed to abnormalities in dopamine and glutamate signaling. Excitatory circuits control the activity of dopamine neurons, and it is thought that abnormalities in these circuits produce the positive, negative and cognitive features of schizophrenia. Glutamate homeostasis refers to the control of glutamate levels in and around excitatory synapses, and we now have evidence that glutamate homeostasis might be important in controlling the circuits involved in schizophrenia. Glutamate transporters control brain glutamate homeostasis, and the major glutamate transporter in the brain is GLT-1, primarily expressed in astrocytes. Others and we have found that GLT-1 is also expressed in excitatory presynaptic terminals. To understand the function of GLT-1 expressed in neurons, we generated a conditional GLT-1 knockout mouse in which we have used synapsin-cre to accomplish the selective inactivation of GLT-1 in neurons. We have performed extensive behavioral phenotyping of this mouse, including testing responses to amphetamine, which are highly modulated by excitatory signaling and therefore likely, we thought, to be affected by glutamate dyshomeostasis. Previous work by many groups has demonstrated the phenomenon of sensitization to amphetamine, in which behavioral or neural (i.e. dopamine release) effects increase with repeated administration. Amphetamine sensitization is thought to model the cellular processes that underlie the positive symptoms of schizophrenia. Remarkably, we found that inactivation of GLT-1 in neurons produced significant decrease in the acute and sensitized locomotor responses to amphetamine. In addition, we have found improved performance of the nGLT-1 KO in novel object recognition and light-dark emergence. Defects on NOR and LDE may reflect impaired working memory and increased anxiety, components of the cognitive and negative domain of symptoms of schizophrenia. These observations have led us to hypothesize that the nGLT-1 KO may demonstrate resilience to the biochemical and circuit disturbances associated with schizophrenia. We hypothesize further that the phenotype that we observe in the nGLT-1 KO partially stems from changes in ambient glutamate within regions of the brain dependent upon neuronal GLT-1 for glutamate homeostasis. We propose to characterize these phenotypes further to establish whether GLT-1 may be valid target for therapeutic drug discovery. Toward that end, we will: 1) characterize the behavioral phenotype of the nGLT-1 KO mouse subjected to subchronic PCP administration to model symptom domains observed in schizophrenia; 2) characterize the biochemical phenotype of nGLT-1 KO mice; 3) determine the effect of neuronal knockout of GLT-1 on glutamate homeostasis in the nucleus reticularis slice preparation.