Optogenetic Analysis of Neostriatal Circuits Engaged by Cholinergic Interneurons

描述（申请人提供）：新纹状体胆碱能（ChAT）中间神经元通过编码外部事件的显著性和强化值，在适应性行为行为的选择和习得中发挥重要作用。这些信息表现在对行为显著性刺激的同步多相群体反应的精确时间结构中。一个重要的问题是要了解这些瞬态信号的中间神经元是如何检测和解码的新纹状体网络。我们在初步实验的基础上提出的假设是，ChAT中间神经元通过尼古丁受体介导的机制来控制纹状体回路的活性，从而激活多种平行的gaba能机制，这些机制在棘投射神经元（SPNs）和其他纹状体神经元中引发动力学上不同的独立抑制反应。这些反应部分源于多种尚未确定类型的GABA能中间神经元的直接激活，这些神经元不同于含有小白蛋白（PV）的快速尖峰（FS）和表达NPY的中间神经元，也可能涉及轴突末端释放GABA的突触前尼古丁促进，并共同为spn， ChAT和其他中间神经元提供选择性的，细胞类型特异性的输入。利用体外光遗传抑制，我们还重现了在行为动物中最常见的ChAT中间神经元的群体反应，包括放电暂停，随后是弱同步反弹兴奋，并证明了这种与行为直接相关的活动模式也涉及上述抑制机制。由于这些反应足以抑制大量spn的动作电位产生，它们可能对基底神经节的功能产生重大影响，并调节正在进行的行为。本实验旨在了解这些gaba能回路的功能组织和机制，并定量表征ChAT中间神经元的强化相关群体反应对体外和体内spn活性的影响。光遗传学方法将用于控制双转基因动物中ChAT和其他中间神经元的活性，从而实现对特定细胞类型的遗传靶向和鉴定。通过识别由ChAT中间神经元激活的gaba能中间神经元，然后通过选择性光遗传激活特定类别的中间神经元群体，并通过检查光遗传抑制各种中间神经元类型对由ChAT诱导的ipsc的影响，确定单个突触前细胞类型对spn和其他神经元中各种gaba能反应成分的贡献，来分析ChAT中间神经元的失突触回路中间神经元。最后，我们将在体内和体外定量表征ChAT中间神经元光遗传复制的暂停兴奋反应对spn的影响。这些实验将描述一个强大的新纹状体回路机制的功能组织，因此将大大促进对基底神经节功能的理解。

DESCRIPTION (provided by applicant): Neostriatal cholinergic (ChAT) interneurons play an important role in the selection and acquisition of adaptive behavioral actions by encoding the salience and reinforcement value of external events. This information is represented in the precise temporal structure of synchronous multiphasic population responses given to the presentation of behaviorally significant stimuli. An important problem is to understand how these transient signals of ChAT interneurons are detected and decoded in the neostriatal network. Our hypothesis, formulated on the basis of preliminary experiments, is that ChAT interneurons control the activity of the striatal circuitry using nicotinic receptor mediated mechanisms to activate multiple parallel GABAergic mechanisms that elicit kinetically distinct independent inhibitory responses in the spiny projection neurons (SPNs) and in other striatal neurons. These responses originate in part from direct activation of multiple as yet unidentified types of GABAergic interneurons that are distinct from the parvalbumin (PV) containing fast spiking (FS) and NPY expressing interneurons, possibly also involve presynaptic nicotinic facilitation of GABA release from axon terminals and together provide selective, cell type specific inputs to SPNs, ChAT and other interneurons. Using in vitro optogenetic inhibition we also reproduced the population response of ChAT interneurons most commonly observed in behaving animals consisting of a pause of firing followed by weakly synchronous rebound-excitation and demonstrated that this behaviorally directly relevant pattern of activity also engages the inhibitory mechanisms described above. Since these responses are sufficient to inhibit action potential generation in large populations of SPNs they are likely to exert significant effects on the functioning of the basal ganglia and regulate ongoing behavior. The proposed experiments aim at understanding the functional organization of these GABAergic circuits and mechanisms and to quantitatively characterize the effects of reinforcement related population responses of ChAT interneurons on the activity of SPNs in vitro and in vivo. Optogenetic methods will be used to control the activity of ChAT and other interneurons in double transgenic animals allowing the genetic targeting and identification of specific cell types. The disynaptic circuits of ChAT interneurons will be analyzed by identifying the GABAergic interneurons activated by ChAT interneurons and then determining the contribution of individual presynaptic cell types to the various GABAergic response components in SPNs and other neurons by selective optogenetic activation of populations of specific classes of interneurons and by examining the effects of optogenetic inhibition of various interneuron types on IPSCs elicited disynaptically by ChAT interneurons. Finally, we will quantitatively characterize the effects of optogenetically reproduced pause-excitation responses of ChAT interneurons on SPNs in vivo and in vitro. These experiments will describe the functional organization of a powerful novel circuit mechanism of the neostriatum and therefore will significantly advance the understanding of the functioning of the basal ganglia.