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突显性和强化值，在选择和获得适应性行为行为中发挥重要作用。这一信息体现在对行为重要刺激的呈现给予同步多相群体反应的精确时间结构中。一个重要的问题是了解这些ChAT中间神经元的瞬时信号是如何在新纹状体网络中被检测和解码的。我们在初步实验的基础上提出的假设是，ChAT中间神经元通过烟碱受体介导的机制控制纹状体回路的活动，激活多个平行的GABA能机制，在棘突投射神经元(SPN)和其他纹状体神经元中引发动态不同的独立抑制反应。这些反应部分源于直接激活多种类型的GABA能中间神经元，这些神经元不同于含有快速峰电位(FS)的小白蛋白(PV)和表达NPY的中间神经元，可能还涉及突触前烟碱促进从轴突终末释放GABA，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输入。利用体外光遗传抑制，我们还复制了在行为动物中最常见的ChAT中间神经元的群体反应，包括放电暂停和弱同步反弹-兴奋，并证明了这种与行为直接相关的活动模式也参与了上述抑制机制。由于这些反应足以抑制大量SPN中动作电位的产生，它们很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能神经元的功能组织和机制，并定量表征ChAT中间神经元的强化相关群体反应在体内外对SPN活性的影响。光遗传学方法将被用来控制双转基因动物中ChAT和其他中间神经元的活动，从而允许遗传靶向和特定细胞类型的鉴定。通过鉴定被ChAT中间神经元激活的GABA能中间神经元，然后通过选择性地激活特定类别的中间神经元群体来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，以及通过检测不同类型的中间神经元对由ChAT中间神经元双突触诱发的IPSCs的光遗传抑制的影响，来分析ChAT中间神经元的双突触回路。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。这些实验将描述新纹状体一种强大的新回路机制的功能组织，因此将极大地促进对基底节功能的理解。 公共卫生相关性：许多重要的神经和精神疾病，包括帕金森氏病和亨廷顿病、精神分裂症、抽动症、强迫症，都是由新纹状体的病理条件引起的。其中有几个直接或间接地与一个主要的神经调制系统-胆碱能中间神经元有关，但关于这些中间神经元的功能作用的关键问题仍未得到回答。该提案将阐明一种新发现的电路机制的组织和功能作用，该机制负责调节这些神经元的强化相关群体反应，这些反应在病理条件下可能直接导致与这些疾病相关的特定行为异常。

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. PUBLIC HEALTH RELEVANCE: Numerous important neurological and psychiatric disorders including Parkinson's and Huntington's diseases, schizophrenia, Tourette's syndrome, obsessive compulsive disorder arise from pathological conditions of the neostriatum. Several of these are directly or indirectly linked to a major neuromodultory system the cholinergic interneurons of this structure, yet critically important issues about the functional role of these interneurons remains unanswered. The proposal will elucidate the organization and functional role of a newly discovered circuit mechanism that is responsible for mediating the reinforcement related population responses of these neurons which under pathological conditions may directly contribute to specific behavioral abnormalities associated with these disorders.