Neuron-glial interactions within the basal ganglia

为了明确成年中枢神经系统基底节(BG)内小胶质细胞的基本特性，我们使用CX3CR1-EGFP转基因小鼠对腹侧被盖区(VTA)、伏隔核(NAC)、黑质致密部(SNC)和黑质网状部(SNR)内的小胶质细胞进行了观察。以前我们发现，与其他BG区域相比，小胶质细胞在VTA中的密度明显较低，并且表现出稀疏的分支。相反，SNR内的小胶质细胞密度非常高，SNR和NAC小胶质细胞都显示出高度分支的形态。在过去的一年里，我们扩展了这些分析，以表明BG小胶质细胞密度与神经元密度或OPC密度之间没有明显的相关性，但在所有被分析的BG核中，小胶质细胞与星形胶质细胞的比例一致。我们还使用VGlut1、2和3的免疫染色来确定小胶质突起分支的复杂性是否与周围组织中兴奋性突触的数量有关。这一分析表明，谷氨酸能突触密度和小胶质组织覆盖率之间并不存在一致的相关性。为了确定这些区域小胶质细胞结构和分布的差异是否伴随着功能状态的差异，我们使用成像、电生理学和转录组测序来定义基底节(BG)小胶质细胞的基本属性，发现不同基底节(BG)核的小胶质细胞解剖特征、溶酶体含量和膜特性显著不同。转录组测序显示了基因表达的区域性差异，包括中脑和皮质小胶质细胞之间的显著差异。总之，这些观察表明，健康大脑中的小胶质细胞沿着一系列不同的功能状态存在，这些数据为确定小胶质细胞对BG回路功能的贡献提供了关键的基础。 我们现在正在使用类似的方法来定义BG小胶质细胞如何受到慢性药物暴露和正常衰老过程的影响。我们正在对长期自我注射生理盐水或可卡因的小鼠的NAC、PFC和VTA中分离的小胶质细胞进行完整的转录组RNAseq。所有组织都被收集起来进行分析和测序，生物信息学分析正在进行中。我们还使用成像、基因表达的RT-PCR分析和小胶质细胞消融来量化衰老诱导的BG小胶质细胞的变化，并分析这些细胞在衰老诱导的认知功能变化中的作用。

In order to define the basic properties of microglia within the basal ganglia (BG) of the adult CNS, we used CX3CR1-EGFP transgenic mice to visualize microglia within the ventral tegmental area (VTA), nucleus accumbens (NAc), substantia nigra pars compacta (SNc), and substantia nigra pars reticulata (SNr). Previously we found that microglia populate the VTA at significantly lower density and exhibit sparse branching compared to other BG regions. In contrast, microglia within the SNr are present at a dramatically high density and both SNr and NAc microglia display highly-ramified morphologies. In the past year, we have expanded these analyses to show that there is no clear correlation between BG microglial density and neuronal density or OPC density but there is a consistent ratio of microglia to astrocytes across all analyzed BG nuclei. We also used immunostaining for VGlut1, 2, and 3 to determine if microglial process branching complexity is related to the number of excitatory synapses within the surrounding tissue. This analysis revealed that there was not a consistent correlation between density of glutamatergic synapses and microglial tissue coverage. To determine if these regional differences in microglial structure and distribution are accompanied by distinctions in functional status, we used imaging, electrophysiology, and transcriptome sequencing to define the basic properties of basal ganglia (BG) microglia and found that microglial anatomical features, lysosome content, and membrane properties differ significantly across BG nuclei. Transcriptome sequencing revealed regional differences in gene expression, including prominent distinctions between midbrain and cortical microglia. Together, these observations demonstrate that microglia in the healthy brain exist along a spectrum of distinct functional states and these data provide a critical foundation for defining microglial contributions to BG circuit function. We are now using similar approaches to define how BG microglia are impacted by chronic drug exposure and the process of normal aging. We are performing whole transcriptome RNAseq of microglia isolated from the NAc, PFC, and VTA of mice following chronic self-administration of saline or cocaine. All tissue is collected for this analysis and sequencing and bioinformatics analysis is ongoing. We are also using imaging, RT-PCR analysis of gene expression, and microglial ablation to quantify aging-induced changes in BG microglia and analyze contributions of these cells to aging-induced alterations in cognitive function.