Functional and transcriptomic profiling of synaptically connected dorsal horn neurons

项目摘要 脊髓中间神经元将有害和无害的感觉输入整合到背角， 脊髓投射神经元尽管背角回路内的特定细胞已经被识别出来， 特征，中间神经元亚型的多样性，以及对其神经化学的有限知识， 和连通性，目前无法进行全面的网络映射。我们的长期目标是 慢性疼痛条件下背角回路的变化。这一探索性项目的目标是 识别和全面表征突触前伤害感受特异性（高- 阈值（HT）和宽动态范围（WDR）投射神经元。我们将采用一种新颖的方法， 结合了以下组件：1）无毒的修饰狂犬病病毒（SiR）的单突触转移， 用于诱导荧光报告基因和钙指标的表达， 神经元2)将使用具有附着背根（DR）的脊髓切片中的钙成像来监测 识别的背角神经元对确定的初级传入输入的反应。3)双膜片钳记录 将与Patch-seq方法集成，用于单细胞 转录组分析。关于抑制性和兴奋性神经元突触互补的信息 对背角投射神经元的影响有限。此外，关于这种互补的差异的数据基于 缺乏投射神经元的功能性（HT，WDR）。这些知识将扩大我们对 兴奋/抑制平衡在特定模式的背角电路会聚投射神经元和 增加用于治疗慢性疼痛的回路特异性神经调节的可能性。中央 该建议假设HT和WDR投射神经元从不同的神经元接收突触输入， 中间神经元的亚群。在目标1中，我们将定义突触连接的中间神经元的亚群， 使用单突触SiR转移的投射神经元，对背根刺激的钙反应成像， 脊髓切片和基于解剖学的神经解剖学分析。在目标2中，我们将检验以下假设： 在HT和WDR投射神经元上突触的中间神经元的亚群可以通过以下来区分： 转录组学分析。在将SiR递送到投射神经元并将其单突触转移到它们的 突触前伴侣，突触对之间的兴奋性和抑制性连接将使用 将采用脊髓切片中的膜片钳电生理学和膜片钳测序方法用于单细胞 记录的神经元的RNAseq转录组学分析。 该项目将产生关于脊髓投射神经元与神经元网络连接的关键新数据。 处理初级传入输入的中间神经元，将为全面的解剖学研究奠定基础。 和背角回路的功能映射及其在慢性疼痛条件下的可塑性。

Project Summary Spinal interneurons integrate noxious and innocuous sensory inputs to the dorsal horn and gate their access to spinal projection neurons. Although specific cells within dorsal horn circuits have been identified and characterized, the diversity of interneuron subtypes, together with limited knowledge on their neurochemistry and connectivity, currently precludes comprehensive network mapping. Our long-term objective is to define changes in dorsal horn circuits under conditions of chronic pain. The objective of this exploratory project is to identify and comprehensively characterize interneurons that are presynaptic to nociceptive-specific (high- threshold, HT) and wide dynamic range (WDR) projection neurons. We will employ a novel approach that combines the following components: 1) Monosynaptic transfer of non-toxic modified rabies virus (SiR) will be used to induce expression of fluorescent reporter genes and calcium indicators in synaptically connected neurons. 2) Calcium imaging in spinal cord slices with attached dorsal roots (DR) will be used to monitor responses of identified dorsal horn neurons to defined primary afferent input. 3) Dual patch-clamp recording from identified synaptically connected neurons will be integrated with Patch-seq methodology for single-cell transcriptome analysis. Information regarding the complement of inhibitory and excitatory neurons synapsing on dorsal horn projection neurons is limited. Further, data on differences in this complement based on the functionality (HT, WDR) of projection neurons is lacking. Such knowledge would expand our understanding of excitatory/inhibitory balance in modality-specific dorsal horn circuits converging on projection neurons and increase the possiblilty of circuit-specific neuromodulation for the treatment of chronic pain. The central hypothesis of this proposal is that HT and WDR projection neurons receive synaptic input from distinct subpopulations of interneurons. In Aim 1, we will define subpopulations of interneurons that synapse on projection neurons using monosynaptic SiR transfer, imaging of calcium responses to dorsal root stimulation in spinal cord slices, and CLARITY-based neuroanatomical analysis. In Aim 2, we will test the hypothesis that subpopulations of interneurons synapsing on HT and WDR projection neurons can be distinguished by transcriptomic analysis. Following delivery of SiR to projection neurons and its monosynaptic transfer to their presynaptic partners, excitatory and inhibitory connections between synaptic pairs will be characterized using patch-clamp electrophysiology in spinal cord slices and patch-seq methodology will be employed for single-cell RNAseq transcriptomic analysis of recorded neurons. This project will generate critical new data on the connectivity of spinal projection neurons to the network of interneurons that process primary afferent inputs and will lay the groundwork for comprehensive anatomical and functional mapping of dorsal horn circuits and their plasticity under chronic pain conditions.