Data_Sheet_1_Synaptic circuits involving gastrin-releasing peptide receptor-expressing neurons in the dorsal horn of the mouse spinal cord.docx

The superficial dorsal horn (SDH) of the spinal cord contains a diverse array of neurons. The vast majority of these are interneurons, most of which are glutamatergic. These can be assigned to several populations, one of which is defined by expression of gastrin-releasing peptide receptor (GRPR). The GRPR cells are thought to be “tertiary pruritoceptors,” conveying itch information to lamina I projection neurons of the anterolateral system (ALS). Surprisingly, we recently found that GRPR-expressing neurons belong to a morphological class known as vertical cells, which are believed to transmit nociceptive information to lamina I ALS cells. Little is currently known about synaptic circuits engaged by the GRPR cells. Here we combine viral-mediated expression of PSD95-tagRFP fusion protein with super-resolution microscopy to reveal sources of excitatory input to GRPR cells. We find that they receive a relatively sparse input from peptidergic and non-peptidergic nociceptors in SDH, and a limited input from A- and C-low threshold mechanoreceptors on their ventral dendrites. They receive synapses from several excitatory interneuron populations, including those defined by expression of substance P, neuropeptide FF, cholecystokinin, neurokinin B, and neurotensin. We investigated downstream targets of GRPR cells by chemogenetically exciting them and identifying Fos-positive (activated) cells. In addition to lamina I projection neurons, many ALS cells in lateral lamina V and the lateral spinal nucleus were Fos-positive, suggesting that GRPR-expressing cells target a broader population of projection neurons than was previously recognised. Our findings indicate that GRPR cells receive a diverse synaptic input from various types of primary afferent and excitatory interneuron, and that they can activate ALS cells in both superficial and deep regions of the dorsal horn.

脊髓背角浅表层（superficial dorsal horn, SDH）包含多种神经元群体，其中绝大多数为中间神经元，且大部分为谷氨酸能神经元。这类神经元可被划分为多个亚群，其中一个亚群以胃泌素释放肽受体（gastrin-releasing peptide receptor, GRPR）的表达为特征。既往研究认为，表达GRPR的神经元属于“三级痒觉感受器”，可将痒觉信息传递至前外侧系统（anterolateral system, ALS）的I层投射神经元。但令人意外的是，我们近期的研究发现，表达GRPR的神经元属于被称为“垂直细胞”的形态学类别——而该类细胞此前被认为是向I层ALS细胞传递伤害性信息的神经元。目前学界对GRPR阳性神经元所参与的突触环路仍知之甚少。本研究将病毒介导的PSD95-tagRFP融合蛋白表达技术与超分辨率显微镜技术相结合，以揭示GRPR阳性神经元的兴奋性输入来源。我们发现，GRPR阳性神经元在SDH内主要接收来自肽能与非肽能伤害性感受器的稀疏输入，同时在其腹侧树突上接收少量来自A类及C类低阈值机械感受器的输入。此外，该类神经元还接收多个兴奋性中间神经元亚群的突触连接，包括表达P物质、神经肽FF、胆囊收缩素、神经激肽B以及神经降压素的中间神经元亚群。我们通过化学遗传学激活GRPR阳性神经元，并识别Fos阳性（即激活的）细胞，以此探究GRPR阳性神经元的下游靶点。研究结果显示，除I层投射神经元外，外侧V层及外侧脊髓核内的大量ALS细胞也呈现Fos阳性，这表明GRPR阳性神经元所靶向的投射神经元群体比此前认知的更为广泛。本研究结果表明，GRPR阳性神经元接收来自多种初级传入纤维与兴奋性中间神经元的多样化突触输入，且其可激活背角浅表层及深层区域内的ALS细胞。