Intersectional genetic dissection of spinal circuits processing pain and itch

描述（由申请人提供）：本联合申请的长期目标是识别处理皮肤躯体感觉信息的脊髓回路，重点关注疼痛和瘙痒通路。整体研究策略采用尖端的交叉遗传操作来标记和消融背侧脊髓中的10多种兴奋性和抑制性中间神经元（IN），再加上行为测试来评估这些细胞类型对门控和转导疼痛，瘙痒和温度的功能贡献。实验方法将使用三组小鼠。第一种是交叉DTR小鼠，其中白喉毒素受体（DTR）蛋白仅在FlpO-和Cre-介导的两个终止盒去除后表达。第二种是Lbx 1FlpO小鼠品系，其中FlpO重组酶的表达仅限于延髓和脊髓背角中的神经元和后脑背侧神经元。第三组包括11个Cre小鼠品系，表达Cre重组酶的兴奋性或抑制性背角INS的各个子集。这些交叉将使研究人员能够消融特定群体的背侧中间神经元，使其特定的贡献，疼痛和瘙痒的途径可以确定。本研究将探讨三个问题：1）控制特定疼痛和瘙痒模式的神经元的分子特性，2）确定异常性疼痛的细胞基础，3）背角中控制疼痛的神经回路的解剖学和功能组织 还很痒 这些分析结合了联合收割机狂犬病病毒逆行追踪、分子神经解剖学电生理记录、遗传操作和行为测试，将首次全面了解处理有害躯体感觉模式的脊髓回路是如何在细胞水平上组织起来的。它们将用于更详细地研究群体编码假设和疼痛门控理论的细胞基础。最后，他们将提供新的见解，发生在不同的感觉方式之间的拮抗作用，当改变时，是疼痛和瘙痒的发病机制的主要因素。

DESCRIPTION (provided by applicant): The long-range goal of this joint application is to identify the spinal circuits that process cutaneous somatic sensory information with a focus on pain and itch pathways. The overall research strategy employs cutting edge intersectional genetic manipulations to mark and ablate more than 10 classes of excitatory and inhibitory interneurons (INs) in the dorsal spinal cord, coupled with behavioral tests to assess the functional contribution that these cell types make to gating and transducing pain, itch and temperature. The experimental approach will utilize three sets of mice. The first is an intersectional DTR mouse in which the diphtheria toxin receptor (DTR) protein is only expressed after FlpO- and Cre- mediated removal of two stop cassettes. The second is the Lbx1FlpO mouse strain, in which the expression of FlpO recombinase is restricted to neurons in the dorsal horn of the medulla and spinal cord and dorsal hindbrain neurons. The third set includes eleven Cre mouse lines that express Cre recombinase in various subsets of excitatory or inhibitory dorsal horn INs. Crosses of these will enable investigators to ablate specific populations of dorsal interneurons so that their specific contributions to pain and itch pathways can be determined. Three issues will be addressed: 1) The molecular identity of neurons that transduce and gate specific pain and itch modalities, 2) a determination of the cellular basis of allodynia, and 3) the anatomical and functional organization of the circuits in the dorsal horn that gate pain and itch. These analyses, which combine rabies virus-based retrograde tracings, molecular neuroanatomy electrophysiological recordings, genetic manipulations and behavioral testing, will provide the first comprehensive picture of how the spinal circuits that process the noxious somatosensory modalities are organized at a cellular level. They will be used to examine in more detail the population-coding hypothesis and the cellular basis for the gate control theory of pain. Finally, they will provide new insights into the antagonistic interactions that occur among different sensory modalities, which when altered are a major factor in the pathogenesis of pain and itch.