Sex-dependent functions of spinal dopamine 1 receptors in neuropathic pain - Chronic pain is more prevalent in women, and the rapidly increasing list of mechanistic sex differences in pain signaling has underscored the importance of considering sexual dimorphism in the development of therapies for chronic pain management in women. Our long-term goal is to investigate sex differences in spinal dopaminergic mechanisms of persistent pain. The objective of this exploratory project is to define dorsal horn (DH) Drd1-expressing neurons and the function of Drd1 in a model of neuropathic pain. Our central hypothesis is that Drd1 participates in nerve injury-induced hypersensitivity in a sexually dimorphic manner due in part to differential expression in DH neuronal subpopulations in male and female mice. Preliminary electrophysiological recordings demonstrate a striking difference in the distribution of firing patterns of Drd1-expressing DH neurons in males and females. This observation suggests sex differences in the expression of Drd1 among subsets of DH neurons that could contribute to sex differences in spinal dopaminergic signaling under conditions of persistent pain. Drd1 is expressed in a heterogeneous population of excitatory and inhibitory DH neurons and partially overlaps with markers of excitatory subpopulations that participate in tactile hypersensitivity. Therefore, excitatory Drd1 neurons may participate in DH circuits, mediating nerve injury-induced hypersensitivity. On the other hand, increased activation of DH Drd1 inhibitory neurons may contribute to disinhibition-mediated hyperexcitability under conditions of persistent pain. We will address the potentially distinct functions of Drd1 in excitatory and inhibitory neurons using intersectional deletion of the receptor. The overall rationale for the proposed research is that it will identify specific mechanisms contributing to sex differences in spinal dopamine signaling, which could be harnessed for more effective chronic pain management in women. In Aim 1, we will identify the pattern of Drd1 distribution among subsets of DH neurons in males and females. We will use fluorescent in situ hybridization analysis to determine the expression of Drd1 in transcriptionally defined subtypes of DH neurons. We will also define the firing patterns and primary afferent inputs of excitatory and inhibitory Drd1 neurons using patch clamp electrophysiology and will label recorded neurons with neurobiotin for neuroanatomical analysis. In Aim 2, we will define the contribution of Drd1 in excitatory and inhibitory DH neurons to nerve injury-induced hypersensitivity in males and females, using an intersectional AAV-mediated CRISPR/Cas9 gene ablation approach; we will determine the functional impact of Drd1 ablation behaviorally. Impact: The proposed project will advance understanding of the mechanisms that underlie sex differences in chronic pain by elucidating the function of Drd1 in nerve injury-induced hypersensitivity in female and male mice. This outcome will provide proof-of concept for comprehensive investigation of sex differences in spinal dopamine signaling, extending to 1) other persistent pain models, 2) the other dopamine receptors, which are also expressed in spinal cord, and 3) the descending dopaminergic inputs, which originate in hypothalamus and have been implicated in pain chronification.
