Accepted

Pain is a multidimensional perception that includes unpleasant somatosensory and affective experiences; however, the underlying neural circuits that mediate different components of pain remain elusive. Although hyperactivity of basolateral amygdala glutamatergic (BLA) neurons is required for the somatosensory and emotional processing of pain, the precise excitatory inputs to BLA neurons and their roles in mediating different aspects of pain are unclear. Here, we identified two discrete glutamatergic neuronal circuits in male mice: a projection from the insular cortex (IC) to BLA neurons, which modulates both the somatosensory and affective components of pain, and a projection from the mediodorsal thalamic nucleus (MD) to BLA neurons, which modulates only the aversive-affective component of pain. Using whole-cell recording and fiber photometry, we found that neurons within the IC→BLA and MD→BLA pathways were activated in mice upon inflammatory pain induced by injection of complete Freund's adjuvant (CFA) into the paw. Optical inhibition of the IC→BLA pathway increased the nociceptive threshold and induced behavioral place preference in CFA mice. In contrast, optical inhibition of the MD→BLA pathway did not affect the nociceptive threshold, but still induced place preference in CFA mice. In normal mice, optical activation of the IC→BLA pathway decreased the nociceptive threshold and induced place aversion, while optical activation of the MD→BLA pathway only evoked aversion. Taken together, our results demonstrate that discrete IC→BLA and MD→BLA pathways are involved in modulating different components of pain, and provide insights into its circuit basis and better our understanding of pain perception.

The human transient receptor potential canonical 5 (TRPC5) is a calcium-permeable, nonselective cation channel expressed in the central and peripheral nervous system and also in other tissues such as the kidney, synovium, and odontoblasts. TRPC5 has been recently confirmed to play a key role in spontaneous, inflammatory mechanical, and cold pain. Although TRPC5 activation is known to be cold sensitive, it is unclear whether this property is intrinsic to the channel protein and whether or to what extent it may be determined by the cellular environment. In this study, we explored the cold sensitivity of human TRPC5 at the single-channel level using transiently transfected HEK293T cells. Upon decreasing the temperature, the channel demonstrated prolonged mean open dwell times and a robust increase in the open probability (P ), whereas the amplitude of unitary currents decreased ~1.5-fold per 10°C of temperature difference. In the absence of any agonists, the temperature dependence of P was sigmoidal, with a steep slope within the temperature range of 16°C-11°C, and exhibited saturation below 8-5°C. Thermodynamic analysis revealed significant changes in enthalpy and entropy, suggesting that substantial conformational changes accompany cold-induced gating. The mutant channel T970A, in which the regulation downstream of G-protein coupled receptor signaling was abrogated, exhibited higher basal activity at room temperature and a less steep temperature response profile, with an apparent threshold below 22°C. An even more pronounced decrease in the activation threshold was observed in a mutant that disrupted the electrostatic interaction of TRPC5 with the endoplasmic reticulum calcium sensor stromal interaction molecule 1. Thus, TRPC5 exhibits features of an intrinsically cold-gated channel; its sensitivity to cold tightly depends on the phosphorylation status of the protein and intracellular calcium homeostasis.