Somatostatin-positive (SOM+) neurons have been proposed as one of the key populations of excitatory interneurons in the spinal dorsal horn involved in mechanical pain. However, the molecular mechanism for their role in pain modulation remains unknown. Here, we showed that the T-type calcium channel Cav3.2 was highly expressed in spinal SOM+ interneurons. Colocalization of Cacna1h (which codes for Cav3.2) and SOMtdTomato was observed in the in situ hybridization studies. Fluorescence-activated cell sorting of SOMtdTomato cells in spinal dorsal horn also proved a high expression of Cacna1h in SOM+ neurons. Behaviorally, virus-mediated knockdown of Cacna1h in spinal SOM+ neurons reduced the sensitivity to light touch and responsiveness to noxious mechanical stimuli in naïve mice. Furthermore, knockdown of Cacna1h in spinal SOM+ neurons attenuated thermal hyperalgesia and dynamic allodynia in the complete Freund’s adjuvant-induced inflammatory pain model, and reduced both dynamic and static allodynia in a neuropathic pain model of spared nerve injury. Mechanistically, a decrease in the percentage of neurons with Aβ-eEPSCs and Aβ-eAPs in superficial dorsal horn was observed after Cacna1h knockdown in spinal SOM+ neurons. Altogether, our results proved a crucial role of Cav3.2 in spinal SOM+ neurons in mechanosensation under basal conditions and in mechanical allodynia under pathological pain conditions. This work reveals a molecular basis for SOM+ neurons in transmitting mechanical pain and shows a functional role of Cav3.2 in tactile and pain processing at the level of spinal cord in addition to its well-established peripheral role.

Background:Lycopene epsilon-cyclase (ε-LCY) is a key enzyme in the carotenoid biosynthetic pathway (CBP) of higher plants. In previous work, we cloned two Ntε-LCY genes from allotetraploid tobacco (Nicotiana tabacum), Ntε-LCY2 and Ntε-LCY1, and demonstrated the overall effect of Ntε-LCY genes on carotenoid biosynthesis and stress resistance. However, their genetic and functional characteristics require further research in polyploid plants.Results: Here, we used CRISPR/Cas9 to obtain Ntε-LCY2 and Ntε-LCY1 mutants in allotetraploid N.tabacum K326. Ntε-LCY2 and Ntε-LCY1 had similar promoter cis-acting elements, including light-responsive elements. The Ntε-LCY genes were expressed in roots, stems, leaves, flowers, and young fruit, and their highest expression levels were found in leaves. Ntε-LCY2 and Ntε-LCY1 genes responded differently to normal light and high light stress. Both the Ntε-LCY2 and the Ntε-LCY1 mutants had a more rapid leaf growth rate, especially ntε-lcy2-1. The expression levels of CBP genes were increased in the ntε-lcy mutants, and their total carotenoid content was higher. Under both normal light and high light stress, the ntε-lcy mutants had higher photosynthetic capacities and heat dissipation levels than the wild type, and this was especially true of ntε-lcy2-1. The reactive oxygen species content was lower in leaves of the ntε-lcy mutants.Conclusion: In summary, the expression patterns and biological functions of the Ntε-LCY genes Ntε-LCY1 and Ntε-LCY2 differed in several respects. The mutation of Ntε-LCY2 was associated with a greater increase in the content of chlorophyll and various carotenoid components, and it enhanced the stress resistance of tobacco plants under high light.