Targeting Tmem63b and Piezo2 in C-fiber low-threshold mechanoreceptors: Limitation of Vglut3-IRES-Cre.

Peripheral somatosensory neurons in the dorsal root ganglia (DRG) transduce mechanical force in the skin and other organs into electrical signals using specialized mechanically activated (MA) ion channels that initiate neuronal activation in response to force. Increasing evidence highlights PIEZO2 as the primary transducer of low-threshold mechanical force in DRG neurons. However, in the absence of Piezo2, mice and humans still respond to noxious painful stimuli like pinch, suggesting that additional MA channel(s) likely exist in DRG neurons. Developing strategies to identify Cre lines and DRG subpopulations that select for non-PIEZO2-expressing neurons is therefore an ongoing effort in the field to discover unknown mechanosensors. Here, we investigated a Vglut3-labeled mouse line as a candidate to identify non-PIEZO2 MA channels in a subtype of DRG neurons called C-fiber low-threshold mechanoreceptors (C-LTMRs). Our study carefully demonstrates that the Vglut3-IRES-Cre mouse line specifically and efficiently labels C-LTMR neurons of the DRG. Electrophysiological recordings using two different in vitro mechanical stimulation assays show that the genetically labeled Vglut3 neurons have robust indentation- and stretch-activated MA currents that are exclusively slowly or ultra-slowly adapting. To determine whether the Vglut3-IRES-Cre mouse line can be used to delete genes of interest and identify the underlying MA ion channels in C-LTMRs, we attempted to generate a Tmem63b conditional knockout using this Cre line but detected incomplete loss of Tmem63b transcript and lack of TMEM63B-dependent effect on C-LTMR MA currents. Together, our results emphasize that although the Vglut3-IRES-Cre line is robust in driving expression of a conditional reporter gene, it is inefficient in deleting genes like Tmem63b as well as Piezo2.