Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits

Abstract

Transient receptor potential channel, TRPM4, the putative molecular substrate for Ca ²⁺-activated nonselective cation current ( I _CAN), is hypothesized to generate bursting activity of pre-Bötzinger complex (pre-BötC) inspiratory neurons and critically contribute to respiratory rhythmogenesis. Another TRP channel, TRPC3, which mediates Na ⁺/Ca ²⁺ fluxes, may be involved in regulating Ca ²⁺-related signaling, including affecting TRPM4/ I _CAN in respiratory pre-BötC neurons. However, TRPM4 and TRPC3 expression in pre-BötC inspiratory neurons and functional roles of these channels remain to be determined. By single-cell multiplex RT-PCR, we show mRNA expression for these channels in pre-BötC inspiratory neurons in rhythmically active medullary in vitro slices from neonatal rats and mice. Functional contributions were analyzed with pharmacological inhibitors of TRPM4 or TRPC3 in vitro as well as in mature rodent arterially perfused in situ brainstem–spinal cord preparations. Perturbations of respiratory circuit activity were also compared with those by a blocker of I _CAN. Pharmacologically attenuating endogenous activation of TRPM4, TRPC3, or I _CAN in vitro similarly reduced the amplitude of inspiratory motoneuronal activity without significant perturbations of inspiratory frequency or variability of the rhythm. Amplitude perturbations were correlated with reduced inspiratory glutamatergic pre-BötC neuronal activity, monitored by multicellular dynamic calcium imaging in vitro. In more intact circuits in situ, the reduction of pre-BötC and motoneuronal inspiratory activity amplitude was accompanied by reduced post-inspiratory motoneuronal activity, without disruption of rhythm generation. We conclude that endogenously activated TRPM4, which likely mediates I _CAN, and TRPC3 channels in pre-BötC inspiratory neurons play fundamental roles in respiratory pattern formation but are not critically involved in respiratory rhythm generation.