Trpm4 ion channels in pre-Botzinger complex interneurons are essential for breathing motor pattern but not rhythm

Inspiratory breathing movements depend on pre-Botzinger complex (preBotC) interneurons that express calcium (Ca2+)-activated nonselective cationic current (I-CAN) to generate robust neural bursts. Hypothesized to be rhythmogenic, reducing I-CAN is predicted to slow down or stop breathing; its contributions to motor pattern would be reflected in the magnitude of movements (output). We tested the role(s) of I-CAN using reverse genetic techniques to diminish its putative ion channels Trpm4 or Trpc3 in preBotC neurons in vivo. Adult mice transduced with Trpm4-targeted short hairpin RNA (shRNA) progressively decreased the tidal volume of breaths yet surprisingly increased breathing frequency, often followed by gasping and fatal respiratory failure. Mice transduced with Trpc3-targeted shRNA survived with no changes in breathing. Patch-clamp and field recordings from the preBotC in mouse slices also showed an increase in the frequency and a decrease in the magnitude of preBotC neural bursts in the presence of Trpm4 antagonist 9-phenanthrol, whereas the Trpc3 antagonist pyrazole-3 (pyr-3) showed inconsistent effects on magnitude and no effect on frequency. These data suggest that Trpm4 mediates I-CAN, whose influence on frequency contradicts a direct role in rhythm generation. We conclude that Trpm4-mediated I-CAN is indispensable for motor output but not the rhythmogenic core mechanism of the breathing central pattern generator.