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

Abstract

Inspiratory breathing movements depend on pre-Bötzinger complex (preBötC) interneurons that express calcium (Ca ²⁺)-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 preBötC 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 preBötC in mouse slices also showed an increase in the frequency and a decrease in the magnitude of preBötC 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.

Author summary

Breathing behavior consists of periodic movements of the chest and airways that ventilate the lungs. The brain must generate a rhythm and form a motor output pattern to make breathing movements happen. Here, we address the ion channel–level neural origins of breathing, particularly the role of a class of transient receptor potential (Trp) channels hypothesized to be rhythmogenic. Using genetic techniques to knockdown these channels in a specialized brainstem site known for its respiratory rhythmogenic role, we surprisingly did not find the expected changes in breathing frequency. Rather, we measured progressive attenuation of breath magnitude, which in some cases led to fatal breathing pathologies. Therefore, the importance of this ion channel class is not respiratory rhythm generation per se but rather governing the motor output pattern. These results cause us to re-evaluate whether rhythm and pattern are discrete neural processes or instead inextricably linked in microcircuits of the central nervous system that generate and control motor behaviors.