The neural basis of breathing rhythm: TRPM4 and TRPC3 ion channels contribute to inspiratory burst generation in Dbx1-derived interneurons of the preBötzinger Complex in mice

Breathing is a primal behavior that emanates from neural rhythms in a region of the ventral-lateral medulla named the preBötzinger Complex (preBötC). Dbx1-derived preBötC neurons comprise the underlying core oscillator. Although we understand the network (preBötC) and cellular (Dbx1) origins of breathing, its molecular (ion channel-level) mechanisms remain unknown. We hypothesized that transient receptor potential (TRP) ion channels are responsible for inspiratory burst generation in Dbx1-derived preBötC neurons (i.e. Dbx1 neurons). In this study, we evaluate the contributions of TRPM4 and TRPC3 channels in inspiratory burst generation using electrophysiological techniques. Pharmacological inhibition of these ion channels in vitro attenuates the drive potentials that underlie inspiratory bursts in Dbx1- preBötC neurons. This suggests that these specific TRP ion channels are important for inspiratory burst generation in Dbx1 preBötC neurons and thus mammalian breathing behavior

Trpm4 Ion Channels in pre-Bo¨tzinger Complex Interneurons Are Essential for Breathing Motor Pattern But Not Rhythm

Inspiratory breathing movements depend on pre-Bötzinger complex (preBötC) interneurons that express calcium (Ca2+)-activated nonselective cationic current (ICAN) to generate robust neural bursts. Hypothesized to be rhythmogenic, reducing ICAN 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 ICAN 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 ICAN, whose influence on frequency contradicts a direct role in rhythm generation. We conclude that Trpm4-mediated ICAN is indispensable for motor output but not the rhythmogenic core mechanism of the breathing central pattern generator

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

Inspiratory breathing movements depend on pre-Bötzinger complex (preBötC) interneurons that express calcium (Ca2+)-activated nonselective cationic current (ICAN) to generate robust neural bursts. Hypothesized to be rhythmogenic, reducing ICAN 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 ICAN 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 ICAN, whose influence on frequency contradicts a direct role in rhythm generation. We conclude that Trpm4-mediated ICAN is indispensable for motor output but not the rhythmogenic core mechanism of the breathing central pattern generator

Hippocampal engrams generate variable behavioral responses and brain-wide network states

Freezing is a defensive behavior commonly examined during hippocampal-mediated fear engram reactivation. How these cellular populations across different environmental demands engage the brain and modulate freezing is unclear. To address this, we optogenetically reactivated a fear engram in the dentate gyrus (DG) subregion of the hippocampus across three distinct contexts in male mice. We found that there were differential amounts of light-induced freezing depending on the size of the context in which reactivation occurred: mice demonstrated robust light-induced freezing in the most spatially restricted of the three contexts but not in the largest. We then utilized graph theoretical analyses to identify brain-wide alterations in cFos expression during engram reactivation across the smallest and largest contexts. Our manipulations induced positive interregional cFos correlations that were not observed in control conditions. Additionally, regions spanning putative “fear” and “defense” systems were recruited as hub regions in engram reactivation networks. Lastly, we compared the network generated from engram reactivation in the small context with a natural fear memory retrieval network. Here, we found shared characteristics such as modular composition and hub regions. By identifying and manipulating the circuits supporting memory function, as well as their corresponding brain-wide activity patterns, it is thereby possible to resolve systems-level biological mechanisms mediating memory’s capacity to modulate behavioral states.</p