Contributions Of The Persistent Sodium And M-Type Potassium Currents In The Prebötzinger Complex For Breathing Rhythm Generation

Vertebrate animals execute different rhythmic motor behaviors such as walking, swallowing, and breathing, and the generation of that rhythmic activity is governed by central pattern generator network (CPGs), which do not require sensory feedback. The central nervous system is endowed with different CPGs are responsible for determining the appropriate sequence of muscle activation that leads to the correct behavior expression. Breathing is an essential rhythmic behavior and drives the gas exchange between the lungs and the ambient air. Unlike most CPGs that are episodic, and generally quiescent in resting conditions, the respiratory network is active continuously throughout life. The normal breathing cycle is composed by two distinct phases: inspiration and expiration. Inspiration is the inexorable phase and happens due to the active recruitment of the diaphragmatic muscle, while expiration happens passively due to the elastic recoil of the diaphragm and thorax. The CPG for inspiratory activity is the preBötzinger complex (preBötC) in the lower brainstem, and it contains essential excitatory interneurons derived from neuronal progenitor cells that express the Developing brain homeobox 1 (Dbx1) transcription factor. However, the ion channel mechanisms involved in initiating and terminating rhythmic burst activity in vivo remain unsolved. Here, I test the ion channels proposed to orchestrate both mechanisms. The first chapter of my thesis evaluates the “pacemaker theory” which suggests that some preBötC neurons express an intrinsic persistent sodium current (INaP) that gives rise to the voltage-dependent bursting-pacemaker activity, thus driving the inspiratory bursts. I knocked out and knocked down the Scn8a that codes for the sodium channel NaV1.6 which gives rise to INaP. I showed that pacemaker activity is no longer present in rhythmic slices of preBötC from neonatal mice; further, juvenile, and adult mice lacking this subunit still generate breathing activity. The second chapter seeks to understand the role of the M-currents in inspiratory burst termination and opioid-induced respiratory depression. Previous experiments showed that pharmacological blockage of voltage-gated potassium channels from the KV7 family prolongs bursts in a rhythmic slice model of breathing. Surprisingly, I found that genetic knockdown of Kcnq2 and Kcnq3 that comprise M-current does not alter ventilation, metabolis and breathing variability of adult mice. I conclude that Scn8a-mediated NaV1.6 channels underlies INaP-driven pacemaker activity in rhythmic slices, and although it contributes to neuronal excitability in preBötC, this feature is dispensable for rhythm generation. Moreover, Kcnq2/Kcnq3-mediated KV7 M-currents are not necessary for inspiration-expiration phase transition in vivo. These results contribute to understanding the role of specific ion channels in breathing control by using cutting-edge genetic tools, while avoiding unwanted side-effects observed in conventional pharmacological approaches. Moreover, this work shows the importance of whole animals to understand the multifaceted origin of vital motor behaviors like breathing, which was a missing information not achieved with the use of reduced preparations.