Effect of altering the gene of ion channels in the brain stem on mice’s breathing patterns

Research has shown that rhythmic neurons depend on a particular class of sodium ion channels called NaV 1.6. Via an RNA sequence study in collaboration with Professor Saha, Dr. Del Negro’s lab was able to find out that the NaV 1.6 genes, Scn8a, which is highly expressed in the respiratory rhythmogenic core neurons (called “Dbx1 neurons” for their genetic origins) in the pre-Bötzinger complex (preBötC) in the lower brain stem. Therefore, we can genetically manipulate Scn8a (NaV 1.6 gene) and test whether those bursting-pacemaker neurons are important for breathing in mice, which is relevant and critical to human breathing and is relevant to the treatment and prevention of breathing disorders.

The questions I will try to answer in the summer of 2019 are related to the sodium channels NaV 1.6. My first question is “How does the short-hairpin RNA (shRNA) targeted to downregulate Scn8a injected in the preBötC affect the NaV 1.6 expression and function?” Following this question is “Does downregulating the expression of Scn8a (hence NaV 1.6) impact breathing in adult mice? More specifically, does NaV 1.6 knockdown via shRNA attenuate breathing, create respiratory pathologies, affect breathing in sleep (i.e., sleep-disordered breathing), or is it perhaps fatal?”
To answer my questions, I will develop Scn8a-targeted shRNA to knockdown the sodium channels in the preBötC, in turn manipulating Dbx1 neurons to downregulate Scn8a gene, thus NaV 1.6 proteins, over time. I will collect and analyze real mice breathing data, perform transcardial perfusions to retrieve the brain stems for sectioning, and image the preBötC to be sure the virus transduced the intended neurons (Dbx1) and affected the ion channels we intended.