RNA Interference Via shRNA Decreases the Number of Transcripts of Scn8a in Glutamatergic Interneurons in the preBötC

Abstract

Rhythmic neural activity that drives the inspiratory phase of the breathing cycle in mammals emanates from glutamatergic interneurons of the pre‐Bötzinger Complex (preBötC). A longstanding question in neuroscience is: what are the underlying mechanisms for inspiratory rhythmogenesis? One proposed mechanism involves the persistent sodium current (INaP), which is the ionic mechanism that underlies the voltage‐dependent bursting‐pacemaker activity of neurons whose rhythmic burst discharges are putatively obligatory for inspiratory rhythm generation. Specifically in brain tissues, the Scn8a gene codes for Nav1.6 ion channels, and those ion channels give rise to INaP.Different genetic tools can help us to understand the role of specific molecules in the generation of motor behaviors, such as breathing. The short hairpin RNA (shRNA) is an artificial RNA molecule, delivered through adeno‐associated virus (AAV), that can inhibit gene expression and promote long‐term knock‐down of the targeted gene.Here we administered Cre‐dependent shRNA to knock‐down Scn8a (thus Nav1.6) in Cre‐expressing glutamatergic neurons in preBötC and quantified mRNA per glutamatergic neuron. We used adult Vglut2Cre mice (10‐14 weeks old) and injected 50 nL of AAVs carrying the shRNA. We used in situ hybridization (RNAscope® Fluorescent Assay) to label mRNA of Scn8a and to identify transduced glutamatergic neurons in the preBötC for RNA quantification at 2 and 6 weeks after injection. Samples were visualized under the Four‐Channel, Upright Confocal Microscope Body Based on Cerna® System (ThorLabs), and images were acquired using the ThorImage®LS Data Acquisition Software, at 40x magnification. The amount of RNA per cell was quantified using the open‐source software Quantitative Pathology(QuPath). We obtained the mean of the number of transcripts per cell ± the standard deviation (SD), and unpaired t‐test was used to evaluate the statistical difference between the shRNA injected mice and their control group (mice whose viral vector contained a non‐targeting shRNA sequence).Our results show that at 2 weeks, the control group had 27.5±9.5 RNA/neuron, whereas the shRNA‐injected had 12.8±3.5, which represents a 53% of decrease in the number of transcripts (P<0.0001). A similar pattern was observed after 6 weeks of the injection in a different cohort, where control animals showed 35.4±6.1 RNA/cells, and the shRNA group only had 24.1±5.2, representing 32% drop in the transcripts for that group (P<0.0001). This initial protocol allowed us to verify that the gene expression has been knocked down at critical time points, encouraging further studies using shRNA to evaluate whether attenuation of Nav1.6 expression in rhythmogenic preBötC neurons impairs breathing, which could resolve the longstanding question regarding the putatively rhythmogenic role of INaP.