CRISPR knockdown of Kcnq3 attenuates the M-current and increases excitability of NPY/AgRP neurons to alter energy balance

Abstract

ObjectiveArcuate nucleus neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons drive ingestive behavior. The M-current, a subthreshold non-inactivating potassium current, plays a critical role in regulating NPY/AgRP neuronal excitability. Fasting decreases while 17β-estradiol increases the M-current by regulating the mRNA expression of Kcnq2, 3, and 5 (Kv7.2, 3, and 5) channel subunits. Incorporating KCNQ3 into heteromeric channels has been considered essential to generate a robust M-current. Therefore, we investigated the behavioral and physiological effects of selective Kcnq3 deletion from NPY/AgRP neurons. MethodsWe used a single adeno-associated viral vector containing a recombinase-dependent Staphylococcus aureus Cas9 with a single-guide RNA to selectively delete Kcnq3 in NPY/AgRP neurons. Single-cell quantitative measurements of mRNA expression and whole-cell patch clamp experiments were conducted to validate the selective knockdown. Body weight, food intake, and locomotor activity were measured in male mice to assess disruptions in energy balance. ResultsThe virus reduced the expression of Kcnq3 mRNA without affecting Kcnq2 or Kcnq5. The M-current was attenuated, causing NPY/AgRP neurons to be more depolarized, exhibit a higher input resistance, and require less depolarizing current to fire action potentials, indicative of increased excitability. Although the resulting decrease in the M-current did not overtly alter ingestive behavior, it significantly reduced the locomotor activity as measured by open-field testing. Control mice on a high-fat diet exhibited an enhanced M-current and increased Kcnq2 and Kcnq3 expression, but the M-current remained significantly attenuated in KCNQ3 knockdown animals. ConclusionsThe M-current plays a critical role in modulating the intrinsic excitability of NPY/AgRP neurons that is essential for maintaining energy homeostasis.