RNA interference of GluN1 inhibits neuronal rhythmogenesis in the adult inferior olive

Abstract

RNA interference (RNAi) to knockdown N-methyl-D-aspartate receptor (NMDAR) function is being investigated to address disorders associated with pathological brain rhythms. A motivating finding has been that pharmacological block of NMDARs inhibited oscillations in neuronal membrane potential that entrain rhythmic bursts of action potentials. To determine whether transient effects of NMDAR antagonist drugs to inhibit neuronal rhythmicity can be stably induced with genetic specificity, we examined the effects of RNAi of GluN1 protein on the subthreshold oscillations (STOs) of neurons in the inferior olive (IO), a pacemaking nucleus necessary for motor and cognitive timing. Western blot of dissociated neurons demonstrated 90% knockdown of GluN1 after a strong in vivo transduction by a dual-microRNA lentiviral vector. GluN1 RNAi in whole-cell-patched IO neurons blocked both membrane depolarization and STOs typically induced by NMDAR activation for up to 54 days without affecting input resistance, membrane capacitance, action potential firing, high-threshold Ca(2+) spikes, the hyperpolarization-activated current Ih, or the activation of the low-threshold Ca(2+) current I(T). Although an off-target effect on Cav3 expression was ruled out also by BlastN query, we found that GluN1 RNAi chronically eliminated I(T)-dependent STOs at resting membrane potential, well below the activation threshold of the NMDAR channel. In the context of a recent report showing that NMDAR activation induces STOs as it strengthens electrical coupling, the long-term block of STOs by GluN1 RNAi may relate to the loss of an essential support mechanism. Lentivector-mediated RNAi of GluN1 provides a novel technique for future investigations of NMDAR involvement in electrical oscillations and behavior.