In vivo silencing of the thalamic CaV3.1 voltage-gated calcium channels demonstrates their region-specific role in anesthetic mediated hypnosis.

Abstract

Although substantial progress has been made in the last three decades towards our understanding of how general anesthetics (GAs) act at the molecular level, much less is known about how GAs cause loss of consciousness at the level of neuronal networks. The role of thalamus as an important brain region in anesthetic-induced hypnosis is relatively well established, but the specific roles of voltage-gated ion channels in different functional regions of the thalamus in anesthetic mechanisms are not well studied. To address this gap in knowledge, we selectively silenced the Cacna1g gene that encodes the low-threshold-activated Ca_V3.1 T-type voltage-gated calcium channel subunit by injecting short-hairpin RNA (shRNA) into midline and intralaminar - nonspecific thalamus (MIT) and sensory - specific ventrobasal (VB) thalamic nuclei in wild-type (WT) mice. Control animals were injected with scrambled shRNA. To validate our silencing approach, we performed patch-clamp experiments in acute thalamic slices ex vivo. In injected animals we determined anesthetic endpoints such as hypnosis measured with loss of righting reflex (LORR) and immobilization measured with loss of withdrawal reflex (LOWR) in vivo after administration of a traditional volatile GA isoflurane. Effective Ca_V3.1 channel knock-down was documented by greatly diminished amplitudes of T-currents and absence of rebound burst firing in our patch-clamp recordings from thalamic slices. We found that knocking down Ca_V3.1 channels in MIT significantly decreased inhaled isoflurane concentration that is required to induce LORR, but it did not affect speed of anesthetic induction and the immobilizing effect of isoflurane. In contrast, knocking down the Ca_V3.1 channel in the VB thalamus did not affect any of the measured anesthetic endpoints. Hence, we concluded that Ca_V3.1 channels in nonspecific MIT thalamus have a preferential role in anesthetic hypnosis when compared to the sensory VB thalamus.