The Serotonergic Dorsal Raphe Promotes Emergence from Propofol Anesthesia in Zebrafish

Abstract

The mechanisms through which general anesthetics induce loss of consciousness remain unclear. Previous studies have suggested that dorsal raphe nucleus serotonergic (DRN^5-HT) neurons are involved in inhalational anesthesia, but the underlying neuronal and synaptic mechanisms are not well understood. In this study, we investigated the role of DRN^5-HT neurons in propofol-induced anesthesia in larval zebrafish (sex undetermined at this developmental stage) using a combination of in vivo single-cell calcium imaging, two-photon laser ablation, optogenetic activation, in vivo glutamate imaging and in vivo whole-cell recording. We found that calcium activity of DRN^5-HT neurons reversibly decreased during propofol perfusion. Ablation of DRN^5-HT neurons prolonged emergence from 30 μM propofol anesthesia, while induction times were not affected under concentrations of 1 μM, 3 μM, and 30 μM. Additionally, optogenetic activation of DRN^5-HT neurons strongly promoted emergence from propofol anesthesia. Propofol application to DRN^5-HT neurons suppressed both spontaneous and current injection-evoked spike firing, abolished spontaneous excitatory postsynaptic currents, and decreased membrane input resistance. Presynaptic glutamate release events in DRN^5-HT neurons were also abolished by propofol. Furthermore, the hyperpolarization of DRN^5-HT neurons caused by propofol was abolished by picrotoxin, a GABA_A receptor antagonist, which shortened emergence time from propofol anesthesia when locally applied to the DRN. Our results reveal that DRN^5-HT neurons in zebrafish are involved in the emergence from propofol anesthesia by inhibiting presynaptic excitatory glutamate inputs and inducing GABA_A receptor-mediated hyperpolarization.

Significance Statement The neural mechanisms of general anesthesia remain unclear. We studied the role of the dorsal raphe nucleus serotonergic (DRN^5-HT) neurons in propofol anesthesia using larval zebrafish, employing in vivo calcium imaging at single-neuron resolution, two-photon ablation, optogenetic activation, and in vivo whole-cell recording. We found that the DRN^5-HT neurons are involved in emergence from anesthesia, but not induction. Propofol suppresses DRN^5-HT activity by inhibiting the activity of DRN^5-HT neurons via GABA_A receptors and blocking presynaptic excitatory glutamate inputs. These findings further support larval zebrafish as an ideal model for investigating the mechanisms of general anesthesia.