Abstract
The mechanisms through which general anesthetics induce loss of consciousness remain unclear. Previous studies have suggested that dorsal raphe nucleus serotonergic (DRN5-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 DRN5-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 DRN5-HT neurons reversibly decreased during propofol perfusion. Ablation of DRN5-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 DRN5-HT neurons strongly promoted emergence from propofol anesthesia. Propofol application to DRN5-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 DRN5-HT neurons were also abolished by propofol. Furthermore, the hyperpolarization of DRN5-HT neurons caused by propofol was abolished by picrotoxin, a GABAA receptor antagonist, which shortened emergence time from propofol anesthesia when locally applied to the DRN. Our results reveal that DRN5-HT neurons in zebrafish are involved in the emergence from propofol anesthesia by inhibiting presynaptic excitatory glutamate inputs and inducing GABAA receptor-mediated hyperpolarization.
Significance Statement The neural mechanisms of general anesthesia remain unclear. We studied the role of the dorsal raphe nucleus serotonergic (DRN5-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 DRN5-HT neurons are involved in emergence from anesthesia, but not induction. Propofol suppresses DRN5-HT activity by inhibiting the activity of DRN5-HT neurons via GABAA receptors and blocking presynaptic excitatory glutamate inputs. These findings further support larval zebrafish as an ideal model for investigating the mechanisms of general anesthesia.
Footnotes
This work was supported by the National Natural Science Foundation of China (82001451 to X.Y., T2293734 and T2293730 to Y.L., 2271285 to Q.Y). The authors appreciate Yongxin Yang for expert assistance with the in vivo whole-cell recording technique. The authors appreciate Changmei Zhang for the suggestions on writing the manuscript. All mentioned individuals are members of Jinlin Du’s lab. The authors also thank Professor Zhe Zhang, Ph.D., from Institute of Neuroscience, State Key Laboratory of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, for providing tetrodotoxin samples in electrophysiology experiment.
↵aThese authors contributed equally to this work.
