Role Of Locus Coeruleus Noradrenergic Neurons In Zebrafishgeneral Anesthesia

Objective and background: Despite nearly 170 years of clinical use, the neural mechanisms by which anesthetics act to induce unconsciousness are still unclear. Recent studies have demonstrated that many arousal-related neuromodulatory nuclei, especially locus coeruleus- norepinephrine(LC-NE) system, play important roles in the emergence from general anesthesia. However, it is unknown about molecular and synaptic mechanisms by which these anesthetics act on these neuromodulator-released neurons.Methods:Through behavioral testing of spontaneous locomotion and electrophysiological recording of brain activities, we first established the anesthetic model in zebrafish larvae via bath application of intravenous anesthetics, propofol or etomidate. Through ablation of the LC neurons via two-photon laser or genetic knockout(KO) of dopamine-β-hydroxylase(DBH), we characterized the role of the LC-NE system in general anesthesia. Furthermore, through in vivo whole-cell recordings of LC neurons, we revealed the molecular and synaptic mechanisms by which propofol or etomidate act on these neurons.Results:1. We successfully established the anesthetic model in zebrafish larvae for the first time through behavioral test, electrophysiological recording of local field potential(LFP) and motoneuron nerves in spinal cord. 2. Ablation of LC neurons via two-photon laser in zebrafish causes faster induction into and delayed emergence from general anesthesia. 3. Depletion of NE in DBH-KO in zebrafish causes faster induction into and delayed emergence from general anesthesia.4. In vivo whole-cell patch-clamp recordings of LC neurons revealed that local puffing of either propofol or etomidate caused an immediate abolishment of spontaneous spike firing and an accompanied hyperpolarization of resting membrane potential(V m) in LC neurons. 5. In vivo whole-cell voltage-clamp recording of LC neurons revealed that local application of propofol or etomidate could totally suppress spontaneous excitatory postsynaptic currents(s EPSCs) and induced a transient tonic outward current. 6. Application of picrotoxin, an antagonist of GABA A receptors, could abolish anesthetic-induced V m hyperpolarization. Conclusions: 1. The LC-NE system in zebrafish is essential for the induction into and emergence from intravenous general anesthesia. 2. Propofol and etomidate may inhibit the release of presynaptic neurotransmitter and act on extrasynaptic GABA A receptors on LC neurons. 3. Zebrafish is an ideal animal model for the investigation of neural circuit mechanisms underlying general anesthesia.