Parafacial GABAergic neurone ablation induces behavioural resistance to volatile anaesthetic-induced hypnosis without reducing sleep.

Abstract

It is hypothesised that general anaesthetics co-opt the neural circuits regulating endogenous sleep and wakefulness to produce hypnosis. To further probe this association, we focused on the GABAergic neurones of the parafacial zone (PZ^GABA), a brainstem site capable of promoting non-rapid eye movement sleep. To determine whether PZ neurones are activated by a hypnotic dose of anaesthetics, c-Fos immunohistochemistry was performed. The behavioural and physiological contributions of PZ^GABA neurones to anaesthetic sensitivity were assessed in mice transfected with an adeno-associated virus (AAV)-driving expression of an mCherry fluorescent control or a caspase that irreversibly eliminates PZ^GABA neurones. EEG-defined sleep was measured in PZ^GABA-ablated and mCherry control mice, as was the homeostatic drive to sleep after sleep deprivation. Consistent with anaesthetic-induced depolarisation, hypnotic doses of isoflurane significantly increased c-Fos expression three-fold in PZ^GABA neurones compared with oxygen-exposed mice. PZ^GABA-ablated mice developed significant and durable behavioural resistance to both isoflurane- and sevoflurane-induced hypnosis, with roughly 50% higher likelihood of intact righting than controls. PZ^GABA-ablated mice emerged from isoflurane significantly faster than mCherry controls with purposeful movements. The degree of anaesthetic resistance was inversely correlated with the number of surviving PZ^GABA neurones. Despite confirming that PZ^GABA ablation reduced the potency of two distinct volatile anaesthetics behaviourally, ablation did not alter the amount of endogenous sleep or wakefulness, nor did it affect the homeostatic sleep drive after sleep deprivation, and it did not produce EEG signatures of anaesthetic resistance during isoflurane exposure. There was an unexpected dissociation in which destruction of up to 70-80% of PZ^GABA neurones was sufficient to alter anaesthetic susceptibility behaviourally without causing insomnia or altering sleep pressure. These findings suggest that PZ^GABA neurones are more critical to drug-induced hypnosis than to the regulation of natural sleep and arousal.