The Involvement of Hypothalamic Sleep Pathways in General Anesthesia: Testing the Hypothesis Using the GABA_AReceptor β₃N265M Knock-In Mouse

Abstract

The GABA A receptor has been identified as the single most important target for the intravenous anesthetic propofol. How effects at this receptor are then translated into a loss of consciousness, however, remains a mystery. One possibility is that anesthetics act on natural sleep pathways. Here, we test this hypothesis by exploring the anesthetic sensitivities of GABAergic synaptic currents in three specific brain nuclei that are known to be involved in sleep. Using whole-cell electrophysiology, we have recorded GABAergic IPSCs from the tuberomammillary nucleus (TMN), the perifornical area (Pef), and the locus ceruleus (LC) in brain slices from both wild-type mice and mice that carry a specific mutation in the GABA A receptor ␤ 3 subunit (N265M), which greatly reduces their sensitivity to propofol, but not to the neurosteroid alphaxalone. We find that this in vivo pattern of anesthetic sensitivity is mirrored in the hypothalamic TMN and Pef nuclei, consistent with their role as direct anesthetic targets. In contrast, anesthetic sensitivity in the LC was unaffected by the ␤ 3 N265M mutation, ruling out this nucleus as a major target for propofol. In support of the hypothesis that orexinergic neurons in the Pef are involved in propofol anesthesia, we further show that these neurons are selectively inhibited by GABAergic drugs in vivo during anesthesia, and that a modulation in the activity of Pef neurons alone can affect loss of righting reflex. Overall, our results support the idea that GABAergic anesthetics such as propofol exert their effects, at least in part, by modulating hypothalamic sleep pathways.

Sleep and general anesthesia

“…74 Inhibiting orexinergic activity was found to prolong emergence from isoflurane anesthesia, 75 but orexins may not be important for the actions of halothane 76 or dexmedetomidine. 77 As we have previously argued, 78 interpretation of such findings is fraught with difficulties due to the behaviourally activating/ sedating effects of neurotransmitter agonists/antagonists, respectively. Clearly, there is a difference as to whether a particular neurotransmitter system can affect anesthesia (by altering the arousal baseline) and whether it is actually relevant to the mechanism of action of these drugs.…”

“…For instance, orexinergic tone appears to be differentially inhibited by propofol and dexmedetomidine, but exogenous orexin can attenuate LORR caused by both. 77 Furthermore, the fact that both drugs appear to enhance VLPO activity and decrease TMN activity 22,74 indicates that studies examining the effects of drugs affecting the histamine system may be behaviourally relevant. Also, in vitro studies looking at neuronal responses to anesthetics can be useful, e.g., the b 3 N265M knockin mouse is behaviourally less sensitive to propofol and etomidate.…”

“…72 Various receptors are known molecular targets of anesthetics (e.g., the expression of the two pore domain acid-sensitive potassium channel-3 (TASK-3) [E, redrawn] 65 or the anesthetic-sensitive point mutation [N265M] in the b 3 subunit of the GABA A receptor [F, redrawn]) 81 and modifying them both increases the anesthetic requirement in vivo. In the case of the point mutant, the reduced sensitivity has been mapped to at least the perifornical area (Pef) and the tuberomammillary nucleus neurons (G, redrawn), 77 but the extent of their involvement and other possible targets within and outside of the sleep circuitry have yet to be determined Sleep and general anesthesia 145 recorded in the LC were still able to be prolonged by the anesthetic. 77 It would be interesting to see whether this test would support anesthetic actions in arousal pathways when applied to other knock-in (e.g., b 2 N265S) 82 or knockout (e.g., two pore domain acid-sensitive potassium channel-3) 65 mice.…”

“…In the case of the point mutant, the reduced sensitivity has been mapped to at least the perifornical area (Pef) and the tuberomammillary nucleus neurons (G, redrawn), 77 but the extent of their involvement and other possible targets within and outside of the sleep circuitry have yet to be determined Sleep and general anesthesia 145 recorded in the LC were still able to be prolonged by the anesthetic. 77 It would be interesting to see whether this test would support anesthetic actions in arousal pathways when applied to other knock-in (e.g., b 2 N265S) 82 or knockout (e.g., two pore domain acid-sensitive potassium channel-3) 65 mice. Another exciting resolution to this problem may be the advance of cell-targeted genetic technologies, particularly those using viral delivery systems.…”

Mechanisms of Anesthetic Emergence: Evidence for Active Reanimation

“…Histaminergic neurons are considered a key in endogenous arousal of the neuronal circuitry; thus, anesthetics produce their hypnotic effects through the neuronal circuitry. One study reported that the lower sensitivity to propofol in genetically mutated mice was mirrored by a reactivity to GABAergic inhibitory postsynaptic currents (IPSCs) in the tuberomammillary nucleus of the hypothalamus [52]. These results indicate that central histaminergic neurons play a crucial role in emergence from general anesthesia via the endogenous arousal neuronal circuitry.…”