Role of endogenous sleep-wake and analgesic systems in anesthesia

Abstract

Classical anesthetics of the gamma-aminobutyric acid type A receptor (GABA(A))-enhancing class (e.g., pentobarbital, chloral hydrate, muscimol, and ethanol) produce analgesia and unconsciousness (sedation). Dissociative anesthetics that antagonize the N-methyl-D-aspartate (NMDA) receptor (e.g., ketamine, MK-801, dextromethorphan, and phencyclidine) produce analgesia but do not induce complete loss of consciousness. To understand the mechanisms underlying loss of consciousness and analgesia induced by general anesthetics, we examined the patterns of expression of c-Fos protein in the brain and correlated these with physiological effects of systemically administering GABAergic agents and ketamine at dosages used clinically for anesthesia in rats. We found that GABAergic agents produced predominantly delta activity in the electroencephalogram (EEG) and sedation. In contrast, anesthetic doses of ketamine induced sedation, followed by active arousal behaviors, and produced a faster EEG in the theta range. Consistent with its behavioral effects, ketamine induced Fos expression in cholinergic, monoaminergic, and orexinergic arousal systems and completely suppressed Fos immunoreactivity in the sleep-promoting ventrolateral preoptic nucleus (VLPO). In contrast, GABAergic agents suppressed Fos in the same arousal-promoting systems but increased the number of Fos-immunoreactive neurons in the VLPO compared with waking control animals. All anesthetics tested induced Fos in the spinally projecting noradrenergic A5-7 groups. 6-hydroxydopamine lesions of the A5-7 groups or ibotenic acid lesions of the ventrolateral periaqueductal gray matter (vlPAG) attenuated antinociceptive responses to noxious thermal stimulation (tail-flick test) by both types of anesthetics. We hypothesize that neural substrates of sleep-wake behavior are engaged by low-dose sedative anesthetics and that the mesopontine descending noradrenergic cell groups contribute to the analgesic effects of both NMDA receptor antagonists and GABA(A) receptor-enhancing anesthetics.

Fig. 1

Effects on the electroencephalogram (EEG) induced by ketamine (A,B) and GABA_A anesthetics (C–F), compared with normal NREM sleep (G) and wakefulness (H). For each state, a 12-second epoch of typical EEG is shown on the left, and the power spectrum for that same epoch on the right. Note that doses of ketamine up to 150 mg/kg (twice that used typically for rat anesthesia; A) increased both delta power (0.5–4.0 Hz, associated with sleep) and theta power (4–7 Hz, associated with arousal and REM sleep) in the EEG, whereas only much higher doses (300 mg/kg) predominantly increased delta power (B). All GABA_A agents induced an EEG pattern dominated by slow, delta waves (C–F). The x-axis in the power spectrum represents the frequency band that was sampled, and the y-axis represents the power in that frequency band, in arbitrary units.

Fig. 2

Ketamine-induced Fos immunoreactivity (black) in the cholinergic cells labeled by choline acetyltransferase immunoreactivity (purple) in the basal forebrain (A,B) and the orexinergic cells in the medial and lateral perifornical (PeriF) fields of the lateral hypothalamus (C,D). Arrows point to double-labeled cells; arrowheads point to single labeling (without Fos expression). Insets show these same cells at higher magnification. Scale bar = 200 μm.

Fig. 3

Ketamine induced Fos immunorectivity in the tuberomam-millary nucleus (TMN; A; section counterstained with light Nissl stain), the dorsal raphe nucleus (DRN) nonserotoninergic cells (B; purple immunohistochemical staining for serotonin), the ventral periaqueductal gray matter (vPAG) dopaminergic cells (C; brown immunohistochemical staining for tyroxine hydroxylase), and the laterodorsal tegmental (LDT) noncholinergic cells (D; purple immunohistochemical staining for choline acetyltransferase). Arrows indicate double labeling; arrowheads indicate single labeling (without Fos expression). Scale bar = 200 μm.

Fig. 4

Effects of systemic anesthetics on Fos expression in the ventrolateral preoptic nucleus (VLPO). Wakefulness (A) and ketamine (B) completely suppress Fos expression in the VLPO, whereas spontaneous sleep (C), muscimol (D), pentobarbital (E), and chlorate hydrate (F) all are associated with Fos expression in the VLPO. OC, optic chiasm. Scale bar = 200 μm.

Fig. 5

Fos induction in the pontine noradrenergic A5, A6, and A7 groups following administration of ketamine (A,A′,A″), chloral hydrate (B,B′,B″), muscimol (C,C′,C″), ethanol (D,D′,D″), or pentobarbital (E,E′,E″). Arrows indicate selected doubled-labeled neuron [tyrosine hydroxylase (TH) and Fos], which are shown at higher magnification in the insets. Note that all groups of anesthetics caused Fos expression in A5–7 noradrenergic neurons, although this was least prominent with pentobarbital [Fos was seen in the LC (A6) but rarely see in A5 and A7 groups]. Scale bar = 200 μm.

Fig. 6

Sections showing selective cell loss in TH-labeled noradrenergic A5–7 groups (A–C; cf Fig. 5) caused by fourth ventricular injection of 6-hydroxydopamine; and complete cell loss in the ventrolateral PAG (but also including the DRN) after ibotenic acid injections at that site (rostral level, D,E; caudal level, F). Arrows indicate the few surviving TH-immunoreactive cells in A–C. The boxed area in D is magnified in E, demonstrating only glial nuclei in the region of the lesion. Scale bar = 200 μm in A (applies to A–C); 200 μm in D (applies to D,F); 400 μm for E.

Fig. 7

Summary diagrams of proposed anesthesia models for GABA_A agonist anesthetics (A) and NMDA antagonist ketamine (B). Red arrows show putative inhibitory pathways; green arrows show putative excitatory pathways. The width of the red and green arrows illustrates the change in firing rate caused by the drugs. Thus a GABA_A agonist will reduce the activity of inhibitory afferents to the VLPO, increasing its firing rate, which in turn uses GABA_A receptors to inhibit the TMN (which is also directly inhibited by the drug); this reduces wakefulness. The GABA_A agonist drug at the same time inhibits the GABAergic vlPAG neurons, which have reduced firing, thus disinhibiting the A5–7 cell groups, which fire faster and have an antinociceptive effect at the level of the spinal dorsal horn. Ketamine at dosages usually used for rat anesthesia directly blocks activation of the VLPO, thus reducing its activity and releasing the TMN to increase wakefulness. However, ketamine blocks activity in the vlPAG, thus disinhibiting the A5–7 noradrenergic neurons to produce an antinociceptive effect at the level of the spinal dorsal horn.