Direct activation of sleep-promoting VLPO neurons by volatile anesthetics contributes to anesthetic hypnosis

Abstract

Background: Despite seventeen decades of continuous clinical use, the neuronal mechanisms through which volatile anesthetics act to produce unconsciousness remain obscure. One emerging possibility is that anesthetics exert their hypnotic effects by hijacking endogenous arousal circuits. A key sleep-promoting component of this circuitry is the ventrolateral preoptic nucleus (VLPO), a hypothalamic region containing both state-independent neurons and neurons that preferentially fire during natural sleep.

Results: Using c-Fos immunohistochemistry as a biomarker for antecedent neuronal activity, we show that isoflurane and halothane increase the number of active neurons in the VLPO, but only when mice are sedated or unconscious. Destroying VLPO neurons produces an acute resistance to isoflurane-induced hypnosis. Electrophysiological studies prove that the neurons depolarized by isoflurane belong to the subpopulation of VLPO neurons responsible for promoting natural sleep, whereas neighboring non-sleep-active VLPO neurons are unaffected by isoflurane. Finally, we show that this anesthetic-induced depolarization is not solely due to a presynaptic inhibition of wake-active neurons as previously hypothesized but rather is due to a direct postsynaptic effect on VLPO neurons themselves arising from the closing of a background potassium conductance.

Conclusions: Cumulatively, this work demonstrates that anesthetics are capable of directly activating endogenous sleep-promoting networks and that such actions contribute to their hypnotic properties.

Figure 1. VLPO c-Fos immunoreactivity was increased following exposure to volatile anesthetics

Panels A-F depict sample coronal sections through the hypothalamus showing staining of c-Fos (brown nuclei) following a 2 hr exposure to (A) oxygen control, dark phase; (B) 0.3% isoflurane, dark phase; (C) 0.6% isoflurane, dark phase; (D) 1.2% isoflurane, dark phase; (E) 1.0% isoflurane at 70 atm, light phase; (F) 1.0% halothane, dark phase. Scale bar in A corresponds to 50 μm and applies to all panels A-F. (G) Bar graph summarizing c-Fos expression in VLPO. Counts are per unilateral VLPO in a 10 μm slice. Cell counts were analyzed using an ANOVA with post-hoc t-tests using the Bonferroni correction for multiple comparisons. Error bars represent SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001 as compared to the non-anesthetized dark phase control; †††, p < 0.001 as compared to 1.2% isoflurane during light phase.

Figure 2. Putative sleep-active VLPO neurons (NA(-)) were depolarized by ex vivo exposure of isoflurane

Panels A and B show sample traces from a NA(+) (A) and a NA(-) (B) VLPO neuron during exposure to norepinephrine (NA) and isoflurane, with insets depicting membrane responses to hyperpolarizing current injections (-100, -80, and -60 pA). The majority of NA(-) neurons (79%) showed clear evidence of a rebound low-threshold spike (B, arrowhead in inset), whereas few of the NA(+) neurons (13%) exhibited this phenomenon. (C) Bar graph depicting change in firing rate relative to baseline. (D) Bar graph depicting change in membrane potential relative to baseline. Recordings were performed on 15 NA(+) and 34 NA(-) neurons and analyzed using two-way ANOVAs with post-hoc t-tests using the Bonferroni correction for multiple comparisons. Error bars represent SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; all comparisons were made against baseline.

Figure 3. Activation of NA(-) VLPO neurons by isoflurane persisted in the presence of synaptic blockade

(A) Sample voltage-clamp traces from VLPO neurons inhibited by norepinephrine (NA(-)) show that both excitatory (downward deflections) and inhibitory (upward deflections) postsynaptic currents were eliminated by the administration of 20 μM DNQX, 100 μM AP5, and 20 μM bicuculline (n=7), or by the replacement of normal artificial cerebrospinal fluid (aCSF) with a mixture where Ca²⁺ content was reduced to 160 μM and Mg²⁺ content increased to 9.68 mM (n=3). (B & C) Isoflurane, at both 240 μM and 480 μM, was effective at increasing the firing rate (B) and depolarizing the resting membrane potential (C) in NA(-) neurons, both in normal aCSF and in preparations where all postsynaptic activity has been abolished. Data were analyzed using two-way ANOVAs with post-hoc t-tests using the Bonferroni correction for multiple comparisons. Error bars represent SEM. n.d., not determined; *, p < 0.05; **, p < 0.01; ***, p < 0.001; all comparisons were made against baseline.

Figure 4. Pre- and postsynaptic effects of isoflurane upon VLPO NA(-) neurons

(A) Sample voltage-clamp traces from a NA(-) neuron clamped at -60 mV showing an inward current during isoflurane exposure, which persisted during synaptic blockade. (B) Average excitatory post synaptic current (EPSC; top) and inhibitory post-synaptic current (IPSC; bottom) traces recorded from a NA(-) neuron (100 traces averaged). Isoflurane (240 μM) increased IPSC half-width, but had no effect on EPSC frequency or amplitude (see Table S1). (C) Administration of 240 μM isoflurane increased inward current in NA(-) cells held at a fixed voltage (-60 mV), an effect that persisted during synaptic blockade with 20 μM DNQX, 100 μM AP5, and 20 μM bicuculline (n=7). Data were analyzed using two-way ANOVAs with post-hoc t-tests using the Bonferroni correction for multiple comparisons. Error bars represent SEM. ***, p < 0.001; all comparisons were made against baseline.

Figure 5. Isoflurane exposure increased the membrane resistance of VLPO NA(-) neurons due to a reduction in potassium conductance

(A) Sample trace from a NA(-) neuron showing membrane voltage response to bath administration of 240 μM isoflurane; membrane resistance was monitored by measuring the magnitude of the downward voltage deflections in response to -30 pA injected current pulses. Exposure to 240 μM isoflurane depolarized the NA(-) neuron and increased membrane resistance. This increase in resistance was maintained when negative current was applied to return the cell to its baseline resting potential in the presence of isoflurane (not shown). (B) Isoflurane significantly increased membrane resistance in all four NA(-) neurons (p < 0.05). (C) Using voltage clamp techniques, the inward current observed during isoflurane exposure was diminished when the neurons were held at E_K (-100 mV). The current produced by isoflurane at -100 mV was not significantly different from 0 pA. Error bars represent SEM. *, p < 0.05; **, p < 0.01.

Figure 6. Mice with targeted VLPO lesions became acutely resistant to isoflurane-induced hypnosis

4× DAPI (A,C) and 20× NeuN (B,D) images of a sample sham (A,B) and lesioned (C,D) animal (see also Figure S1). Lesion efficacy was assessed via cell counts of NeuN-positive cells; the white boxes in (B) and (D) are 250 μm tall by 400 μm wide and correspond to the location used for counting. Two animals out of 17 in the lesion cohort that had less than 80% cell loss in the VLPO compared to shams were excluded from analysis (n=7 in the sham group). Average neuronal loss in VLPO for lesioned animals was 91% ± 2%. (E) Dose-response curves showing the fraction of unconscious animals as judged by a loss of righting reflex in response to stepwise increases in isoflurane concentrations at 6 and 24 days post-surgery; the horizontal axis is displayed on a logarithmic scale. VLPO lesions produced a significant rightward shift of the induction dose-response curve at day 6, and a significant leftward shift at day 24 (Table S2). (E, Insert) Bracketed ED₅₀ values as calculated by averaging the last concentration at which an animal had an intact righting reflex with the first concentration at which an animal lost its righting reflex. Error bars represent SEM. *, p < 0.05; ***, p < 0.001.