Glucose Induces Slow-Wave Sleep by Exciting the Sleep-Promoting Neurons in the Ventrolateral Preoptic Nucleus: A New Link between Sleep and Metabolism

Abstract

Sleep-active neurons located in the ventrolateral preoptic nucleus (VLPO) play a crucial role in the induction and maintenance of slow-wave sleep (SWS). However, the cellular and molecular mechanisms responsible for their activation at sleep onset remain poorly understood. Here, we test the hypothesis that a rise in extracellular glucose concentration in the VLPO can promote sleep by increasing the activity of sleep-promoting VLPO neurons. We find that infusion of a glucose concentration into the VLPO of mice promotes SWS and increases the density of c-Fos-labeled neurons selectively in the VLPO. Moreover, we show in patch-clamp recordings from brain slices that VLPO neurons exhibiting properties of sleep-promoting neurons are selectively excited by glucose within physiological range. This glucose-induced excitation implies the catabolism of glucose, leading to a closure of ATP-sensitive potassium (KATP) channels. The extracellular glucose concentration monitors the gating of KATP channels of sleep-promoting neurons, highlighting that these neurons can adapt their excitability according to the extracellular energy status. Together, these results provide evidence that glucose may participate in the mechanisms of SWS promotion and/or consolidation.

Significance statement: Although the brain circuitry underlying vigilance states is well described, the molecular mechanisms responsible for sleep onset remain largely unknown. Combining in vitro and in vivo experiments, we demonstrate that glucose likely contributes to sleep onset facilitation by increasing the excitability of sleep-promoting neurons in the ventrolateral preoptic nucleus (VLPO). We find here that these neurons integrate energetic signals such as ambient glucose directly to regulate vigilance states accordingly. Glucose-induced excitation of sleep-promoting VLPO neurons should therefore be involved in the drowsiness that one feels after a high-sugar meal. This novel mechanism regulating the activity of VLPO neurons reinforces the fundamental and intimate link between sleep and metabolism.

Keywords: glucose; homeostasis; hypothalamus; polysomnography; preoptic nucleus; sleep.

Figure 1.

Microinjections of glucose into the VLPO increase sleep. A, Camera lucida drawings of frontal sections illustrating all injection sites (one open circle per animal). The four different sections are evenly spaced (120 μm intervals) throughout the rostrocaudal extent of the VLPO. Bregma coordinates range approximately from 0.26 to −0.22 mm according to the Franklin and Paxinos atlas (2007). B, Hypnograms of a representative animal illustrating the organization of the sleep-waking cycle during the 2 h after a bilateral injection of vehicle (VEH; top) or glucose 5 mm (bottom) into the VLPO. Recordings began immediately after the injection procedure once the animals were returned to their home barrels and reconnected to the recording setup. C, Quantification of SWS durations (left), SWS latencies (middle), and PS (right) durations during the 2 h after bilateral glucose or VEH injection (n = 7). Individual data for each mouse injected bilaterally with glucose or VEH (gray dots and lines) are displayed and data are presented as mean ± SEM (black bars). *p < 0.05, Wilcoxon test.

Figure 2.

Density of c-Fos-labeled neurons in anterior hypothalamic regions after glucose infusion into the VLPO. A, Schema illustrating the VLPO and surrounding brain areas examined for c-Fos counting, including the VLPO (red), MCPO (blue), LPOA (orange), and MPOA (green). B, Photomicrographs of sections stained against c-Fos after unilateral infusions of glucose (left) and VEH (right) and counterstained with neutral red in a representative animal (c-Fos-labeled cells are shown in the bottom left inset at a higher magnification Scale bar, 10 μm. C–F, Quantification of the density of c-Fos-labeled neurons in the VLPO (C), MCPO (D), LPOA (E), and MPOA (F) of animals that received glucose in one hemisphere and VEH in the other hemisphere (n = 7). Individual data for each hemisphere injected with glucose or VEH (gray dots and lines) are displayed and data are presented as mean ± SEM (black bars). *p < 0.05, Wilcoxon test: ns.

Figure 3.

Effect of glucose applications on putative sleep-promoting VLPO neurons. A, Reversible inhibitory effect of decreasing extracellular glucose concentration from 5 to 1 mm on the spontaneous firing activity of a VLPO neuron recorded in loose-cell-attached configuration. B, Quantification of the excitatory effect of glucose (from 1 to 5 mm) on the spontaneous firing activity of glucose-responsive VLPO cells (n = 8). Data are presented as mean ± SEM. **p < 0.01, Wilcoxon test: ns. C, Dose–response relationship of the excitatory effect of glucose on glucose-sensitive VLPO cells. Data fit a Hill curve with an EC₅₀ of 4.06 mm and are presented as mean ± SEM with group sizes for each glucose concentration (1, 2.5, 5, 10, 25 mm). D, Reversible inhibitory effect of bath-applied NA (100 μm; right) on the spontaneous firing activity of the same cell shown in A characterized by a multipolar shape (left). Scale bar, 20 μm. E, Current-clamp recording of the same cell shown in A characterized by the presence of a potent LTS (*).

Figure 4.

Absence of glucose effect on identified non-sleep-promoting neurons. A, Absence of effect of acute increase in extracellular glucose concentration on the spontaneous firing activity of a VLPO neuron. B, Spontaneous firing activity of VLPO neurons showing insensitivity to changes in extracellular glucose concentration for 1 mm (n = 6), 2.5 mm (n = 5), 10 mm (n = 8), and 25 mm (n = 5) glucose. Data are presented as mean ± SEM. Mann–Whitney test: ns. C, Reversible excitatory effect of bath-applied NA (100 μm) to the same cell shown in A. D, Current-clamp recording of VLPO cell devoid of LTS characterized by a bipolar shape (insert) Scale bar, 20 μm.

Figure 5.

Glucose-induced excitation implies the catabolism of glucose in neurons. A, Excitatory effect of glucose on the spontaneous firing activity of identified sleep-active cells under synaptic uncoupling conditions (low Ca²⁺/high Mg²⁺) (n = 4). Data are presented as mean ± SEM. *p < 0.05, permutation test. B, Effect of the application of a nonmetabolizable glucose analog (2-DG) on the spontaneous firing activity of identified putative sleep-promoting and glucose-excited neurons (n = 4). Data are presented as mean ± SEM. *p < 0.05, permutation test: ns. C, Effect of the blockade of lactate transporters (4-CIN) and glucokinase inhibition (alloxan) on the glucose-induced increase in resting membrane potential of identified sleep-active neurons. Data are presented as mean ± SEM with group sizes for each condition. **p < 0.01, Mann–Whitney test: ns. D, Gel electrophoresis of scRT-PCR products of three characterized sleep-promoting cells expressing GK mRNAs, with the third neuron expressing GK and GLUT3 mRNAs. MW, Molecular weight (100 bp ladder).

Figure 6.

Characterization of K_ATP channels in VLPO neurons. A, Current-clamp recording of a sleep-promoting neuron identified by the presence of a potent LTS (*) and a multipolar shape (insert) Scale bar, 20 μm. B, Effect of pinacidil (500 μm), diazoxide (300 μm), and tolbutamide (500 μm) on the stationary currents recorded at −65 mV for the same cell shown in A. C, Effect of diazoxide (blue) and tolbutamide (red) on the I–V relationship of the same neuron shown in A and B. I–V plot of the net K_ATP-related current (black), which reverses near the K⁺ equilibrium potential. D, Gel electrophoresis of scRT-PCR products of a sleep-promoting cell revealing the expression of Kir6.2 but not SUR1 or SUR2 mRNAs. MW, Molecular weight (100 bp ladder).

Figure 7.

Extracellular glucose level monitors K_ATP channel opening state in sleep-promoting cells. A–B, Top, Representative traces of stationary currents recorded at −65 mV in 2.5 mm (A) and 10 mm (B) glucose and during diazoxide (300 μm) and tolbutamide (500 μm) application. Note the difference in currents induced by diazoxide and tolbutamide compared with the initial current. Bottom, Currents recorded during 100 ms voltage steps from −65 to −75 mV at times indicated by a–d in recordings illustrated above. C, Effect of diazoxide and tolbutamide applications on holding current of LTS neurons held at −65 mV. Data are presented as changes induced by pharmacological treatments from the initial value of the holding current at cell opening. Data are presented as mean ± SEM; n = 8 per condition. *p < 0.05, **p < 0.01, Wilcoxon test vs initial current at cell opening; #p < 0.05, Mann–Whitney test 2.5 mm vs 10 mm glucose.