Activation of the GABAergic Parafacial Zone Maintains Sleep and Counteracts the Wake-Promoting Action of the Psychostimulants Armodafinil and Caffeine

Abstract

We previously reported that acute and selective activation of GABA-releasing parafacial zone (PZ^Vgat) neurons in behaving mice produces slow-wave-sleep (SWS), even in the absence of sleep deficit, suggesting that these neurons may represent, at least in part, a key cellular substrate underlying sleep drive. It remains, however, to be determined if PZ^Vgat neurons actively maintain, as oppose to simply gate, SWS. To begin to experimentally address this knowledge gap, we asked whether activation of PZ^Vgat neurons could attenuate or block the wake-promoting effects of two widely used wake-promoting psychostimulants, armodafinil or caffeine. We found that activation of PZ^Vgat neurons completely blocked the behavioral and electrocortical wake-promoting action of armodafinil. In some contrast, activation of PZ^Vgat neurons inhibited the behavioral, but not electrocortical, arousal response to caffeine. These results suggest that: (1) PZ^Vgat neurons actively maintain, as oppose to simply gate, SWS and cortical slow-wave-activity; (2) armodafinil cannot exert its wake-promoting effects when PZ^Vgat neurons are activated, intimating a possible shared circuit/molecular basis for mechanism of action; (3) caffeine can continue to exert potent cortical desynchronizing, but not behavioral, effects when PZ^Vgat neurons are activated, inferring a shared and divergent circuit/molecular basis for mechanism of action; and 4) PZ^Vgat neurons represent a key cell population for SWS induction and maintenance.

Figure 1

Example 12 h recordings showing hypnograms and corresponding cortical EEG delta power, theta/delta ratio and EMG activity following injection of vehicle, CNO 0.3 mg/kg, armodafinil 90 mg/kg, CNO 0.3 mg/kg+armodafinil 90 mg/kg, caffeine 20 mg/kg and CNO 0.3 mg/kg+caffeine 20 mg/kg at 10:00 A.M. in a PZ^Vgat-hM3Dq mouse. Red, wakefulness; green, SWS; blue, REM sleep.

Figure 2

Sleep-wake and EEG responses to injections of vehicle, CNO (0.3 mg/kg), armodafinil (90 mg/kg) and CNO+armodafinil (0.3 mg/kg and 90 mg/kg, respectively) in PZ^Vgat-hM3Dq mice. (a) Hourly amount of the vigilance stages and (a2-3) sleep latencies, n=9 mice. (b) Amount of the vigilance stages during the 2 h post injection (10:00–12:00), the rest of the light period (12:00–19:00), the following dark period (19:00–7:00) and the first 3 h of the light period the next day (07:00–10:00), n=9 mice. (c) Power spectrum changes (%±SEM) over baseline during the 3 h post-injection period for vehicle injection as compared with the 1 h post-injection period for CNO, armodafinil and CNO+armodafinil administration, and the quantitative changes (%±SEM) in power for the delta (δ: 0.5–5 Hz), theta (θ: 5–9 Hz), sigma (σ: 9–15 Hz), beta (β: 15–30 Hz), low gamma (lγ: 30–60 Hz) and high gamma (hγ: 60–120 Hz) frequency bands, n=8 mice. Black star, p<0.05 as compared with control injection; orange star, p<0.05 as compared with CNO injection; light blue star, p<0.05 as compared with armodafinil injection, two-way ANOVA corrected with a Bonferroni’s multiple comparisons test. (d) Raw EEG/EMG examples showing SWS following injection of CNO or CNO+armodafinil from the same mouse.

Figure 3

Sleep-wake and EEG responses to injections of vehicle, CNO (0.3 mg/kg), armodafinil (180 mg/kg) and CNO+armodafinil (0.3 mg/kg and 180 mg/kg, respectively), in PZ^Vgat-hM3Dq mice. (a) Hourly amount of the vigilance stages, n=9 mice. (b) Power spectrum changes (%±SEM) over baseline during the 3 h post-injection period for vehicle injection as compared with the 1 h post-injection period for CNO, armodafinil and CNO+armodafinil administration and the quantitative changes (%±SEM) in power for the delta (δ: 0.5–5 Hz), theta (θ: 5–9 Hz), sigma (σ: 9–15 Hz), beta (β: 15–30 Hz), low gamma (lγ: 30–60 Hz) and high gamma (hγ: 60–120 Hz) frequency bands, n=6 mice. Black star, p<0.05 as compared with control injection; orange star, p<0.05 as compared with CNO injection, two-way ANOVA corrected with a Bonferroni’s multiple comparisons test.

Figure 4

Sleep-wake and EEG responses to injections of vehicle, CNO (0.3 mg/kg), caffeine (20 mg/kg) and CNO+caffeine (0.3 mg/kg and 20 mg/kg, respectively), in PZ^Vgat-hM3Dq mice. (a) Hourly amount of the vigilance stages and (a2-3) sleep latencies, n=15 mice. (b) Amount of the vigilance stages during the 2-h post injection (10:00-12:00), the rest of the light period (12:00-19:00), the following dark period (19:00–7:00) and the first 3 h of the light period the next day (7:00–10:00), n=15 mice. (c) Power spectrum changes (%±SEM) over baseline during the 3 h post-injection period for vehicle injection as compared with the 1 h post-injection period for CNO, caffeine and CNO+caffeine administration and the quantitative changes (%±SEM) in power for the delta (δ: 0.5–5 Hz), theta (θ: 5–9 Hz), sigma (σ: 9–15 Hz), beta (β: 15–30 Hz), low gamma (lγ: 30–60 Hz) and high gamma (hγ: 60–120 Hz) frequency bands, n=10 mice. Black star, p<0.05 as compared with control injection; orange star, p<0.05 as compared with CNO injection; light green star, p<0.05 as compared with caffeine injection, two-way ANOVA corrected with a Bonferroni’s multiple comparisons test. (d) Raw EEG/EMG examples showing SWS following injection of CNO or CNO+caffeine.

Figure 5

Sleep-wake and EEG responses to injections of vehicle, CNO (0.3 mg/kg), caffeine (5 mg/kg) and CNO+caffeine (0.3 mg/kg and 5 mg/kg, respectively), in PZ^Vgat-hM3Dq mice. (a) Hourly amount of the vigilance stages, n=9 mice. (b) Power spectrum changes (%±SEM) over baseline during the 3-h post-injection period for vehicle injection as compared with the 1-h post-injection period for CNO, caffeine and CNO+caffeine administration and the quantitative changes (%±SEM) in power for the delta (δ: 0.5–5 Hz), theta (θ: 5–9 Hz), sigma (σ: 9–15 Hz), beta (β: 15–30 Hz), low gamma (lγ: 30–60 Hz) and high gamma (hγ: 60–120 Hz) frequency bands, n=5 mice. Black star, p<0.05 as compared with control injection; orange star, p<0.05 as compared with CNO injection, two-way ANOVA corrected with a Bonferroni’s multiple comparisons test.