Dopaminergic role in stimulant-induced wakefulness

Abstract

The role of dopamine in sleep regulation and in mediating the effects of wake-promoting therapeutics is controversial. In this study, polygraphic recordings and caudate microdialysate dopamine measurements in narcoleptic dogs revealed that the wake-promoting antinarcoleptic compounds modafinil and amphetamine increase extracellular dopamine in a hypocretin receptor 2-independent manner. In mice, deletion of the dopamine transporter (DAT) gene reduced non-rapid eye movement sleep time and increased wakefulness consolidation independently from locomotor effects. DAT knock-out mice were also unresponsive to the normally robust wake-promoting action of modafinil, methamphetamine, and the selective DAT blocker GBR12909 but were hypersensitive to the wake-promoting effects of caffeine. Thus, dopamine transporters play an important role in sleep regulation and are necessary for the specific wake-promoting action of amphetamines and modafinil.

Fig. 1.

Effect of modafinil andd-amphetamine on wakefulness and caudate DA efflux in narcoleptic hypocretin receptor 2 mutant dogs. Systemic administration of modafinil (5 mg/kg, i.v.) and d-amphetamine (0.1 mg/kg, i.v.) equipotently increased time spent awake. The most pronounced effect on wakefulness was observed during the first hour after injection [inset; *p < 0.01 for both treatments compared with the respective vehicle treatment (white bars) by Student's t test;n = 4 per group; mean ± SEM]. Microdialysis experiments demonstrated that administration of amphetamine and modafinil significantly increased extracellular DA levels (p < 0.05;d-amphetamine or modafinil relative to vehicle treatment; repeated-measures ANOVA with Bonferroni's–Dunn'spost hoc comparisons; n = 4 per group; mean ± SEM). The baseline DA concentrations (at time 0) for modafinil and d-amphetamine sessions were 17.2 ± 3.1 and 17.6 ± 3.5 nm (mean ± SEM), respectively, and did not differ statistically among treatments.VEH, 1 ml of 100% DMSO; d-AMP,d-amphetamine.

Fig. 2.

Entrained circadian rhythms of wheel-running activity in wild-type (DAT +/+) and homozygous DAT knock-out (DAT −/−) mice. Representative raster plots (top 2 panels) show nocturnal consolidation of wheel-running and a lack of wheel-running activity during the light phase in both DAT +/+ and DAT −/− mice. Group mean ± SEM waveforms are shown for DAT +/+ (filled triangles; n = 7), DAT −/− (filled circles; n= 6), and heterozygous DAT +/− (open squares;n = 4) mice in the bottom panel.Tick marks in the raster plots denote when wheel-running activity was present in 5 min bins. Light–dark bar at the top denotes lighting schedule. Twenty-four hour activity patterns of DAT −/− mice differ significantly from those of DAT +/+ mice (p < 0.02; two-way repeated-measures ANOVA), *p < 0.05; DAT−/− versus DAT +/+; Bonferroni's–Dunn's post hoccomparisons.

Fig. 3.

Mean ± SEM wake bout duration during 24 hr baseline recordings in wild-type (+/+; n = 14), heterozygous (+/−; n = 11), and homozygous DAT knock-out (n = 14) mice. One-way ANOVA indicated significant genotype effect [p < 0.014; df = (2,36)]. *p < 0.05 versus DAT +/+; Student's t test.

Fig. 4.

Effect of GBR12909 (20 mg/kg) on sleep states in wild-type (+/+) and DAT knock-out (−/−) mice. Data are the percentages of time (mean ± SEM in 2 hr bins) in NREM, REM, and wake in the 24 hr pretreatment and 24 hr posttreatment periods. Open circles, Vehicle (0.25% methylcellulose vehicle); filled circles, GBR12909 (20 mg/kg).Arrows indicate injection. Light–dark bars at the bottom of thepanels indicate lighting schedule. Two-way repeated-measures ANOVA indicated postinjection treatment × time interactions in DAT +/+ (p < 0.001 for wake; p < 0.003 for NREM; p < 0.011 for REM) but not DAT −/− (p > 0.1; all states) mice. Sample sizes (+/+, −/−): vehicle (9, 8); GBR12909 (14, 6).

Fig. 5.

Effect of modafinil (90 mg/kg) on sleep states in wild-type (+/+) and DAT knock-out (−/−) mice. Data are the percentages of time (mean ± SEM in 2 hr bins) in NREM, REM, and wake in the 24 hr pretreatment and 24 hr posttreatment periods. Open circles, Vehicle (0.25% methylcellulose vehicle); filled circles, modafinil (90 mg/kg).Arrows indicate injection. Light–darkbars at the bottom of the panels indicate lighting schedule. Two-way repeated-measures ANOVA indicated postinjection treatment × time interactions for wake, NREM, and REM sleep in both DAT +/+ (p < 0.001 for wake, NREM, and REM) and DAT −/− (p = 0.007, 0.004, and 0.007, respectively) mice, but increased wake after modafinil administration was only observed in DAT +/+ mice. Sample sizes (+/+, −/−): vehicle (9, 8); modafinil (10, 9).

Fig. 6.

Cumulative effects of GBR12909 (20 mg/kg) and of modafinil (90 mg/kg) on wake in wild-type (+/+) and DAT knock-out (−/−) mice. The cumulative change in wake time (mean ± SEM) in the posttreatment period relative to the corresponding period of the baseline 24 hr pretreatment period is shown for vehicle (open circles) and drug treatment (filled circles). Note large increase in wakefulness in DAT +/+ but not DAT −/− animals. Two-way repeated-measures ANOVA within each genotype indicated significant treatment effect and treatment × time interaction in DAT +/+ but not in DAT −/−. *p < 0.05 drug versus vehicle; Bonferroni's–Dunn's post hoc comparisons. Sample sizes (+/+, −/−): vehicle (9, 8); GBR12909, (14, 6); modafinil (10, 9).

Fig. 7.

Response to wake-promoting therapeutics in DAT knock-out (−/−; open bars) and wild-type (filled bars) mice. Data are reported as the cumulative change in time awake (mean ± SEM) 5 hr after treatment relative to corresponding baseline 24 hr earlier. Two-way ANOVA indicated significant genotype × treatment interaction in comparisons of individual pharmacological treatments versus vehicle (p < 0.05, all treatments).VEH, 0.25% methylcellulose vehicle;METH, methamphetamine; GBR, GBR12909.¹p < 0.001;²p < 0.025 between groups;³p < 0.002 relative to vehicle; Student's t test. Sample sizes (+/+, −/−): vehicle (9, 8); methamphetamine, 2 mg/kg (14, 10); GBR12909, 20 mg/kg (14, 6); modafinil, 90 mg/kg (10, 9) and 300 mg/kg (14, 6); caffeine, 2.5 mg/kg (10, 8), 10 mg/kg (12, 7), and 20 mg/kg (8, 0).