Manipulation of adenosine kinase affects sleep regulation in mice

Abstract

Sleep and sleep intensity are enhanced by adenosine and its receptor agonists, whereas adenosine receptor antagonists induce wakefulness. Adenosine kinase (ADK) is the primary enzyme metabolizing adenosine in adult brain. To investigate whether adenosine metabolism or clearance affects sleep, we recorded sleep in mice with engineered mutations in Adk. Adk-tg mice overexpress a transgene encoding the cytoplasmic isoform of ADK in the brain but lack the nuclear isoform of the enzyme. Wild-type mice and Adk(+/-) mice that have a 50% reduction of the cytoplasmic and the nuclear isoforms of ADK served as controls. Adk-tg mice showed a remarkable reduction of EEG power in low frequencies in all vigilance states and in theta activity (6.25-11 Hz) in rapid eye movement (REM) sleep and waking. Adk-tg mice were awake 58 min more per day than wild-type mice and spent significantly less time in REM sleep (102 ± 3 vs 128 ± 3 min in wild type). After sleep deprivation, slow-wave activity (0.75-4 Hz), the intensity component of non-rapid eye movement sleep, increased significantly less in Adk-tg mice and their slow-wave energy was reduced. In contrast, the vigilance states and EEG spectra of Adk(+/-) and wild-type mice did not differ. Our data suggest that overexpression of the cytoplasmic isoform of ADK is sufficient to alter sleep physiology. ADK might orchestrate neurotransmitter pathways involved in the generation of EEG oscillations and regulation of sleep.

Figure 1.

EEG power density in microvolt square per hertz (0.75–25 Hz) in NREM sleep (NREMS), REM sleep (REMS), and waking in WT (black dots; n = 14) and Adk-tg mice (white dots; n = 11) during the 24 h baseline. Shown are the mean values of the parietal (PAR) and frontal (FRO) EEG. Triangles, Genotype differences (p < 0.05 for NREMS PAR and waking; p < 0.01 for NREMS FRO and REMS; Bonferroni's adjustment for multiple comparisons).

Figure 2.

Representative 5 s raw parietal EEG and EMG traces during NREM sleep, REM sleep, and waking in WT, Adk^+/−, and Adk-tg mice.

Figure 3.

Time course of the vigilance states NREM sleep, REM sleep, waking, and REM sleep as percentage of total sleep time (REMS/TST) in WT and Adk-tg mice during the 24 h baseline (BL) (left panels) and 18 h recovery (Rec.) (right panels) after 6 h SD (gray area). Shown are mean 2 h values. Triangles, Genotype differences (p < 0.05, unpaired t test; ANOVA “genotype” by repeated “2 h interval”: NREMS BL, F_(11,253) = 5.67, p < 0.0001, and Rec., F_(8,184) = 2.14, p = 0.034; REMS BL, F_(11,253) = 2.54, p = 0.0046, and Rec., F_(8,184) = 1.81, p = 0.078, “genotype” F_(1,23) = 26.82, p < 0.0001, followed by Tukey–Kramer “genotype” p < 0.0001; REMS/TST BL, F_(11,253) = 1.14, p = 0.33, “genotype” F_(1,23) = 20.96, p = 0.0001 followed by Tukey–Kramer “genotype” p < 0.0001, and Rec., F_(8,184) = 2.04, p = 0.0437; waking BL, F_(11,253) = 5.25, p < 0.0001, and Rec., F_(8,184) = 1.91, p = 0.06).

Figure 4.

Left, Number of NREM sleep episodes in WT (n = 14) and Adk-tg mice (n = 11) during the 24 h baseline. Mean values are expressed as percentage of the total number of NREM sleep episodes. Numbers below the x-axis are duration range in seconds. Triangles, Genotype differences (p < 0.005, Tukey–Kramer; ANOVA “genotype” by “episode duration” F_(1,7) = 14.02, p < 0.0001). Right, Activity intensity (defined as infrared counts/number of 1 min epochs with counts >0; mean 1 h values; average of 10 consecutive baseline days) in WT (n = 11) and Adk-tg mice (n = 12). Triangles, Genotype differences (p < 0.05, unpaired t test; ANOVA “genotype” by repeated “1 h interval” F_(23,483) = 4.32, p < 0.0001).

Figure 5.

A, Time course of SWA in NREM sleep in the parietal (PAR) and frontal (FRO) EEG in WT and Adk-tg mice during 18 h recovery after 6 h SD (gray area). Mean 2 h values are expressed as percentage of the mean 24 h baseline SWA in NREM sleep of the corresponding EEG. Genotype differences (p < 0.001, Tukey–Kramer; ANOVA “genotype” F_(1,24) = 29.04, p < 0.0001 for PAR, and F_(1,24) = 14.23, p < 0.0009 for FRO; interaction “genotype” by “2 h interval” F_(8,183) = 1.06, p = 0.39 for PAR, and F_(8,183) = 1.55, p = 0.14 for FRO). Differences from corresponding baseline interval in Adk-tg (open squares below the curves; p < 0.05, paired t test; ANOVA “condition” by “2 h interval” F_(8,180) = 32.81 and 46.08, p < 0.0001 for PAR and FRO, respectively) and WT mice (filled squares; p < 0.0001, paired t test; F_(8,234) = 48.32 and 46.76, p < 0.0001 for PAR and FRO). B, Time course of SWA during the 2 min before and after transitions from waking to NREM sleep. Mean 20 s values computed for the first 6 h interval after SD and the corresponding baseline interval are expressed as percentage of the mean 24 h baseline SWA in NREM sleep. Triangles, Genotype differences during recovery (p < 0.05, unpaired t test; ANOVA “genotype” by “20 s epoch”: F_(11,252) = 2.01, p = 0.0276 for PAR, and F_(11,252) = 2.73, p = 0.0024 for FRO).

Figure 6.

Time course of specific frequency bands during the 2 min before and after transitions from NREM to REM sleep in WT and Adk-tg mice. SFA (10.25–15 Hz) in the frontal EEG (left panel) and theta frequency activity (7.25–11 Hz) in the parietal EEG (right panel). Mean 20 s values for the 24 h baseline are expressed as percentage of the mean 24 h baseline SFA or theta activity in NREM sleep. Triangles, Genotype differences (SFA, p < 0.0005, unpaired t test; ANOVA “genotype” by “20 s epoch”: F_(11,252) = 10.64 and 41.49, p < 0.0001 for SFA and theta activity, respectively).

Figure 7.

Time course of EEG power in waking in three frequency bands in the parietal EEG of WT and Adk-tg mice during the 6 h SD (gray area) and first 6 h recovery. Mean 2 h values are expressed as percentage of the mean 24 h baseline power in the corresponding frequency band in waking. Triangles, Genotype differences (p < 0.05, unpaired t test; ANOVA “genotype” by “2 h interval” F_(5,114) = 2.46, p = 0.037, for 2.25–3.5 Hz band, F_(5,114) = 15.67 and 6.95, p < 0.0001, for 6.25–9 and 11.25–15 Hz band, respectively). Asterisks, Difference between the first and last 2 h SD interval (p < 0.05, paired t test).

Figure 8.

Time course of NREM sleep (NREMS), REM sleep (REMS), and SWA (EEG power between 0.75 and 4 Hz) in NREM sleep in the parietal (PAR) and frontal (FRO) EEG in WT (black dots; n = 14) and Adk^+/− mice (white dots; n = 15) during the 24 h baseline. The white and black bars indicate the 12 h light and dark period. Data represent 2 h means; SWA is expressed as percentage of the 24 h mean SWA in NREM sleep.

Figure 9.

Top, Western blot of ADK protein levels from the right hemisphere of adult WT, Adk^+/−, and Adk-tg mice (numbers 1–6 correspond to individual mice). Bottom, Corresponding quantitative analysis of the ADK levels of n = 6 mice per genotype (O.D., optical density). ADK was first normalized to equal loading according to the α-tubulin standard and then to the ADK level of WT mice (=100%). Differences from WT: **p < 0.001, unpaired t test.