In this study, we found abnormalities in the sleep architecture of DD mice. Wakefulness was reduced and highly fragmented, indicating that DD mice cannot properly maintain wakefulness. Our observations are in line with the previously reported hypoactive phenotype of DD mice  and the critical roles of the dopaminergic system in maintaining wakefulness [2,3,4,5,6,7,8,9]. Importantly, loss of VTA dopaminergic neurons did not affect the time spent in wakefulness according to a viral vector-mediated ablation study . Thus, dopaminergic neurons in the other areas might contribute to maintaining wakefulness.
Unexpectedly, we also found that REM sleep is drastically reduced in DD mice. Considering that REM sleep typically follows deep NREM sleep, reduced REM sleep might be a result of fragmented NREM sleep in DD mice. However, it should be noted that dopamine D2 receptor knockout mice also exhibit highly fragmented NREM sleep in the dark phase and yet the amount of REM sleep is unaffected . Genetic activation of dopaminergic neurons in the VTA or dorsal raphe does not increase the time spent in REM sleep [3,4,5]. On the other hand, dopaminergic neurons in the VTA are active during REM sleep according to fiber photometry recordings  and the dopamine concentration in the nucleus accumbens and prefrontal cortex, where the VTA dopaminergic neurons massively project, is increased during REM sleep according to measurements by microdialysis . In addition, several drugs that are considered to antagonize dopamine receptors reduce the time spent in REM sleep . Thus, dopamine might contribute to the positive regulation of REM sleep through unknown mechanisms. We also found abnormalities in the EEG power spectrum of DD mice, implying that dopamine also contributes to the patterns of neuronal synchrony and/or oscillation.
One technical limitation of this study is that there is a possibility that the daily l-DOPA treatment and/or consecutive intraperitoneal injections in DD mice might have had some effect on sleep. However, 50 mg/kg l-DOPA injection does not affect the amount of each vigilance state in WT mice . In addition, at the timing of EEG/EMG recordings (48 h after the last 50 mg/kg l-DOPA injection), dopamine level in the striatum of DD mice is expected to be largely depleted according to our previous study (extracellular dopamine [fmol/10 min] 24 h after the last 50 mg/kg l-DOPA injection: WT 39.23 ± 3.76, DD mice 0.62 ± 0.12; 72 h after the last 50 mg/kg l-DOPA injection: WT 43.55 ± 4.80, undetectable in DD mice) . Another technical issue is that the transgenic expression of tyrosine hydroxylase in DD mice might affect the level of other neurotransmitters such as noradrenaline, although we previously confirmed that noradrenaline level in the striatum of DD mice was unaffected (extracellular noradrenaline [fmol/10 min] 72 h after the last 50 mg/kg l-DOPA injection: WT 0.51 ± 0.07, DD 0.67 ± 0.09) .
In summary, by direct measurement of EEG/EMG and analysis of sleep architecture in DD mice, this study further supports the current understanding of the critical roles of dopaminergic system in maintaining wakefulness and implicates its overlooked effects on the positive regulation of REM sleep.