(A–C) Typical sections from the Rosa26-Dlx5/6-Cre reporter mouse were stained with mouse polyclonal antibodies against β-gal to visualize Cre-expressing neurons indirectly. (A) Robust expression of β-gal is seen in the striatum of the reporter mouse. (B) At a single-cell level, double immunofluorescence for β-gal (green) and ChAT (magenta) on an adjacent section to (A) shows that cholinergic interneurons in the striatum also express β-gal. The arrows in the upper and lower panels of (B) indicate neurons with dual immunolabeling for β-gal and ChAT. (C) In the classical arousal/sleep-related cell groups of the basal forebrain and anterior hypothalamus, i.e., the nucleus of the horizontal limb of the diagonal band of Broca (HDB), the substantia inominata (SI) or the ventrolateral preoptic area (VLPO), only moderate immunoreactivity for β-gal is detected in the HDB. β-Gal immunolabeling is absent in neurons of the SI and VLPO. Scale bars: 500 µm in (A), 20 µm in (B), and 250 µm in (C). (D and E) The BG-A_2AR KO mice and WT littermates were treated with vehicle or caffeine (2 or 30 mg/kg, i.p.). Time course (D) and total time (E) of wakefulness during the first 3 h after caffeine injection (arrows) were assessed with EEG/EMG recordings. Data are presented as the mean ± S.E.M. (n=4–5). * P<0.05; ** P<0.01; compared with vehicle treatment within corresponding genotype. ## P<0.01; compared with corresponding WT littermates. †† P<0.01; compared between caffeine doses.

Rest-activity assessment was performed based on the amount of LMA in 50 s bins for 3 h after caffeine (2, 10 or 30 mg/kg) treatment. Time spent in inactivity (A and B) or high LMA (ambulation; C and D) is presented as percentage of total time (3 h). Data are presented as mean ± S.E.M. (n=8, per genotype and drug dose rest-activity assessment). * P<0.05; ** P<0.01; compared to vehicle treatment within corresponding genotypes. # P<0.05; ## P<0.01; compared with corresponding WT littermates. †† P<0.01; compared between caffeine doses.

(A and B) Typical sections of conditional A_2AR KO mice after injection with mCherry-expressing AAVs (WT; left photomicrograph) and AAV carrying Cre recombinase (NAc-A_2AR KO; right photomicrograph) were stained with a goat polyclonal antibody against A_2AR (Santa Cruz) to visualize the presence (black circle) or loss (red dashed circle) of A_2ARs in the NAc. Immunostaining with a rabbit polyclonal antibody against D₂R (Millipore) confirms the integrity of the NAc (black and blue circles) in the WT and NAc-A_2AR KO mice (B). (C and D) The NAc-A_2AR KO and WT mice were treated with caffeine (15 mg/kg, i.p.) or modafinil (45 mg/kg, i.p.). The time course (C) and total time (D) of wakefulness for the first 3 h after caffeine treatment were assessed from EEG/EMG recordings, as was modafinil-induced wakefulness for 3 h (right panel in D). Arrows in (C) indicate the time of caffeine injection. (E and F) Typical examples of EEG, EMG, LMA and hypnograms after administration of caffeine (15 mg/kg, i.p., upper panels), vehicle for caffeine administration (middle panels) or modafinil (45 mg/kg, i.p., lower panels) in a WT (E) and NAc-A_2AR KO (F) mouse. Arrows in (C, E and F) indicate the time of injection. Data are presented as the mean ± S.E.M. (n=4–5). * P<0.05; ** P<0.01; compared with vehicle treatment within corresponding AAV injection. ## P<0.01; compared with AAV treatment. Scale bars: 500 µm in (A) and (B).

(A) Generation of AAV vectors that contained short-hairpin RNA specific for the A_2AR (shA_2AR) and the red fluorescent protein mCherry as a reporter gene. (B) AAV-shA_2AR vectors were stereotaxically injected into the A_2AR-positive core and shell of the nucleus accumbens (NAc) as well as into the olfactory tubercle (OLT), caudate-putamen (CPu), and globus pallidus (GP). (C and D) Typical sections from rats injected bilaterally with AAV carrying control shRNA (shCTRL) or shA_2AR into the NAc were stained with mouse monoclonal antibody against A_2AR. Immunoreactivity for A_2AR is depleted selectively in the NAc of the AAV-shA_2AR-treated rats (D, dashed red circle), but it is unaffected in the AAV-shCTRL-treated rats (C, red circle). (E and F) NeuN-staining confirms the integrity of NAc neurons (green circles) in AAV-shCTRL (E) and AAV-shA_2AR injected rats (F). Scale bars: 500 µm in (C) and (E); also apply to (D) and (F), respectively.

(A) The loss of the arousal response to caffeine (15 mg/kg, i.p.) in rats that had received AAV-shA_2AR injections (black circles) into the NAc correlated closely with the knockdown of A_2ARs in the NAc, whereas there was very little variation in caffeine-induced wakefulness in control animals injected with shCTRL (white triangles). (B) Caffeine-induced wakefulness for 3 h in AAV-untreated rats, and in rats that received injection of AAV-shCTRL into the NAc or injection of AAV-shA_2AR into the NAc shell and core, OLT, CPu, and GP. Caffeine was given at 15 mg/kg, i.p. (C and D) Typical sections from two rats injected bilaterally with AAV carrying shA_2AR into the shell or core of the NAc that were stained with mouse monoclonal antibody against A_2AR showed dominant depletion of A_2ARs either in the shell (D, right photomicrograph) or the core (C, left photomicrograph) of the NAc. Adjacent sections to ‘C’ and ‘D’ were stained with rabbit polyclonal antibody against mCherry (Clontech) to confirm the extent to which neural cells in the NAc were transfected with AAV-shA_2AR (C and D, right photomicrographs). The red circles in the photomicrographs in ‘D’ outline the entire NAc including both the core and shell region, whereas the green circles in the photomicrographs in ‘C’ delineate the core of the NAc. The polysomnographic recordings in ‘C’ and ‘D’ show typical examples of EEG, EMG, LMA and hypnograms after administration of caffeine at a dose of 15 mg/kg (upper polysomnographic displays) or vehicle (lower polysomnographic displays) in two rats with AAV-shA_2AR infections of the shell (D) or the core (C) of the NAc. Data are presented as mean ± S.E.M. (n=5–6 per AAV-treatment). ** P<0.01; compared with AAV-untreated or AAV-shCTRL-injected rats, assessed by one-way ANOVA. Scale bars: 300 µm in (C) and (D).

Endogenous somnogens such as adenosine and prostaglandin D₂ (PGD₂) promote sleep by activating sleep-promoting neurons of the ventrolateral preoptic area (VLPO) (Saper et al., 2005; Saper et al., 2010; Urade, 2011) which, in a putative flip-flop arrangement, inhibit the arousal-promoting regions, including the lateral hypothalamus (LHA), the tuberomammillary hypothalamic nucleus (TMN), and the locus coeruleus (LC) in the brainstem. Adenosine acting at A_2ARs on medium spiny neurons in the shell of the NAc is hypothesized to exert inhibitory effects on the arousal systems via indirect (GABAergic and glutamatergic) pathways. Caffeine blocks the A_2ARs in the NAc shell, thereby removing the restraint on the arousal systems to promote wakefulness.