Regulation of dopaminergic transmission and cocaine reward by the Clock gene

Abstract

Although there are clear interactions between circadian rhythms and drug addiction, mechanisms for such interactions remain unknown. Here we establish a role for the Clock gene in regulating the brain's reward circuit. Mice lacking a functional Clock gene display an increase in cocaine reward and in the excitability of dopamine neurons in the midbrain ventral tegmental area, a key brain reward region. These phenotypes are associated with increased expression and phosphorylation of tyrosine hydroxylase (the rate-limiting enzyme in dopamine synthesis), as well as changes in several genes known to regulate dopamine activity in the ventral tegmental area. These findings demonstrate the involvement of a circadian-associated gene, Clock, in regulating dopamine function and cocaine reward.

Fig. 1.

Clock mutant mice are hyperactive. Rearing behavior (a) and locomotor activity (b) were measured in a novel environment over 2 hours with the number of beam breaks measured every 15 min. (c and d) Activity throughout the light/dark cycle was measured for 24 h, with activity recorded every 30 min. The solid dark bars indicate the dark cycle. All data points are significant (a and b) (P < 0.05 by ANOVA, n = 6). The total activity over 24 h is also significantly different for both rears and ambulations (P < 0.05 by ANOVA, n = 6). Clk, Clock mutants; WT, wild-type mice.

Fig. 2.

Clock mutant mice sensitize to cocaine. (a and b) Clock mutants (Clk) and wild-type (WT) controls were habituated to the locomotor activity boxes for 4 days with saline injections. On days 5–9, animals were given 10 mg/kg cocaine or saline i.p., and locomotor activity was measured for 10 min. Plotted are the results from a challenge cocaine injection on day 10. *, P < 0.05 by ANOVA, n = 6–8. (c) Clock mutants have an increased preference for cocaine. Clock mutants and WT controls were tested for bias on day 1, conditioned on days 2–4, and tested for preference for cocaine on day 5 by using an unbiased protocol at 2.5, 5, and 10 mg/kg. *, P < 0.05 by ANOVA, n = 11–14.

Fig. 3.

CLOCK is expressed in dopamine neurons, and TH levels and phosphorylation are increased in Clock mutants. (a) Sections containing the VTA were labeled with antibodies against CLOCK (red) and TH (green). Fluorescence was integrated by using confocal microscopy. Results are representative of multiple sections obtained throughout the anterior–posterior axis of the VTA of five mice (data not shown). (b–g) Clock mutants have increased TH protein and mRNA levels in the VTA. mRNA levels were determined from VTA and substantia nigra (SN) tissue punches from Clock mutants (Clk) and wild-type (WT) controls by real-time PCR (n = 5) (b) and in situ hybridization (n = 8). (c) Protein levels and phosphorylation (d–g) were determined by Western blot analysis (representative blots are shown in d and f, n = 5). In all cases, GAPDH was used as a control. *, P < 0.05 by ANOVA.

Fig. 4.

Dopamine cell-firing rates and bursting are increased in Clock mutant mice. (a)(Left) Representative recording of a mouse dopamine neuron. (Right) Example of an averaged dopamine triphasic waveform. (b) Clock mutant mice (Clk) (n = 24) exhibited higher basal dopamine firing rates compared with wild-type (WT) (n = 26) mice (P < 0.03). Squares represent the mean ± SEM of each group. (c) Clock mutant mice exhibited more burst events per 10 sec compared with wild-type controls (P < 0.01). Each vertical bar represents the mean ± SEM of each group. The percentage of bursting activity, i.e., spikes emitted in bursts, was greater compared with wild-type mice (Clk, 23.5 ± 4.3 vs. 10.6 ± 3.6 in WT; P < 0.03). Clock mutant mice had larger burst sizes (number of spikes/burst) compared with controls (2.8 ± 0.1 vs. 2.0 ± 0.1; P < 0.008).