The dopamine D3 receptor is part of a homeostatic pathway regulating ethanol consumption

Abstract

We recently identified a homeostatic pathway that inhibits ethanol intake. This protective pathway consists of the scaffolding protein RACK1 and brain-derived neurotrophic factor (BDNF). RACK1 translocates to the nucleus after exposure of neurons to ethanol and increases expression of BDNF (McGough et al., 2004). We also found that increasing the levels of BDNF via systemic administration of RACK1 expressed as a Tat-fusion protein (Tat-RACK1) reduces ethanol consumption, whereas reduction of BDNF levels augments this behavior (McGough et al., 2004). Based on these results, we hypothesized that activation of the BDNF receptor TrkB is necessary for the effects of BDNF on ethanol intake and that gene products downstream of BDNF negatively regulate ethanol consumption. Here, we show that inhibition of the BDNF receptor TrkB increases voluntary ethanol consumption in wild-type mice but not in mice lacking one copy of the BDNF gene (BDNF(+/-)). We also find that increases in the levels of BDNF, mediated by ethanol or RACK1, lead to increased dorsal striatal levels of the dopamine D3 receptor (D3R), a gene downstream of BDNF, via activation of the TrkB receptor. Finally, we show that the Tat-RACK1-mediated reduction of ethanol consumption is attenuated by coinjection with either the Trk inhibitor K252a or the dopamine D3R-prefering antagonist U-99194A [5, 6-dimethoxy-2-(di-n-propylamino)indan], suggesting that activation of the BDNF pathway via RACK1 leads to increased expression of the dopamine D3R, which in turn mediates the attenuation of ethanol consumption.

Figure 1.

a, K252a attenuates the Tat–RACK1 effect on voluntary ethanol intake in mice. Tat–RACK1 (4 mg/kg), Tat–RACK1 (4 mg/kg) plus K252a (25 μg/kg), or vehicle was administered at 3 P.M., and the amount of ethanol consumed overnight was recorded. Data are presented as mean ± SEM grams/kilogram of body weight. n = 12. *p < 0.01 compared with vehicle. b, c, Activation of the BDNF receptor TrkB regulates ethanol consumption. The Trk receptor inhibitor K252a (5 and 25 μg/kg) or vehicle was administered at 3 P.M., and the amount of ethanol consumed overnight was recorded. Data are presented as mean ± SEM grams/kilogram of body weight. b, Inhibition of the Trk receptor increases ethanol intake in BDNF^+/+ mice. n = 12. *p < 0.05 compared with saline. c, K252a has no effect on ethanol intake in BDNF^+/− mice (n = 9).

Figure 2.

Tat-RACK1 and BDNF induce D₃R expression in the striatum via activation of the TrkB receptor. a, Striatal slices dissected from five rats were preincubated without (lanes 1 and 2) or with 200 nm K252a (lane 3) for 30 min before addition of 50 ng/ml BDNF (lanes 2 and 3) for 1 h. D₃R expression was analyzed by RT-PCR with GPDH control. The histogram depicts the mean ratios of D₃R to GPDH ± SD. n = 5. **p < 0.01 compared with control, or **p < 0.01 comparing BDNF plus K252a with BDNF alone. b, Striatal slices dissected from four rats were treated with 1 μm Tat–RACK1 for the indicated times. D₃R expression was analyzed by RT-PCR with GPDH control. The histogram depicts the mean ratios of D₃R to GPDH ± SD. n = 3. c, Striatal slices dissected from 10 rats were preincubated without or with 200 nm K252a (lanes 3 and 4) for 30 min before addition of 1 μm Tat–RACK1 (lanes 2 and 4) for 4 h. D₃R expression was analyzed by RT-PCR with GPDH control. The histogram depicts the mean ratios of D₃R to GPDH ± SD. n = 6. **p < 0.01 compared with control, or ^##p < 0.01 comparing Tat-RACK1 plus K252a with Tat-RACK1 alone.

Figure 3.

a, b, Tat–RACK1 increases D₃R expression in striatum in vivo. Six hours after intraperitoneal injection of vehicle or Tat–RACK1 (4 mg/kg), bilateral tissue punches of the striatum were taken and homogenized for RNA isolation as described in Materials and Methods. Expression of D₃R (a), D₁R, and D₂R (b) were analyzed by RT-PCR. The histograms depict the mean ratios of D₃R, D₁R, or D₂R to GPDH (±SD). n = 12 (4 mice used per treatment). *p < 0.05 compared with vehicle. c, Microinjection of Tat–RACK1 into striatum increases D₃R expression in vivo. Four rats were used for injection with PBS (1 μl per side) into one side of the dorsal striatum and Tat–RACK1 (1 μm, 1 μl per side) into the opposite side. Four hours after the microinjection, bilateral tissue punches of the striatum were dissected and homogenized for RNA isolation. Expression of D₃R (c), D₁R, and D₂R (d) were analyzed by RT-PCR. The histogram depicts the mean ratios of D₃R or D₁R, D₂R to GPDH ± SD. n = 4. **p < 0.01 compared with vehicle. e, Intradorsal striatum injection of Tat–RACK1 reduces ethanol consumption in rats. PBS (1 μl per side) or Tat–RACK1 (1 μm, 1 μl per side) was administered at 9:00 A.M. The rats were tested in the self-administration chambers at 1:00 P.M. for 1 h. The data are expressed in mean ± SEM of number of presses in 1 h. n = 11. *p < 0.05 compared with PBS.

Figure 4.

a, b, Ethanol induces D₃R expression in the striatum via activation of the TrkB receptor. a, Striatal slices dissected from five rats were treated with 100 mm ethanol for the indicated times. D₃R expression was analyzed by RT-PCR with GPDH control. The histogram depicts the mean ± SD ratios of D₃R to GPDH from triplicate experiments (n = 3). b, Striatal slices dissected from nine rats were preincubated without or with 200 nm K252a (lanes 3 and 4) for 30 min before the addition of 100 mm (lanes 2 and 4) ethanol for 3 h. D₃R expression was analyzed by RT-PCR with GPDH control. The histogram depicts the mean ± SD ratios of D₃R to GPDH. n = 6. **p < 0.01 compared with control, or ^##p < 0.01 comparing ethanol plus K252a with ethanol alone. c, d, Ethanol self-administration increases D₃R expression in the striatum in vivo. C57BL/6J mice were allowed continuous access to ethanol for 4 weeks using the two-bottle choice procedure as described in Materials and Methods (ethanol group). Age-matched control mice (water group) were exposed to water only for the same time period. Three hours after the start of the dark cycle, bilateral tissue punches of the striatum were taken and homogenized for RNA isolation. Expression of D₃R (c), D₁R, and D₂R (d) were analyzed by RT-PCR. The histogram depicts the mean ratios of D₃R, D₁R, or D₂R to GPDH, ± SD. n = 8 (4 mice per group). *p < 0.05 compared with the water group.

Figure 5.

The D₃R-preferring antagonist U-99194A, but not the D₂R antagonist eticlopride, reduces the effect of Tat–RACK1 on ethanol (EtOH) consumption in mice. Tat–RACK1 or saline was administered at 3:00 P.M. followed at 6:00 P.M. by injection of the dopaminergic antagonists. The amount of ethanol consumed in 4 h between 6:00 and 10:00 P.M. was recorded. Data are presented as mean ± SEM grams/kilogram of body weight. a, The dopamine D₃R antagonist U-99194A (20 mg/kg) attenuates the ability of Tat–RACK1 (2 mg/kg) to decrease drinking (saline, n = 5; Tat–RACK1, n = 7; Tat–RACK1 plus U-99194A, n = 6; U-99194A, n = 6). *p < 0.05 compared with saline; **p < 0.01 compared with Tat-RACK1 alone. b, The significant decrease induced by Tat–RACK1 (2 mg/kg) injection is not altered by eticlopride (0.1 mg/kg) injection (saline, n = 6; Tat–RACK1, n = 6; Tat–RACK1 plus eticlopride, n = 7; eticlopride, n = 6). *p < 0.05 compared with saline.