Anterior thalamic paraventricular nucleus is involved in intermittent access ethanol drinking: role of orexin receptor 2

Abstract

The paraventricular nucleus of the thalamus (PVT) has been shown to participate in hedonic feeding and is thought to influence drug seeking. This understudied nucleus contains anterior (aPVT) and posterior (pPVT) subregions, which receive dense projections from hypothalamic orexin/hypocretin (OX) but exhibit anatomical and functional differences. This study sought to characterize in Long-Evans rats the involvement of these PVT subregions and their OX receptor activity in consumption of the drug, ethanol. Compared with those maintained on water and chow only (water group), rats trained to drink pharmacologically relevant levels of ethanol (ethanol group) showed increased neuronal activation in the PVT, specifically the aPVT but not pPVT, as indicated by c-Fos immunoreactivity. Similar results were obtained in rats administered ethanol via oral gavage, indicating that this site-specific effect was due to ethanol exposure. In support of the involvement of OX, the ethanol group also showed increased mRNA levels of this neuropeptide in the hypothalamus and of OX 2 receptor (OX2R) but not OX 1 receptor (OX1R), again in the aPVT but not pPVT. Similarly, ethanol gavage increased double labeling of c-Fos with OX2R but not OX1R, specifically in the aPVT. Evidence directly supporting a role for aPVT OX2R in ethanol consumption was provided by results with local injections, showing ethanol intake to be enhanced by OX-A or OX-B in the aPVT but not pPVT and reduced by a local antagonist of OX2R but not OX1R. These results focus attention on the aPVT and specifically its OX2R in mediating a positive feedback relationship with ethanol intake.

Keywords: c-Fos; emotional behavior; immunohistochemistry; intermittent access; microinjection; rat.

Figure 1

Intermittent access induces high and stable levels of 20% ethanol drinking in Long-Evans rats. A. Average daily ethanol intake across the duration of the experiment (Set 1, n = 8). B. Hourly ethanol intake, assessed during Week 3 of the experiment (Set 1, n = 8). The greatest ethanol intake occurred during the first 30 minutes of access.

Figure 2

Intermittent-access 20% ethanol drinking alters emotional behavior after 30 minutes of access. A. The Ethanol compared to Water group showed reduced anxiety in an elevated plus maze, as indicated by increased time and number of entries in the open arm, but no difference in spontaneous locomotor behavior, as assessed by closed and total arm entries (Set 1, n = 8/group; Week 5 of ethanol access). B. The Ethanol group showed increased novelty-induced locomotor activity in an open field, as indicated by ambulatory counts, distance, and time (Set 2, n = 8/group; Week 4). C. The Ethanol and Water groups showed no significant differences in locomotor activity in a familiar open field, as assessed by ambulatory counts, distance, and time (Set 1, n = 8/group; Week 4 of ethanol access). ***p < 0.001, **p < 0.01, *p < 0.05, ^#p = 0.06 vs. Water Group.

Figure 3

Photomicrographs showing increased number c-Fos-positive cells (red) in the anterior paraventricular thalamus (aPVT) but not the posterior paraventricular thalamus (pPVT) 90 minutes after the start of access to 20% ethanol compared to water (Set 2, n = 8/group; Week 6 of access). Scale bars = 50 µm.

Figure 4

Photomicrographs showing increased number c-Fos-positive cells (red) in the anterior paraventricular thalamus (aPVT) but not the posterior paraventricular thalamus (pPVT) 90 minutes after oral 20% ethanol (1.50 g/kg/gavage) compared to water gavage (Set 3, n = 6/group; 11 treatments). Similar results were obtained when comparing the High to Low Ethanol groups (0.75 g/kg) and the Water to Control groups (images of Low Ethanol and Control groups not shown). Scale bars = 50 µm.

Figure 5

Ethanol drinking increases gene expression of orexin in the perifornical lateral hypothalamus and the orexin 2 receptor in the anterior paraventricular thalamus, as assessed by quantitative real-time polymerase chain reaction 30 minutes after the start of access to 20% ethanol compared to water (Set 1, n = 8/group; Week 7 of ethanol access). Target gene expression was quantified relative to cyclophilin using the relative quantification method. Abbreviations: aPVT, anterior paraventricular thalamus; pPVT, posterior paraventricular thalamus; OX, orexin/hypocretin; OX1R, orexin 1 receptor; OX2R, orexin 2 receptor; PFLH, perifornical lateral hypothalamus. **p < 0.01, *p < 0.05 vs. Water group.

Figure 6

Photomicrographs showing greater double-labeling of c-Fos immunoreactivity with the orexin 2 receptor (OX2R) but not orexin 1 receptor (OX1R) in the anterior paraventricular thalamus (aPVT) 90 minutes after oral 20% ethanol (1.50 g/kg/gavage) compared to water gavage (Set 3, n = 6/group; 11 treatments). Similar results were obtained when comparing the High to Low Ethanol groups (0.75 g/kg) and the Water to Control groups (images of Low Ethanol and Control groups not shown). c-Fos-positive cells are labeled in red, and receptor-positive cells are labeled in green. Images on the far right are higher magnifications of image marked with a white square. Arrowheads indicate double-labeled neurons. Scale bars = 50 µm.

Figure 7

Injection of orexin-A (OX-A) and orexin-B (OX-B) in the anterior paraventricular thalamus (aPVT) but not posterior paraventricular thalamus (pPVT) increases intermittent-access 20% ethanol drinking. A. Ethanol drinking in rats injected in the aPVT with 0.3 µl OX-A (1.0, 0.5 nmol) or saline vehicle (Set 4, n = 8). B. Drinking in rats injected in the aPVT with 0.3 µl OX-B (1.0, 0.5 nmol) or saline vehicle (Set 4, n = 8). C. Ethanol drinking in rats injected in the pPVT with OX-A or saline vehicle (Set 4, n = 7). D. Drinking in rats injected in the pPVT with OX-B or saline vehicle (Set 4, n = 7). Effects were no longer evident at four hours post-injection (data not shown). *p < 0.05, ^#p = 0.06 vs. vehicle.

Figure 8

Injection of orexin-A (OX-A) in the anterior paraventricular thalamus (aPVT) has no effect on intermittent-access 2% sucrose drinking but injection in the posterior paraventricular thalamus (pPVT) increases sucrose intake. A. Sucrose drinking in rats injected in the aPVT with 0.3 µl OX-A (1.0 nmol) or saline vehicle (Set 5, n = 10). B. Drinking in rats injected in the pPVT with OX-A or vehicle (Set 5, n = 10). Effects were no longer evident at four hours post-injection (data not shown). *p < 0.05 vs. vehicle.

Figure 9

Blockade of orexin 2 receptor (OX2R) but not orexin 1 receptor (OX1R) activity in the anterior paraventricular thalamus (aPVT) but not posterior paraventricular thalamus (pPVT) decreases intermittent-access 20% ethanol drinking. A. Ethanol drinking in rats injected in the aPVT with 0.3 µl of the OX1R antagonist SB 334867 (10 nmol) or dimethyl sulfoxide vehicle (Set 5, n = 6). B. Drinking in rats injected in the aPVT with 0.3 µl of the OX2R antagonist TCS OX2 29 (10 nmol) or saline vehicle (Set 5, n = 6). C. Ethanol drinking in rats injected in the pPVT with SB 334867 or vehicle (Set 5, n = 6). D. Drinking in rats injected in the pPVT with TCS OX2 29 or vehicle (Set 5, n = 6). Effects were no longer evident at four hours post-injection (data not shown). *p < 0.05 vs. vehicle.