Environmental enrichment alters nicotine-mediated locomotor sensitization and phosphorylation of DARPP-32 and CREB in rat prefrontal cortex

Abstract

Exposure within an environmental enrichment paradigm results in neurobiological adaptations and decreases the baseline of locomotor activity. The current study determined activation of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32) and CREB (cAMP response element binding protein), and locomotor activity in rats raised in enriched (EC), impoverished (IC), and standard (SC) conditions following repeated administration of nicotine or saline. In the saline-control group, the basal phosphorylation state of DARPP-32 at Threonine-34 site (pDARPP-32 Thr34) in the prefrontal cortex (PFC) was lower in EC compared to IC and SC rats, which was positively correlated with their respective baseline activities. While nicotine (0.35 mg/kg, freebase) produced locomotor sensitization across all housing conditions when the nicotine-mediated locomotor activity was expressed as a percent change from their respective saline control, EC rats displayed greater sensitization to nicotine than IC and SC rats. Consistent with the behavioral findings, repeated nicotine injection increased pDARPP-32 Thr34 in PFC of EC and IC rats and in nucleus accumbens of EC rats; however, the magnitude of change from saline control in nicotine-induced enhancement of pDARPP-32 Thr34 in PFC was strikingly increased in EC rats relative to IC rats. Moreover, EC rats had lower basal phosphorylation levels of CREB at serine 133 in PFC and nucleus accumbens compared to IC and SC rats, whereas the nicotine-induced increase in phosphorylated CREB-Ser133 was more pronounced in PFC of EC rats relative to IC and SC rats. Collectively, these findings suggest innovative insights into advancing our understanding of the molecular mechanisms of enrichment-induced changes in the motivational effects of nicotine, and aiding in the identification of new therapeutic strategies for tobacco smokers.

Figure 1. Environmental enrichment diminishes baseline activity during the habituation and saline baseline sessions.

Panels A, C, and E show the total horizontal activity (mean ± SEM) across the two 60-min habituation periods and 60-min session post-saline injection. Panels B, D, and F show the time course of the total horizontal activity (mean ± SEM) during each 5-min interval across the two 60-min habituation periods and 60-min session post saline injection. Total horizontal activity revealed a significant effect of housing condition (F_{(2, 70)} = 46.48, p<0.001), day (F_{(1, 70)} = 317.41, p<0.001) and time (F_{(11, 770)} = 148.57, p<0.001), and a significant housing condition × day × time interaction (F_{(22, 770)} = 2.48, p < 0.001). Overall total horizontal activity was lower in EC than in IC or SC (p<0.001, Bonferroni t-test). # p<0.001 denotes difference between EC and IC or SC groups (n = 22–27 rats/group).

Figure 2. The time-course data during the behavioral sensitization phase.

EC, IC or SC rats were administrated nicotine (Nic, 0.35 mg/kg, s.c.) or saline (Sal) on Days 1–15. Panels A and B show the total horizontal activity (mean ± SEM) in saline control and nicotine-treated groups during the 30 min pre-injection habituation period. Panels C and D shows the total horizontal activity (mean ± SEM) during the 60 min following saline or nicotine injection with robust behavioral sensitization observed under all housing conditions (panel D). Total horizontal activity after a subcutaneous injection of saline or nicotine revealed a significant effect of housing condition (F_{(2, 67)} = 104.29, p<0.001), treatment (F_{(1, 67)} = 333.57, p<0.001) and day (F_{(7, 469)} = 139.97, p<0.001). A significant interaction of housing condition × treatment × day (F_{(14, 469)} = 2.76, p<0.05) and treatment × day (F_{(7, 469)} = 115.42, p<0.05) were found. n = 12 rats/group.

Figure 3. The time-course data for total horizontal activity during day 1 and day 15 of the behavioral sensitization phase.

Panels A and B show the total horizontal activity (mean ± SEM) across the 60-min session. Data are presented as percent of their saline controls for respective housing condition. Panels C and D show the time course of the total horizontal activity (mean ± SEM) during each 5-min interval. * p<0.05 between EC and IC or SC groups. n = 12 rats/group.

Figure 4. Levels of pDARPP-32 Thr34 and total DARPP-32 in prefrontal cortex, nucleus accumbens and striatum in EC, IC, and SC rats.

Top panels show representative immunoblots of pDARPP-32 Thr34 and total DARPP-32 in prefrontal cortex (PFC, A), nucleus accumbens (NAc, B) and striatum (STR, C) in nicotine or saline-treated rats. Bottom panels show the ratio of levels of pDARPP-32 Thr34 to total DARPP-32 in PFC (D), NAc (E) and STR (F) of nicotine (0.35 mg/kg) or saline-treated rats. Data are presented as the percentage of pDARPP-32 Thr34 to total DARPP32 densitometry values of immunoreactivity. Histobars represent means and bars are SEM. Total DARPP-32 and pDARPP-32 Thr34 were run at the same time with the same loading amount of protein. No statistically significant differences were found with total DARPP-32 levels in any of the groups. *p<0.05 denotes difference between the nicotine- and saline-treated groups. #p<0.05 denotes difference between housing groups. n = 10 rats/group.

Figure 5. Levels of pCREB and total CREB in prefrontal cortex, nucleus accumbens, and striatum in EC, IC, and SC rats.

Top panels show representative Western blots of pCREB and CREB in prefrontal cortex (PFC, A), nucleus accumbens (NAc, B) and striatum (STR, C). Bottom panels show the ratio of phosphorylated CREB levels to total CREB in PFC (D), NAc (E) and STR (F) of nicotine (0.35 mg/kg) or saline-treated rats. Data are presented as the percentage of pCREB to total CREB densitometry values of immunoreactivity. Histobars represent means and bars are SEM. Total CREB and pCREB were run at the same time with the same loading amount of protein. No statistically significant differences were found with total CREB levels in any of the groups. *p<0.05 denotes difference between the nicotine- and saline-treated groups. #p<0.05 denotes difference between housing groups. n = 10 rats/group.

Figure 6. Correlations of the phosphorylation state of DARPP-32 at Thr34 in PFC with locomotor activity for EC, IC, and SC saline-treated rats.

Locomotor activity counts were collected from behavioral testing on day 15. Total horizontal activity is presented as mean values of each rat within the 60-min session. Phosphorylation levels of DARPP-32 at Thr34 are presented as the percentage of pDARPP-32 Thr34 to total DARPP-32 densitometry values of immunoreactivity. Dashed lines represent the 95% confidence interval of the linear regression fit (solid line). n = 10 rats/group.