DCC-related developmental effects of abused- versus therapeutic-like amphetamine doses in adolescence

Abstract

The guidance cue receptor DCC controls mesocortical dopamine development in adolescence. Repeated exposure to an amphetamine regimen of 4 mg/kg during early adolescence induces, in male mice, downregulation of DCC expression in dopamine neurons by recruiting the Dcc microRNA repressor, microRNA-218 (miR-218). This adolescent amphetamine regimen also disrupts mesocortical dopamine connectivity and behavioral control in adulthood. Whether low doses of amphetamine in adolescence induce similar molecular and developmental effects needs to be established. Here, we quantified plasma amphetamine concentrations in early adolescent mice following a 4 or 0.5 mg/kg dose and found peak levels corresponding to those seen in humans following recreational and therapeutic settings, respectively. In contrast to the high doses, the low amphetamine regimen does not alter Dcc mRNA or miR-218 expression; instead, it upregulates DCC protein levels. Furthermore, high, but not low, drug doses downregulate the expression of the DCC receptor ligand, Netrin-1, in the nucleus accumbens and prefrontal cortex. Exposure to the low-dose regimen did not alter the expanse of mesocortical dopamine axons or their number/density of presynaptic sites in adulthood. Strikingly, adolescent exposure to the low-dose drug regimen does not impair behavioral inhibition in adulthood; instead, it induces an overall increase in performance in a go/no-go task. These results show that developmental consequences of exposure to therapeutic- versus abused-like doses of amphetamine in adolescence have dissimilar molecular signatures and opposite behavioral effects. These findings have important clinical relevance since amphetamines are widely used for therapeutic purposes in youth.

Keywords: Netrin-1; Prefrontal cortex; cognitive control; guidance cues.

FIGURE 1

Peak plasma concentrations achieved by an intraperitoneal injection of 0.5 or 4.0 mg/kg of d-amphetamine (AMPH) correspond to those measured in humans in therapeutic and recreational settings, respectively. A, Diagram describing AMPH regimen and the different time points for plasma collection. B, and C, Bioanalysis of d-amphetamine in plasma observed over time after a single intraperitoneal injection of 0.5 or 4 mg/kg dose (n = 4 per time point). B, Plasma concentration of AMPH showed a maximum concentration of 97 ± 21 ng/mL 5 minutes after low-dose injection. C, Plasma concentration of AMPH showed a maximum concentration of 1300 ± 79 ng/mL 5 minutes after high-dose injection

FIGURE 2

Exposure to a therapeutic-like dose of amphetamine in adolescence upregulates DCC protein expression without altering miR-218 or Dcc mRNA levels in the ventral tegmental area (VTA) and does not change Netrin-1 in dopamine terminal regions. A, Timeline of treatment and experimental procedures. B, Locomotor activity during the 90-minute test performed after each treatment injection. C, DCC expression is significantly increased in the VTA 1 week after the treatment with 0.5 mg/kg of amphetamine in adolescence. Inset: levels of DCC protein expression in the VTA in animals treated with 4 mg/kg of amphetamine, using the exact same schedule. In contrast to animals exposed to 4 mg/kg (D and E insets), animals treated with 0.5 mg/kg of amphetamine showed no changes in D, Dcc mRNA or E, miR-218 in the VTA compared with saline controls. F, and G, Netrin-1 expression in nucleus accumbens (NAcc) and prefrontal cortex (PFC) 1 week after termination of treatment with 0.5 or 4.0 mg/kg of amphetamine. No changes were observed in Netrin-1 expression levels in the F, NAcc or in the G, PFC in mice exposed to 0.5 mg/kg of amphetamines in comparison with saline-treated ones. Mice exposed to 4 mg/kg of amphetamine during adolescence showed a downregulation on Netrin-1 protein expression in the F, NAcc and the G, PFC compared with saline-treated controls. *Significantly different from saline group, P < .05. The data presented in the insets are reproduced from Cuesta et al. All data are shown as mean ± SEM

FIGURE 3

Low amphetamine doses in adolescence do not alter mesocortical dopamine connectivity in adulthood. A, Timeline of treatment and experimental procedures. B, Schematic representation of the regions of interest in the medial prefrontal cortex outlined according to the Mouse Brain Atlas (Paxinos and Franklin, 2008). The cingulate (Cg1), prelimbic (PrL), and infralimbic (IL) subregions of the medial prefrontal cortex were analyzed. C, Volume, D, total number, and E, density of the TH-positive fiber innervation to the inner layers of the medial prefrontal cortex. Mice treated with 0.5 mg/kg of AMPH in adolescence do not show significant differences to their saline counterparts. All data are shown as mean ± SEM (n = 4-6 per group)

FIGURE 4

Low doses of amphetamine in adolescence improve overall cognitive performance in adulthood. A, Timeline of treatment and experimental procedures. B, Diagram of the go/no-go task adapted for mice. (C-E) There are no differences in the number of C, commission errors, D, omission errors, or E, “hits” between animals treated with 0.5 mg/kg or saline during adolescence. However, amphetamine exposure during adolescence induce a significant increase in the correct response rate across the test, when compared with saline-treated ones F. All data are shown as mean ± SEM (n = 8-9 per group)