Non-Contingent Exposure to Amphetamine in Adolescence Recruits miR-218 to Regulate Dcc Expression in the VTA

Abstract

The development of the dopamine input to the medial prefrontal cortex occurs during adolescence and is a process that is vulnerable to disruption by stimulant drugs such as amphetamine. We have previously linked the amphetamine-induced disruption of dopamine connectivity and prefrontal cortex maturation during adolescence to the downregulation of the Netrin-1 receptor, DCC, in dopamine neurons. However, how DCC expression in dopamine neurons is itself regulated is completely unknown. MicroRNA (miRNA) regulation of mRNA translation and stability is a prominent mechanism linking environmental events to changes in protein expression. Here, using male mice, we show that miR-218 is expressed in dopamine neurons and is a repressor of DCC. Whereas Dcc mRNA levels increase from early adolescence to adulthood, miR-218 exhibits the exact opposite switch, most likely maintaining postnatal Dcc expression. This dynamic regulation appears to be selective to Dcc since the expression of Robo 1, the other guidance cue receptor target of miR-218, does not vary with age. Amphetamine in adolescence, but not in adulthood, increases miR-218 in the VTA and this event is required for drug-induced downregulation of Dcc mRNA and protein expression. This effect seems to be specific to Dcc because amphetamine does not alter Robo1. Furthermore, the upregulation of miR-218 by amphetamine requires dopamine D2 receptor activation. These findings identify miR-218 as regulator of DCC in the VTA both in normal development and after drug exposure in adolescence.

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

“…In concordance with our and other studies, we define early adolescence in mice as the period between the day of weaning and PND 32 . While this range is not an absolute margin, but an age during which mice exhibit distinct neurobehavioral characteristics, this definition seems now to be a consensus in the rodent literature .…”

“…One week after completing the saline or amphetamine treatment regimens, different cohorts of mice were rapidly decapitated, and their brains were flash‐frozen in 2‐methylbutane (Fisher Scientific, Hampton, New Hampshire) chilled with dry ice. Bilateral punches of the VTA, NAcc, and PFC were excised from 1‐mm thick coronal slices starting from sections corresponding to plate 55 (−2.92 mm, anterior/posterior relative to Bregma) and 15 (1.94 mm, anterior/posterior relative to Bregma), respectively, of Paxinos and Franklin and processed for western blot as before . Briefly, protein samples (15 μg) were separated on a 10% SDS‐PAGE and transferred to a nitrocellulose membrane that was incubated overnight at 4°C with antibodies against DCC (1:1000, Cat#554223, BD Pharmingen, Mississauga, ON, Canada), Netrin‐1 (1:750 dilution, Cat#NB100‐1605, Novus Biologicals, Littleton, Colorado) and β‐actin (1:15000, Sigma‐Aldrich, Oakville, ON, Canada).…”

MiR-218: A Molecular Switch and Potential Biomarker of Susceptibility to Stress

“…Determining whether the source of miR-218 in blood is derived at least in part from the mPFC is a provocative and timely question, considering that miRNAs are secreted by cells into the extracellular space 16,45 . In comparison to other tissues, miR-218 is highly enriched in the brain 46 , mostly in the cortex, and cerebellum, with low but detectable levels in midbrain and striatal regions 26,40 . Our previous findings demonstrate that miR-218 alterations observed in susceptible mice are mPFC specific: mice susceptible to CSDS exhibit reduced levels of miR-218 in the mPFC, but not in the VTA 7 , a brain region highly involved in susceptibility to CSDS 47 .…”

“…A scrambled LNA oligonucleotide sequence was used as control (Antscrambled). Ant-miR-218 or Ant-scrambled were dissolved in sterile PBS (Sigma-Aldrich, Oakville, ON, Canada) at a final concentration of 0.3 mM, as indicated by 25,26 . We confirmed the efficacy and specificity of Ant-miR-218 ( Supplementary Figure 1) by quantitative PCR as in 26 .…”

“…Reduced expression of miR-218 in the mPFC is associated with depression in humans and susceptibility to CSDS in adult mice 7 . To address whether downregulation of mPFC levels of miR-218 directly causes increased susceptibility to CSDS, we used nucleic acid (LNA)-modified antisense oligonucleotides (antagomiRs) that hybridize miR-218 (Ant-miR-218) with high specificity in vivo, leading to its degradation 26 (Figure 1a; Supplementary Table 1; Supplementary Figure 1). For control micro-infusions, we used LNA-modified antisense oligonucleotides Circulating miR-218 as biomarker of stress susceptibility 14 containing a scrambled sequence (Ant-scrambled).…”