Amphetamine in adolescence disrupts the development of medial prefrontal cortex dopamine connectivity in a DCC-dependent manner

Abstract

Initiation of drug use during adolescence is a strong predictor of both the incidence and severity of addiction throughout the lifetime. Intriguingly, adolescence is a period of dynamic refinement in the organization of neuronal connectivity, in particular medial prefrontal cortex (mPFC) dopamine circuitry. The guidance cue receptor, DCC (deleted in colorectal cancer), is highly expressed by dopamine neurons and orchestrates their innervation to the mPFC during adolescence. Furthermore, we have shown that amphetamine in adolescence regulates DCC expression in dopamine neurons. Drugs in adolescence may therefore induce their enduring behavioral effects via DCC-mediated disruption in mPFC dopamine development. In this study, we investigated the impact of repeated exposure to amphetamine during adolescence on both the development of mPFC dopamine connectivity and on salience attribution to drug context in adulthood. We compare these effects to those induced by adult exposure to an identical amphetamine regimen. Finally, we determine whether DCC signaling within dopamine neurons is necessary for these events. Exposure to amphetamine in adolescence, but not in adulthood, leads to an increase in the span of dopamine innervation to the mPFC, but a reduction of presynaptic sites present on these axons. Amphetamine treatment in adolescence, but not in adulthood, also produces an increase in salience attribution to a previously drug-paired context in adulthood. Remarkably, DCC signaling within dopamine neurons is required for both of these effects. Drugs of abuse in adolescence may therefore induce their detrimental behavioral consequences by disrupting mesocortical dopamine development through alterations in the DCC signaling cascade.

Figure 1

Exposure to amphetamine in adolescence results in altered dopamine connectivity in the adult medial prefrontal cortex. (a) Timeline of treatment in adolescence and experimental procedures. n=5–6 per group. (b) Regions of interest in the medial prefrontal cortex (mPFC) were outlined according to the Mouse Brain Atlas (Paxinos and Franklin, 2008) and are highlighted in red. The cingulate, prelimbic, and infralimbic subregions of the mPFC were analyzed. (c) Left panel: micrograph of a coronal section of the pregenual mPFC at a low magnification ( × 5) showing an overlay of the contours traced to delineate subregions of interest. Scale bar=500 μm. Right panel: micrograph of a coronal section of the pregenual mPFC a high magnification ( × 100) showing the tyrosine hydroxylase (TH)-positive varicosities. Scale bar=10 μm. (d) Left panel: volume of the TH-positive fiber innervation (mean±SEM) to the inner layers of the mPFC. Mice treated with AMPH in adolescence show increased fiber volume relative to their saline counterparts (significant main effect of treatment, p=0.0075). Middle panel: density of TH-positive varicosities to the inner layers (mean±SEM) of the mPFC. Mice treated with AMPH in adolescence show decreased varicosity density relative to their saline counterparts (significant main effect of treatment, p=0.01). Right panel: total number of TH-positive varicosities to the inner layers of the mPFC (mean±SEM). Mice treated with AMPH in adolescence show decreased total number of varicosities relative to their saline counterparts (significant main effect of treatment, p=0.03). (e) Schematic representation of fiber expanse and varicosity density in the mPFC of adult mice exposed to AMPH or saline in adolescence. A putative axon is superimposed over both schematics. (f) Timeline of treatment in adolescence and analysis of netrin-1 expression by qPCR. n=5–6 per group. (g) Netrin-1 mRNA expression normalized to GAPDH in the mPFC. There were no differences in relative Netrin-1 mRNA expression in the mPFC between mice that received amphetamine (AMPH) or saline in adolescence (from PND22±1—PND31±1) when examined 1 week later (PND38±1).

Figure 2

Exposure to amphetamine during adulthood does not alter dopamine connectivity in the medial prefrontal cortex. (a) Timeline of treatment in adulthood and experimental procedures, n=8 per group. (b) Regions of interest in the medial prefrontal cortex (mPFC) were outlined according to the Mouse Brain Atlas (Paxinos and Franklin, 2008) and are highlighted in blue. The cingulate, prelimbic, and infralimbic subregions of the mPFC were analyzed. (c) Left panel: volume of the TH-positive fiber innervation (mean±SEM) to the inner layers of the mPFC. There were no differences in the volume of TH-positive fiber innervation to the mPFC inner layers between mice that received AMPH or saline in adulthood. Right panel: density of TH-positive varicosities to the inner layers (mean±SEM) of the mPFC. The density of TH-positive varicosities to the mPFC inner layers was not different between mice that received AMPH or saline in adulthood. (d) Schematic representation of fiber expanse and varicosity density in the mPFC of adult mice exposed to AMPH or saline in adulthood. A putative axon is superimposed over both schematics.

Figure 3

DCC signaling within dopamine neurons is necessary for the effects of amphetamine in adolescence on dopamine connectivity in the medial prefrontal cortex. (a) Timeline of treatment in adolescence and experimental procedures for dcc^lox/loxDAT^Cre mice. n=5–9 per group. (b) Regions of interest in the medial prefrontal cortex (mPFC) were outlined according to the Mouse Brain Atlas (Paxinos and Franklin, 2008) and are highlighted in green. The cingulate, prelimbic, and infralimbic subregions of the mPFC were analyzed. (c) Left panel: volume of the TH-positive fiber innervation (mean±SEM) to the inner layers of the mPFC. There were no differences in the volume of TH-positive fiber innervation to the mPFC inner layers between dcc^lox/loxDAT^Cre mice that received AMPH or saline in adolescence. Right panel: density of TH-positive varicosities to the inner layers (mean±SEM) of the mPFC. The density of TH-positive varicosities to the mPFC inner layers was not different between dcc^lox/loxDAT^Cre mice that received AMPH or saline during adolescence.

Figure 4

Exposure to amphetamine in adolescence does not result in altered dopamine connectivity in the nucleus accumbens in adulthood. (a) The nucleus accumbens (NAcc) was outlined according to the Mouse Brain Atlas (Paxinos and Franklin, 2008) and highlighted in red. (b) Top panel: micrograph of a coronal section of the pregenual NAcc at a low magnification ( × 5) showing an overlay of the contour traced to delineate this area. Scale bar=500 μm. Bottom panel: micrograph of a coronal section of the pregenual NAcc a high magnification ( × 100) showing the tyrosine hydroxylase (TH)-positive varicosities. Scale bar=10 μm. (c) Left panel: volume of the TH-positive fiber innervation (mean±SEM) to the NAcc. There were no differences in the volume of TH-positive fiber innervation to the NAcc between mice that received amphetamine (AMPH) or saline in adolescence (from PND22±1—PND31±1) when examined in adulthood (PND75±15). Right panel: density of TH-positive varicosities to the NAcc (mean±SEM). The density of TH-positive varicosities to the NAcc was not different between mice that received AMPH or saline in adolescence. (d) Left panel: volume of the TH-positive fiber innervation (mean±SEM) to the NAcc. There were no differences in the volume of TH-positive fiber innervation to the NAcc between mice that received AMPH or saline in adulthood (from PND75±15—PND84±15) when examined after an approximately 6-week interval following treatment termination (PND120±15). Right panel: density of TH-positive varicosities to the NAcc (mean±SEM). The density of TH-positive varicosities to the NAcc was not different between mice that received AMPH or saline in adulthood. n=4–5 for all groups.

Figure 5

Amphetamine exposure in adolescence, but not in adulthood, leads to augmented salience attribution and requires dcc in dopamine neurons. Salience attribution was quantified by calculating the total locomotor activity across a 15-min test of conditioned locomotor activity (mean±SEM). (a) Mice exposed to amphetamine (AMPH) during adolescence (from PND 22±1 to PND 31±1) display augmented salience attribution when re-exposed to the previously drug-paired chamber in adulthood (PND 75±15), in comparison with their saline controls (n=5 per group; * indicates p=0.006). (b) Mice exposed to AMPH (n=7) or saline (n=9) during adulthood (from PND 75±15 to PND 845±1) show no differences in salience attribution when re-exposed to a previously drug-paired chamber approximately 6 weeks (∼PND 120±15) after treatment termination. (c) dcc^lox/loxDAT^Cre mice (n=8 per group) exposed to AMPH or saline during adolescence (from PND 22±1 to PND 31±1) show no differences in salience attribution when re-exposed to a previously drug-paired chamber in adulthood (PND 75±15).