Effect of environmental enrichment on dopamine and serotonin transporters and glutamate neurotransmission in medial prefrontal and orbitofrontal cortex

Abstract

Recent studies have reported that rats raised in an enriched condition (EC) have decreased dopamine transporter (DAT) function and expression in medial prefrontal cortex (mPFC), as well as increased d-amphetamine-induced glutamate release in nucleus accumbens compared to rats raised in an isolated condition (IC). In these previous studies, DAT function and expression were evaluated using mPFC pooled from four rats for each condition to obtain kinetic parameters due to sparse DAT expression in mPFC. In contrast, accumbal glutamate release was determined using individual rats. The current study extends the previous work and reports on the optimization of DAT and serotonin transporter (SERT) functional assays, as well as cell surface expression assays using both mPFC and orbitofrontal cortex (OFC) from individual EC or IC rats. In addition, the effect of d-amphetamine on glutamate release in mPFC and OFC of EC and IC rats was determined using in vivo microdialysis. Results show that environmental enrichment decreased maximal transport velocity (Vmax) for [(3)H]dopamine uptake in mPFC, but increased Vmax for [(3)H]dopamine uptake in OFC. Corresponding changes in DAT cell surface expression were not found. In contrast, Vmax for [(3)H]serotonin uptake and cellular localization of SERT in mPFC and OFC were not different between EC and IC rats. Further, acute d-amphetamine (2mg/kg, s.c.) increased extracellular glutamate concentrations in mPFC of EC rats only and in OFC of IC rats only. Overall, these results suggest that enrichment produces long-lasting alterations in mPFC and OFC DAT function via a trafficking-independent mechanism, as well as differential glutamate release in mPFC and OFC. Rearing-induced modulation of DAT function and glutamate release in prefrontal cortical subregions may contribute to the known protective effects of enrichment on drug abuse vulnerability.

Keywords: Dopamine transporter; Enrichment; Glutamate; Medial prefrontal cortex; Orbitofrontal cortex; Serotonin transporter.

Fig. 1. Environmental enrichment differentially alters maximal velocity (V_max) of [³H]DA uptake in medial prefrontal cortex (mPFC) and orbitofrontal cortex (OFC)

Data (mean ± S.E.M) for maximal velocity of [³H]DA uptake (V_max, top panel) and affinity of DA for DAT (K_m, bottom panel) are expressed as pmol/min/mg protein and nM, respectively. * p< 0.05, different from IC group. Both mPFC and OFC were obtained from individual rats in the EC and IC conditions. For the EC group, n = 8 for mPFC and n = 9 for OFC. For the IC group, n = 9 for mPFC and OFC.

Fig. 2. Environmental enrichment does not alter DAT immunoreactivity in total (I), non-biotinylated (II, intracellular) or biotinylated (III, cell surface) fractions from mPFC (left panel) or OFC (right panel)

Subcellular fractions of mPFC and OFC from EC and IC rats were assayed using biotinylation and immunoblotting. Top panels show representative immunoblots of DAT expression from mPFC and OFC of EC and IC rats. Immunoreactive bands were within the linear range of detection. Bottom panels show mean ± SEM densitometry values for DAT immunoreactivity. β-Actin was used to monitor protein loading. Both mPFC and OFC were obtained from individual rats within the EC and IC conditions. For EC and IC groups, n = 8 rats for mPFC and OFC.

Fig. 3. Environmental enrichment does not alter V_max or K_m values for [³H]5-HT uptake in mPFC or OFC obtained from EC and IC rats

Data (mean ± S.E.M) for V_max (top panel) and K_m (bottom panel) are presented as pmol/min/mg protein and nM, respectively. Both mPFC and OFC were obtained from individual rats in the EC and IC conditions. For EC and IC groups, n = 9 rats for mPFC and OFC.

Fig 4. Environmental enrichment did not alter SERT immunoreactivity in mPFC or OFC from EC and IC rats

Top panel shows representative immunoblots of SERT expression in mPFC and OFC from EC and IC groups. Immunoreactive bands were within the linear range of detection. Bottom panel shows mean ± SEM of densitometry values for SERT immunoreactivity. β-Actin was used to monitor protein loading. Both mPFC and OFC were obtained from individual rats within the EC and IC conditions. For EC and IC groups, n = 8 rats for mPFC and OFC.

Fig 5. Histological placements of microdialysis probes in mPFC and OFC

Locations of microdialysis probe placements in mPFC and OFC as indicated by vertical lines. Numbers to the right indicate distance in mm from bregma according to Paxinos and Watson (1998). The active probe protruded 2 mm below the distal end of the guide cannulae for mPFC (top panels) and OFC (bottom panels). Probe placements for EC (left panels) and IC rats (right panels) are identified in two separate sections of both mPFC and OFC. Probe placements in EC and IC rats indicated by dark black and gray vertical lines, respectively.

Fig 6. Environmental enrichment modulated the effect of amphetamine on extracellular glutamate concentrations in mPFC and OFC

Top panel shows the time course of the effect of acute d-amphetamine (AMPH, 2 mg/kg, s.c.) on extracellular glutamate concentrations in mPFC of EC and IC rats. Bottom panel shows the time course of the effect of acute d-amphetamine (AMPH, 2 mg/kg, s.c.) on extracellular glutamate concentrations in OFC of EC and IC rats. After collection of basal samples, rats were injected with saline (SAL), as indicated by the left arrow, and then 60 min later, AMPH was injected, as indicated by the right arrow. Data are mean ± SEM percent change from control. For the EC group, n = 6 for mPFC and n = 4 for OFC. For the IC group, n = 5 for mPFC and OFC.