Mu opioid receptor modulation of somatodendritic dopamine overflow: GABAergic and glutamatergic mechanisms

Abstract

Mu opioid receptor (MOR) regulation of somatodendritic dopamine neurotransmission in the ventral tegmental area (VTA) was investigated using conventional microdialysis in freely moving rats and mice. Reverse dialysis of the MOR agonist DAMGO (50 and 100 microm) into the VTA of rats produced a concentration-dependent increase in dialysate dopamine concentrations. Basal dopamine overflow in the VTA was unaltered in mice lacking the MOR gene. However, basal gamma-aminobutyric acid (GABA) overflow in these animals was significantly increased, whereas glutamate overflow was decreased. Intra-VTA perfusion of DAMGO into wild-type (WT) mice increased dopamine overflow. GABA concentrations were decreased, whereas glutamate concentrations in the VTA were unaltered. Consistent with the loss of MOR, no effect of DAMGO was observed in MOR knockout (KO) mice. These data provide the first direct demonstration of tonically active MOR systems in the VTA that regulate basal glutamatergic and GABAergic neurotransmission in this region. We hypothesize that increased GABAergic neurotransmission following constitutive deletion of MOR is due to the elimination of a tonic inhibitory influence of MOR on GABAergic neurons in the VTA, whereas decreased glutamatergic neurotransmission in MOR KO mice is a consequence of intensified GABA tone on glutamatergic neurons and/or terminals. As a consequence, somatodendritic dopamine release is unaltered. Furthermore, MOR KO mice do not exhibit the positive correlation between basal dopamine levels and the glutamate/GABA ratio observed in WT mice. Together, our findings indicate a critical role of VTA MOR in maintaining an intricate balance between excitatory and inhibitory inputs to dopaminergic neurons.

Figure 1

Pharmacological validation of the separation of glutamate (A) and GABA (B) by the CE-LIFD technique employed. Glutamate and GABA uptake inhibitors (tPDC and NO-711, respectively) were administered via reverse dialysis in rats. Microdialysis samples were collected every 10 min. Ordinate: GABA and glutamate concentration expressed as percentage (mean ± SEM) of the baseline values (n = 3 animals per group). Abscissa: time in min. * denotes a significant difference from baseline for glutamate and ⁺ denotes a significant difference from baseline for GABA (p ≤ 0.05, Tukey test).

Figure 2

Concentration-dependent increases in somatodendritic dopamine overflow following reverse dialysis of the MOR agonist DAMGO into the VTA in rats. A – Time course of dialysate dopamine levels before and following reverse dialysis of DAMGO (50 and 100 μM, black circles and black squares, respectively) into the VTA. Ordinate: dopamine concentration in nM. Data are expressed as the means ± S.E.M., n -number of animals in each experimental group (6–7 animals per group). Abscissa: time in min. Microdialysis samples were collected every 10 min. * and ** denote significant differences between basal and drug-evoked levels for two concentrations of DAMGO accordingly (p ≤ 0.05, Tukey test). B – Bar graphs of AUC values for the dopamine response to DAMGO expressed as means ± S.E.M.

Figure 3

Somatodendritic dopamine overflow during reverse-dialysis of the MOR agonist DAMGO into the VTA of WT and MOR KO mice. A – Time course of dialysate dopamine levels before and following reverse dialysis of DAMGO (50 μM; black circles – WT, white circles – KO) into the VTA. Data are expressed as the means ± S.E.M., n - number of animals in each experimental group (5 animals per group). Other details as in Fig. 2..B – Bar graphs of basal VTA dopamine levels in WT and MOR KO mice expressed as the means ± S.E.M. C – Bar graphs of AUC values for the dopamine response to DAMGO in WT and MOR KO mice expressed as the means ± S.E.M. * denotes a significant difference in DAMGO-induced dopamine responses between WT and KO animals (p ≤ 0.05).

Figure 4

GABA neurotransmission during retro-dialysis of the MOR agonist DAMGO into the VTA of WT and MOR KO mice. A and B – Time course of dialysate GABA levels before and following reverse dialysis of 50 and 100 μM DAMGO into the VTA (black circles – WT, white circles – KO). Data are expressed as the means ± S.E.M., n - number of animals in each experimental group (5 animals per group). Other details as in Fig. 2..C – Bar graphs of basal VTA GABA levels in WT and MOR KO mice expressed as the means ± S.E.M. D – Bar graphs of AUC values for the GABA response to DAMGO in WT and MOR KO mice expressed as the means ± S.E.M. * denotes a significant difference in basal GABA levels between WT and KO animals (p ≤ 0.05).

Figure 5

Glutamate neurotransmission during retro-dialysis of the MOR agonist DAMGO into the VTA of WT and MOR KO mice. A and B – Time course of dialysate glutamate levels before and following reverse dialysis of 50 and 100 μM DAMGO (black circles – WT, white circles – KO) into the VTA. Data are expressed as the means ± S.E.M., n - number of animals in each experimental group (5 animals per group). Other details as in Fig. 2..C – Bar graphs of basal VTA glutamate levels in WT and MOR KO mice expressed as the means ± S.E.M. D – Bar graphs of AUC values for the glutamate response to DAMGO in WT and MOR KO mice expressed as the means ± S.E.M. * denotes a significant difference in basal glutamate levels between WT and KO animals (p ≤ 0.05).

Figure 6

Correlation analysis of the relationship between basal glutamate/GABA ratio and dialysate dopamine concentrations in the VTA of WT (A) and MOR KO (B) mice. Each graph shows regression lines, regression equation, and correlation coefficient. Significant positive correlation between basal glutamate/GABA ratio and dopamine levels was observed only in WT (n = 6, r = 0.83, P < 0.05) but not in MOR KO animals (n = 5, r = 0.53, P > 0.05).