Reduced inhibitory action of a GABAB receptor agonist on [3H]-dopamine release from rat ventral tegmental area in vitro after chronic nicotine administration

Abstract

Background: The activation of GABAB receptors in the ventral tegmental area (VTA) has been suggested to attenuate the rewarding properties of psychostimulants, including nicotine. However, the neurochemical mechanism that underlie this effect remains unknown. Since GABAB receptors modulate the release of several neurotransmitters in the mammalian brain, we have characterised the effect of the GABAB receptor agonist baclofen on the release of [3H]-dopamine ([3H]-DA) from VTA slices of naive rats and of rats pre-treated with nicotine.

Results: In naive rats, baclofen concentration-dependently inhibited the electrically evoked release of [3H]-DA from the isolated VTA (EC50 = 0.103 microM, 95% CI = 0.043-0.249), without affecting the basal [3H]-monoamine overflow. This effect was mediated by activation of GABAB receptors as it was blocked by the selective receptor antagonist CGP55845A. Chronic administration of nicotine (0.4 mg kg(-1), s.c., for 14 days) affected neither the basal nor the electrically evoked release of [3H]-DA from VTA slices. However, the inhibitory effect of baclofen (10 microM) on the stimulated [3H]-monoamine overflow was abolished in rats pre-treated with nicotine as compared to saline-injected controls.

Conclusions: Our results demonstrate that GABAB receptor activation reduces the release of DA from the rat VTA. In addition, a reduced sensitivity of VTA GABAB receptors appears to develop after chronic exposure to nicotine. The resulting disinhibition of VTA DA neurones might therefore contribute to the sensitised dopaminergic responses observed in the rat mesocorticolimbic system following repeated administration of nicotine.

Figure 2

Calcium-dependence and tetrodotoxin (TTX) sensitivity of the electrically evoked release of [³H]-DA from ventral tegmental area slices. Electrical stimulation (20 mA, 2 Hz, 4 min) occurred 14 min (S1) and 59 min (S2) after the beginning of sample collection. Slices were perfused with buffer alone (control), calcium-free medium containing 1 mM EGTA (Ca²⁺-free), or buffer containing 1 μM TTX for 15 min before and during S1 (black bar). After 18 min from the beginning of sample collection, all samples were superfused with normal buffer. Values represent mean ± s.e.mean (n = 3 rats).

Figure 3

Baclofen-mediated inhibition of the electrically evoked [³H]-DA release from ventral tegmental area slices of naïve rats (EC₅₀ = 0.103 μM, 95% CI = 0.043–0.249). Baclofen (0.01–100 μM) was added to the superfusion buffer 36 min before and during S2. Values represent mean ± s.e.mean (n = 4–7 rats).

Figure 4

Reversal of baclofen-mediated inhibition of the evoked [³H]-DA release from VTA slices by the GABA_B antagonist CGP55845A. Baclofen (Bacl, 10 μM) and/or CGP55845A (CGP, 1 μM) were added to the superfusion buffer 36 min before and during S2. Drug effects were assessed by comparing the ratio S2/S1 in the presence and absence of the drug, respectively. Values represent mean ± s.e.mean (n = 6–10 rats). *p < 0.05 vs control (one-way ANOVA, Dunnett's post hoc test).

Figure 5

Effect of nicotine pre-treatment on basal and electrically evoked release of [³H]-DA from VTA slices. The rats received daily subcutaneous injections of nicotine (0.4 mg kg^-1, n = 6) or saline (n = 6) for 14 consecutive days and the experiments were performed 24 hours after the last injection. An electrical stimulation (20 mA, 2 Hz, 4 min, black bar) was applied to the VTA slices 14 min after the beginning of sample collection. The data are expressed as fractional [³H]-DA release and represented as mean ± s.e.mean.

Figure 6

Effect of baclofen on the electrically evoked release of [³H]-DA from VTA slices of rats pretreated with nicotine or saline. The rats received daily subcutaneous injections of nicotine (0.4 mg kg^-1, n = 5) or saline (n = 5) for 14 consecutive days and the experiments were performed 24 hours after the last injection. Two electrical stimulations (20 mA, 2 Hz, 4 min) were applied to the slices 14 min (S1) and 59 min (S2) after the beginning of sample collection. Baclofen (10 μM) was added to the superfusion buffer 36 min before and during S2. Control tissue was perfused with buffer alone during both S1 and S2. Data are expressed as mean ± s.e.mean and compared by ANOVA for repeated measures. **p < 0.01 vs saline-control (Bonferroni post-test).

Figure 1

Photomicrograph of a representative coronal section of rat brain labelled with a polyclonal antibody directed against tyrosine hydroxylase. The picture shows the orientation of the cuts (dark lines) for the dissection of the ventral tegmental area (VTA). The basal cerebral peduncle (Cp) was used as a visual landmark to obtain the coronal section containing the VTA. To remove the substantia nigra (pars compacta, SNC, and reticulata, SNR) from this slice, two transversal cuts were made along the medial lemniscus (Ml) on either side. Finally, the VTA was dissected out by making a horizontal cut 1.5 mm dorsal to the ventral edge of the section and stripped of the interpeduncular nucleus (IP) by cutting 0.5 mm above the same edge. Scale bar is 1 mm.