Presynaptic GABAB autoreceptor regulation of nicotinic acetylcholine receptor mediated [(3)H]-GABA release from mouse synaptosomes

Abstract

Activation of nicotinic acetylcholine receptors (nAChRs) can elicit neurotransmitter release from presynaptic nerve terminals. Mechanisms contributing to cell-and-terminal specific regulation of nAChR-mediated neurotransmitter exocytosis are not fully understood. The experiments discussed here examine how activation of GABAB auto- and hetero-receptors suppress nAChR-mediated release of [(3)H]-GABA and [(3)H]-dopamine ((3)H-DA) from mouse striatal synaptosomes. Activation of presynaptic GABAB receptors with (R)-baclofen decreased both [(3)H]-GABA and [(3)H]-DA release evoked by potassium depolarization. However, when nAChRs were activated with ACh to evoke neurotransmitter release, (R)-baclofen had no effect on [(3)H]-DA release, but potently inhibited ACh-evoked [(3)H]-GABA release. Inhibition of nAChR-evoked [(3)H]-GABA release by (R)-baclofen was time sensitive and the effect was lost after prolonged exposure to the GABAB agonist. The early inhibitory effect of GABAB activation on ACh-evoked [(3)H]-GABA release was partially attenuated by antagonists of the phosphatase, calcineurin. Furthermore, antagonists of protein kinase C (PKC) prevented the time-dependent loss of the inhibitory (R)-baclofen effect on [(3)H]-GABA release. These results suggest that α4β2*-nAChRs present on GABAergic nerve terminals in the striatum are subject to functional regulation by GABAB autoreceptors that is apparently cell-type specific, since it is absent from DAergic striatal nerve terminals. In addition, the functional modulation of α4β2*-type nAChRs on striatal GABAergic nerve terminals by GABAB autoreceptor activation is time-sensitive and appears to involve opposing actions of calcineurin and PKC.

Keywords: Calcineurin; GABA(B) receptor; Neurotransmitter release; Nicotinic receptor; Protein kinase C.

Figure 1

Application (10 minutes) of (R)-baclofen elicits a concentration-dependent decrease in the amount of [³H]-GABA released by depolarization with 10mM K⁺. (*)-denotes p<0.05, Dunnett’s post-hoc analysis.

Figure 2

Effect of 100μM (R)-baclofen on [³H]-GABA (a) and [³H]-DA release (b) evoked by increasing concentrations of K⁺. Neurotransmitter release was measured under control conditions (black bars) and with a 30 second exposure to 100μM (R)-baclofen (gray bars). Inhibition of both [³H]-GABA and [³H]-DA release by (R)-baclofen is apparently voltage dependent as strong depolarization (20mM K⁺) overcomes inhibition of neurotransmitter release. (*)-denotes p<0.05, student’s t-test.

Figure 3

Inhibition of [³H]-GABA release evoked by 10μM ACh with 100μM (R)-baclofen is time-dependent. The inhibitory effect of (R)-baclofen decreases over time with constant drug application, with a t_1/2 of 3.3 minutes. (*)-denotes p<0.05, Dunnett’s post-hoc analysis.

Figure 4

Application of (R)-baclofen inhibits [³H]-GABA release evoked by 10μM ACh in a concentration-dependent fashion when applied 30 seconds prior to stimulation with ACh. (*)-denotes p<0.05, Dunnett’s post-hoc analysis.

Figure 5

Effect of 100μM (R)-baclofen on ACh-evoked [³H]-GABA (a) and [³H]-DA (b) release. (a) [³H]-GABA release evoked by ACh under control conditions (black circles) and with a 30 second exposure to 100μM (R)-baclofen (white circles). Pretreatment with 100μM (R)-baclofen significantly decreased ACh-evoked [³H]-GABA release, and had no appreciable effect on ACh-evoked [³H]-DA release.

Figure 6

(a) 4μM CGP54626 completely eliminates the inhibitory effect of 100μM (R)-baclofen on [³H]-GABA release evoked by 30μM ACh from striatal synaptosomes, but has no effect on ACh-evoked [³H]-GABA release on its own (b).

Figure 7

100μM (R)-baclofen inhibits [³H]-GABA release evoked by ACh from synaptosomes prepared from interpeduncular nucleus (a), midbrain (b), cortex (c), and hippocampus (d). (*)-denotes p<0.05, 2-way ANOVA.

Figure 8

Inhibition of calcineurin with 100nM cypermethrin does not affect (R)-baclofen inhibition of [³H]-GABA release evoked by 10mM K⁺ (a), but incompletely prevents (R)-baclofen inhibition of [³H]-GABA release evoked by ACh (b). An additional calcineurin antagonist (FK506; 1μM) also prevents (R)-baclofen inhibition of ACh-evoked [3H]-GABA release to a similar extent as 100nM cypermethrin (c). (*)-denotes p<0.05, student’s t-test, (***)-denotes p=0.0003, paired t-test).

Figure 9

Loss of (R)-baclofen inhibition of nAChR agonist-evoked [³H]-GABA release after 5 minutes of continuous (R)-baclofen (100μM) exposure is prevented by antagonism of PKC with chelerythrine (10μM, 5 minutes prior to (R)-baclofen application). (*)-denotes p<0.05, student’s t-test.