Multiple muscarinic acetylcholine receptor subtypes modulate striatal dopamine release, as studied with M1-M5 muscarinic receptor knock-out mice

Abstract

A proper balance between striatal muscarinic cholinergic and dopaminergic neurotransmission is required for coordinated locomotor control. Activation of striatal muscarinic acetylcholine receptors (mAChRs) is known to modulate striatal dopamine release. To identify the mAChR subtype(s) involved in this activity, we used genetically altered mice that lacked functional M1-M5 mAChRs [knock-out (KO) mice]. In superfused striatal slices from wild-type mice, the non-subtype-selective muscarinic agonist oxotremorine led to concentration-dependent increases in potassium-stimulated [3H]dopamine release (by up to 60%). The lack of M1 or M2 receptors had no significant effect on the magnitude of these responses. Strikingly, oxotremorine-mediated potentiation of stimulated striatal [3H]dopamine release was abolished in M4 receptor KO mice, significantly increased in M3 receptor-deficient mice, and significantly reduced (but not abolished) in M5 receptor KO mice. Additional release studies performed in the presence of tetrodotoxin suggested that the dopamine release-stimulating M4 receptors are probably located on neuronal cell bodies, but that the release-facilitating M5 and the release-inhibiting M3 receptors are likely to be located on nerve terminals. Studies with the GABA(A) receptor blocker bicuculline methochloride suggested that M3 and M4 receptors mediate their dopamine release-modulatory effects via facilitation or inhibition, respectively, of striatal GABA release. These results provide unambiguous evidence that multiple mAChR subtypes are involved in the regulation of striatal dopamine release. These findings should contribute to a better understanding of the important functional roles that the muscarinic cholinergic system plays in striatal function.

Fig. 1.

Effect of oxotremorine on potassium-stimulated [³H]dopamine release in striatal slices from M₁–M₄ mAChR KO mice and corresponding WT controls. Striatal slices that had been preincubated with [³H]dopamine were depolarized with 20 mm KCl, and the resulting [³H]dopamine outflow was quantitated in the absence and in the presence of the indicated concentrations of oxotremorine. Data are expressed as the percentage increase in [³H]dopamine release above control levels (no oxotremorine). The WT curves shown inC and D are identical. Because the M₃ and M₄ mAChR KO mice were both 129/SvEv × CF₁ hybrids (50 of 50), only one WT strain of the same genetic background was tested in parallel with these mutant mice. Eachdata point represents the mean ± SEM from 6-11 independent experiments (mice). Asterisks indicate significant differences between responses in KO versus WT preparations (*p < 0.05; **p < 0.01; Student's t test followed by the Holm correction for multitesting adjustment).

Fig. 2.

Effect of TTX on oxotremorine-mediated modulation of potassium-stimulated [³H]dopamine release in striatal slices from WT and M₃ receptor KO mice. In the presence of TTX (600 nm), oxotremorine had no significant effect on stimulated [³H]dopamine release in WT preparations (A) but induced a significant enhancement in [³H]dopamine output in striatal slices from M₃ receptor KO mice (B). Each bar represents the mean ± SEM of S₂/S₁ values from six or seven independent experiments (mice). Micromolar concentrations are shown.Asterisks indicate significant differences from the control group (no oxotremorine) (*p < 0.05; **p < 0.01; one-way ANOVA followed by Dunnett's test).

Fig. 3.

Effect of TTX on oxotremorine-mediated modulation of potassium-stimulated [³H]dopamine release in striatal slices from M₅ receptor KO mice. In the presence of TTX (600 nm), oxotremorine induced a decrease in potassium-stimulated [³H]dopamine release in striatal slices from M₅ receptor KO mice (A). This effect was blocked by the GABA_A receptor antagonist bicuculline methochloride (B). Each bar represents the mean ± SEM of S₂/S₁ values from eight independent experiments (mice). Micromolar concentrations are shown. Asterisks indicate significant differences from the control group (no drug) (*p < 0.05; **p < 0.01; one-way ANOVA followed by Dunnett's test).

Fig. 4.

Effect of the GABA_Areceptor antagonist bicuculline methochloride on oxotremorine-mediated enhancement of potassium-stimulated [³H]dopamine release in striatal slices from WT mice. When administered alone, bicuculline methochloride (100 μm) mimicked the release-facilitating effect of oxotremorine; however, when coadministered with oxotremorine, bicuculline methochloride did not further enhance [³H]dopamine output. Eachbar represents the mean ± SEM of S₂/S₁ values from eight independent experiments (mice). Micromolar concentrations are shown.Asterisks indicate significant differences from the control group (no drug) (** p < 0.01; one-way ANOVA followed by Dunnett's test).