Characterization of central inhibitory muscarinic autoreceptors by the use of muscarinic acetylcholine receptor knock-out mice

Abstract

Forebrain muscarinic acetylcholine (ACh) receptors (mAChRs; M1-M5) are predicted to play important roles in many fundamental central functions, including higher cognitive processes and modulation of extrapyramidal motor activity. Synaptic ACh levels are known to be regulated by the activity of presynaptic muscarinic autoreceptors mediating inhibition of ACh release. Primarily because of the use of ligands with limited receptor subtype selectivity, classical pharmacological studies have led to conflicting results regarding the identity of the mAChR subtypes mediating this activity in different areas of the brain. To investigate the molecular identity of hippocampal, cortical, and striatal inhibitory muscarinic autoreceptors in a more direct manner, we used genetically altered mice lacking functional M2 and/or M4 mAChRs [knock-out (KO) mice]. After labeling of cellular ACh pools with [3H]choline, potassium-stimulated [3H]ACh release was measured in superfused brain slices, either in the absence or the presence of muscarinic drugs. The nonsubtype-selective muscarinic agonist, oxotremorine (0.1-10 microm), inhibited potassium-stimulated [3H]ACh release in hippocampal, cortical, and striatal slices prepared from wild-type mice by up to 80%. This activity was totally abolished in tissues prepared from M2-M4 receptor double KO mice. Strikingly, release studies with brain slices from M2 and M4 receptor single KO mice indicated that autoinhibition of ACh release is mediated primarily by the M2 receptor in hippocampus and cerebral cortex, but predominantly by the M4 receptor in the striatum. These results, together with additional receptor localization studies, support the novel concept that autoinhibition of ACh release involves different mAChRs in different regions of the brain.

Fig. 1.

Separation of radiolabeled choline and ACh by reverse-phase HPLC followed by liquid scintillation spectrometry.A, Standards. The radiochromatogram shown here was obtained after injection of a solution (100 μl) that contained 11,590 dpm [³H]choline and 24,850 dpm [¹⁴C]ACh. When the two radiolabeled compounds were injected alone, the retention times were similar to those observed in the coinjection experiments (data not shown). B, Representative radiochromatogram showing KCl-dependent [³H]choline and [³H]ACh release from striatal slices from WT mice. Superfused striatal slices prepared from M₄ receptor WT mice were prelabeled with [³H]choline and stimulated with KCl as described in Materials and Methods. The superfusion medium contained 100 μm physostigmine. The depicted radiochromatogram was obtained after injection of 100 μl of the fraction (fraction 5) collected immediately after K⁺ stimulation (total [³H] content of the 100 μl aliquot: 3223 dpm). Note that >90% of the [³H] outflow represents authentic [³H]ACh.

Fig. 2.

Effect of oxotremorine on potassium-stimulated [³H]ACh release in hippocampal slices from M₂–M₄ receptor double KO, M₂receptor single KO, and M₄ receptor single KO mice (A–C, bottom panels) and their corresponding WT control mice (A–C, top panels). Each bar represents the mean ± SEM of S2/S1 values from 6–11 independent experiments (mice). Concentrations shown are micromolar. Asterisks indicate significant differences from the control group (no drug) (*p < 0.05; **p < 0.01).

Fig. 3.

Effect of oxotremorine on potassium-stimulated [³H]ACh release in cortical slices from M₂–M₄ receptor double KO, M₂receptor single KO, and M₄ receptor single KO mice (A–C, bottom panels) and their corresponding WT control mice (A–C, top panels). Each bar represents the mean ± SEM of S2/S1 values from 6–11 independent experiments (mice). Concentrations shown are micromolar. Asterisks indicate significant differences from the control group (no drug) (*p < 0.05; **p < 0.01).

Fig. 4.

Effect of oxotremorine on potassium-stimulated [³H]ACh release in striatal slices from M₂–M₄ receptor double KO, M₂receptor single KO, and M₄ receptor single KO mice (A–C, bottom panels) and their corresponding WT control mice (A–C, top panels). Each bar represents the mean ± SEM of S2/S1 values from 6–11 independent experiments (mice). Concentrations shown are micromolar. Asterisks indicate significant differences from the control group (no drug) (*p < 0.05; **p < 0.01).

Fig. 5.

Expression of M₂ muscarinic receptors in cholinergic terminals in the striatum of M₄ receptor WT and M₄ receptor KO mice. Striatal slices (∼50-mm-thick) were prepared from M₄ receptor WT or M₄receptor KO mice, and the M₂ muscarinic receptor and the VAChT were visualized via confocal immunofluorescence microscopy (see Materials and Methods for details). M₂ receptors (green) colocalize with VAChT (red) in some cholinergic terminals in both M₄ receptor WT and KO mice. Colocalization is visualized asyellow in the merged images (arrows). Scale bars, 2 μm.

Fig. 6.

Localization of M₄ muscarinic receptors to cholinergic terminals in mouse striatum. Striatal slices (∼50-mm-thick) were prepared from M₄ receptor WT mice, and the M₄ muscarinic receptor and the VAChT were visualized via confocal immunofluorescence microscopy (see Materials and Methods for details). M₄ receptors (green) colocalize with VAChT (red) in some cholinergic terminals. Colocalization is visualized as yellow in the merged images (arrows). Scale bar, 2 μm.