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, 5 (4), 318-24

M4 mAChR-mediated Modulation of Glutamatergic Transmission at Corticostriatal Synapses

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M4 mAChR-mediated Modulation of Glutamatergic Transmission at Corticostriatal Synapses

Tristano Pancani et al. ACS Chem Neurosci.

Abstract

The striatum is the main input station of the basal ganglia and is extensively involved in the modulation of motivated behavior. The information conveyed to this subcortical structure through glutamatergic projections from the cerebral cortex and thalamus is processed by the activity of several striatal neuromodulatory systems including the cholinergic system. Acetylcholine potently modulates glutamate signaling in the striatum via activation of muscarinic receptors (mAChRs). It is, however, unclear which mAChR subtype is responsible for this modulatory effect. Here, by using electrophysiological, optogenetic, and immunoelectron microscopic approaches in conjunction with a novel, highly selective M4 positive allosteric modulator VU0152100 (ML108) and M4 knockout mice, we show that M4 is a major mAChR subtype mediating the cholinergic inhibition of corticostriatal glutamatergic input on both striatonigral and striatopallidal medium spiny neurons (MSNs). This effect is due to activation of presynaptic M4 receptors, which, in turn, leads to a decrease in glutamate release from corticostriatal terminals. The findings of the present study raise the interesting possibility that M4 mAChR could be a novel therapeutic target for the treatment of neurological and neuropsychiatric disorders involving hyper-glutamatergic transmission at corticostriatal synapses.

Figures

Figure 1
Figure 1
VU0152100 potentiates CCh-induced reduction of EPSC amplitude at corticostriatal synapses in MSNs. (A,B) Representative traces of averaged EPSCs showing the effects of 1 μM CCh (A1), 3 μM CCh (B1), 1 μM CCh with 5 μM VU0152100 (A2), and 1 μM CCh with 5 μM VU0152100 (B2) on EPSCs. (C,D) Bar graphs summarizing the potentiation effect of VU0152100 on the CCh-induced reduction of EPSC amplitude by 1 μM CCh (C) and 3 μM CCh (D). Data are presented as mean ± SEM (**p < 0.01, t test). Insets show the effects of VU0152100 + CCh on PPR (inset in C: 5 μM VU0152100 with 1 μM CCh; inset in D: 5 μM VU0152100 with 3 μM CCh, *p < 0.05, paired t test).
Figure 2
Figure 2
Loss of CCh effect on EPSCs in M4 KO mice. (A,B) Representative traces of averaged EPSCs showing the effects of 3 μM CCh (A) and 100 μM CCh (B) on EPSCs in WT mice (A1 and B1) and M4 KO mice (A2 and B2). (C) Bar graph summarizing the effects of 3 μM and 100 μM CCh on EPSC amplitude in WT and M4-KO mice. Bars represent the percentage inhibition compared to baseline shown as mean ± SEM (**p < 0.01, ***p < 0.001, t test).
Figure 3
Figure 3
M4-mediated depression of EPSCs is due to decrease in glutamate release by activation of presynaptic M4 mAChRs. (A,B) Representative traces of averaged EPSCs showing the effects of 3 μM CCh in the presence of 5 μM VU1052100 in D1 (A) and non-D1 (B) MSNs. (C,D) Bar graphs summarizing the potentiation effect of VU0152100 on the CCh-induced reduction of EPSC amplitude in D1 (C) and non-D1 (D) MSNs. Insets show the effect of 3 μM CCh + 5 μM VU0152100 on PPR in D1 (C) and non-D1 (D) MSNs. Bars represent the percentage inhibition compared to baseline shown as mean ± SEM (*p < 0.05, paired t test; **p < 0.01, t test). (E) Representative traces of averaged EPSCs (left) and summary bar graph (right) showing the effect of 3 μM CCh on EPSC amplitude in MSNs recorded with patch pipets with and without GDP-β-S (*p < 0.05, t test). (F) Electron micrographs showing M4-immunoreactive terminals (Te) forming asymmetric axo-spinous synapses with unlabeled and labeled spines (an unlabeled terminal also shown, u.Te). Scale bars: 0.5 μm. (G) Representative traces of averaged EPSCs (left) and summary bar graph (right) showing the effect of 3 μM CCh on EPSC amplitude in the presence of the cocktail of D1/D2 antagonists (*p < 0.05, paired t test).
Figure 4
Figure 4
M4-PAM enhances EPSC depression induced by synchronized activation of cholinergic interneurons (ChIs). (A) A representative trace of ChI firing in a slice taken from a ChAT-ChR2-EYFP mouse during control and light stimulation (473 nm, 22 mW, 500 ms, *p < 0.05, paired t test). (B) Bar graph summarizing the effect of light stimulation on firing rate of ChIs. (C) Representative traces of averaged EPSCs showing the effect of light (C1), light + VU0152100 (5 μM, C2) and light + VU0152100 (5 μM) + scopolamine (20 μM, C3) on EPSC amplitude. (D) Bar graph summarizing the effect on EPSC amplitude in the following conditions, light stimulation, light with VU0152100, and light with VU0152100 and scopolamine. Data are shown as mean ± SEM (**p < 0.01, t test).

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