Heterogeneity of release-regulating muscarinic receptors in rat sympathetic neurons: evidence for inhibitory presynaptic M₁ receptors

Of the 5 known subtypes of mAChRs, M₂, M₃, and M₄ have been reported to act as inhibitory presynaptic receptors in the nervous system, in general, and in sympathetic neurons, in particular. M₁ receptors, in contrast, have rather been viewed as facilitatory presynaptic receptors. In superior cervical ganglion (SCG) neurons, M₁ receptors are well known to inhibit KCNQ channels. Previously, we were able to show that non-presynaptic M₁ receptors in SCG neurons enhance noradrenaline release through an inhibition of KCNQ channels. However, M₁ receptors also mediate an inhibition of voltage-activated Ca²⁺ channels, which represents the predominant mechanism of presynaptic inhibition. Hence, presynaptic M₁ receptors may exert inibitory presynaptic modulation. To test this possibility, we performed experiments on rat superior cervical ganglion neurons. In primary cultures tritium overflow was assayed to investigate the release of [³H]noradrenaline, and the perforated patch-clamp technique was employed to record Ca²⁺ currents. The muscarinic agonist oxotremorine M transiently enhanced ³H outflow and reduced electrically evoked release, once the stimulatory effect had faded. The stimulatory effect was enhanced by pertussis toxin and was abolished by blocking M₁ receptors, by opening KCNQ channels, and by preventing action potential propagation. The inhibitory effect, in contrast, was not altered by preventing action potentials or by opening KCNQ channels, but was reduced by pertussis toxin. The inhibition remaining after pertussis toxin treatment was abolished by blockage of M₁ receptors or inhibition of phospholipase C. When [³H]noradrenaline release was triggered independently of voltage-activated Ca²⁺ channels, oxotremorine M failed to cause any inhibition. The inhibition of Ca²⁺ currents by oxotremorine M was reduced by pertussis toxin and then abolished by the blockage of M₁ receptors. This demonstrates that M₁, in addition to M₂, M₃, and M₄, receptors mediate presynaptic inhibition in sympathetic neurons using phospholipase C to close voltage-activated Ca²⁺ channels. In addition, our results contradict the widely accepted concept that all inhibitory presynaptic receptors restrict transmitter release through a direct inhibition of Ca²⁺ channels via G protein βγ subunits and offer an alternative mechanism.