Neuromedin U receptor 2 modulates climbing fiber-Purkinje cell synaptic transmission through presynaptic protein kinase A signaling cascades in mouse cerebellar slices.

OBJECTIVE

The central neuromedin U receptor 2 (NMUR2) participates in regulating neuronal activity and synaptic transmission. This study aimed to explore the functional role of NMUR2 in climbing fiber-Purkinje cell synaptic transmission in mouse cerebellar slices.

METHODS

Climbing fiber-Purkinje cell excitatory postsynaptic currents (EPSCs) and miniature excitatory postsynaptic currents (mEPSCs) were recorded from Purkinje cells using an Axopatch 700B patch-clamp amplifier. Western blot, glutamate sensor fluorescence imaging, and immunohistochemistry were applied to measure phosphorylated protein kinase A (PKA) levels, climbing fiber terminal glutamate release, and NMUR2 expression, respectively.

RESULTS

Bath application of the selective NMUR2 agonist CPN-219 reduced the amplitude and area under the curve of climbing fiber-Purkinje cell EPSCs and increased the paired-pulse ratio ( N 2/ N 1). The CPN-219-induced suppression of EPSCs was reversed by an adenylyl cyclase activator and occluded by an AC inhibitor. The CPN-219-mediated inhibitory effect on EPSCs was completely abolished by bath perfusion of the PKA inhibitor, but not by intracellular PKA inhibition. Western blot analysis revealed that CPN-219 decreased phosphorylated PKA levels in cerebellar molecular layer tissue. Furthermore, CPN-219 markedly attenuated Ca 2+ transients and complex spike activity in Purkinje cells, suppressed climbing fiber-evoked glutamate fluorescent signals, and decreased mEPSC frequency, with no significant alteration in mEPSC amplitude. Moreover, immunohistochemical staining showed NMUR2 immunoreactivity distributed throughout the somata and dendritic arbors of Purkinje cells.

CONCLUSION

These results indicate that NMUR2 activation suppresses climbing fiber-Purkinje cell synaptic transmission in mouse cerebellar slices predominantly via a mechanism consistent with a presynaptic adenylyl cyclase-PKA signaling cascade.