Knockouts reveal overlapping functions of M2 and M4 muscarinic receptors and evidence for a local glutamatergic circuit within the laterodorsal tegmental nucleus

Kohlmeier KA, Ishibashi M, Wess J, Bickford ME, Leonard CS. Knockouts reveal overlapping functions of M-2 and M-4 muscarinic receptors and evidence for a local glutamatergic circuit within the laterodorsal tegmental nucleus. J Neurophysiol 108: 2751-2766, 2012. First published September 5, 2012; doi:10.1152/jn.01120.2011.-Cholinergic neurons in the laterodorsal tegmental (LDT) and peduncolopontine tegmental (PPT) nuclei regulate reward, arousal, and sensory gating via major projections to midbrain dopamine regions, the thalamus, and pontine targets. Muscarinic acetylcholine receptors (mAChRs) on LDT neurons produce a membrane hyperpolarization and inhibit spike-evoked Ca2+ transients. Pharmacological studies suggest M-2 mAChRs are involved, but the role of these and other localized mAChRs (M1--M-4) has not been definitively tested. To identify the underlying receptors and to circumvent the limited receptor selectivity of available mAChR ligands, we used light- and electron-immunomicroscopy and whole cell recording with Ca2+ imaging in brain slices from knockout mice constitutively lacking either M-2, M-4, or both mAChRs. Immunomicroscopy findings support a role for M-2 mAChRs, since cholinergic and noncholinergic LDT and pedunculopontine tegmental neurons contain M-2-specific immunoreactivity. However, whole cell recording revealed that the presence of either M-2 or M-4 mAChRs was sufficient, and that the presence of at least one of these receptors was required for these carbachol actions. Moreover, in the absence of M-2 and M-4 mAChRs, carbachol elicited both direct excitation and barrages of spontaneous excitatory postsynaptic potentials (sEPSPs) in cholinergic LDT neurons mediated by M-1 and/or M-3 mAChRs. Focal carbachol application to surgically reduced slices suggest that local glutamatergic neurons are a source of these sEPSPs. Finally, neither direct nor indirect excitation were knockout artifacts, since each was detected in wild-type slices, although sEPSP barrages were delayed, suggesting M 2 and M 4 receptors normally delay excitation of glutamatergic inputs. Collectively, our findings indicate that multiple mAChRs coordinate cholinergic outflow from the LDT in an unexpectedly complex manner. An intriguing possibility is that a local circuit transforms LDT muscarinic inputs from a negative feedback signal for transient inputs into positive feedback for persistent inputs to facilitate different firing patterns across behavioral states.