Hippocampal networks are particularly susceptible to dysfunction in many neurodegenerative diseases and neuropsychiatric disorders including Alzheimer's disease, Lewy body dementia, and schizophrenia. CA1, a major output region of the hippocampus, receives glutamatergic input from both hippocampal CA3 and entorhinal cortex, via the Schaffer collateral (SC) and temporoammonic (TA) pathways, respectively. SC and TA inputs to CA1 are thought to be differentially involved in the retrieval of previously stored memories versus the encoding of novel information, and switching between these two crucial hippocampal functions is thought to critically depend on acetylcholine (ACh) acting at muscarinic receptors. In this study, we aimed to determine the roles of specific subtypes of muscarinic receptors in mediating the neuromodulatory effects of ACh on glutamatergic synaptic transmission in the SC and TA pathways of CA1. Using selective pharmacological activation of M1 or M4 receptors along with extracellular and intracellular electrophysiology recordings from adult rat hippocampal slices, we demonstrate that activation of M1 receptors increases spontaneous spike rates of neuronal ensembles in CA1 and increases the intrinsic excitability of pyramidal neurons and interneurons. Selective activation of M4 receptors inhibits glutamate release in the SC pathway, while leaving synaptic transmission in the TA pathway comparatively intact. These results suggest specific mechanisms by which M1 and M4 activation may normalize CA1 circuit activity following disruptions of signaling that accompany neurodegenerative dementias or neuropsychiatric disorders. These findings are of particular interest in light of clinical findings that xanomeline, an M1/M4 preferring agonist, was able to improve cognitive and behavioral symptoms in patients with Alzheimer's disease or schizophrenia.

Abnormal hippocampal activity has been linked to impaired cognitive performance in Alzheimer's disease and schizophrenia, leading to a hypothesis that normalization of this activity may be therapeutically beneficial. Our work suggests that one approach for hippocampal normalization may be through activation of the M4 muscarinic acetylcholine receptor. We used a brain penetrant M4 muscarinic acetylcholine receptor selective activator, PT-3763, to show dose-dependent attenuation of field potentials in Schaffer collateral (CA3-CA1) and recurrent associational connections (CA3-CA3) ex vivo in hippocampal slices. In vivo, systemic administration of PT-3763 led to attenuation of glutamate release in CA3 as measured by amperometry and to a dose-dependent decrease in population CA1 pyramidal activity as measured by fiber photometry. This decrease in population activity was also evident with a localized administration of the compound to the recorded site. Finally, PT-3763 reversed scopolamine-induced deficit in Morris water maze. Our results suggest that M4 muscarinic acetylcholine receptor activation may be a suitable therapeutic treatment in diseases associated with hyperactive hippocampal activity.

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Preclinical and clinical data suggest that muscarinic acetyl­choline receptor activation may be therapeutically beneficial for the treatment of schizophrenia and Alzheimer’s diseases. This is best exemplified by clinical observations with xanomeline, the efficacy of which is thought to be mediated through co-activation of the M1 and M4 muscarinic acetyl­choline receptors (mAChRs). Here we examined the impact of treatment with xanomeline and compared it to the actions of selective M1 and M4 mAChR activators on in vivo intra­cellular signaling cascades in mice, including 3′-5′-cyclic adenosine monophosphate response element binding protein (CREB) phosphorylation and inositol phosphate-1 (IP1) accumulation in the striatum, hippocampus, and prefrontal cortex. We additionally assessed the effects of xanomeline on hippocampal electro­physiological signatures in rats using ex vivo recordings from CA1 (Cornu Ammonis 1) as well as in vivo hippocampal theta. As expected, xanomeline’s effects across these readouts were consistent with activation of both M1 and M4 mAChRs; however, differences were observed across different brain regions, suggesting non-uniform activation of these receptor subtypes in the central nervous system. Interestingly, despite having nearly equal in vitro potency at the M1 and the M4 mAChRs, during in vivo assays xanomeline produced M4-like effects at significantly lower brain exposures than those at which M1-like effects were observed. Our results raise the possibility that clinical efficacy observed with xanomeline was driven, in part, through its non-uniform activation of mAChR subtypes in the central nervous system and, at lower doses, through preferential agonism of the M4 mAChR.