Cholinergic modulation of hippocampal CA1 pyramidal cell excitability in Arx^GCG+7 mice

Developmental and Epileptic Encephalopathies (DEE) are rare neurodevelopmental disorders defined by seizures, developmental delays, and abnormal EEG patterns. Infantile Spasms Syndrome (ISS), the most common DEE, was one of the first linked to single-gene variants, including polyalanine expansions in the Aristaless Related Homeobox (ARX) gene. The Arx^GCG+7 mouse, which models the human mutation, exhibits behavioral phenotypes, epileptiform hippocampal activity, and reduced cholinergic input to the hippocampus. The role of the disrupted cholinergic signaling has not been explored in this model and may be contributing to hippocampal dysfunction and DEE pathogenesis. Hence, we investigated the impact of the ARX GCG expansion on the septo-hippocampal network by first reproducing the changes in Choline acetyltransferase (ChAT) protein levels in the medial septum (MS) and diagonal band of Broca (DBB). Next, we performed ex vivo patch-clamp recordings in CA1 pyramidal neurons and measured responses to cholinergic agonists. Finally, we assessed acetylcholine receptor expression by western blot across the hippocampus to assess for homeostatic changes in receptors. Our data demonstrated a ∼ 60 % reduction in ChAT expression in the MS and DBB of Arx^GCG+7 mice. The Arx^GCG+7 CA1 pyramidal neurons exhibited increased firing activity and reduced sag potential. Despite reduced ChAT, carbachol- and pilocarpine-induced firing responses were preserved, while nicotinic receptor-mediated excitation was abolished. Carbachol-induced depolarization block threshold was elevated in mutants. Molecular analysis revealed increased muscarinic receptor 1 expression and decreased β4 nicotinic receptor expression in CA1.Together, these findings demonstrate histological, electrophysiological, and molecular alterations in the septo-hippocampal network of Arx^GCG+7 mice. Together, they suggest impaired cholinergic signaling contributes to ISS pathogenesis and highlight the cholinergic system as a potential therapeutic target.