The Function And Molecular Mechanisms Of Cholinergic Circuits In RTT-like Behaviors

Mutations in the X-linked MECP2 gene cause Rett syndrome（RTT）,an autism spectrum disorder characterized by impaired social interactions,motor abnormalities,cognitive defects and a high risk of epilepsy.Currently,however,there is no treatment available for RTT due to its specific neurodevelopmental phenotypes.Thus,understanding the pathogenesis of various RTT phenotypes is necessary and urgent for developing effective treatments.Decreases in cholinergic markers have been correlated with clinical severity in RTT patients.Cholinergic neurons in the brain include projection neurons that innervate distal areas and interneurons that are interspersed among their cellular targets.The BF cholinergic neurons project diffusely to the entire cortex and hippocampus,mediating the processes of attention,arousal and even mood regulation.Cholinergic interneurons in the striatum appear to act in motor regulation,associative plasticity and reward-dependent learning.The link between cholinergic systems and specific RTT phenotypes led us to hypothesize that dysfunction in distinct cholinergic circuits should be involved in divergent RTT phenotypes.In this study,we showed that conditional deletion of Mecp2 from cholinergic neurons resulted in the exhibition of several RTT-like phenotypes such as impaired social interaction,altered anxiety-related behavior,fear deficit and epilepsy susceptibility,suggesting the involvement of the cholinergic systems in the pathogenesis of RTT.To determine the population of cholinergic neurons that contribute to the behavioral features observed in Chat-Mecp2^-/y mice,we injected the AAV-FLEX-Mecp2-GFP（AAV8/Mecp2）vector into different brain areas（basal forebrain or striatum）to specifically re-express Mecp2 in cholinergic neurons.The regional re-expression data indicate that impaired social interaction,altered anxiety-related behavior and epilepsy susceprtibility in Chat-Mecp2^-/y mice arises at least in part from the lack of MeCP2 expression in basal forebrain cholinergic neurons and fear deficit in striatal cholinergic neurons.We next sought to find the mechanism underlying how the distinct cholinergic circuits regulate various RTT phenotypes.Part 1:Basal forebtain:we found that choline acetyltransferase（ChAT）expression was decreased in the BF and that α7 nicotine acetylcholine receptor（nAChR）signaling was strongly impaired in the hippocampus of Chat-Mecp2^-/y mice to an extent sufficient to produce neuronal hyperexcitation and increase seizure susceptibility.Application of PNU282987 or nicotine in the hippocampus rescued these phenotypes in Chat-Mecp2^-/y mice.Part 2:Striatum:The spontaneous firing of striatal cholinergic neurons was decreased and chemogenetic activation of these neurons reversed the impaired fear memory in Chat-Mecp2^-/y mice.We further found that the activity of striatal cholinergic neurons in Chat-Mecp2^-/y mice was mediated by an enhanced inhibitory transmission via a2-containing GABAA receptors.Down-regulation of a2-GABAA receptors in cholinergic neurons completely rescued fear memory.Furthermore,we demonstrated that cholinergic interneurons can regulate striatal output through modulating excitatory synaptic transmission and optogenetic activation of this circuit improved fear memory in Chat-Mecp2^-/y mice.