Dissection Of The Basal Forebrain Cholinergic Neurons Circuit In Aging Mice

The basal forebrain cholinergic neurons(BFCNs)play an essential role in functions such as attention,sleep-wake control,and cognitive memory.Different subpopulations of BFCNs have tremendous difference in terms of developmental,connectivity and function.Studies have found that natural aging and neurodegenerative diseases lead to different levels of changes in molecules,cells and physiological functions of cholinergic neurons.However,the structural changes and mechanisms of the neural circuits,especially the long-range connection of BFCNs during aging remains unclear.In this thesis,combined with virus tracing and fluorescence micro-optical sectioning tomography(fMOST),the mechanism of BFCNs vulnerability was analyzed.The main research are as follows:(1)A pipeline for multi-sample imaging and analyzing was established,including multi-sample embedding and batch sections staining methods.In order to reduce individual differences on structure dissection,an array embedding method was proposed.The embedding mold was designed to realize the consistency of multiple sample positions and improves the cutting flatness.Combined with fMOST system,the whole brain threedimensional data of 32 mice were collected simultaneously.The preparation and imaging time required for a single mouse brain was about 9～12 hours.For sections obtained after imaging,a multi-slice staining box was developed to achieve a slice integrity rate of 98.5%during multi-round staining.This method provides a basis for analyzing cell morphology and molecular phenotype in multiple samples.(2)The region preferrd loss of cholinergic neurons was discovered.Different stages of aging were identified in mice by detecting their motor abilities,cognitive abilities and brain volume.The changes of neurons,glias and vessels in the basal forebrain of mice aged 3～6 months,10～14 months and 18～26 months were analyzed.Results showed that the cholinergic neurons in the vertical limb of the diagonal band(VDB)were vulnerable.The number of BFCNs in 26-month-old mice was significantly reduced by 17%.The volume of BFCNs retained in this region decreased.Astrocytes in the basal forebrain proliferated,while microglias and blood vessels did not change significantly.These indicates that the loss of cholinergic neurons during aging presents subregional preference.(3)The target-preferred lesions of BFCNs long-range connection circuits were discovered.Combined with monosynaptic retrograde tracing,the whole brain input distribution of BFCNs in 3-and 26-month-old mice was obtained.It showed that the input from hippocampal CA1/CA3,lateral hypothalamus and ventral tegmental area was significantly reduced.Anterograde tracing showed that there were different degrees of damage in the long-range projection of BFCNs in aging mice,the most serious being the hippocampus,in which the reduction of cholinergic fibers was CA1(60%)>CA3(46%)>dentate gyrus(35%).In addition,axonal swelling of BFCNs was mainly distributed in the ventral CA1,indicating the long-range connectivity of BFCNs in aging has target-preferred lesions in ventral CA1.(4)The loss of BFCNs was related to the axon projection path.With monosynaptic retrograde tracing,it was found that the cholinergic neurons projected to ventral CA1 were mainly distributed in the medial septum(MS),with a few located in VDB.There was no difference in the expression of p16 protein and glials in these two regions.Furthermore,the axon projection paths of cholinergic neurons in MS and VDB were reconstructed,respectively.The axons of cholinergic neurons located in the posterior part of VDB showed the highest density of swelling compared to MS.This unique projection path may make it more vulnerable to aging.In summary,combined with fMOST,a multi-sample imaging and staining method was established,and the lesions in the basal forebrain during aging was explored.It was found that the loss of cholinergic neurons has a subregional preference.The heterogeneity of BFCNs circuit lesions was analyzed,and the mechanism of BFCNs selective vulnerability in aging was revealed based on neuronal target and swelling density.These provides an anatomical basis for understanding the selective vulnerability of cholinergic neurons during aging,and provides new ideas for further research on the treatment of degenerative diseases.