Molecularly Defined And Functionally Distinct Cholinergic Neurons In The Medial Septal Nucleus

Background:Neurons within an organism can be grouped into different types—neurons with similarstructure and function belong to the same type.Categorizing neurons into types greatly reduces the complexity of investigating the organization and function,enabling a deeper understanding of the dynamic changes of neuronal function under healthy and diseased conditions.With the development of science and technology,the traditional neuron classification methods can no longer meet our current needs for human brain neuron classification.The basal forebrain usually referred to as a group of structures concentrated on the anterior medial and ventral sides of the cerebral hemispheres provides the main source of acetylcholine in the mammalian neocortex.Cholinergic neurons in the basal forebrain regulate a variety of physiological functions including arousal,reward,plasticity,learning memory,and attention.Cholinergic neurons in the Medial septum nucleus(MS)of the basal forebrain project extensively to various hippocampal subregions and play a very important role in memory consolidation and attention.Recent studies have demonstrated that the electrophysiology and morphology of cholinergic neurons in the MS region of the basal forebrain are heterogeneous.However,there are few studies on the categorical labeling of cholinergic neurons in this region.In addition,the circuitry and function of these heterogeneous cholinergic neurons remain unclear.Objective:(1)To investigate the classification specific marker molecules of cholinergic neurons in the medial septal nucleus.(2)To determine the anatomical structure,physiological characteristics,synaptic circuits and function of molecularly defined cholinergic neurons in the medial septal nucleus.Methods:The basal forebrain cholinergic neurons were identified as inhibitory neurons by immunofluorescence staining.Using markers of different inhibitory neurons,two distinct populations of cholinergic neurons were identified in the medial septal nucleus.One group was cholinergic neurons that express Calbindin-D28K(D28K+cholinergic neurons),and the other group was cholinergic neurons that do not express D28K(D28K-cholinergic neurons).A Triple Enzymatic Recombination Mutant(TERM)mouse expressing three recombinant enzymes was constructed by the principle of site-specific recombination technology.D28K+cholinergic neurons were labeled by injection of CRE and FLP recombinase-dependent recombinant adeno-associated virus into the medial septal nucleus of TERM mice.D28K-cholinergic neurons were labeled by injection of recombinant adeno-associated virus dependent on CRE and DRE recombinases expressing green fluorescence.These two cholinergic neurons in the medial septal nucleus were identified to form functionally distinct synaptic connections with two physiologically distinct neuronal types in the hippocampus by anterograde monosynaptic transneuronal tracing combined with patch-clamp technique.To analyze the anatomical morphology and electrophysiological characteristics of different cholinergic neurons in the medial septal nucleus by combining neuronal remodeling and patch-clamp technique.Virus injection combined with a patch-clamp technique was used to reveal the characteristics of neural projections and synaptic connections of different cholinergic neurons in the medial septal nucleus.Virus injection combined with single-cell RNA sequencing,CRISPR-Cas9 functional screening,patch-clamp technique,chemogenetics technology,it was confirmed that D28K+cholinergic neurons express kcnhl and 31 other genes,while D28K-cholinergic neurons express cacnalh and 22 other genes.The circuits and functions of different cholinergic neurons were screened and determined by virus injection combined with CRISPR-Cas9 gene function screening,patch-clamp technique,chemical genetics technology,optogenetic technology,and behavioral detection.Results:Two distinct populations of cholinergic neurons,expressing Calbindin-D28K(D28K+)and not expressing Calbindin-D28K(D28K-),were found in the medial septal nucleus.D28K+cholinergic neurons were found to exhibit a bipolar morphology with a relatively low firing frequency,while D28K-neurons showed a multilevel morphology accompanied by a higher firing frequency and burst firing characteristics.D28K+neurons formed synaptic connections with pyramidal neurons in the ventral CA1 hippocampus(VPNs),which were mediated by both Acetylcholine(Ach)and GABA,and D28K-neurons form synaptic connections with granule neurons in the dorsal dentate gyrus(DGNs),mediated by Ach only.Kcnhl and cacnalh genes mediate the electrophysiological properties and behavior of D28K+and D28K-cholinergic neurons,respectively.The application of chemogenetics and optogenetics revealed that the D28K+→VPNs circuitry regulates anxiety-like behavior,whereas the D28K-→DGNs circuitry encodes spatial memory.Conclusion:(1)We found D28K+and D28K-cholinergic neuron subtypes.(2)We showed that D28K+and D28K-cholinergic neurons have unique anatomical morphology and electrophysiological signatures.(3)We demonstrated that D28K+cholinergic neurons form synaptic connections with VPNs,whereas D28K-forms synaptic connections with DGNs.(4)We reported that D28K+→VPNs circuitry regulates anxiety-like behavior,whereas D28K-→DGNs circuitry encodes spatial memory.