Transcriptome Of Cholinergic Neurons In The Nucleus Of The Diagonal Band Of Mice

Cholinergic neurons are a kind of important modulating neurons in the central nervous system,which distribute in several key brain regions and dominate the functions of learning,cognition,sleep and fear.Abnormalities in cholinergic circuits can cause a variety of neurological disorders.Previous studies have shown that the cholinergic neurons in the basal forebrain participate in different functional circuits through complex projections,which are significantly different from the cholinergic neurons such as the motor nuclei,and the mechanism at the gene level is still unclear.Single-cell sequencing is the analysis of genetic information at the level of a single cell or a single cell population,which can analyze the diversity of cell morphology and function from the perspective of transcriptome.In this paper,transcriptome information of cholinergic neurons in basal forebrain was obtained by using single cell sequencing technology,and compared with the cholinergic neurons in midbrain and brainstem,the genetic mechanism of their morphological and functional differences was preliminarily analyzed.In this study,Chat-ires-Cre×Ai47 transgenic mice were used that specifically express green fluorescent protein in cholinergic neurons.The specificity of the fluorescence was verified with immunohistostainning to determine the neurons and the boundary of the brain regions.The preparation method of cell suspension was optimized in the parameters during the whole process,including sectioning parameters,incubation temperature,oxygen saturation and blow strength during digestion.These adjustments improved the activity of neurons,while kept the cell viability at about 80%.Then,I digested and isolated the fluorescent cells in the selected brain region,constructed the c DNA library with Smart-seq2 scheme,and sequenced with Illumina.The results showed that the matching efficiency to the gene pool was 95%,with an average of 19,157 genes per sample.With the enrichment analysis of differential genes with GO and KEGG,I found that the different genes of cholinergic neurons in the diagonal band mainly related with the formation of axons and dendrites,synaptic connections of neurons and the construction of neural circuits,compared with those in laterodorsal tegmental nucleus and hypoglossal nucleus.Moreover,I found that Lhx2,Lhx6,Lhx8,Six3 and Vax1 expressed differently among theseregions.Futhermore,comparing with the transcriptome data of cholinergic neurons in the laterodorsal tegmental nucleus,Robo3,Robo4,Mybpc1,Ptprd,Kndc1 were found to be significantly different,which were related to the morphology and neural circuits.Comparing with the cholinergic neurons in the hypoglossal nucleus,I found that Mbp、Mobp、Cnp、Prkacb,Syt11 were significantly different,which were involved in the process of neuronal morphology and axon guidance.These results indicate that the complex morphological structure of basal forebrain neurons is related to the expression of Robo3,Robo4,Mybpc1,Ptprd,Kndc1,Mbp,Mobp,Cnp,Prkacb and Syt11.This study provided the genetic data of cholinergic neurons in the basal forebrain,motor nucleus,and midbrain,which was helpful to understand the differentiation regulatory mechanism of the same type of neurons in different brain regions from the transcriptional level.