Deciphering the Heterogeneity of the Internal Environment of Hippocampal Neurons during Maturation by Raman Spectroscopy

[Image: see text] Hippocampal neurons are sensitive to changes in the internal environment and play a significant role in controlling learning, memory, and emotions. A remarkable characteristic of the aging brain is its ability to shift from a state of normal inflammation to excessive inflammation. Various cognitive abilities of the elderly may suffer from serious harm due to the change in the neural environment. Hippocampal neurons may have various subsets involved in controlling their internal environment at different stages of development. Developmental differences may eventually result from complex changes in the dynamic neuronal system brought on by metabolic changes. In this study, we used an in vitro hippocampal neuron model cultured in C57BL/6J mice in conjugation with Raman spectroscopy to examine the relative alterations in potential biomarkers, such as levels of metabolites in the internal environment of hippocampal neurons at various developmental stages. The various differentially expressed genes (DEGs) of hippocampal neurons at various developmental stages were simultaneously screened using bioinformatics, and the biological functions as well as the various regulatory pathways of DEGs were preliminarily analyzed, providing an essential reference for investigating novel therapeutic approaches for diseases that cause cognitive impairment, such as Alzheimer’s disease. A stable hippocampal neuron model was established using the GIBCO C57BL/6J hippocampal neuron cell line as a donor and in vitro hippocampal neuron culture technology. The Raman peak intensities of culture supernatants from the experimental groups incubated for 0, 7, and 14 days in vitro(DIV) were examined. The GEO database was used to screen for different DEGs associated with various developmental stages. The data was then analyzed using a statistical method called orthogonal partial least squares discriminant analysis (OPLS-DA). The levels of ketogenic and glycogenic amino acids (such as tryptophan, phenylalanine, and tyrosine), lipid intake rate, glucose utilization rate, and nucleic acid expression in the internal environment of hippocampal neurons were significantly different in the 14 DIV group compared to the 0 DIV and 7 DIV groups (P < 0.01). The top 10 DEGs with neuronal maturation were screened, and the results were compared to the OPLS-DA model’s analysis of the differential peaks. It was found that different genes involved in maturation can directly relate to changes in the body’s levels of ketogenic and glycogenic amino acids (P < 0.01). The altered expression of the maturation-related genes epidermal growth factor receptor, protein tyrosine kinase 2-beta, discs large MAGUK scaffold protein 2, and Ras protein-specific guanine nucleotide releasing factor 1 may be connected to the altered uptake of ketogenic and glycogenic amino acids and nucleic acids in the internal environment of neurons at different developmental stages. The levels of ketogenic, glycogenic amino acids, and lipid intake increased while glucose utilization decreased, which may be related to mature neurons’ metabolism and energy use. The decline in nucleic acid consumption could be connected to synaptic failure. The Raman spectroscopy fingerprint results of relevant biomarkers in conjugation with multivariable analysis and biological action targets suggested by differential genes interpret the heterogeneity of the internal environment of mature hippocampal neurons in the process of maturation, open a new idea for exploring the dynamic mechanism of the exchange energy metabolism of information molecules in the internal environment of hippocampal neurons, and provide a new method for studying this process.