Biology of Astrocytes Research Group
Research topics
Astrocytes are known for their diverse and essential functions, such as neurotransmitter recycling, regulation of synaptic transmission, and control of brain metabolism. These tasks are modulated by systemic signals, including hormones. Our team has discovered that astrocytes are a direct transcriptional target of glucocorticoids in the brain, and we have identified metabolic pathways in astrocytes that are controlled through activation of the glucocorticoid receptor.
Moreover, we have demonstrated that this signaling pathway is necessary for the central effects of acute and chronic stress. Interestingly, many members of the glucocorticoid receptor–dependent pathway have consistently been identified as dysregulated in transcriptomic studies of brain samples from patients with various psychiatric disorders.
In our current research, we analyze the involvement of selected pathways and genes in the neurobiological correlates of disturbances specific to defined brain circuits associated with psychiatric phenotypes.
Transgenic Mouse Models
In our research, we use advanced transgenic mouse models that allow precise genetic manipulations in selected cell populations and at defined developmental stages or during adulthood. We apply conditional gene knockouts using the Cre-ERT2/loxP system, which enables temporally and spatially controlled gene inactivation.
To deliver genetic material, we also use viral vectors. Additionally, through in utero electroporation, we can introduce genetic manipulations into specific cell types not only in mice but also in other species.
Imaging
We use modern in vivo imaging techniques that allow real-time monitoring of glial and neuronal cell activity in behaving animals. We employ one-photon and two-photon microscopy to image calcium signaling in astrocytes.
Additionally, using fiber photometry, it is possible to record cell activity in real time while simultaneously analyzing the behavior of individual animals. We also perform long-term in vitro imaging of cells to monitor physiological and metabolic changes.
Omics
We have optimized protocols for isolating various cell types from the mouse brain, enabling precise molecular analyses. We have also developed effective methods for isolating astrocyte nuclei from freshly frozen human samples. RNA-seq and single-nucleus RNA-seq analyses allow for deep profiling of gene expression in selected cell populations.
Behavior
In our research, we use automated systems to track spontaneous behavior in multiple animals simultaneously. We work with rodent stress models, such as chronic social defeat stress and early maternal separation.
We analyze depressive phenotypes using a social interaction test in three chambers, sucrose preference tests, and dark–light box tests. We also conduct cognitive tests, such as the Y-maze or elevated plus maze, and we analyze circadian rhythms using running-wheel cages.
Cell Cultures
We use primary cultures of mouse astrocytes and neurons, as well as human astrocytes derived from induced pluripotent stem cells (iPSCs). We also perform co-cultures of neurons and astrocytes, which allow us to analyze interactions between these cell types in controlled in vitro conditions.
Metabolic Measurements
For cellular metabolism analysis, we use the Seahorse system, which enables measurement of glycolytic rate, oxygen consumption, and energy substrate utilization. We also use the TMRE indicator to assess mitochondrial membrane potential, as well as live-cell imaging to measure glycolytic rate. In addition, we perform long-term imaging of genetically encoded metabolic indicators both in vitro and in vivo.
Molecular Genetics
In our laboratory, we routinely use classical gene-cloning methods, including Gateway, Gibson, and Golden Gate techniques. We also use bacterial artificial chromosomes and viral vectors for genetic material delivery. We conduct gene editing using the CRISPR/Cas9 method, as well as gene knockdown using shRNA and siRNA.
Standard Analytical Methods
To assess protein and RNA expression, we use immunocytochemistry, immunohistochemistry, Western blotting, Southern blotting, and Golgi staining. We also perform ELISA analyses and RNAscope, which enables precise localization and quantitative assessment of RNA transcripts in tissues.