Morphotype-Specific Calcium Signaling in Human Microglia
Morphology-Specific Calcium Signaling Characteristics in Human Microglia
Background and Research Objectives
Microglia are the primary immune cells of the central nervous system (CNS), involved in almost all physiological and pathological processes, including development, synaptic transmission, neuroplasticity, sleep, trauma, glioblastoma, and neurodegenerative diseases. Additionally, microglia monitor their microenvironment by sensing danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). Microglia detect changes in intracellular calcium ion concentrations through the expression of numerous genes encoding different membrane receptors (known as the “microglial sensome”), triggering the production and release of cytokines and other inflammatory factors, as well as cell proliferation, differentiation, migration, and phagocytosis. In mouse models, microglial calcium transient signals are closely related to neuronal network activity and exhibit distinct spatial compartmentalization characteristics. However, the calcium signaling characteristics of human microglia remain unknown, and most studies are limited to cultured primary cells or induced pluripotent stem cell (iPSC)-derived microglia-like cells.
Paper Source and Authors
This study was conducted by Sofia Nevelchuk et al., with authors primarily from the Department of Neurophysiology, Institute of Physiology, Eberhard Karls University of Tübingen, the Hertie Institute for Clinical Brain Research, University of Tübingen, and the Max-Planck-Institute for Biological Intelligence in Martinsried. The paper was published in the Journal of Neuroinflammation in 2024.
Research Methods and Workflow
Preparation and Cultivation of Tissue Samples
Research samples were obtained from cortical tissue excised during surgery from six patients, which were subsequently used to prepare organotypic cortical slices. These slices were preserved in cold artificial cerebrospinal fluid and transferred to culture membranes within 1-2 hours, followed by cultivation with human cerebrospinal fluid (hCSF). Cultivation was performed using 1.5mm long neural stem cell culture medium, with hCSF replaced every 3 days. During the study, slices were transduced with lentiviral vectors expressing three different colored fluorescent proteins (mCherry, mVenus, mTurquoise2) and the newly developed genetically encoded calcium indicator mcyrfp1-caneon to label and analyze microglial calcium signals.
Development and Performance Evaluation of Novel Calcium Indicator
To monitor calcium signals in human microglia, the research team developed a new genetically encoded ratiometric calcium indicator, mcyrfp1-caneon. This indicator combines the yellow-green fluorescent protein mNeonGreen with the red fluorescent protein mcyrfp1, enabling calcium imaging in both single-photon and two-photon modes. Compared to the currently popular GCaMPs, caneon offers higher fluorescent protein brightness and only two calcium binding sites, providing better linearity and reduced calcium buffering load.
Calcium Signal Detection and Data Analysis
The study used MATLAB’s Begonia framework for calcium signal activity pixel detection and analysis. The fluorescence values of each pixel were converted into binary time series, active pixels were identified using empirically determined thresholds, and these pixels were grouped to define regions of activity (ROA). The average fluorescence values of caneon and mcyrfp1 were then calculated at each time point, and the ratio of calcium transient changes (δR/R) was calculated and analyzed.
Main Research Findings
Calcium Signal Characteristics in Microglia of Different Morphologies
This study found significant calcium signal compartmentalization characteristics in human microglia, with distinct calcium signal characteristics in cells of different morphologies. The study described calcium signal characteristics in detail from the following aspects:
Basal Calcium Levels: The study showed significant differences in basal calcium levels among microglia of different morphologies in humans. Ramified microglia had the lowest basal calcium levels, while amoeboid microglia showed significantly elevated basal calcium levels.
Calcium Signal Distribution and Frequency: Calcium signals were mostly confined to the process areas in ramified cells, while in amoeboid cells, most calcium signals were distributed in the cell body. Further analysis revealed that process calcium transients in ramified cells were characterized by high amplitude and short duration, while calcium transients in amoeboid cells had high amplitude but long-lasting characteristics.
Characteristics of Regions of Activity (ROAs): In ramified cells, calcium signal activity areas were smaller and mainly concentrated in cellular processes; in amoeboid cells, activity areas were larger and primarily located in the cell body. The amplitudes and AUC of calcium transients differed among the three cell morphologies, with particularly notable differences between ramified and amoeboid cells.
Dynamic Characteristics of Calcium Signals
- Frequency and Oscillatory Behavior: In ramified and hypertrophic cells, calcium signal frequencies were relatively low, while amoeboid cells frequently exhibited calcium signal oscillations with a frequency of approximately 12.33×10^-3 s^-1.
- Association between Movement and Calcium Transients: Many ramified and hypertrophic cells showed significant process motility, with corresponding areas displaying local calcium transients; although no cell body displacement was observed during the 15-minute recording period.
Research Conclusions and Value
This study deeply analyzed the calcium signal characteristics of human microglia (including ramified, hypertrophic, and amoeboid morphologies) in their native environment using a new genetically encoded calcium indicator and advanced calcium signal detection and analysis techniques. The discovered different compartmentalization characteristics of calcium signals provide important clues for understanding the specific functions of microglia in physiological and pathological processes, offering significant guidance for future research on neurological diseases. Additionally, new technical approaches, such as RGB labeling and the novel calcium indicator mcyrfp1-caneon, provide powerful tools for further research on human microglia. This study fills a gap in our understanding of the dynamic changes in calcium signals in human microglia and demonstrates their functional heterogeneity in different morphologies.
Research Highlights
Novel Genetically Encoded Calcium Indicator: Developed mcyrfp1-caneon, a new ratiometric calcium indicator suitable for single-photon and two-photon calcium imaging, featuring high brightness and good linearity, serving as an important tool for existing human microglial calcium imaging research.
Calcium Signal Compartmentalization Characteristics: Revealed significant compartmentalization characteristics of calcium signals in human microglia and differences in calcium signals between different morphologies, aiding in understanding microglial functional responses in various environments.
Tissue Sample and Cultivation Techniques: Successfully preserved the three main morphologies of microglia using fresh post-operative human cortical tissue combined with human cerebrospinal fluid for organotypic culture, providing an important in vitro research model.
Limitations of the Study
Despite the significant progress in analyzing calcium signals in human microglia, this study has certain limitations. As tissue samples were sourced from epilepsy or tumor surgeries, they may not fully represent normal healthy tissue. Additionally, the expression of genetically encoded calcium indicators requires organotypic slice culture, which may alter cellular functional characteristics.
This study achieved important breakthroughs in technology and methodology, providing valuable data and methodological support for future functional research on human microglia.