Microglia, the resident immune cells of the brain, play a complex role in health and disease. They actively survey the brain parenchyma by physically interacting with other cells and structurally shaping the brain. Yet, the mechanisms underlying microglial motility and significance for synapse stability, especially in the hippocampus during adulthood, remain widely unresolved. Here, we investigated the effect of neuronal activity on microglial motility and the implications for the formation and survival of dendritic spines on hippocampal CA1 neurons in vivo. We used repetitive two-photon in vivo imaging in the hippocampus of awake and anesthetized mice to simultaneously study the motility of microglia and their interaction with dendritic spines. We found that CA3 to CA1 input is sufficient to modulate microglial process motility. Simultaneously, more dendritic spines emerged in mice after awake compared to anesthetized imaging. Interestingly, the rate of microglial contacts with individual dendritic spines and dendrites was associated with the stability, removal, and emergence of dendritic spines. These results suggest that microglia might sense neuronal activity via neurotransmitter release and actively participate in synaptic rewiring of the hippocampal neural network during adulthood. Further, this study has profound relevance for hippocampal learning and memory processes.

Glioblastoma (GB) cells infiltrate the brain parenchyma and colonize distant regions, driving recurrence and therapy resistance. Here, we examined dynamic microglial responses to infiltrating tumor cells during GB progression. Three-photon imaging in an autochthonous, immunocompetent GB mouse model enabled visualization of microglia-GB interactions at the far infiltration zone (FIZ) in the corpus callosum (CC). GB infiltration speed varied by anatomical location and tumor microtube (TM) number. Microglia increased surveillance in sparsely infiltrated areas but reduced it with higher GB density, revealing a biphasic response. Directional migration toward GB cells was restricted to microglial subsets within a defined spatial range, indicating heterogeneous reactivity. CX3CR1 deficiency enhanced microglial reactivity while limiting GB cell migration. Microglia depletion with the CSF1R inhibitor PLX5622 reduced GB cell migration and constrained TM plasticity. Thus, microglia respond to GB cell infiltration in a stage-dependent manner and critically modulate dissemination at the FIZ.

Microglia, the resident immune cells of the brain, play a complex role in health and disease. They actively survey the brain parenchyma by physically interacting with other cells and structurally shaping the brain. Yet, the mechanisms underlying microglia motility and their significance for synapse stability, especially during adulthood, remain widely unresolved. Here we investigated the impact of neuronal activity on microglia motility and its implication for synapse formation and survival. We used repetitive two-photon in vivo imaging in the hippocampus of awake mice to simultaneously study microglia motility and their interaction with synapses. We found that microglia process motility depended on neuronal activity. Simultaneously, more dendritic spines emerged in awake compared to anesthetized mice. Interestingly, microglia contact rates with individual dendritic spines were associated with their stability. These results suggest that microglia are not only sensing neuronal activity, but participate in synaptic rewiring of the hippocampus during adulthood, which has profound relevance for learning and memory processes.

Microglia are highly motile cells that play a crucial role in the central nervous system in health and disease. Here we show that actin depolymerizing factors ADF and Cofilin1 (Cfl1) are key factors of microglia integrity and function. We found a profound morphological phenotype in absence of ADF and Cfl1 in microglia. In vivo two-photon imaging of microglia with ADF/Cfl1-KO revealed reduced microglial fine processes motility and impaired microglia migration towards a laser-induced lesion. We found increased accumulation of stabilized F-actin and altered microtubule dynamics in ADF/Cfl1-KO microglia, indicating that ADF/Cfl1 are necessary for microglial cytoskeleton dynamics. Interestingly, microglial ADF/Cfl1-deficiency decreased learning and memory, suggesting that impaired microglial cytoskeleton dynamics affect neuronal functions relevant for cognition. Our results reveal a fundamental role of ADF/Cfl1 in microglia function and underscore the importance of these innate immune cells for higher cognitive functions.

Modern fluorescence and multiphoton microscopy workflows operate within a heterogeneous ecosystem of file formats, partially overlapping metadata standards, and reader-specific conventions. In practice, this frequently leads to silent axis misinterpretations, loss or corruption of physical voxel size information, and laboratory-specific glue code that is fragile, poorly documented, and difficult to reproduce. OMIO, short for Open Microscopy Image I/O, addresses these issues by providing a lightweight, policy-driven image I/O layer for Python that enforces a canonical, OME-compatible data representation at the API boundary. The central contribution of OMIO is the explicit separation of low-level format access from semantic normalization. Existing reader libraries are used as interchangeable backends for extracting pixel data and available metadata, while OMIO enforces axis conventions, metadata interpretation, and fallback decisions in a centralized and auditable policy layer. This design allows heterogeneous microscopy inputs to be converted into a stable representation without propagating backend-specific assumptions into downstream analysis code. The core design principles of OMIO include canonical axis semantics (TZCYX), robust metadata normalization with explicit and auditable fallbacks, memory-aware operation via optional Zarr-based backends, and workflow-level semantics that extend beyond individual files to folder stacks and BIDS-like project structures. This architecture allows OMIO to orchestrate existing reader libraries into a coherent and reproducible I/O pipeline without replacing or duplicating their functionality. OMIO is implemented as an open-source and community-oriented system in which support for additional file formats and metadata conventions can be added incrementally through modular reader backends. By encouraging the contribution of example datasets, backend extensions, and feature requests, OMIO is designed to evolve alongside emerging acquisition systems while preserving strict semantic guarantees at the interface level. The resulting standardized OME-TIFF outputs are immediately suitable for downstream quantitative analysis and interactive inspection in scientific Python workflows, including workflows based on ImageJ and Napari.

The interaction of glioblastoma (GB) and microglia is critical due to its implications for tumor progression, immune response modulation, and potential therapeutic strategies. However, the role of microglia in GB pathogenesis remains unclear, especially regarding the in vivo dynamics of their interplay. Performing three-photon imaging in an autochthonous, immunocompetent mouse GB model, we examined tumor/microglia dynamics within previously inaccessible regions at the GB far infiltration zone in the corpus callosum. Initially, microglia increased tissue surveillance upon encountering GB-cells in sparsely infiltrated areas. In contrast, when GB-cell density increased, microglia reduced surveillance, suggesting a biphasic response to tumor invasion. Additionally, microglia were not uniformly attracted to infiltrating GB-cells; only a subset moved directionally toward them within a defined spatial range. This study provides insight into the heterogeneity of the immune response to tumor invasion and the dynamics of microglia-GB interactions in vivo.