Biological tissues require branched cellular architectures to maximize spatial coverage while minimizing redundancy. Yet, how cells decode local spatial information to collectively tile territories without a global blueprint remains a key open question. Here, we develop a biophysical theory of interacting branched cells, and show that coupling their growth to short-range repulsion drives efficient tiling with minimal territorial overlap. Our model predicts that the same local mechanism simultaneously suppresses long-range density fluctuations, driving the cellular collective toward hyperuniformity. We confirm these theoretical predictions with experiments on microglial patterning in the developing retina, and show that perturbations resulting in limited cell growth disrupt both tiling and fluctuation suppression. Our results reveal that two seemingly distinct optimization principles of biological patterning, large-scale regularity and efficient tiling, are intimately linked and can arise from a single growth-repulsion feedback, suggesting a general principle for self-organized tissue coverage.

Anesthesia recovery is critical for resuming normal physiological and neuronal functions; however, the mechanisms involved remain elusive. Here, we identify a female-selective corticosterone-mediated microglia-neuron interaction during ketamine anesthesia recovery, absent in males. This microglia-neuron interaction induces plastic and functional neuronal changes, as evidenced by increased mEPSC frequency, which was occluded upon microglia depletion. We showed that this process is driven through up-regulation of the stress-responsive co-chaperone Fkbp5 mRNA and its protein, Fkbp51, in female microglia. Fkbp5/Fkbp51 is a key intermediary in a corticosteroid-induced stress response, and its involvement points toward a critical interface between endocrine signaling and microglia. To counteract the observed ketamine anesthesia-mediated increase in blood corticosterone during recovery, we removed the primary source of corticosterone by adrenalectomy. Close microglia-neuron interaction was reduced and increased again following corticosterone injection. Our findings identify a sex-specific microglia-mediated mechanism of neuronal plasticity during anesthesia recovery, driven by corticosterone, thereby enhancing our understanding of sex differences in brain function.

Abstract In branched cells, including neurons and glia, intracellular organelles are distributed across complex cellular arbors where their spatial arrangement supports transport, signaling, and compartmentalized function. Although intracellular organelle organization is increasingly recognized as an important feature of cellular state and function, existing approaches assess organelle abundance or spatial position without accounting for the branching architecture that shapes cellular function. Here, we introduce the chromatic topological morphology descriptor ( chromatic TMD ), a framework that quantitatively resolves intracellular organization in relation to branching morphology. Applied to reconstructed microglia with annotated lysosomal and mitochondrial compartments across retinal layers and after optic nerve crush injury, chromatic TMD identifies distinct organelle-specific spatial programs: CD68 + -endosomal-lysosomes undergo layer-dependent branch-specific redistribution, revealing selective intracellular reorganization after injury, whereas mitochondrial organization remains closely coupled to branching morphology. These findings establish intracellular organization as an additional layer of cellular architecture that can be systematically analyzed across branched neural cells.

Purpose:Optic nerve crush (ONC) is a model for studying optic nerve trauma. Unilateral ONC induces massive retinal ganglion cell (RGC) degeneration in the affected eye, leading to vision loss within a month. A common assumption has been that the non-injured contralateral eye is unaffected due to the minimal retino-retinal projections of the RGCs at the chiasm. Yet, recently, microglia, the brain-resident macrophages, have shown a responsive phenotype in the contralateral eye after ONC. Whether RGC loss accompanies this phenotype is still controversial. Methods:Using the available RGCode algorithm and developing our own RGC-Quant deep-learning-based tool, we quantify RGC's total number and density across the entire retina after ONC. Results:We confirm a short-term microglia response in the contralateral eye after ONC, but this did not affect the microglia number. Furthermore, we cannot confirm the previously reported RGC loss between naïve and contralateral retinas 5 weeks after ONC induction across the commonly used Cx3cr1creERT2 and C57BL6/J mouse models. Neither sex nor the direct comparison of the RGC markers Brn3a and RBPMS, with Brn3a co-labeling, on average, 89% of the RBPMS+-cells, explained this discrepancy, suggesting that the early microglia-responsive phenotype does not have immediate consequences on the RGC number. Conclusions:Our results corroborate that unilateral optic nerve injury elicits a microglial response in the uninjured contralateral eye but without RGC loss. Therefore, the contralateral eye should be treated separately and not as an ONC control.

Prenatal immune challenges pose significant risks to human embryonic brain and eye development. However, our knowledge about the safe usage of anti-inflammatory drugs during pregnancy is still limited. While human induced pluripotent stem cells (hIPSC)-derived brain organoid models have started to explore functional consequences upon viral stimulation, these models commonly lack microglia, which are susceptible to and promote inflammation. Furthermore, microglia are actively involved in neuronal development. Here, we generate hIPSC-derived microglia precursor cells and assemble them into retinal organoids. Once the outer plexiform layer forms, these hIPSC-derived microglia (iMG) fully integrate into the retinal organoids. Since the ganglion cell survival declines by this time in 3D-retinal organoids, we adapted the model into 2D and identify that the improved ganglion cell number significantly decreases only with iMG presence. In parallel, we applied the immunostimulant POLY(I:C) to mimic a fetal viral infection. While POLY(I:C) exposure alters the iMG phenotype, it does not hinder their interaction with ganglion cells. Furthermore, iMG significantly enhance the supernatant’s inflammatory secretome and increase retinal cell proliferation. Simultaneous exposure with the non-steroidal anti-inflammatory drug (NSAID) ibuprofen dampens POLY(I:C)-mediated changes of the iMG phenotype and ameliorates cell proliferation. Remarkably, while POLY(I:C) disrupts neuronal calcium dynamics independent of iMG, ibuprofen rescues this effect only if iMG are present. Mechanistically, ibuprofen targets the enzymes cyclooxygenase 1 and 2 (COX1/PTGS1 and COX2/PTGS2) simultaneously, from which iMG mainly express COX1. Selective COX1 blockage fails to restore the calcium peak amplitude upon POLY(I:C) stimulation, suggesting ibuprofen’s beneficial effect depends on the presence and interplay of COX1 and COX2. These findings underscore the importance of microglia in the context of prenatal immune challenges and provide insight into the mechanisms by which ibuprofen exerts its protective effects during embryonic development.

The shape of a cell contains essential information about its function within the biological system. Segmenting these structures from large-scale 3D microscopy images is challenging, limiting clinical insights especially for microglia, immune-associated cells involved in neurodegenerative diseases. Existing segmentation methods mainly focus on cell bodies, struggle with overlapping structures, perform poorly on noisy images, require hyperparameter tuning for each new dataset, or rely on tedious semi-automated approaches. We introduce trAIce3D, a deep-learning architecture designed for precise microglia segmentation, capturing both somas and branches. It employs a two-stage approach: first, a 3D U-Net with vision transformers in the encoder detects somas using a sliding-window technique to cover the entire image. Then, the same architecture, enhanced with cross-attention blocks in skip connections, refines each soma and its branches by using soma coordinates as a prompt and a 3D window around the target cell as input. Training occurs in two phases: self-supervised Soma Segmentation, followed by prompt-based Branch Segmentation, leveraging pre-trained weights from the first phase. Trained and evaluated on a dataset of 41,230 microglial cells, trAIce3D significantly improves segmentation accuracy and generalization, enabling scalable analysis of complex cellular morphologies. While optimized for microglia, its architecture can extend to other intricate cell types, such as neurons and astrocytes, broadening its impact on neurobiological research.

Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanometer synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS uses fixation-compatible extracellular labeling and optical imaging, including stimulated emission depletion or expansion microscopy, to comprehensively delineate cellular structures. It enables three-dimensional reconstruction of single synapses and mapping of synaptic connectivity by identification and analysis of putative synaptic cleft regions. Applying CATS to the mouse hippocampal mossy fiber circuitry, we reconstructed and quantified the synaptic input and output structure of identified neurons. We furthermore demonstrate applicability to clinically derived human tissue samples, including formalin-fixed paraffin-embedded routine diagnostic specimens, for visualizing the cellular architecture of brain tissue in health and disease.

Environmental cues influence the highly dynamic morphology of microglia. Strategies to characterize these changes usually involve user-selected morphometric features, which preclude the identification of a spectrum of context-dependent morphological phenotypes. Here we develop MorphOMICs, a topological data analysis approach, which enables semiautomatic mapping of microglial morphology into an atlas of cue-dependent phenotypes and overcomes feature-selection biases and biological variability. We extract spatially heterogeneous and sexually dimorphic morphological phenotypes for seven adult mouse brain regions. This sex-specific phenotype declines with maturation but increases over the disease trajectories in two neurodegeneration mouse models, with females showing a faster morphological shift in affected brain regions. Remarkably, microglia morphologies reflect an adaptation upon repeated exposure to ketamine anesthesia and do not recover to control morphologies. Finally, we demonstrate that both long primary processes and short terminal processes provide distinct insights to morphological phenotypes. MorphOMICs opens a new perspective to characterize microglial morphology.

Cerebral organoids differentiated from human-induced pluripotent stem cells (hiPSC) provide a unique opportunity to investigate brain development. However, organoids usually lack microglia, brain-resident immune cells, which are present in the early embryonic brain and participate in neuronal circuit development. Here, we find IBA1(+) microglia-like cells alongside retinal cups between week 3 and 4 in 2.5D culture with an unguided retinal organoid differentiation protocol. Microglia do not infiltrate the neuroectoderm and instead enrich within non-pigmented, 3D-cystic compartments that develop in parallel to the 3D-retinal organoids. When we guide the retinal organoid differentiation with low-dosed BMP4, we prevent cup development and enhance microglia and 3D-cysts formation. Mass spectrometry identifies these 3D-cysts to express mesenchymal and epithelial markers. We confirmed this microglia-preferred environment also within the unguided protocol, providing insight into microglial behavior and migration and offer a model to study how they enter and distribute within the human brain.

Balanced and dynamic mitochondrial networks are essential for cell survival and function. Mitochondrial networks remodel their connectivity, content and subcellular localization to support optimized energy production under conditions of increased stress. In vivo , stressors can arise from the environment, such as in neuronal injury, or from mutation-induced cellular dysfunction. Cells programmed to identify and respond to these stress signals, like microglia, rely on optimized mitochondrial function, however we know very little about mitochondrial networks of microglia in vivo or their adaptation to environmental or cellular stressors. Here, we define the mitochondrial networks of retinal microglia in physiological conditions in vivo and evaluate network alterations by taking advantage of a microglia-selective mitochondria-labeled mouse model. First, we demonstrate significant differences in the mitochondrial networks of microglia in vivo and in vitro. Then, we induced neuronal injury in the in vivo environment using optic nerve crush, where responsive microglia exhibit more fragmented mitochondrial networks with increased content and perinuclear localization, supporting a state of increased cellular stress. Surprisingly, when we selectively increase cellular stress by knocking out the mitochondria-associated gene uncoupling protein 2 (UCP2), only male UCP2 KO microglia establish a hyperfused mitochondrial network after injury, indicating sex differences in microglial stress mitigation. Ovariectomy in UCP2 KO females elicits a shift toward the male hyperfused mitochondrial phenotype suggesting that circulating estrogens are a contributing factor to the differences in microglial stress mitigation.

G protein-coupled receptors (GPCRs) regulate processes ranging from immune responses to neuronal signaling. However, ligands for many GPCRs remain unknown, suffer from off-target effects or have poor bioavailability. Additionally, dissecting cell type-specific responses is challenging when the same GPCR is expressed on different cells within a tissue. Here, we overcome these limitations by engineering DREADD-based GPCR chimeras that bind clozapine-N-oxide and mimic a GPCR-of-interest. We show that chimeric DREADD-β2AR triggers responses comparable to β2AR on second messenger and kinase activity, post-translational modifications, and protein-protein interactions. Moreover, we successfully recapitulate β2AR-mediated filopodia formation in microglia, an immune cell capable of driving central nervous system inflammation. When dissecting microglial inflammation, we included two additional DREADD-based chimeras mimicking microglia-enriched GPR65 and GPR109A. DREADD-β2AR and DREADD-GPR65 modulate the inflammatory response with high similarity to endogenous β2AR, while DREADD-GPR109A shows no impact. Our DREADD-based approach allows investigation of cell type-dependent pathways without known endogenous ligands. Understanding the function of GPCRs requires stimulation with their specific ligands. Here, the authors design chemogenetic G-protein coupled receptors that allows for the study of receptors without knowing the immediate ligand, and demonstrate its use for the β2-adrenergic receptor in microglia.

To understand how potential gene manipulations affect in vitro microglia, we provide a set of short protocols to evaluate microglia identity and function. We detail steps for immunostaining to determine microglia identity. We describe three functional assays for microglia: phagocytosis, calcium response following ATP stimulation, and cytokine expression upon inflammatory stimuli. We apply these protocols to human induced-pluripotent-stem-cell (hiPSC)-derived microglia, but they can be also applied to other in vitro microglial models including primary mouse microglia. For complete details on the use and execution of this protocol, please refer to Bartalska et al. (2022).1.

Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanoscopic synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS leverages fixation-compatible extracellular labeling and advanced optical readout, in particular stimulated-emission depletion and expansion microscopy, to comprehensively delineate cellular structures. It enables 3D-reconstructing single synapses and mapping synaptic connectivity by identification and tailored analysis of putative synaptic cleft regions. Applying CATS to the hippocampal mossy fiber circuitry, we demonstrate its power to reveal the system’s molecularly informed ultrastructure across spatial scales and assess local connectivity by reconstructing and quantifying the synaptic input and output structure of identified neurons.

Brain organoids differentiated from human induced pluripotent stem cells provide a unique opportunity to investigate the development, organization and connectivity of neurons in a complex cellular environment. However, organoids usually lack microglia, brain-resident immune cells which are both present in the early human embryonic brain and participate in neuronal circuit development. Here, we find that microglia innately develop in unguided retinal organoid differentiation between week 3 and 4 in 2.5D culture and appear later in floating, non-pigmented, 3D-cystic compartments. We enriched for cystic structures using a low-dosed BMP4 application and performed mass spectrometry, thus defining the protein composition of microglia-containing compartments. We found that cystic compartments expressed both mesenchymal and epithelial markers with microglia enriched in the mesenchymal region. Interestingly, microglia-like cells started to express the border-associated macrophage marker CD163. The preferential localization of human microglia to a mesenchymal compartment provides insight into the behavior and migration of microglia. The model will ultimately allow detailed study of these enigmatic cells and how they enter and distribute within the human brain.

Enzymatic digestion of the extracellular matrix with chondroitinase-ABC reinstates juvenile-like plasticity in the adult cortex as it also disassembles the perineuronal nets (PNNs). The disadvantage of the enzyme is that it must be applied intracerebrally and it degrades the ECM for several weeks. Here, we provide two minimally invasive and transient protocols for microglia-enabled PNN disassembly in mouse cortex: repeated treatment with ketamine-xylazine-acepromazine (KXA) anesthesia and 60-Hz light entrainment. We also discuss how to analyze PNNs within microglial endosomes-lysosomes. For complete details on the use and execution of this protocol, please refer to Venturino et al. (2021).

Microglia contribute to tissue homeostasis in physiological conditions with environmental cues influencing their ever-changing morphology. Strategies to identify these changes usually involve user-selected morphometric features, which, however, have proved ineffective in establishing a spectrum of context-dependent morphological phenotypes. Here, we have developed MorphOMICs, a topological data analysis approach to overcome feature-selection-based biases and biological variability. We extracted a spatially heterogeneous and sexually-dimorphic morphological phenotype for seven adult brain regions, with ovariectomized females forming their own distinct cluster. This sex-specific phenotype declines with maturation but increases over the disease trajectories in two neurodegeneration models, 5xFAD and CK-p25. Females show an earlier morphological shift in the immediately-affected brain regions. Finally, we demonstrate that both the primary- and the short terminal processes provide distinct insights to morphological phenotypes. MorphOMICs maps microglial morphology into a spectrum of cue-dependent phenotypes in a minimally-biased and semi-automatic way.

Adeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells, allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy, likely due to the cells' heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding by introducing four point mutations (K531E, R576Q, K493S, and K459S), resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette for use in combination with the Cx3cr1(CreERT2) mouse line. Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency.

Perineuronal nets (PNNs), components of the extracellular matrix, preferentially coat parvalbumin-positive interneurons and constrain critical-period plasticity in the adult cerebral cortex. Current strategies to remove PNN are long-lasting, invasive, and trigger neuropsychiatric symptoms. Here, we apply repeated anesthetic ketamine as a method with minimal behavioral effect. We find that this paradigm strongly reduces PNN coating in the healthy adult brain and promotes juvenile-like plasticity. Microglia are critically involved in PNN loss because they engage with parvalbumin-positive neurons in their defined cortical layer. We identify external 60-Hz light-flickering entrainment to recapitulate microglia-mediated PNN removal. Importantly, 40-Hz frequency, which is known to remove amyloid plaques, does not induce PNN loss, suggesting microglia might functionally tune to distinct brain frequencies. Thus, our 60-Hz light-entrainment strategy provides an alternative form of PNN intervention in the healthy adult brain.