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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.

Flickering light is a new promising, fully non-invasive brain stimulation technique that utilizes intermittent sensory stimulation to induce brainwave synchronization (entrainment). While the effects of 40 Hz externally induced neural entrainment have been extensively described, little is known about 60 Hz entrainment in humans. This study presents preliminary observations on the neural and somatic response to flickering 60 Hz light in healthy volunteers over a 3-week period. Fourteen volunteers were randomized to receive either 60 Hz flickering white light or constant light as sham (30-min sessions, 3 weeks, 5 days/week on weekdays). Neural entrainment was assessed with EEG on days 1, 5 and 19. Salivary cortisol and C-reactive protein (CRP) levels, measured with ELISA, assessed the somatic response to stimulation. Side effects and well-being were monitored via questionnaires. EEG recordings showed neural entrainment and synchrony in response to 60 Hz flickering light across multiple cortical regions, including occipital, central, temporal, and frontal areas. The entrainment power and synchronization between different cortical regions declined significantly by day 19 compared to day 1, indicating possible neural habituation. Cortisol and CRP salivary levels were unchanged, and minor side effects were reported with equal frequency in the active and sham groups. Our findings show that 60 Hz flickering light can induce significant neural entrainment and synchrony in healthy adults and is well tolerated. The decline in entrainment strength and neural synchrony observed with repeated 60 Hz stimulations suggests plastic changes in the cortex. To the best of our knowledge, this is the first study to characterize neural and somatic responses to repeated 60 Hz flickering visual stimuli. Given the well-known connection between 60 Hz brain oscillations and cognition, neuroplasticity, and their role in neuropsychiatric disorders, additional research in both preclinical and clinical settings is warranted.

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.

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. Colombo et al. build a morphological spectrum of over 40,000 microglia across development and disease with a topological data analysis approach that allows mapping of new conditions along these sex-region-specific and brain-region-specific atlases.

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. Mapping fixed brain samples with extracellular labeling and optical microscopy reveals synaptic connections.

The dynamics of vestibular afferent responses are thought to be strongly influenced by presynaptic properties. In this paper, by performing whole-cell perforated-patch experiments in the frog utricle, we characterized voltage-dependent currents and voltage responses to current steps and 0.3-100 Hz sinusoids. Current expression and voltage responses are strongly related to hair cell type. In particular, voltage responses of extrastriolar type eB (low pass, -3 dB corner at 52.5 ± 12.8 Hz) and striolar type F cells (resonant, tuned at 60 ± 46 Hz) agree with the dynamics (tonic and phasic, respectively) of the afferent fibers they contact. On the other hand, hair cell release (measured with single-sine membrane ΔCm measurements) was linearly related to Ca in both cell types, and therefore did not appear to contribute to dynamics differences. As a tool for quantifying the relative contribution of basolateral currents and other presynaptic factors to afferent dynamics, the recorded current, voltage and release data were used to build a NEURON model of the average extrastriolar type eB and striolar type F hair cell. The model contained all recorded conductances, a basic mechanosensitive hair bundle and a ribbon synapse sustained by stochastic voltage-dependent Ca channels, and could reproduce the recorded hair cell voltage responses. Simulated release obtained from eB-type and F-type models display significant differences in dynamics, supporting the idea that basolateral currents are able to contribute to afferent dynamics; however, release in type eB and F cell models does not reproduce tonic and phasic dynamics, mainly because of an excessive phase lag present in both cell types. This suggests the presence in vestibular hair cells of an additional, phase-advancing mechanism, in cascade with voltage modulation.

Doc number: P313

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.

Prenatal immune challenges pose significant risks to human embryonic brain and eye development. However, we still lack knowledge about the safe usage of anti-inflammatory drugs during pregnancy. 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. Yet, microglia are susceptible to and promote inflammation and will influence the effects. Here, we generate hIPSC-derived microglia precursor cells and assemble them into retinal organoids to dissect their interplay with the developing nervous system and expose them to the immunostimulant poly(I:C). We first identified successful hIPSC-derived microglia (iMG) integration into the retinal organoid at the time when the outer plexiform layer forms. To improve ganglion cell surveillance, we adapted the retinal organoid model and found that the ganglion cell number significantly decreased only with iMG presence. While poly(I:C) exposure alters the iMG phenotype, it does not hinder their interaction with ganglion cells. Furthermore, iMG significantly enhances the supernatant’s inflammatory secretome and increases retinal cell proliferation. Simultaneous exposure with the non-steroidal anti-inflammatory drug (NSAID) ibuprofen dampens the 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. This effect depends on cyclooxygenase 1 in microglia and cyclooxygenase 2, common NSAID targets. 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.