@article{21245, abstract = {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.}, author = {Alamalhoda, MohammadAmin and Leesch, Friederike and Giovanetti, Francesca and Dunne, Eoghan and Pilloni, Giuseppina and Caffrey, Mark and O’Keeffe, Jack and Venturino, Alessandro and Ferretti, Maria Teresa}, issn = {1932-6203}, journal = {PLOS One}, number = {10}, publisher = {Public Library of Science}, title = {{Exploring neural entrainment and synchrony in response to repeated 60 Hz flickering white light in healthy volunteers}}, doi = {10.1371/journal.pone.0332310}, volume = {20}, year = {2025}, } @article{19593, abstract = {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.}, author = {Hübschmann, Verena and Korkut, Medina and Venturino, Alessandro and Maya-Arteaga, Juan Pablo and Siegert, Sandra}, issn = {1742-2094}, journal = {Journal of Neuroinflammation}, number = {1}, publisher = {Springer Nature}, title = {{Microglia determine an immune-challenged environment and facilitate ibuprofen action in human retinal organoids}}, doi = {10.1186/s12974-025-03366-x}, volume = {22}, year = {2025}, } @article{14257, abstract = {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.}, author = {Michalska, Julia M and Lyudchik, Julia and Velicky, Philipp and Korinkova, Hana and Watson, Jake and Cenameri, Alban and Sommer, Christoph M and Amberg, Nicole and Venturino, Alessandro and Roessler, Karl and Czech, Thomas and Höftberger, Romana and Siegert, Sandra and Novarino, Gaia and Jonas, Peter M and Danzl, Johann G}, issn = {1546-1696}, journal = {Nature Biotechnology}, pages = {1051--1064}, publisher = {Springer Nature}, title = {{Imaging brain tissue architecture across millimeter to nanometer scales}}, doi = {10.1038/s41587-023-01911-8}, volume = {42}, year = {2024}, } @article{14363, abstract = {Mitochondrial networks remodel their connectivity, content, and subcellular localization to support optimized energy production in conditions of increased environmental or cellular stress. Microglia rely on mitochondria to respond to these stressors, however our knowledge about mitochondrial networks and their adaptations in microglia in vivo is limited. Here, we generate a mouse model that selectively labels mitochondria in microglia. We identify that mitochondrial networks are more fragmented with increased content and perinuclear localization in vitro vs. in vivo. Mitochondrial networks adapt similarly in microglia closest to the injury site after optic nerve crush. Preventing microglial UCP2 increase after injury by selective knockout induces cellular stress. This results in mitochondrial hyperfusion in male microglia, a phenotype absent in females due to circulating estrogens. Our results establish the foundation for mitochondrial network analysis of microglia in vivo, emphasizing the importance of mitochondrial-based sex effects of microglia in other pathologies.}, author = {Maes, Margaret E and Colombo, Gloria and Schoot Uiterkamp, Florianne E and Sternberg, Felix and Venturino, Alessandro and Pohl, Elena E. and Siegert, Sandra}, issn = {2589-0042}, journal = {iScience}, number = {10}, publisher = {Elsevier}, title = {{Mitochondrial network adaptations of microglia reveal sex-specific stress response after injury and UCP2 knockout}}, doi = {10.1016/j.isci.2023.107780}, volume = {26}, year = {2023}, } @article{11478, abstract = {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.}, author = {Bartalska, Katarina and Hübschmann, Verena and Korkut, Medina and Cubero, Ryan J and Venturino, Alessandro and Rössler, Karl and Czech, Thomas and Siegert, Sandra}, issn = {2589-0042}, journal = {iScience}, number = {7}, publisher = {Elsevier}, title = {{A systematic characterization of microglia-like cell occurrence during retinal organoid differentiation}}, doi = {10.1016/j.isci.2022.104580}, volume = {25}, year = {2022}, } @article{11995, abstract = {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.}, author = {Schulz, Rouven and Korkut, Medina and Venturino, Alessandro and Colombo, Gloria and Siegert, Sandra}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses}}, doi = {10.1038/s41467-022-32390-1}, volume = {13}, year = {2022}, } @article{12244, abstract = {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.}, author = {Colombo, Gloria and Cubero, Ryan J and Kanari, Lida and Venturino, Alessandro and Schulz, Rouven and Scolamiero, Martina and Agerberg, Jens and Mathys, Hansruedi and Tsai, Li-Huei and Chachólski, Wojciech and Hess, Kathryn and Siegert, Sandra}, issn = {1546-1726}, journal = {Nature Neuroscience}, keywords = {General Neuroscience}, number = {10}, pages = {1379--1393}, publisher = {Springer Nature}, title = {{A tool for mapping microglial morphology, morphOMICs, reveals brain-region and sex-dependent phenotypes}}, doi = {10.1038/s41593-022-01167-6}, volume = {25}, year = {2022}, } @unpublished{11950, abstract = {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.}, author = {Michalska, Julia M and Lyudchik, Julia and Velicky, Philipp and Korinkova, Hana and Watson, Jake and Cenameri, Alban and Sommer, Christoph M and Venturino, Alessandro and Roessler, Karl and Czech, Thomas and Siegert, Sandra and Novarino, Gaia and Jonas, Peter M and Danzl, Johann G}, booktitle = {bioRxiv}, title = {{Uncovering brain tissue architecture across scales with super-resolution light microscopy}}, doi = {10.1101/2022.08.17.504272}, year = {2022}, } @article{10565, abstract = {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).}, author = {Venturino, Alessandro and Siegert, Sandra}, issn = {2666-1667}, journal = {STAR Protocols}, number = {4}, publisher = {Elsevier}, title = {{Minimally invasive protocols and quantification for microglia-mediated perineuronal net disassembly in mouse brain}}, doi = {10.1016/j.xpro.2021.101012}, volume = {2}, year = {2021}, } @article{9642, abstract = {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.}, author = {Venturino, Alessandro and Schulz, Rouven and De Jesús-Cortés, Héctor and Maes, Margaret E and Nagy, Balint and Reilly-Andújar, Francis and Colombo, Gloria and Cubero, Ryan J and Schoot Uiterkamp, Florianne E and Bear, Mark F. and Siegert, Sandra}, issn = {2211-1247}, journal = {Cell Reports}, number = {1}, publisher = {Elsevier}, title = {{Microglia enable mature perineuronal nets disassembly upon anesthetic ketamine exposure or 60-Hz light entrainment in the healthy brain}}, doi = {10.1016/j.celrep.2021.109313}, volume = {36}, year = {2021}, }