Calcium Signaling in the Cerebellar Radial Glia and Its Association with Morphological Changes during Zebrafish Development

Radial glial cells are a distinct non-neuronal cell type that, during development, span the entire width of the brain walls of the ventricular system. They play a central role in the origin and placement of neurons, since their processes form structural scaffolds that guide and facilitate neuronal migration. Furthermore, glutamatergic signaling in the radial glia of the adult cerebellum (i.e., Bergmann glia), is crucial for precise motor coordination. Radial glial cells exhibit spontaneous calcium activity and functional coupling spread calcium waves. However, the origin of calcium activity in relation to the ontogeny of cerebellar radial glia has not been widely explored, and many questions remain unanswered regarding the role of radial glia in brain development in health and disease. In this study we used a combination of whole mount immunofluorescence and calcium imaging in transgenic (gfap-GCaMP6s) zebrafish to determine how development of calcium activity is related to morphological changes of the cerebellum. We found that the morphological changes in cerebellar radial glia are quite dynamic; the cells are remarkably larger and more elaborate in their soma size, process length and numbers after 7 days post fertilization. Spontaneous calcium events were scarce during the first 3 days of development and calcium waves appeared on day 5, which is associated with the onset of more complex morphologies of radial glia. Blockage of gap junction coupling inhibited the propagation of calcium waves, but not basal local calcium activity. This work establishes crucial clues in radial glia organization, morphology and calcium signaling during development and provides insight into its role in complex behavioral paradigms.

Ca2+ oscillations, waves, and networks in islets from human donors with and without type 2 diabetes

Pancreatic islets are highly interconnected structures that produce pulses of insulin and other hormones, maintaining normal homeostasis of glucose and other nutrients. Normal stimulus-secretion and intercellular coupling are essential to regulated secretory responses and these hallmarks are known to be altered in diabetes. In the present study, we used calcium imaging of isolated human islets to assess their collective cell behavior. The activity occurred in the form of calcium oscillations, was synchronized across different regions of islets through calcium waves, and was glucose-dependent: higher glucose enhanced the activity, elicited a greater proportion of global calcium waves, and led to denser and less fragmented functional networks. Hub regions were identified in stimulatory conditions, and they represented the most active islet regions. Moreover, calcium waves were found to be initiated in different subregions and the roles of initiators and hubs did not overlap. In type 2 diabetes, glucose-dependence was retained, but a reduced activity, locally restricted waves, and more segregated networks were detected compared with control islets. Interestingly, hub regions seemed to suffer the most by losing a disproportionately large fraction of connections. These changes affected islets from donors with diabetes in a heterogeneous manner.

The people behind the papers – Donald Ready and Henry Chang

ABSTRACT Coordinating contractility across tissues is key for maintaining the fidelity of morphogenetic processes. A new paper in Development explains how cytosolic calcium waves in the interommatidial cells, the pigment-secreting cells in the Drosophila eye, lead to remodelling of the retinal floor, by activating contraction of the basal actomyosin stress fibres. We caught up with the authors, Professor Donald Ready and Associate Professor Henry Chang, both from Purdue University, to find out more about this story.

What can astrocytes compute?

I demonstrate theoretically that calcium waves in astrocytes can compute anything neurons can. A foundational result in neural computation was proving the firing rate model of neurons defines a universal function approximator. In this work I show a similar proof extends to a model of calcium waves in astrocytes, which I confirm in a series of computer simulations. I argue the major limit in astrocyte computation is not their ability to find approximate solutions, but their computational complexity. I suggest some initial experiments that might be used to confirm these predictions.

P13.12 Effect of tumor treating fields on tumor microtubes in glioma

BACKGROUND Tumor microtubes (TMs) are ultralong membrane protrusions of tumor cells in astrocytic gliomas, including glioblastomas. TMs are used as routes for brain invasion and for cells to interconnect over long distances resulting in a functional network that allows multicellular communication. This network mediates resistance against the cytotoxicity of radiation and chemotherapy. One explanation for TM network protection is a better homeostasis of calcium ions that would otherwise increase to toxic intracellular levels in response to these therapies. Our working hypothesis is that interfering with the integrity of the glioblastoma cell network is key to a potential breakthrough in glioma therapy. Many cellular structures are polarized and composed of charged elements and are thus potential subjects to electrical forces; this might also influence the complex intercellular calcium waves (ICWs) that are characteristic for glioma networks. We were therefore interested in the effect of TTF on glioma network maintenance. MATERIAL AND METHODS To examine the effect of TTF on glioma TMs we have established a 2D in vitro glioma model using glioblastoma stem cells (GBSCs) grown in high-glucose medium and a 3D model using glioma tumor organoids. Both models reliably reproduce functionality and complexity of morphological features we observe in our mouse model. We analyzed the disruption of tumor network complexity and disruption of functionality by measuring intercellular calcium waves. Tumor cell death and proliferation was investigated in the 2D in vitro glioma model using the inovitroTM-System. RESULTS A peculiar “cricked-TM” phenotype that rarely (0.2% ±0.14) occurred under standard or control conditions was observed in TTF-treated cells (16.22% ±5.12). Cell number was reduced by 75% in two lines of GBSCs after 5 days of TTF exposure; predominantly TM-rich GBSCs (> 4 TMs) were affected. This reduction in tumor cell number corresponded with an increase in cell death (0.3% ±0.09 in untreated cells; 1.4% ±0.45 at day 5 of TTF exposure). The frequency of intercellular calcium transients, a measurement for calcium wave frequency in the glioma networks, was instantly reduced after TTF exposure to 58% ±20.42 of control levels in the primary GBSC 2D culture, and to 57.78% ±12.34 in tumor organoids derived from 3 glioblastoma patients. CONCLUSION This data suggests a potential effect of TTF application on tumor cell networks, at least in vitro. Interestingly, particularly those glioblastoma cells that have so far been proven to be resistant to radio- and chemotherapy appeared to be affected. We will confirm the observed effects of TTFs on tumor cell calcium signaling in our in vivo chronic cranial window mouse model. We anticipate that the results of our project will provide important insights into the underlying mechanism of TTF therapy.

Mechanosensory trichome cells evoke a mechanical stimuli-induced immune response in plants

Perception of pathogen-derived ligands by corresponding host receptors is a pivotal strategy in eukaryotic innate immunity. In plants, this is complemented by circadian anticipation of infection timing, promoting basal resistance even in the absence of pathogen threat. Here, we report that trichomes, hair-like structures on the epidermis, directly sense external mechanical forces caused by raindrops to anticipate waterborne infections in Arabidopsis thaliana. Exposure of leaf surfaces to mechanical stimuli initiates the concentric propagation of intercellular calcium waves away from trichomes to induce defence-related genes. Propagating calcium waves enable effective immunity against pathogenic microbes through the calmodulin-binding transcription activator 3 (CAMTA3) and mitogen-activated protein kinases. We propose a novel layer of plant immunity in which trichomes function as mechanosensory cells to detect potential risks.

NMDA receptor inhibition increases, synchronizes, and stabilizes the collective pancreatic beta cell activity: Insights through multilayer network analysis

NMDA receptors promote repolarization in pancreatic beta cells and thereby reduce glucose-stimulated insulin secretion. Therefore, NMDA receptors are a potential therapeutic target for diabetes. While the mechanism of NMDA receptor inhibition in beta cells is rather well understood at the molecular level, its possible effects on the collective cellular activity have not been addressed to date, even though proper insulin secretion patterns result from well-synchronized beta cell behavior. The latter is enabled by strong intercellular connectivity, which governs propagating calcium waves across the islets and makes the heterogeneous beta cell population work in synchrony. Since a disrupted collective activity is an important and possibly early contributor to impaired insulin secretion and glucose intolerance, it is of utmost importance to understand possible effects of NMDA receptor inhibition on beta cell functional connectivity. To address this issue, we combined confocal functional multicellular calcium imaging in mouse tissue slices with network science approaches. Our results revealed that NMDA receptor inhibition increases, synchronizes, and stabilizes beta cell activity without affecting the velocity or size of calcium waves. To explore intercellular interactions more precisely, we made use of the multilayer network formalism by regarding each calcium wave as an individual network layer, with weighted directed connections portraying the intercellular propagation. NMDA receptor inhibition stabilized both the role of wave initiators and the course of waves. The findings obtained with the experimental antagonist of NMDA receptors, MK-801, were additionally validated with dextrorphan, the active metabolite of the approved drug dextromethorphan, as well as with experiments on NMDA receptor KO mice. In sum, our results provide additional and new evidence for a possible role of NMDA receptor inhibition in treatment of type 2 diabetes and introduce the multilayer network paradigm as a general strategy to examine effects of drugs on connectivity in multicellular systems.