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11.07.2020, Saturday 09:00 - 11:30 Hall K Technical Workshop
Date
11.07.2020, Saturday
Session Time
09:00 - 11:30
Session Description
The past 4 decades highlighted the importance of glia, shifting the neurocentric vision of structure, function and pathology of nervous system to a more holistic perspective. In this view, the demand of technologies targeting the selective monitoring and control of glia is emerging as a challenge across Neuroscience, Engineering and Material Science. This workshop aims to present the most promising advances in unconventional tools & methods, that enabled to reach unprecedented insights into the 4 dimensional (4D) nature of spatio-temporal neuron-glial interactions, in the healthy and pathological brain. Specifically, we will show how 1)(bio)materials as silicon nanowire, graphene or hydrogels can provide transition from 2D to 3D models, mimicking in-vivo structure, where to study healthy or reactive glial cells in vitro 2)the use of nanostructured device allows stimulation and recording of astrocytes and neurons at different spatiotemporal scale 3)computational approaches and control theory can reverse-engineer neuron-glial interactions 4)combination of opto/chemogenetics, CLARITY and two-photon imaging reveals clues on significance of astrocytic domains in memory consolidation 5)photonic and spectroscopic methods uncover the signature of microglial cells in pain. The forum, presented by scientists from 5 different countries, will provide a multidisciplinary perspective on tools, potentially enabling to extend our knowledge on cognitive functions, pain and Alzheimer diseases
Technical Workshop

Chairperson introduction (ID 5641)

Lecture Time
09:00 - 09:03
Technical Workshop

Multiscale excitation and recording of astrocytes signalling and conductance by nanostructured devices (ID 571)

Lecture Time
09:03 - 09:23

Abstract

Abstract Body

The past four decades demonstrated that the expression and function of proteins forming ion channels, receptors and water channels in astrocytes are critical in brain function & dysfunction. However, most of the tools and approaches used to probe and sense astrocytes are derived from those developed to study neurons. The goal of our research is to provide insight on astrocytes physiology and biophysics by using multifunctional materials and nanotechnological approaches targeted to record and manipulate structural and functional properties of ion channels, calcium signalling and water permeability. The presentation will show the results of our recents works demonstrating that 1)the growth on nano-biomaterials induces astrocytes differentiation that is accompanied with upregulated expression at the process microdomains and increased function of K+channel Kir4.1 and Aquaporin-41; 2)the use of carbon-based and silicon based device enable the electrical stimulation of intracellular Ca2+([Ca2+]i) and the extracellular recording of transmembrane voltage oscillations in the slow frequency range in differentiated astrocytes in vitro2; 3) infrared laser pulse provide the possibility to stimulate water transport, [Ca2+]iand whole-cell conductance mediated via Transient Receptor Potential Ankirin 1 (TRPA1) and TRP Vanilloid-4 (TRPV4)3.

Acknowledgments:The work is supported by AFOSR-Biophysics Programme with the projects ASTROMAT, ASTRONIR, 3D NEUROGLIA. The speaker is grateful to all the co-authors and Co-PIs that contributed to the results presented.1Saracino E., Maiolo L., et al., Adv Biosystem, 2020, in press; Saracino, Guarino et al., J Mater Chem B 2Borrachero-Conejo et al., Adv Healt. Mater, 2020 3Borrachero-Conejo A., Adams W. et al., Faseb J, under review

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Technical Workshop

Exposure to graphene oxide alters astrocyte homeostasis and astrocyte-to-neuron communication. (ID 570)

Lecture Time
09:23 - 09:43

Abstract

Abstract Body

Aims: There is an increasing interest toward the use of graphene (G) and G-related materials (GRMs) for biomedical applications, especially for targeting the central nervous system. Within the European Graphene Flagship, we previously studied the interaction between GRMs and primary neurons (Bramini et al, ACSNano, 2016). We subsequently characterized the impact of GRMs on astroglial cells, addressing the mechanisms of G flake internalization together with the possible inflammatory responses.

Methods: We cultured primary rat cortical astrocytes and exposed them to pristine G (GR) and graphene oxide (GO) flakes. The biological interaction between flakes and astrocytes was studied through confocal and electron microscopy, biochemistry and molecular biology, live calcium imaging and electrophysiology.

Results: Astrocytes internalized GR/GO through the endo-lysosomal pathway, acquiring a differentiated phenotype associated with cytoskeletal rearrangements. Proteomic and lipidomic analyses unveiled alterations in intracellular Ca2+ homeostasis and cholesterol metabolism, which were particularly intense in cells exposed to GO. Indeed, GO exposure impaired spontaneous and evoked astrocyte Ca2+ signals and induced a marked increase in membrane cholesterol levels. Moreover, GO internalization led to the upregulation of inward-rectifying K+ channels and of Na+-dependent glutamate uptake. Interestingly, GO-pretreated astrocytes induced an increase in intrinsic excitability and in the density of GABAergic synapses of co-cultured primary neurons.

Conclusions: Our results indicate that graphene nanomaterials profoundly affect astrocyte physiology in vitro with consequences for neuronal network activity. This work supports the view that GO-based materials could be of great interest to address pathologies of the central nervous system associated with astrocyte dysfunctions.

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Technical Workshop

Lessons learned from a tissue engineered 3D model of the glial scar (ID 643)

Abstract

Abstract Body

The diseases of the central nervous system (CNS) can have devastating consequences and repercussions at the individual, societal and economical level.

The CNS has a poor regenerative capacity with the astrocytic scar tissue that is formed in the aftermath of an insult being blamed for the little functional recovery that is observed. Although its formation is paramount for our survival, the scar tissue is seen as a hostile territory and a physical barrier for regeneration. But while inevitable, the scar is a dynamic structure, about which the physicality has been largely neglected. How do astrocytes respond to the tissue alterations that occur upon an injury? How does matrix rigidity impact their response? How are these changes orchestrated as disease progresses? How do these alterations contribute to the unbalancing of the forces that inhibit remyelination and axonal growth?

To understand when and how the changes in a CNS lesion environment occur and how these condition the progress of the tissue response and ultimately of the disease, requires a systematic approach, as scar formation results from a plethora of events. We proposed the development of a tissue engineered glial scar model to investigate the astrogliosis process. With this “tool box” we expect to unveil molecular mechanisms that rule not only the astrogliosis dynamics, and contribute to the design of new therapeutic strategies, but also contribute to a better understanding of the CNS homeostasis.

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Technical Workshop

Reverse engineering neuron-glial interactions: An overarching approach from cellular biophysics to medicine (ID 646)

Lecture Time
10:03 - 10:23
Technical Workshop

Exploring the structure, neuronal content, and functional significance of astrocytic domains of behaving animals with CLARITY and 2-photon imaging (ID 649)

Abstract

Abstract Body

Astrocytes have a significant role in modulating neuronal activity and even behavior as shown by recent ground-breaking research. Intriguingly, astrocytes cover discrete physical domains, with minimal overlap between their fine processes, yet a possible functional significance of this tightly-controlled spatial organization was never directly explored. First, we virally expressed fluorophores in thousands of astrocytes in mouse brains, and cleared the tissue using the CLARITY technique, enabling us to image large hippocampi volumes. Non-truncated astrocytes were segmented and their elaborate morphology was reconstructed, revealing novel correlations between their location in the hippocampal lamina and various anatomical characteristics. Next, in addition to astrocyte tagging, we simultaneously expressed a fluorophore in the nuclei of pyramidal neurons in dorsal CA1 to determine the excitatory neuronal content distribution in astrocyte domains. Finally, we investigated the spatial distribution of distinct inhibitory neuronal populations (Parvalbumin, Somatostatin, or VIP) relative to astrocytes, and found that the minimal distance between astrocyte somata to Somatostatin somata was significantly smaller compared to their distance from Parvalbumin or VIP somata. To study the real-time functionality of astrocyte domains in the dorsal hippocampus, we have used 2-photon imaging of astrocytic and neuronal activity in mice navigating a virtual reality maze. We provide the first comprehensive quantification of 3D hippocampal astrocytic domains and their neuronal contents, breaking new ground to studying the functional interactions between astrocytes and neurons in and beyond the hippocampus.

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Technical Workshop

The colour of pain: Technology advances allowing the future objective quantification (ID 569)

Lecture Time
10:43 - 11:03