Tommaso Dalla Tor

Universitè de Bordeaux

(FP1)

Analysis of endocannabinoid dynamics in astrocytes

The existence of astroglial CB1 receptors has raised many questions about their contribution to synaptic functions, indeed it has been observed and demonstrated that the activation of astroglial CB1 receptors mediates fundamental effects for synaptic communication and modulation. In the brain, eCB production has been always attributed to neurons, in a process involving postsynaptic depolarization and calcium increases. However, astrocytes express all the machinery to produce eCBs, although their role in eCB production and release has been largely ignored. Therefore, in this project, we wanted to analyze the production of endocannabinoids by astrocytes in order to better understand how these molecules can modulate the communication between astrocytes and neurons. In order to do so, we successfully expressed and validated GRABeCB2.0, a fluorescent biosensor consisting of a human CB1 receptor coupled to a GFP protein, which increases its fluorescence right after the receptor binds to cannabinoids and changes its conformation. In this study, we were able to find an effective protocol to study the dynamics of eCBs in astrocytes in vitro, using in vitro fluorescence imaging. Indeed, our data suggest that astrocytes present spontaneous endocannabinoid activity.

Karan Gupta

UniPD

(FP2)

3D brain model for prediction of TBI and its surgical approaches

Currently, we are working on a brain model to investigate the TBI that may lead to brain hemorrhage during several impact conditions. The on-going data is validated with the experimentally performed analysis of the brain in different impact directions. Further implementation of this research is to create a virtual simulation where the surgeons can easily watch the surgery steps to cure and diagnose the hemorrhage and other related injuries without any prior rehearsal. This project will impact other severely impacted regions and make the workflow of surgeons effortless.

Matteo Bruzzone

UniPD

(FP3)

Inferring brain-wide modules from databases of anatomically reconstructed neurons

Despite the development of techniques to generate everyday more detailed anatomical reconstructions of neuronal circuits, unravelling the connectivity patterns is still a heavy workload. In fact, retrieving connectivity schemes across the whole brain requires an accessible model organism, high-resolution acquisitions, careful cell skeletonization followed by annotation and proofreading of the synaptic contacts. This results in a time-demanding process. Theoretical approaches can help in exploring the available datasets. Here, we propose a method to infer the connectivity patterns between anatomically reconstructed neurons and, based on this, to reveal brain-wide modules at different levels of organization.

Sandra Beriain

Universitè de Bordeaux

(FP4)

Endocannabionid system in Müller glial cells

Cannabinoids are organic substances found in marijuana but also could be synthase endogenously as endocannabinoids. Endocannabinoids are secreted by different cells as intercellular lipophilic messenger that activated cannabinoids receptor (CB1 and CB2). The endogenous cannabinoids, the enzyme involved in the synthesis and degradation and the specific receptors form the endocannabinoid system. In previous studies done, it was demonstrated that synthetic cannabinoid induce neuroprotection in the retina as well as a hypotensor effect in glaucoma disease. However, the specific role of the endocannabinoid system in the retina is unknown. In this work, we study the presence of cannabinoid receptor 1 (CB1) and the endocannabinoid production in the principal glia of the retina, Müller cells, using in vitro fluorescence imaging.

Samuel Leitao

EPFL
(CH)

(FP5)

Correlative 3D Imaging of Single Cells using Scanning Ion Conductance (SICM) and Super-Resolution Microscopy

Samuel M. Leitao¹, V. Navikas², K. S. Grussmayer², A. Descloux², B. Drake¹, K. Yserentant³, P. Werther⁴, D.-P. Herten³, R. Wombacher^4,5, A. Radenovic², and G. E. Fantner^1
1Laboratory for Bio- and Nano-Instrumentation, Institute of Bioengineering, School of Engineering, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland.
²Laboratory of Nanoscale Biology, EPFL, Lausanne, Switzerland.
³College of Medical and Dental Sciences, Medical School & School of Chemistry, University of Birmingham, Birmingham, United Kingdom.
⁴Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany.
⁵Department of Chemical Biology, Max Planck Institute for Medical Research, Heidelberg, Germany.

The evolution of the 3D morphology is at the center of many relevant biological processes ranging from cellular differentiation to cancer invasion and metastasis. Microscopy techniques, such as super-resolution optical microscopy, electron microscopy, and atomic force microscopy (AFM), have been applied to image the structure of cells in great detail. The major challenge is to obtain 3D information at nanometer resolution without affecting the viability of the cells and avoiding interference with the process. Here, we show a scanning ion conductance microscope (SICM) as an alternative approach for high-speed and long-term nanoscale imaging of mammalian cells [1]. By implementing advances in nanopositioning, nanopore fabrication, microelectronics, and controls engineering we developed a microscopy method that can resolve spatiotemporally diverse 3D processes on the cell membrane at sub-5 nm axial resolution, without probe contact. Additionally, we combine 3D surface data from SICM with volumetric super-resolution optical fluctuation imaging (SOFI), to generate correlative high-resolution information of cell morphology (Figure 1). To demonstrate the capabilities of our method, we employ correlative SICM/SOFI microscopy for visualizing the membrane and cytoskeleton dynamics in live cells with subdiffraction-resolution [2]. The combination of the multimodal SICM and flexible SOFI approach has the potential to become a routine live-cell imaging modality that will offer new insights into cell-cell interactions, infection, and neurobiology. Ultimately, we applied our combined system to study neural regeneration, by 3D imaging of primary neurons from M.domestica.

References
[1] Samuel M. Leitao et al. Time-resolved scanning ion conductance microscopy for three-dimensional tracking of nanoscale cell surface dynamics. ACS Nano (2021), 15, 11, 17613–17622.
[2] Vytautas Navikas, Samuel M. Leitao et al. Correlative 3D imaging of single cells using super-resolution and scanning ion-conductance microscopy. Nature Communications 12, 4565 (2021).

Ksenia Orekhova

UniPD

(FP6)

Stereological assessment of immunohistochemical markers in the auditory nuclei of bottlenose dolphins

The importance of assessing the neurochemical processes in the cetacean brain to monitor their cognitive health and model neurodegenerative diseases in humans is increasingly evident, but most investigations rely on semi-quantitative data. Stereological analyses on serial thick sections generate rigorous quantitative estimates of identifiable cell types according to their morphology and expression of molecular markers. This study counted cell numbers in serial sections of a bottlenose dolphin brain, quantifying the expression of SMI312, β-amyloid peptide, Iba1, GFAP, TDP43 and fibronectin in the inferior colliculus (IC) and ventral cochlear nucleus (VCN) using StereoInvestigator® software. Comparisons were drawn to human brains of healthy individuals and chronic traumatic encephalopathy cases. Cell counts yielded coefficients of error <10% for most of the tested markers. The immunoreactivity of fibronectin in the dolphin resembled the pattern in a human chronic encephalopathy brain. This may mean that neurochemical compensation for insults such as hypoxia may constitute a noxious response in humans, while being physiological in dolphins. 

Clara Zaccaria

UniTN
(IT)

(FP7)

A platform for single cell optogenetics to study synaptic engrams in vitro

Storage of memory relies on the correlated firing of a specific set of simultaneously activated cells (neuronal engram) in networks of interconnected neurons, whose connecting synapses are strengthened by long-term potentiation (LTP) mechanisms. Synapses are expected to be involved in these processes (synaptic engram) and might contribute to distinct aspects of the neuronal engram. Optogenetic tools, together with a fluorescence probe allowing activity mapping of synaptic engram, and a digital light processor device (DLP), provide the access to visualize/reactivate with spatiotemporal accuracy selected populations of potentiated spines connecting engram neurons. The DLP platform has 3 LED sources and a digital micromirror device. It is optically aligned to a confocal spinning-disk microscope, projecting on the sample plane 460 nm light patterns with 3+/-1um resolution. This device was used to apply LTP-like patterned light illumination on targeted neurons in cultures transfected with SynActive (SA), an approach combining Arc mRNA targeting elements with a protein tag for activity-dependent protein production in spines, to show synaptic potentiation.

Sebastiano Antonio Rizzo

Cardiff Universtiy – Wales (UK)

(FP8)

Glucose-delivering materials to rescue neurons in oxygen-glucose deprivation: a strategy to improve neural transplantation in neurodegenerative diseases?

Sebastiano Antonio Rizzo¹, Oliver Bartley² , Anne Rosser², Ben Newland^1
1 – School of Pharmacy and Pharmaceutical Sciences, Cardiff University, CF10 3NB, Wales, UK
² – Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, CF10 3AX Wales, UK

Cell therapies hold the potential to halt or reverse several pathologies, including Parkinson’s Disease (1). Despite showing promise, cell death post transplantation remains a limitation and recent findings have suggested that a paucity of glucose (glc) is playing a crucial role in graft death (2, 3).
To improve graft survival, this work aimed to formulate glucose-delivering materials by encapsulating glc into a variety of photocrosslinkable acrylate monomers, which allowed us to produce bulk (1.5 x 2.0 mm) and injectable microspheres (120-180 µm), then screened for glc release.
To mimic the prolonged graft ischemia, and assess glc impact on hypoxic neurons, iPSCs derived neurons (dopaminergic, medium spiny and cortical), foetal ventral mesencephalon tissue and SH-SY5Y cells were cultured up to 96 hours either at 21% or 0.1% pO2 at different glc concentrations.
To date we have shown that: 1) glc paucity, rather than oxygen deprivation, negatively affects neurons viability; 2) glc can be released in a sustained manner (>7 days) and, although showing a brief release, 3) in vitro, the microspheres where still able to rescue cell viability from OGD induced death.

Acknowledgements and Funding
This work was supported by the School of Pharmacy and Pharmaceutical Sciences, Cardiff University and the Academy of Medical Sciences via the Springboard Fund.

References
1. Parmar M, Torper O, Drouin-Ouellet J. Cell-based therapy for Parkinson’s disease: A journey through decades toward the light side of the Force. European Journal of Neuroscience 2019;49:463-471.
2. Newland H, Eigel D, Rosser AE, Werner C, Newland B. Oxygen producing microscale spheres affect cell survival in conditions of oxygen-glucose deprivation in a cell specific manner: implications for cell transplantation. Biomaterial Science 2018;6:2571-2577.
3. Rizzo SA, Bartley O, Rosser AE, Newland B. Oxygen-glucose deprivation in neurons: implications for cell transplantation therapies. Progress in Neurobiology 2021;205:102126.

Benedetto Ardini

PoliMI
(IT)

(FP9)

A multimodal widefield hyperspectral microscope

Fluorescence and Raman spectroscopy are complementary methods for investigating the properties of materials since they address different transitions of molecules. However, due to the huge difference in the typical cross sections of the two effects, fluorescence has been always considered a hindrance for Raman, and no combined instrument exists. In addition, typical Raman microscopes rely on point-by-point scanning and dispersive spectrometers: this results in long acquisition times (∼5h for 0.1MP images), which can be reduced only by sacrificing the spatial resolution. We have developed an innovative Fourier-transform hyperspectral microscope capable of disentangling Raman and fluorescence signals only by changing the sampling approaches. The microscope is based on an ultrastable and compact common path interferometer that enables multimodal high-throughput, wide-field imaging, greatly reducing the acquisition time. Our hyperspectral microscope is capable of performing sequentially high-spatial resolution (<1 μm) Raman mapping and spectrally resolved fluorescence imaging in ∼30min for 0.1MP image. The details of the instrument and applications will be presented.

Davide Colaianni

UniPD

(P1)

miR-210 knock-out leads to retinal degeneration in Drosophila melanogaster and mice

miR-210 is one of the most evolutionary conserved microRNAs and is involved in many physiological and pathological conditions. Recently, several studies have suggested critical roles of miRNAs in visual system development and functioning as well as in eye diseases, both in Drosophila melanogaster and mammals. In particular, miR-210 has been linked to corneal epithelial repair and found to be elevated in primary open-angle glaucoma patients. In fruit flies, the loss of miR-210 results in a progressive retinal degeneration, which seems to be related with lipid droplets accumulation and alterations in lipid metabolism. The first morphological (confocal immunofluorescence and transmission electron microscopy) and molecular (gene expression analysis) characterization of the retina of miR-210 knock-out mice show retinal stress as well as photoreceptor degeneration similar to those described in miR-210 knock-out flies, but no evidences suggest an involvement of lipid metabolism in the pathological phenotype. Further characterization of these models will pave the way for a complete understanding of the functional role of miR-210 in the maintenance of the proper homeostasis of the visual system.

Heather Strelevitz

Univerity of Trento

(P2)

The Honeybee PER Technique: Improvements from Automation.

Honeybees (Apis mellifera) are a well-loved model within the neurosciences, prized for their remarkable cognitive abilities, rich social lives, and simple central nervous system. To evaluate bees’ capacity for learning and memory, the proboscis extension reflex (PER) uses classical conditioning to pair an unconditioned sucrose reward (US) with a conditioned stimulus (CS) until the presentation of the CS alone will elicit the proboscis extension. However, the manual stimulus presentation and behavioral scoring introduces variation which may lead to vastly different outcomes. We present an automated PER technique, from the mechanized stimulus delivery to the video recording analysis by a neural network. It is flexible for presenting a range of stimuli and has a success rate similar to previously reported data. This novel tool standardizes PER so we may take fuller advantage of the method’s scientific power, and more reliably pair it with other techniques including imaging.

Angelica Casotto

UniPD

(P3)

GBA2 dysfunction in Hereditary Spastic Paraplegia impairs neuronal function and morphology

Hereditary Spastic Paraplegia (HSP) is a heterogeneous group of inherited neurodegenerative and neurodevelopmental disorders. The primary clinical manifestation is spastic paraparesis, but they can also show other various neurological and systemic symptoms.
Mutations in several different genes were associated to HSP. Among them, the mutations in the GBA2 gene, encoding non-lysosomal enzyme glucocerebrosidase, cause a particular form of HSP with ataxia. GBA2 mutations are loss of function and cause the reduction of GBA2 enzymatic activity and GBA2 mislocalization. These lead to the alteration of the levels of GBA2 substrates, glucosylceramide and cholesterol-β-D-glucoside (β-GlcChol). β-GlcChol is a glycosylated cholesterol that has only been recently found in mammalian tissues, and its homeostasis is believed to be very important for neuronal health.
Interestingly, cerebellar neurons in which GBA2 was inhibited present reduced neurite outgrowth at the early stages of the development, but no information are available at later stages of their maturation.
Here, we characterize the arborization of GBA2-inhibited mice cerebellar neurons via confocal imaging at day in vitro 9-14, to possibly correlate morphological defects to neuronal signalling impairment. To identify functional defects, Ca2+ imaging experiments were performed on mature cerebellar neurons by stimulating neurons with non-toxic glutamate concentrations (10uM). Biochemical experiments were also performed to match morphological and functional features of neurons to GBA2 level, GBA2 activity, and to β-GlcChol lipid content in mature neurons. Moreover, the activity and level of GBA1, which is the lysosomal glucocerebrosidase performing cholesterol transglycosylation, were also characterized.
Our results suggest that GBA2 inhibition has a subtle impact on mature neurons at the morphological levels, while they present impaired calcium signalling in physiological conditions. The molecular mechanisms that link β-GlcChol depletion and glucocerebrosidases function to dysfunction in mature neurons remain to be elucidated and will be the object of further investigations.

Tommaso Dalla Tor

Universitè de Bordeaux

(P4)

Analysis of endocannabinoid dynamics in astrocytes

The existence of astroglial CB1 receptors has raised many questions about their contribution to synaptic functions, indeed it has been observed and demonstrated that the activation of astroglial CB1 receptors mediates fundamental effects for synaptic communication and modulation. In the brain, eCB production has been always attributed to neurons, in a process involving postsynaptic depolarization and calcium increases. However, astrocytes express all the machinery to produce eCBs, although their role in eCB production and release has been largely ignored. Therefore, in this project, we wanted to analyze the production of endocannabinoids by astrocytes in order to better understand how these molecules can modulate the communication between astrocytes and neurons. In order to do so, we successfully expressed and validated GRABeCB2.0, a fluorescent biosensor consisting of a human CB1 receptor coupled to a GFP protein, which increases its fluorescence right after the receptor binds to cannabinoids and changes its conformation. In this study, we were able to find an effective protocol to study the dynamics of eCBs in astrocytes in vitro, using in vitro fluorescence imaging. Indeed, our data suggest that astrocytes present spontaneous endocannabinoid activity.

Sandra Beriain

Universitè de Bordeaux

(P5)

Endocannabionid system in Müller glial cells

Cannabinoids are organic substances found in marijuana but also could be synthase endogenously as endocannabinoids. Endocannabinoids are secreted by different cells as intercellular lipophilic messenger that activated cannabinoids receptor (CB1 and CB2). The endogenous cannabinoids, the enzyme involved in the synthesis and degradation and the specific receptors form the endocannabinoid system. In previous studies done, it was demonstrated that synthetic cannabinoid induce neuroprotection in the retina as well as a hypotensor effect in glaucoma disease. However, the specific role of the endocannabinoid system in the retina is unknown. In this work, we study the presence of cannabinoid receptor 1 (CB1) and the endocannabinoid production in the principal glia of the retina, Müller cells, using in vitro fluorescence imaging.

Sebastiano Antonio Rizzo

Cardiff Universtiy – Wales (UK)

(P6)

Glucose-delivering materials to rescue neurons in oxygen-glucose deprivation: a strategy to improve neural transplantation in neurodegenerative diseases?

Sebastiano Antonio Rizzo¹, Oliver Bartley², Anne Rosser², Ben Newland<sup>1

1</sup> – School of Pharmacy and Pharmaceutical Sciences, Cardiff University, CF10 3NB, Wales, UK
² – Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, CF10 3AX Wales, UK

Cell therapies hold the potential to halt or reverse several pathologies, including Parkinson’s Disease (1). Despite showing promise, cell death post transplantation remains a limitation and recent findings have suggested that a paucity of glucose (glc) is playing a crucial role in graft death (2, 3).
To improve graft survival, this work aimed to formulate glucose-delivering materials by encapsulating glc into a variety of photocrosslinkable acrylate monomers, which allowed us to produce bulk (1.5 x 2.0 mm) and injectable microspheres (120-180 µm), then screened for glc release.
To mimic the prolonged graft ischemia, and assess glc impact on hypoxic neurons, iPSCs derived neurons (dopaminergic, medium spiny and cortical), foetal ventral mesencephalon tissue and SH-SY5Y cells were cultured up to 96 hours either at 21% or 0.1% pO2 at different glc concentrations.
To date we have shown that: 1) glc paucity, rather than oxygen deprivation, negatively affects neurons viability; 2) glc can be released in a sustained manner (>7 days) and, although showing a brief release, 3) in vitro, the microspheres where still able to rescue cell viability from OGD induced death.

Acknowledgements and Funding
This work was supported by the School of Pharmacy and Pharmaceutical Sciences, Cardiff University and the Academy of Medical Sciences via the Springboard Fund.

References
1. Parmar M, Torper O, Drouin-Ouellet J. Cell-based therapy for Parkinson’s disease: A journey through decades toward the light side of the Force. European Journal of Neuroscience 2019;49:463-471.
2. Newland H, Eigel D, Rosser AE, Werner C, Newland B. Oxygen producing microscale spheres affect cell survival in conditions of oxygen-glucose deprivation in a cell specific manner: implications for cell transplantation. Biomaterial Science 2018;6:2571-2577.
3. Rizzo SA, Bartley O, Rosser AE, Newland B. Oxygen-glucose deprivation in neurons: implications for cell transplantation therapies. Progress in Neurobiology 2021;205:102126.

Federico Antonio Giacomazzi

CIMEC

(P7)

Trail pheromone-mediated-communication in the ant Crematogaster scutellaris

Chemical communication is essential for ant’s colony organization and survival. Actions like trail-following, recruitment, orientation, signaling of the reproductive state and evaluation of food resources are coordinated by social (pheromones) and non-social (environmental) odors. Given the cruciality of detection and processing of chemical signals by the ant’s olfactory system, it is our interest to understand how social and non-social trails are processed in the brain, and whether there are specific  neural circuits dedicated for each type. To this aim we are studying the trail-following system in the ant C. scutellaris.
We identified 2-tridecanol as a main component of the trail pheromone for this ant. In this stage of the project, by performing behavioural experiments with artificial odors we are investigating the threshold concentration at which this compound is detected. This will allow us to perform functional studies on the neuronal circuits for trail pheromone processing, by means of electroantennography and two-photon calcium imaging on the antennal lobes, the primary olfactory area in insects brain.

Matteo Corti

PoliMI

(P8)

A multimodal widefield hyperspectral microscope

Fluorescence and Raman spectroscopy are complementary methods for investigating the properties of materials since they address different transitions of molecules. However, due to the huge difference in the typical cross sections of the two effects, fluorescence has been always considered a hindrance for Raman, and no combined instrument exists. In addition, typical Raman microscopes rely on point-by-point scanning and dispersive spectrometers: this results in long acquisition times (∼5h for 0.1MP images), which can be reduced only by sacrificing the spatial resolution. We have developed an innovative Fourier-transform hyperspectral microscope capable of disentangling Raman and fluorescence signals only by changing the sampling approaches. The microscope is based on an ultrastable and compact common path interferometer that enables multimodal high-throughput, wide-field imaging, greatly reducing the acquisition time. Our hyperspectral microscope is capable of performing sequentially high-spatial resolution (<1 μm) Raman mapping and spectrally resolved fluorescence imaging in ∼30min for 0.1MP image. The details of the instrument and applications will be presented.