Periodic Reporting for period 1 - NeuroExcell (Modulation of neuronal Signaling by Brain Extracellular Space Structure and Dynamics)
Reporting period: 2023-09-29 to 2025-09-28
The brain’s extracellular space (ECS) is a dynamic microenvironment that critically regulates neuronal signaling, molecular diffusion, and homeostasis. Despite its importance, the ECS remains poorly understood because current imaging methods cannot capture its nanoscale structure or dynamics across intact brain tissue. This knowledge gap limits our understanding of fundamental brain function and how extracellular processes contribute to neurological disorders.
NeuroExcell addresses this challenge by applying Super-resolution Shadow Imaging (SUSHI)—a breakthrough technique enabling direct visualization of the ECS at nanometer resolution in entire mouse brain hemispheres. The project will produce the first comprehensive nanoscale maps of the ECS, revealing how its structure and dynamics vary across brain regions and how they relate to extracellular matrix composition and protein distribution. Live-tissue imaging will further uncover how ECS organization influences diffusional processes and neuronal signaling.
By combining advanced fluorescence imaging, electrophysiology, computational modeling, and biochemistry, NeuroExcell will uncover the physiological roles of the ECS with unprecedented detail.
The project’s impact will be twofold. Scientifically, it will fill a critical gap in current brain maps, offering a new framework for understanding intercellular communication and glymphatic function. Technologically, it will establish SUSHI as a powerful tool for large-scale super-resolution imaging in living tissue. Beyond scientific advances, NeuroExcell aligns with European priorities in brain health and innovation, contributing to new strategies for tackling neurodegenerative and psychiatric disorders. The project will also strengthen the researcher’s independence and leadership through advanced training in a multidisciplinary environment, fostering long-term excellence in neuroscience research.
NeuroExcell addresses this challenge by applying Super-resolution Shadow Imaging (SUSHI)—a breakthrough technique enabling direct visualization of the ECS at nanometer resolution in entire mouse brain hemispheres. The project will produce the first comprehensive nanoscale maps of the ECS, revealing how its structure and dynamics vary across brain regions and how they relate to extracellular matrix composition and protein distribution. Live-tissue imaging will further uncover how ECS organization influences diffusional processes and neuronal signaling.
By combining advanced fluorescence imaging, electrophysiology, computational modeling, and biochemistry, NeuroExcell will uncover the physiological roles of the ECS with unprecedented detail.
The project’s impact will be twofold. Scientifically, it will fill a critical gap in current brain maps, offering a new framework for understanding intercellular communication and glymphatic function. Technologically, it will establish SUSHI as a powerful tool for large-scale super-resolution imaging in living tissue. Beyond scientific advances, NeuroExcell aligns with European priorities in brain health and innovation, contributing to new strategies for tackling neurodegenerative and psychiatric disorders. The project will also strengthen the researcher’s independence and leadership through advanced training in a multidisciplinary environment, fostering long-term excellence in neuroscience research.
The work focused on improving the preparation and maintenance of hippocampal organotypic slice cultures to ensure their long-term viability and physiological relevance in vitro. Specific optimization steps were implemented to enhance tissue health and preserve cellular morphology over extended culture periods, enabling more reliable experimental outcomes for subsequent imaging and manipulation.
High-resolution imaging experiments were carried out using a STED (Stimulated Emission Depletion) microscopy setup to investigate the dynamics of the brain extracellular space (ECS) within these organotypic hippocampal slices.
Time-lapse recordings were performed to capture real-time changes in ECS structure and diffusion properties under various experimental conditions.To probe the influence of neuronal activity on ECS dynamics, optogenetic approaches were employed. Constructs expressing light-sensitive modulators of ion channels were introduced to selectively alter neuronal signaling. By combining optogenetic stimulation with advanced live imaging, it was possible to monitor how controlled modulation of neuronal activity affects the organization and temporal behavior of the extracellular environment.
Main technical and scientific outcomes include:
- Development of an improved protocol for maintaining healthy hippocampal organotypic slices over extended in vitro periods.
- Successful implementation of time-lapse STED imaging to study extracellular space dynamics at subcellular resolution.
- Establishment of an experimental platform combining optogenetic manipulation with high-resolution imaging to investigate the relationship between neuronal signaling and extracellular space properties.
- Generation of quantitative data describing how neuronal modulation impacts ECS diffusion characteristics and microstructural organization.
High-resolution imaging experiments were carried out using a STED (Stimulated Emission Depletion) microscopy setup to investigate the dynamics of the brain extracellular space (ECS) within these organotypic hippocampal slices.
Time-lapse recordings were performed to capture real-time changes in ECS structure and diffusion properties under various experimental conditions.To probe the influence of neuronal activity on ECS dynamics, optogenetic approaches were employed. Constructs expressing light-sensitive modulators of ion channels were introduced to selectively alter neuronal signaling. By combining optogenetic stimulation with advanced live imaging, it was possible to monitor how controlled modulation of neuronal activity affects the organization and temporal behavior of the extracellular environment.
Main technical and scientific outcomes include:
- Development of an improved protocol for maintaining healthy hippocampal organotypic slices over extended in vitro periods.
- Successful implementation of time-lapse STED imaging to study extracellular space dynamics at subcellular resolution.
- Establishment of an experimental platform combining optogenetic manipulation with high-resolution imaging to investigate the relationship between neuronal signaling and extracellular space properties.
- Generation of quantitative data describing how neuronal modulation impacts ECS diffusion characteristics and microstructural organization.
The project has successfully advanced the understanding of the brain extracellular space (ECS) and its dynamic properties under physiological and controlled experimental conditions. Using organotypic brain slice cultures as a model system, we characterized the spatial and temporal changes in ECS parameters in response to neuronal activity and network modulation. The implementation of optogenetic stimulation allowed precise manipulation of neuronal signaling, enabling us to correlate specific activity patterns with alterations in ECS geometry, diffusion properties, and molecular exchange.
Potential Impacts:
Scientific and Technological Impact
Establishes a novel experimental platform combining organotypic cultures with real-time imaging and optogenetics for ECS research.
Provides quantitative data supporting theoretical models of ECS dynamics, improving the accuracy of computational simulations.
Enables further exploration of neuro-glial interactions and volume transmission mechanisms in intact tissue environments.
Key Needs for Further Uptake and Success:
Further Research:
Deeper investigation of ECS remodeling under pathological conditions and in vivo validation of the current findings.
Demonstration and Validation:
Scaling up to multi-modal imaging platforms and integration with electrophysiological readouts to confirm causality between neuronal firing and ECS changes.
Potential Impacts:
Scientific and Technological Impact
Establishes a novel experimental platform combining organotypic cultures with real-time imaging and optogenetics for ECS research.
Provides quantitative data supporting theoretical models of ECS dynamics, improving the accuracy of computational simulations.
Enables further exploration of neuro-glial interactions and volume transmission mechanisms in intact tissue environments.
Key Needs for Further Uptake and Success:
Further Research:
Deeper investigation of ECS remodeling under pathological conditions and in vivo validation of the current findings.
Demonstration and Validation:
Scaling up to multi-modal imaging platforms and integration with electrophysiological readouts to confirm causality between neuronal firing and ECS changes.