Periodic Reporting for period 1 - SynECS (Combining carbon nanotubes and gold nanorods to investigate the extracellular space around synapses during neuronal communication)
Periodo di rendicontazione: 2019-01-01 al 2020-12-31
The brain extracellular space (ECS) is a complex network of biomolecules that constitutes a key microenvironment for cellular communication, homeostasis, and clearance of toxic metabolites. In the brain, its spatial organization varies during sleep, development, and aging, and it is probably altered in neuropsychiatric and degenerative diseases. Although the ECS has recently emerged as key modulator of neural function, the interplay between this unique microenvironment and neuronal signalling at nanoscale temporal and spatial resolution still represent a knowledge frontier in brain research. SynECS proposed an innovative nanotechnological approach to unveil the specific biophysical characteristics of the ECS around synapses. Using the movements of carbon nanotubes in the intricate maze of the ECS channels, we managed to reconstruct super-resolved maps and local diffusivity values around identified synaptic areas. Interestingly, the nanotubes modified their diffusional rates accordingly to stimulation or inhibition of neuronal firing, suggesting that a molecular change of the local ECS around synapses was induced after application of the chemical stimuli. These results are an important step to underpin the activity of brain circuits in both physiological and pathological conditions and for a broader understanding of chemical-based neuronal communication and synaptic connectivity.
SynECS started with the development of an innovative microscopy technique to correctly visualize synapses within a thick brain slice. This technique, called HiLo microscopy, was key to correlate morphological and diffusional properties of the brain ECS around fluorescently labelled synaptic regions and was used to achieve simultaneous visible and near infrared images. HiLo microscopy is based on speckle illumination and allow acquisition of optically sectioned images from a standard widefield microscope.
HiLo imaging was indeed used to correlate the position of single-walled carbon nanotubes (SWCNTs) with fluorescently-labelled synaptic regions. SWCNTs are thin and long carbon-based nanowires with excellent mechanical and photophysical properties. Indeed, these nanoprobes have attracted particular attention for deep-tissue microscopy, due to their unique brightness, photostability, and spectral imaging range in the near infrared region. SWCNTs were initially tracked in organotypic and acute brain slices up to 100 μm deep using a widefield microscopy approach at millisecond timescale. Super-localization analysis showed that the ECS is a maze of polymorphic channels with widths in the order of 50-500 nm. Importantly, tracking of individual SWCNTs could also provide simultaneous measurements of the local ECS diffusivity environment. Indeed, specific rheological properties of the space, ranging from low to high local diffusivity, were measured in brain slices. SWCNT trajectories were also correlated with the synaptic extracellular environment, showing a modulation of the diffusional NT rates depending on different conditions of neuron stimulation.
SWCNT super-localization analysis was also applied to a mouse model of α-synuclein-induced neurodegeneration. Our group firstly performed an integrative study on pathological animals, exploring the extracellular microenvironment as a whole in adult brain tissue and giving novel insights on Parkinson’s disease effects in the brain. The study revealed poor correlation between local ECS width and nanoscale diffusion, suggesting that the diffusive inhomogeneities were not only driven by geometrical factors, but also by the molecular composition of the space. Importantly, our study pointed out to hyaluronan as major actor for the local variations in ECS diffusivity properties, opening up new hypothesis for the connection between ECS and brain pathologies.
SynECS was supported by a strong international visibility throughout the 2 years of funding. The project was presented in 4 different international conferences, including Photonics West and SPIE photonics Europe, the largest biophotonics, biomedical optics, and imaging conferences in the World. Moreover, results from the project have been already published in 3 different high-impact journals (including Nature Communication). Two additional publications are on current preparation for imminent submission.
HiLo imaging was indeed used to correlate the position of single-walled carbon nanotubes (SWCNTs) with fluorescently-labelled synaptic regions. SWCNTs are thin and long carbon-based nanowires with excellent mechanical and photophysical properties. Indeed, these nanoprobes have attracted particular attention for deep-tissue microscopy, due to their unique brightness, photostability, and spectral imaging range in the near infrared region. SWCNTs were initially tracked in organotypic and acute brain slices up to 100 μm deep using a widefield microscopy approach at millisecond timescale. Super-localization analysis showed that the ECS is a maze of polymorphic channels with widths in the order of 50-500 nm. Importantly, tracking of individual SWCNTs could also provide simultaneous measurements of the local ECS diffusivity environment. Indeed, specific rheological properties of the space, ranging from low to high local diffusivity, were measured in brain slices. SWCNT trajectories were also correlated with the synaptic extracellular environment, showing a modulation of the diffusional NT rates depending on different conditions of neuron stimulation.
SWCNT super-localization analysis was also applied to a mouse model of α-synuclein-induced neurodegeneration. Our group firstly performed an integrative study on pathological animals, exploring the extracellular microenvironment as a whole in adult brain tissue and giving novel insights on Parkinson’s disease effects in the brain. The study revealed poor correlation between local ECS width and nanoscale diffusion, suggesting that the diffusive inhomogeneities were not only driven by geometrical factors, but also by the molecular composition of the space. Importantly, our study pointed out to hyaluronan as major actor for the local variations in ECS diffusivity properties, opening up new hypothesis for the connection between ECS and brain pathologies.
SynECS was supported by a strong international visibility throughout the 2 years of funding. The project was presented in 4 different international conferences, including Photonics West and SPIE photonics Europe, the largest biophotonics, biomedical optics, and imaging conferences in the World. Moreover, results from the project have been already published in 3 different high-impact journals (including Nature Communication). Two additional publications are on current preparation for imminent submission.
The results and their interpretation placed the conducted research beyond the state-of-the-art in different fields of science. SWCNTs could indeed characterize the brain ECS of adult mice with nanometer resolution up to 100 μm deep in brain tissues at millisecond timescale, opening up novel possibilities for in vivo brain imaging. SWCNTs could also reveal the local rheological properties and dimensions of the ECS around synapses, leading to the definition of a novel synaptic extracellular environment. Moreover, our group firstly performed an integrative study on pathological animals, exploring the extracellular microenvironment as a whole in adult brain tissue and giving novel insights on the role of ECS in Parkinson’s disease.
The implications of our work in understanding the brain ECS is significant. As mentioned before, the ECS environment still represent a knowledge frontier in brain research and the spatiotemporal profile of molecular mobility in relation to the nanoscopic ECS dimensions is still poorly understood. Our results paved the way for the understanding of the interplay between this unique microenvironment and neuronal signalling in both healthy and pathological conditions. This is an important step to underpin the activity of brain circuits and for a broader understanding of chemical-based neuronal communication and synaptic connectivity. Furthermore, information on the ECS network at the nanoscale will foster novel strategies for genetic manipulation and pharmacological delivery in both healthy and pathological conditions. The possibility to probe adult brain tissues opens up new opportunities on the exploration of the ECS in aging and age-related brain disorders.
The implications of our work in understanding the brain ECS is significant. As mentioned before, the ECS environment still represent a knowledge frontier in brain research and the spatiotemporal profile of molecular mobility in relation to the nanoscopic ECS dimensions is still poorly understood. Our results paved the way for the understanding of the interplay between this unique microenvironment and neuronal signalling in both healthy and pathological conditions. This is an important step to underpin the activity of brain circuits and for a broader understanding of chemical-based neuronal communication and synaptic connectivity. Furthermore, information on the ECS network at the nanoscale will foster novel strategies for genetic manipulation and pharmacological delivery in both healthy and pathological conditions. The possibility to probe adult brain tissues opens up new opportunities on the exploration of the ECS in aging and age-related brain disorders.