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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - DNA (Dendritic integration by nanoscale neuroanatomy)

Teaser

Summary

How single neurons integrate and process incoming synaptic signals to generate a physiologically meaningful output remains a fundamental question in neuroscience. Long standing theoretical work predicts that nanoscale morphology of dendritic spines, the sites of excitatory synapses where the neuronal communication occur, plays a major role in synaptic function and dendritic integration of synaptic inputs. Moreover, brain disorders of neurodevelopment and ageing, which poses a substantial societal burden are increasingly associated with deficits in dendritic spines.
My overall goal in the Marie SÅ‚odowska-Curie Actions EF project-DNA was to examine how nanoscale dendritic spine morphology directly influences synaptic function and dendritic integration of pyramidal neurons over brain development. This has been a technically challenging question to address: electron microscopy allows the visualisation of spine ultrastructure, but it only provides a static snapshot; although, conventional light microscopy enables live cell imaging, its spatial resolution is diffraction limited. I proposed to use state-of-the-art superresolution technique, STED microscopy that allows live cell imaging with high spatial resolution in combination with physiological methods.
I find that the spine morphology and diffusion coupling of spines to the dendrite are dynamically regulated over brain development. In particular there is an age specific transient increase in diffusion coupling of spines coordinated with a widening and shortening of their necks. Together with my neurocomputational collaborator, I am currently developing a computational model based on the morphology data to further investigate how the microphysiology of dendritic spines and the integration of inputs onto multiple spines might be influenced by spine morphology over brain development.

Work performed

To visualise dendritic spines with STED microscopy, I used acute brain slices from mice where the pyramidal cells were volume labelled with the fluorescent protein, yellow fluorescent protein (YFP). I focused on three specific age groups: young [postnatal day(P) 15-18], intermediate (P35-38), and old (P60-65) on the basis of what is known about the gross development of dendrites, spine densities and somatic physiology of layer 5 pyramidal cells in the somatosensory cortex, which is the sensory area in mouse cortex that processes sensory input arising from whiskers. I also examined the diffusion coupling of spines to the dendrite by monitoring the recovery of the YFP signal after photobleaching it. To look at how spine morphology may influence the integration of multiple synaptic inputs along the dendrite, I explored how holographic photolysis, an optical method that allows stimulation of multiple synapses, which my secondment host specialises in can be applied with STED microscopy.
I find that the developmental trajectory of dendritic spine morphology and the kinetics of their diffusion coupling are dynamically regulated over brain development. Interestingly, there is a transient intermediate age at which the diffusion kinetics are faster between the spine and the dendrite coinciding with more spines having shorter and wider spine necks. This time period of more permissive spineâ€“dendrite crosstalk might be a way in which synapses undergo more cooperative and complex interactions between neighbouring synapses, thereby possibly boosting the computational power of dendrites. The relative autonomy of spines in younger animals might be necessary for discriminating synapses based on varying synaptic efficacies, thus facilitating competitive mechanisms that refine synaptic connectivity during development. While the relative autonomy in older age group might reflect the already stabilised input specific-synaptic connections.
I am currently developing and optimising a MCell based computational model based on my morphology data in collaboration with a neurocomputational partner. The model allows the simulation of the movements and reactions of molecules within the spine and its dendritic milieu to predict the influence of spine morphology on its microphysiology. We will also use the model to predict how spine morphology changes over brain development might influence dendritic integration, i.e. integration of inputs onto multiple spines. Spine morphology analyses together with the MCell based computational model of spine morphology is currently being prepared into a manuscript.
I also conceived a detailed optical path with my secondment host to incorporate holographic photolysis with the STED microscope, which can be implemented and used in the future to investigate the influence of spine morphology on dendritic integration and test the predictions of the neurocomputational model.

Final results

It has been long hypothesised that the nanoscale morphology of dendritic spines plays a key role in synaptic function and integration. As such understanding how spine morphology influences synaptic function is central to deciphering the complexities of neuronal processing. Here, I characterised the developmental trajectory of spine morphology and how it influences diffusion coupling of spines. The neurocomputational model based on this data provides a powerful means by which we can simulate how spine morphology influences its function, and how spines along the dendrite with differing morphologies that can become co-active together in response to a natural stimulus influence dendritic integration. Given that a number of disorders of neurodevelopment and ageing display abnormalities in dendritic spine morphology and/or density, this basic characterisation of their normal developmental trajectory provides a reference framework to better understand the diseased state. Furthermore, our neurocomputational model of dendritic spines provides a powerful means by which morphological deficits of dendritic spines associated with each disorder can be modelled to better understand how this influence synaptic function. In the long term the implementation of holographic photolysis with STED microscopy will enable us to experimentally test the predictions of the model on the integration of multiple synaptic inputs.