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 spine–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.
