The core of the work entailed the establishment of an all-optical approach for mapping higher-order thalamocortical feedback inputs onto cortical pyramidal neurons using the mouse whisker primary somatosensory cortex as a model system.
To this end, we sparsely expressed a genetically-encoded glutamate indicator in cortical pyramidal neurons. We densely expressed the red-light activatable opsin in the higher-order thalamus, followed by cranial window implantation for subsequent in vivo imaging.
We optically stimulated dense axonal projections originating from the higher-order thalamus expressing the light-sensitive opsin through the microscope objective, upon which we near-simultaneously monitored the released glutamate by imaging of the indicator expressed on the post-synaptic pyramidal cells using two-photon laser-scanning microscopy. This identified the location of functional synaptic connections.
We developed an automatized analysis pipeline for high-throughput synaptic mapping analysis. In order to probe functional connectivity longitudinally over several weeks, we refined our imaging and stimulation protocol ensuring neuronal health and integrity. Our findings show that inputs from the higher-order thalamus connect to some dendritic branches of L2/3 pyramidal neurons but not others suggesting a converging organization. Based on this first part, we currently continue investigating the spatial arrangement of other cortical converging pathways. Next, we used our methodology to identify the active pathways in response to sensory-evoked activity. Data from this second part of the project are still being acquired and analyzed.
So far, we have presented this methodology at conferences and included data in two different research grant applications but have not been further disseminated yet.
