We organized our original proposal into four work packages, the first of which included initialization goals. We extended the scope of these goals to include customization and optimization of surgical techniques developed in other laboratories, upgrading of existing microscopy equipment in the lab to enable the monitoring of multiple cortical areas simultaneously, and the training of other members of the laboratory in the execution of these experiments.
Our second work package included the functional mapping of two cortical areas, and assessment of the border zone between the two. We performed these experiments at the border between primary visual cortex and the Lateral-Medial higher visual area in mice. We located this border with widefield calcium imaging and followed up with cellular-resolution two-photon calcium imaging (Figure 1.) During these experiments animals were head-fixed, but free to locomote on a cylindrical treadmill and passively viewed visual stimuli. We found with both methods that the spatial extent of the retinotopic transition zone is on the scale of dendritic arbor widths, meaning that it is a fairly sharp border topographically. Next we measured the precision of the border in terms of tuning for simple visual features. These experiments revealed that the gradient of visual tuning preferences at the border is gradual. We further adopted more unbiased analytical approaches using classification of neurons into areas based on statistical features their activity, and explicit measurement of the correlations between pairs of neurons. Most of these analyses revealed activity across two cortical areas to be highly correlated, and the border between them to be gradual. In the process of these experiments we found that the topographic precision of the border, as well as other important features of activity depend on cortical layer. Cortical layers are composed of neurons that differ in many ways. Importantly for us, they differ in dendritic morphology and physiology, and our observations suggested that some of these differences may have a strong impact on cortico-cortical communication. This has led us to investigate the relationship between cortico-cortical communication and dendritic recruitment.
Having found that activity in related cortical areas is highly correlated, but with important differences between layers, we re-focused the experiments of our third work package to investigate how apical dendritic excitability influences cortico-cortical communication. In order to achieve our goals we further customized a two-photon microscope to perform dual-color volume imaging. We assayed several indicator combinations as well as excitation sources. After optimizing our conditions we were able to achieve imaging of glutamatergic inputs to apical and basal dendrites simultaneously with imaging of calcium signals in both dendritic and somatic compartments. For glutamate imaging we used a novel glutamate indicator (SF-iGluSnFR-A184S, Marvin, et al. 2018) while for calcium imaging we used the red indicator jRGeco1a (Dana et al 2015). This allowed us to investigate how input– output relationships depend on conditions that we found to modulate cortico-cortical communication in the experiments above.
We have presented this work at two conferences: the 2018 EMBO Workshop on Dendritic Anatomy, Molecules, and Function in Heraklion, Greece and the Society for Neuroscience Annual Meeting in San Diego, USA in November 2018.
