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Neuronal and dendritic recruitment on neocortical area borders

Periodic Reporting for period 1 - DENDRITESONBORDERS (Neuronal and dendritic recruitment on neocortical area borders)

Reporting period: 2016-09-01 to 2018-08-31

One of the most striking features of cortex is the electrically active dendritic arbors of its neurons. Besides serving as a scaffold for synaptic inputs, dendrites can amplify inputs and act as computational subunits within a neuron. However, the relationship between dendritic processing and information flow through cortical networks remains to be understood in vivo. Decades of progress in neurophysiology has lead to a growing understanding of the richness of dendritic mechanisms and their powerful impact on cellular output. However our understanding derives largely from in vitro studies and extensions in vivo are surprisingly sparse in the literature. Because of this basic lack of information we still cannot identify general principles or rules that govern the relationship between dendritic processing and neural computation in vivo. This limits our capacity to predict the consequences of therapeutic interventions and it limits our capacity to explain the neural bases of behaviour. Both of these objectives are of critical importance to society. Therapeutic approaches usually target subcellular mechanisms. If we cannot predict the impact of subcellular manipulations at the level of neural circuits, we cannot rationally design novel therapies. This limits our ability to fight diseases in the brain. Failure of prediction also limits our ability to relate neural computation to behaviour, the ultimate output of the brain. A full understanding of how behaviour is generated remains a goal of vital importance to society and could unlock vast unknown possibilities in scientific and technological advancement.
Our overall goal is to relate sub-cellular processes like synaptic integration to computations performed by large populations of neurons. In service of this larger goal, we focused in this project on dendritic integration in sensory neocortex because it presents experimental advantages as well as room for growth in this conceptual direction. Specifically, we have investigated the relationship between cortico-cortical communication, dendritic excitability, and brain state, a term used to refer to brain-wide regulatory processes like arousal. We focused on a specific cell type, one that represents one of the output channels of neocortex. We first investigated how correlated activity across cortical areas depends on sensory stimulation and brain state, and next investigated how the dendrites of these neurons are recruited under the same conditions. As a follow-up, we are currently performing experiments that bring these two sets of observations together.
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.
The experiments of work package 3 explained above required the development of dual color imaging techniques which extended beyond the state-of-the-art. Similarly, the completion of the last work package of our proposal required further development beyond the current state-of-the-art in two photon optogenetics. Functional connectivity mapping across cortical areas requires an imaging as well as a photostimulation field-of-view larger than what was possible with existing technology. In order to improve conditions, we partnered with Bruker Nanosystems, and have been working together to finalize the development of a prototype microscope with a field-of-view large enough to execute this experiment. We have now obtained proof-of-concept results with this new tool and are executing these experiments to complete the project.
We expect these final experiments to conclude this investigation, with the ultimate product indicating the importance of dendritic excitability in the regulation of cortico-cortical communication. Our findings have important implications for moment-to-moment computation in the brain, as well as longer timescale processes involving learning. We hope that the insights obtained here improve our predictive capacities in future studies designing novel therapeutic approaches, as well as our understanding of the relationship between neural activity and behaviour.