Advanced Expansion Microscopy Imaging System for Brain Microglia

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Interactions between the nervous and immune systems are critical for normal brain development and function. Microglia, the brain's immune cells, directly interact with neurons during brain development to control neuronal numbers and connections. Microglia also shape neuronal connections required for learning and memory in the adult brain. Imaging microglial interactions and phagocytosis (pruning) of neuronal synapses has been extremely limited due to the small size of synapses (<1um3) and the low throughput abilities of standard confocal microscopy. We have recently overcome the first limitation using Expanded Microscopy (ExM) that expands the tissue ~4x while preserving biomolecules of interest. By combining ExM of brain tissues with staining for microglia (P2YR12), activation state (CD68 lysosomal activation) and markers of synapses from different neuronal subtypes (e.g.serotonergic, GABAergic), we will be able to identify and quantify microglial pruning of specific neuronal circuits in development and adulthood. ExM is revolutionary in that it is compatible with high throughput imaging platforms, such as the Zeiss Cell Discoverer 7 (CD7). Thus, we are requesting funds for a novel, high throughput imaging setup with optimized objectives for ExM. The CD7 is 30x faster than confocal imaging and significantly easier for novice users. Hundreds of microglia can be imaged and synaptic phagocytosis quantified within hours, compared to the current rate of weeks. This facilitates the efficient, high confidence, quantitative analysis of microglial pruning across brain regions, sexes, ages and treatments. Currently, there is no viable alternative at our institution for ExM high throughput analysis of microglial phagocytosis. This state-of-the-art technique can only be fully utilized by with the proposed imaging setup and will establish UBC as a leader in microglial biology. This equipment is vital for three NSERC-related research programs, all of which aim to establish how immune cells contribute to normal brain function. 1) Ciernia's work will establish how innate immune cells in the brain control brain development 2) Galea's work will establish whether estradiol modulates neuroinflammation to impact cognition, and 3) Winstanley's work will establish the contribution of neuroinflammation within the prefrontal cortex on higher-order cognition. The co-applicants have the scientific expertise and operating cost support to fully utilize the proposed imaging setup. Together Ciernia, Galea and Winstanley have 39 trainees that will have access to this equipment, with half of these trainees directly engaged in NSERC-related work. HQP will acquire skills in ExM tissue preparation, staining, image acquisition, analysis, statistics and manuscript preparation, all important for highly competitive careers in and outside of academia. HQP in all three NSERC funded research programs will directly benefit from this new approach to analyzing microglial pruning.