Neurones, blood vessels and glial cells interact at the neurovascular unit (NVU) interface. The development of the NVU and neuronal networks coincide during short but critical developmental periods, suggesting that their development and maturation may be intimately linked from a functional perspective. In the neonatal mouse retina, spontaneous waves of electrical activity consisting of intense bursting activity propagate across the retinal ganglion cell layer, coinciding with the expansion of the superficial vascular plexus during the first postnatal week. At the same time, autofluorescent clusters cell complexes (CCCs) appear under the leading edge of the plexus, disappearing once the plexus reaches the retinal periphery, around postnatal day 8. We hypothesise that these newly discovered transient CCCs are linked to the onset of retinal waves. We propose that they contain cells responsible for the generation of local activity hotspots, which leads to local hypoxia and results in the need for local metabolic energy, supplied by oxygen provided by the growing blood vessels. Once blood vessels reach the activity hotspots, cells responsible for triggering the waves are eliminated via microglia- the resident immune cells of the CNS. The origin of the autofluorescence in the CCCs may reflect metabolic stress as the cells become phagocytosed by microglia. By exploiting the autofluorescent nature of the CCCs, we performed fluorescence-activated cell sorting (FACS) followed by single-cell RNA sequencing (scRNA-Seq) to elucidate the genomic identity of the CCCs. Cluster analysis showed that the CCCs highly express multiple microglial markers, including, P2RY12, SALL1 and TGFBR1, as well as various neuronal markers. This strongly suggests that the CCCs reflect microglia in the process of cellular phagocytosis. Tracking the dynamic microglial behaviour in different compartments of the living retina during the first postnatal week, we can start to understand the function of microglia during this critical period of retinal development.

Using fluorescent time-lapse imaging of microglia in live retinas derived from CX3CR-GFP transgenic mice, we investigate how microglia interact with their neighbours under different pharmacological conditions. EGFPFluro-4(470 nm)16 filter is used to visualise the microglia, CY5 (635 nm)16 is used to visualise the blood vessels stained with isolectin B4 (IB4 594) and finally, DAPI (400 nm)16 filter is used to indicate the position of the cluster cells. Custom-coded algorithms in ImageJ enhance image quality, segment and mask microglial structures, and quantify changes in cell spatiotemporal features. Custom-written Matlab scripts extract crucial information for tracking complex microglial behaviours over time, such as cell area, number of branch points and velocity. Statistical comparisons of these behaviours are made between cells at varying distances from the CCCs.

By looking at various morphological microglial characteristics, we can establish their role during the period of retinal waves and angiogenesis by studying their phagocytic activity and motility at different retinal eccentricities, within and outside the vascular plexus, and within or outside CCCs areas.