The role of neuronal activity on retinal angiogenesis and blood-retina barrier (BRB) maturation - PROJECT SUMMARY Reciprocal interactions among neuroglial and vascular components of the postnatal retina are critical for proper angiogenesis and the establishment of the blood-retinal barrier (BRB). Although neuronal or glial cells-derived signals that promote angiogenesis and BRB maturation are being elucidated, we still do not understand: a) how neuronal synaptic activity in the retina influences these processes; b) which neurotransmitter(s) contribute to these processes; and c) whether neurotransmitters act on endothelial cells (ECs) directly or indirectly, via astrocytes or Müller cells. The superficial plexus vasculature develops (P1-P8) during the spontaneous cholinergic wave of neural activity (P0-P10) and photoactivation of Opn4+ retinal ganglion cells (RGC). In contrast, the deep plexus vasculature and BRB development span cholinergic and glutamatergic waves of neural activity (P10-P14), glutamatergic synaptic activity of photoreceptors (P10 onwards) as well as photoactivation of Opn4+ RGCs. Transient pharmacological blockade of cholinergic wave impairs deep plexus angiogenesis and BRB maturation; however, the role of extracellular glutamate in these processes is unknown. In preliminary findings, we have primarily used three mouse strains to assess the effects of synaptic glutamate release on retinal angiogenesis and the BRB: a) Vglut1 -/-, that lack glutamate release, b) Gnat1-/-, in which rod photoreceptors remain depolarized, thus release excess glutamate, and c) Chrnb2-/-, where cholinergic wave is abolished and glutamatergic wave begins early (P8). We have found that synaptic glutamate release is a positive regulator of deep plexus angiogenesis and BRB maturation. This effect is mediated by inducing expression of Norrin in Müller glia and Norrin/b-catenin pathway activation in ECs. Based on these preliminary data, we hypothesize that released glutamate levels are sensed by Müller cells, which in turn operate as transducers to induce expression of angiogenic and BRB-forming factors and promote deep plexus angiogenesis and BRB maturation. We will test this hypothesis through three aims. First, we will examine how modulation of glutamate levels [Vglut1-/- mice (no glutamate release), Gnat1-/- mice (high glutamate release), and Chrnb2-/- mice (early onset of glutamatergic wave)] regulates retinal angiogenesis and structural and functional BRB integrity. Next, we will determine whether Müller cells respond to extracellular glutamate levels by inducing expression of angiogenic and barriergenic factors. We will examine Müller cell response [cell number, expression of GLAST (Slc1a3; a major glutamate transporter), glutamine synthetase (enzyme responsible for converting glutamate to glutamine) and Norrin (the major Wnt ligand expressed by Müller cells)] in Vglut1-/-, Gnat1-/- and Chrnb2-/- mice. Finally, we will test if activation of endothelial Norrin/b-catenin signalling that promotes angiogenesis and BRB maturation can rescue deficits in Vglut1-/- mice. These studies will provide a mechanistic understanding of how glutamatergic synaptic activity regulates angiogenesis and BRB in the developing retina and provide new insights into effects of excitotoxicity on neurodegenerative diseases in the retina.
