Background: Src is a non-receptor tyrosine kinase that tightly modulates neuronal function in the CNS. Results: AMPAR-mediated signaling regulates Src activation and retinal cell death through the canonical nitric oxide signaling pathway. Conclusion: Nitric oxide coupling to Src transduces AMPARs signaling in retinal neurons. Significance: Src activity may integrate death-associated signaling in retinal neurons.

Ascorbate, the reduced form of vitamin C, is highly concentrated in the central nervous system (CNS), including the retina, where it plays important physiological functions. In the CNS, the plasma membrane transporter sodium vitamin C co-transporter 2 (SVCT2) is responsible for ascorbate transport in neurons. The neurotransmitter dopamine (DA), acting through D- and D-like receptor subfamilies and classically coupled to adenylyl cyclase, is known to modulate synaptic transmission in the retina. Here, we reveal that DA controls the release of ascorbate from retinal neurons. Using primary retinal cultures, we show that this DA effect is dose-dependent, occurring by the reversal of the SVCT2, and could be elicited by brief and repetitive pulses of DA. The DA effect in inducing ascorbate release occurs by the activation of DR and is independent of PKA. Moreover, the exchange protein directly activated by cAMP type 2 (EPAC2) is present in retinal neurons and its specific knockdown using shRNAs abrogates the DR-induced ascorbate release. Confirming the physiological relevance of this pathway, activation of DR or EPAC2 also triggered ascorbate release ex vivo in acute preparations of the intact retina. Overall, DA plays pivotal roles in regulating ascorbate homeostasis through an unanticipated signaling pathway involving DR/adenylyl cyclase/cAMP/EPAC2, thereby suggesting that vitamin C might fine-tune dopaminergic neurotransmission in the retina.

Dopamine and glutamate are critical neurotransmitters involved in light-induced synaptic activity in the retina. In brain neurons, dopamine D1 receptors (D1Rs) and the cytosolic protein tyrosine kinase Src can, independently, modulate the behavior of NMDA-type glutamate receptors (NMDARs). Here we studied the interplay between D1Rs, Src and NMDARs in retinal neurons. We reveal that dopamine-mediated D1R stimulation provoked NMDAR hypofunction in retinal neurons by attenuating NMDA-gated currents, by preventing NMDA-elicited calcium mobilization and by decreasing the phosphorylation of NMDAR subunit GluN2B. This dopamine effect was dependent on upregulation of the canonical D1R/adenylyl cyclase/cAMP/PKA pathway, of PKA-induced activation of C-terminal Src kinase (Csk) and of Src inhibition. Accordingly, knocking down Csk or overexpressing a Csk phosphoresistant Src mutant abrogated the dopamine-induced NMDAR hypofunction. Overall, the interplay between dopamine and NMDAR hypofunction, through the D1R/Csk/Src/GluN2B pathway, might impact on light-regulated synaptic activity in retinal neurons.