Abstract
BackgroundDeveloping retinas display retinal waves, the patterned spontaneous activity essential for circuit refinement. During the first postnatal week in rodents, retinal waves are mediated by synaptic transmission between starburst amacrine cells (SACs) and retinal ganglion cells (RGCs). The neuromodulator adenosine is essential for the generation of retinal waves. However, the cellular basis underlying adenosine's regulation of retinal waves remains elusive. Here, we investigated whether and how the adenosine A2A receptor (A2AR) regulates retinal waves and whether A2AR regulation of retinal waves acts via presynaptic SACs.Methodology/Principal FindingsWe showed that A2AR was expressed in the inner plexiform layer and ganglion cell layer of the developing rat retina. Knockdown of A2AR decreased the frequency of spontaneous Ca2+ transients, suggesting that endogenous A2AR may up-regulate wave frequency. To investigate whether A2AR acts via presynaptic SACs, we targeted gene expression to SACs by the metabotropic glutamate receptor type II promoter. Ca2+ transient frequency was increased by expressing wild-type A2AR (A2AR-WT) in SACs, suggesting that A2AR may up-regulate retinal waves via presynaptic SACs. Subsequent patch-clamp recordings on RGCs revealed that presynaptic A2AR-WT increased the frequency of wave-associated postsynaptic currents (PSCs) or depolarizations compared to the control, without changing the RGC's excitability, membrane potentials, or PSC charge. These findings suggest that presynaptic A2AR may not affect the membrane properties of postsynaptic RGCs. In contrast, by expressing the C-terminal truncated A2AR mutant (A2AR-ΔC) in SACs, the wave frequency was reduced compared to the A2AR-WT, but was similar to the control, suggesting that the full-length A2AR in SACs is required for A2AR up-regulation of retinal waves.Conclusions/SignificanceA2AR up-regulates the frequency of retinal waves via presynaptic SACs, requiring its full-length protein structure. Thus, by coupling with the downstream intracellular signaling, A2AR may have a great capacity to modulate patterned spontaneous activity during neural circuit refinement.
Highlights
During a critical period in the developing retina, immature retinal ganglion cells (RGCs) spontaneously fire periodic bursts of action potentials that propagate across the retina, encompassing hundreds to thousands of cells [1,2]
We found that A2A receptor (A2AR) immunoreactivity was localized to the IPL and the GCL but not the neuroblast layer (NBL) or other regions of the postnatal rat retina (Fig. 1A and C), consistent with A2AR expression in the GCL and amacrine cell layer in the developing ferret retina [20]
Further immunostaining in the dissociated starburst amacrine cells (SACs) confirmed the colocalization of A2AR and ChAT immunoreactivities (Fig. 1E), indicating that A2AR is expressed in SACs
Summary
During a critical period in the developing retina, immature retinal ganglion cells (RGCs) spontaneously fire periodic bursts of action potentials that propagate across the retina, encompassing hundreds to thousands of cells [1,2]. A general adenosine receptor antagonist, aminophylline, which blocks waves [3,21], was later found to act as a GABAAR agonist mediating tonic activation that can alter the correlation structure of stage-II waves [14,15]. These results suggest that revisiting the pharmacological results is necessary to verify the role of adenosine signaling in regulating retinal waves. We investigated whether and how the adenosine A2A receptor (A2AR) regulates retinal waves and whether A2AR regulation of retinal waves acts via presynaptic SACs
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