Retinal circadian clock enhances GABA feedback to cones at night

Abstract

Purpose: Because GABA_A receptor (GABA_AR) expression and activity increase when intracellular cAMP increases (Luscher et al, 2011) and the retinal circadian clock, which activates the dopamine D₂Rs of cones in the day, increases cAMP in cones at night by not activating the D₂Rs (Ribelayga et al, 2008), we studied whether the clock increases the GABA_AR expression and activity of cones at night compared to the day.

Methods: Using whole-cell patch-clamp recording, the responses of cones in intact goldfish retinas to GABA puffed onto their terminals were studied in the day and night during synaptic blockade with 20 mM Mg²⁺ (in the presence of TPMPA, a GABA_CR antagonist). Rabbit and goldfish retinal sections were processed in an identical manner with specific antibodies against the GABA_AR β2/3 subunit (rabbits: bd-17; fish: 62-3G1).

Results: Cone responses to GABA puffs were clearly evident at night in the dark and in the day in the dark in the presence of spiperone, a D₂R antagonist that increases cAMP in cones, but were minimal in the day in the dark (control). Gabazine, a GABA_AR antagonist, increased the input resistance of fish cones at night, but had minimal effect in the day. Mid-mesopic light stimulation (which normally occurs at dawn and dusk) for 10 min at night increased cone light response size by 60%, an effect that was blocked by spiperone but which occurred following both spiperone and gabazine.<br /> Single label immuno-EM and double labeling with cell type-specific antibodies (cones: PNA (rabbits), zpr-1 (fish); ON-BCs: Goa;HCs: calbindin) revealed that GABA_AR antibody labeling was located in cone terminals at the HC to cone synapse following maintained (30 min) darkness at night (control) and following maintained darkness in the day with spiperone, but not in the day following maintained illumination or darkness, and not on HC or cone-BC dendrites following darkness at night or following darkness in the day with spiperone.

Conclusions: The results suggest that GABA_ARs are expressed in cone pedicles at the HC to cone synapse and functional to a greater extent at night in the dark than in the day in the dark or light, because the retinal clock, by not activating D₂Rs at night, elevates cAMP in cones. The findings also suggest that inhibitory GABA_AR-mediated feedback, by reducing cone light response size in the dark at night, acts as a circadian pathway that speeds up the transitions to cone vision at dawn and to rod vision at dusk.