Abstract
Purpose: :
Photoreceptors receive a negative feedback signal from horizontal cells (HCs), establishing the center-surround receptive field organization of the visual system. The mechanism mediating the feedback is still under great debate. One of the hypothesis is that a change in extracellular pH mediates the feedback, but the small size of the synaptic cleft has prevented testing if a pH change actually occurs. The goal of this study is to test if pH at cone terminals really changes in a light-dependent manner.
Methods: :
A transgenic zebrafish line expressing a fusion of pH-sensitive GFP and the α2Δ4 subunit of the L-type Ca channel (named CalipHluorin) in cones was generated. Flat-mount retinas were imaged using 2-photon microscope, and the fluorescence intensity of CalipHluorin indicated the pH in the synaptic cleft.
Results: :
A 5% increase of CalipHluorin fluorescence was detected immediately after light stimulation, indicating alkalization of the synaptic cleft in the light. The signal increased with light intensity, saturated in bright light, and decayed in ~0.5 sec in darkness. CalipHluorin was expressed in cone terminals and inner segments, but light only affected the fluorescence signal at the terminals.The signal was blocked by the pH buffer HEPES. Consistent with HCs being the source of the signal, the pH change was blocked by the glutamate receptor antagonist DNQX, which blocks receptors on HCs but not photoreceptors. The pH change decreased with decreasing light spot diameter and was undetectable when the spot barely covered the imaged area. However, stimulation with an annulus, positioned completely outside the imaged area generated a significant pH change, indicating that laterally projecting neurons must mediate the response.
Conclusions: :
According to the pH hypothesis, light-elicited HC hyperpolarization should alkalize the synaptic pH, potentiating Ca channels in cone terminals, thereby increasing neurotransmitter release. It is known that buffering pH blocks feedback and artificially elevating pH mimics feedback, but whether light actually causes a change in pH is previously unknown. Here we have shown that synaptic pH is indeed regulated by light. The magnitude, direction, kinetics, and spatial dependency of the pH signal are consistent with the notion that a change in pH mediates lateral inhibition.
Keywords: horizontal cells • pH