Abstract
Purpose::
The objective is to understand mechanisms underlying vesicular release of glutamate from photoreceptors to hyperpolarizing bipolar cells in darkness and in light, and the impact of glutamate receptor desensitization on HBC postsynaptic currents.
Methods::
Dual patch clamp recordings were made from rod/HBC pairs under dark-adapted conditions (with infrared guidance). Ribbon synaptic contacts between rods/cones and HBCs were studied by serial reconstruction of electron micrographs of the outer plexiform layer.
Results::
In darkness, rod-dominated HBCs (HBCRs) exhibited many large miniature excitatory postsynaptic currents (mEPSCs). Light evoked a sustained outward current and significantly reduced the mEPSC frequency. A depolarizing (hyperpolarizing) voltage clamp step in the rod elicited a large transient inward current in the HBCR at the step onset (offset) (rod elicited excitatory postsynaptic currents, or reEPSCs). Application of cyclothiazide, an AMPA receptor desensitization blocker, prolonged the reEPSCs by about 58±7 msec, but exerted little actions on the amplitude of HBCR light responses. On the other hand, cyclothiazide greatly enhanced the glutamate-induced postsynaptic currents in HBCRs. By counting the number of ribbon synaptic contacts in the outer plexiform layer and measuring the dendritic coverage of HBCs, we found that each HBCR received photoreceptor inputs from about 100 releasing sites. By dividing the total charge of dark inward current by the charge of the average mEPSC, we estimated on average a HBC was bombarded by 485±23 mEPSCs per second in darkness. This suggests that on average about 5 mEPSCs per second (average interval of 200 msec) were generated at each releasing site. Since the average mEPSC interval is substantially longer than the time of recovery from AMPA receptor desensitization, mEPSCs in darkness are not significantly desensitized. This may explain why cyclothiazide exerts little action on HBCR light-evoked current response.
Conclusions::
Although HBCs are tonically depolarized by continuous vesicular glutamate release from rods and cones in darkness, vesicles to be released are spread over many release sites so that the release frequency at each site is low enough to avoid mutual desensitization.
Keywords: retina: distal (photoreceptors, horizontal cells, bipolar cells) • receptors: pharmacology/physiology • excitatory neurotransmitters