We examined the contribution of calcium-induced calcium release (CICR) to synaptic transmission from rod photoreceptor terminals. Whole-cell recording and confocal calcium imaging experiments were conducted on rods with intact synaptic terminals in a retinal slice preparation from salamander. Low concentrations of ryanodine stimulated calcium increases in rod terminals, consistent with the presence of ryanodine receptors. Application of strong depolarizing steps (−70 to −10 mV) exceeding 200 ms or longer in duration evoked a wave of calcium that spread across the synaptic terminals of voltage-clamped rods. This secondary calcium increase was blocked by high concentrations of ryanodine, indicating it was due to CICR. Ryanodine (50 μM) had no significant effect on rod calcium current (I ca ) although it slightly diminished rod light-evoked voltage responses. Bath application of 50 μM ryanodine strongly inhibited light-evoked currents in horizontal cells. Whether applied extracellularly or delivered into the rod cell through the patch pipette, ryanodine (50 μM) also inhibited excitatory post-synaptic currents (EPSCs) evoked in horizontal cells by depolarizing steps applied to rods. Ryanodine caused a preferential reduction in the later portions of EPSCs evoked by depolarizing steps of 200 ms or longer. These results indicate that CICR enhances calcium increases in rod terminals evoked by sustained depolarization, which in turn acts to boost synaptic exocytosis from rods.

Presynaptic inhibition is a major mechanism for regulating synaptic transmission in the CNS and adenosine inhibits Ca(2+) currents (I(Ca)) to reduce transmitter release at several synapses. Rod photoreceptors possess L-type Ca(2+) channels that regulate the release of L-glutamate. In the retina, adenosine is released in the dark when L-glutamate release is maximal. We tested whether adenosine inhibits I(Ca) and intracellular Ca(2+) increases in rod photoreceptors in retinal slice and isolated cell preparations. Adenosine inhibited both I(Ca) and the [Ca(2+)]i increase evoked by depolarization in a dose-dependent manner with approximately 25% inhibition at 50 microM. An A2-selective agonist, (N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine) (DPMA), but not the A1- or A3-selective agonists, (R)-N(6)-(1-methyl-2-phenylethyl)adenosine and N(6)-2-(4-aminophenyl)ethyladenosine, also inhibited I(Ca) and depolarization-induced [Ca(2+)]i increases. An inhibitor of protein kinase A (PKA), Rp-cAMPS, blocked the effects of DPMA on both I(Ca) and the depolarization-evoked [Ca(2+)]i increase in rods. The results suggest that activation of A2 receptors stimulates PKA to inhibit L-type Ca(2+) channels in rods resulting in a decreased Ca(2+) influx that should suppress glutamate release.

Activation of D 2 -like dopamine receptors in rods with quinpirole stimulates L-type calcium currents (I Ca

Adenosine is released from retina in darkness; photoreceptors possess A2 adenosine receptors, and A2 agonists inhibit L-type Ca2+ currents (ICa) in rods. We therefore investigated whether A2 agonists inhibit rod inputs into second-order neurons and whether selective antagonists to A1, A2A, or A3 receptors prevent Ca2+ influx through rod ICa. [Ca2+]i changes in rods were assessed with fura-2. ICa in rods and light responses of rods and second-order neurons were recorded using perforated patch-clamp techniques in the aquatic tiger salamander retinal slice preparation. Consistent with earlier results using the A2 agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA), the A2A agonist CGS-21680 significantly inhibited ICa and depolarization-evoked [Ca2+]i increases in rods. The A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and A2A antagonist, ZM-241385, but not the A3 antagonist, VUF-5574, inhibited effects of adenosine on Ca2+ influx in rods. DPCPX and ZM-241385 also inhibited effects of CGS-21680, suggesting they both act at A2A receptors. Both A2 agonists, CGS-21680 and DPMA, reduced light-evoked currents in second-order neurons but not light-evoked voltage responses of rods, suggesting that activation of A2 receptors inhibits transmitter release from rods. The inhibitory effects of CGS-21680 on both depolarization-evoked Ca2+ influx and light-evoked currents in second-order neurons were antagonized by ZM-241385. By itself, ZM-241385 enhanced the light-evoked currents in second-order neurons, suggesting that endogenous levels of adenosine inhibit transmitter release from rods. The effects of these drugs suggest that endogenous adenosine activates an A2-like adenosine receptor on rods leading to inhibition of ICa, which in turn inhibits l-glutamate release from rod photoreceptors.

(Bader et al. 1982). In cones, the current flux through I Cl(Ca) is at least eightfold greater than the current through I Ca (Barnes and Hille 1989). The chloride equilibrium potential (E Cl ) of salamander rods is about -20 mV . In olfactory receptors, E Cl is also positive to the cell's resting potential and acts to boost the receptor potential (Kleene and Gesteland 1991). In rods, depolarizing responses to darkness would also presumably be boosted by activation of I Cl(Ca) . In addition, activation of I Cl(Ca) at the dark resting potential (around -45 mV) generates a Cl Ϫ efflux . The resulting reduction in [Cl Ϫ ] i has the unusual effect of inhibiting the open channel probability of single Ca 2ϩ channels in photoreceptor terminals . This may promote a negative feedback interaction whereby activation of I Ca leads to a Ca 2ϩ influx that activates a Cl Ϫ efflux, which in turn feeds back to inhibit I Ca . When I Ca is enhanced by quinpirole, this negative feedback interaction may help to account for the paradoxical finding that, although activation of D2/D4 dopamine receptors enhances I Ca , quinpirole nonetheless inhibits synaptic transmission from rods Witkovsky et al. 1989). Is this feedback interaction restricted to conditions where I Ca has been enhanced (e.g., with quinpirole) or does it regulate I Ca under normal operating conditions at the rod synapse? To address this question, we combined electrophysiology with Ca 2ϩ and Cl Ϫ imaging techniques to assess the reciprocal interactions between Ca 2ϩ influx and Cl Ϫ efflux under physiological conditions. The results show an intimate relationship between the two that support the hypothesis of a feedback interaction between I Ca and I Cl(Ca) operating near the dark potential and defines mechanisms that contribute to maintenance of a positive value for E Cl in rod photoreceptors. M E T H O D S Tissue preparationLarval tiger salamanders (Ambystoma tigrinum, 18 -25 cm) were cared for according to institutional guidelines. Retinal slices were prepared according to methods pioneered by Werblin (Werblin 1978) and Wu (Wu 1987). Salamanders were pithed and decapitated, an eye was enucleated, and the front of the eye was removed. The resulting eyecup was cut into three or four pieces, and a single piece was placed vitreal surface down onto a piece of filter paper (Millipore 2 ϫ 5 mm, Type GS, 0.2-m pores). After the retina adhered to the filter paper, the retina was isolated under chilled amphibian superfusate and cut into 125-m slices using a razor blade tissue chopper (Stoelting, Wood Dale, IL). The slices were rotated 90°to view the retinal layers when placed under a water immersion objective (60ϫ, 1.0 NA) on an upright fixed stage microscope (EF 600, Nikon). For electrophysiological experiments, all procedures were performed under infrared illumination. For imaging experiments, slices were prepared under a dissecting lamp in visible light.