Reciprocal Interactions Between Calcium and Chloride in Rod Photoreceptors

Abstract

(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.

Synaptic transmission at retinal ribbon synapses

“…In contrast, the Cl − efflux which accompanies activation of I Cl(Ca) depletes intracellular Cl − and thus inhibits I Ca through direct effects of Cl − on Ca 2+ channel open probability . In support of such a feedback relationship between I Ca and I Cl(Ca) (Thoreson et al, 2003a). In cones, E Cl appears to be near or slightly below the dark resting potential (Kaneko and Tachibana, 1986;Thoreson and Burkhardt, 1991;Kraaij et al, 2000;Thoreson and Bryson, 2005), whereas in rods it is 25 mV positive to the dark resting potential .…”

“…4 which shows the change in I Ca from rod photoreceptors recorded when using a rod voltage response to light as the command waveform. At a dark potential of −44 mV in salamander rods, I Ca attains <20% of its peak activity (Thoreson et al, 2003a). The probability that calcium channels remain open diminishes as the membrane hyperpolarizes in response to light and I Ca becomes vanishingly small as the membrane potential passes below −55 mV.…”

“…Cl − flux can also alter membrane potential and a balance between these two effects may help to stabilize the tonic activation level of I Ca (Thoreson et al, 2003a). To better understand these simultaneous feedback interactions, we consider the case of rods where E Cl is positive to the dark membrane potential (Bader et al, 1982;Somlyo and Walz, 1985;Burkhardt et al, 1991;Thoreson et al, , 2003a.…”

“…Activation of D 2 dopamine receptors stimulates I Ca but nonetheless inhibits glutamate release from rods, at least partly as the result of a feedback loop involving the activation of I Cl(Ca) (Thoreson et al, , 2003a. Invoking the same sort of feedback loop, one would predict that inhibition of I Ca by A 2 receptors should stimulate glutamate release, but instead A 2 receptor activation inhibits synaptic transmission.…”

“…In rod terminals, E Cl is well above the resting potential and thus the opening of calcium-activated chloride channels or the chloride channels associated with plasma membrane glutamate transporters produces a chloride efflux (Thoreson et al, , 2003aRabl et al, 2003). By altering the rate of glutamate uptake, changes in intraterminal chloride levels might therefore alter the strength of synaptic output from photoreceptors.…”