STARBURST AMACRINE CELL DENDRITES IN THE RABBIT RETINA EXPRESS BOTH THE Na–K–Cl and K–Cl COTRANSPORTERS.

Abstract

Abstract: : Purpose: Recent evidence indicates that directionally–selective (DS) light responses occur first in the retina in the dendrites of starburst amacrine cells (SACs), interneurons presynaptic to DS ganglion cells. However, the mechanism that underlies the DS responses of SAC dendrites is unclear. The Na–K–Cl (NKCC) and K–Cl (KCC) cotransporters mediate the depolarizing and hyperpolarizing effects of GABA, respectively. Because blockade of the Na–K–Cl and K–Cl cotransporters by bumetanide (BMN) and furosemide (FUR), respectively, eliminates the directional responses of DS ganglion cells and SAC dendrites, it has been suggested that the differential distribution of the Na–K–Cl and K–Cl cotransporters on SAC dendrites underlies their DS responses (Gavrikov et al., 2003, PNAS). We therefore studied whether SAC dendrites express both types of chloride cotransporter. Methods: Whole–cell patch clamp recordings of DAPI–stained rabbit displaced SACs were obtained and the effects of BMN and FUR studied. The identity of SACs was confirmed with biocytin injections. Expression of NKCC2 was also assessed immunohistochemically. Results: Because BMN and FUR are lipid soluble, we examined whether the cotransporters are expressed by the SACs themselves by studying the effects of BMN and FUR when the drugs were bath applied, compared to when they were introduced into the cells during whole–cell recording. FUR (25 µM) depolarized the SACs to the same extent when it was bath applied, compared to when it was introduced into the cells via the recording pipettes and BMN (10 µM) hyperpolarized the SACs to the same extent following both application techniques. DAPI and choline acetyltransferase labeled SACs showed specific NKCC2 labeling. The proximal dendrites of these cells were also clearly labeled. Conclusions:The finding that both FUR and BMN had the same effects when they were applied through the recording pipette, compared to when they were bath applied, indicates that the SAC dendrites express both types of chloride cotransporter, and that the cotransporters are tonically and highly active. The finding that FUR and BMN had opposite effects on the SACs further suggests that the Na–K–Cl and K–Cl cotransporters work in opposition to generate the directional responses of SAC dendrites and that the differential distribution of the two cotransporters along SAC dendrites mediates their directional light responses. Localization of NKCC2 on SACs dendrites also provides the first anatomical evidence to support this model.

Keywords: amacrine cells • ion transporters • retina: proximal (bipolar, amacrine, and ganglion cells)