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FJ Rosenstein, RL Chappell; Identification of Isolated Skate Retinal Bipolar Cells with ON- and OFF-like Properties . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3766.
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Purpose: To obtain direct physiological evidence concerning the presence of both ON-like and OFF-like bipolars in cell populations isolated from the all-rod retina of the skate (Raja erinacea). Methods: Isolated cells were maintained in a skate-modified L-15 culture medium (Malchow and Ripps. 1990. PNAS 87:8945) supplemented with 5 µg/ml insulin, 12.5 µg/ml taurine, 4.41 µg/ml trimethylamine N-oxide (TMAO), 0.5 µg/ml linoleic acid-oleic acid-albumin (albumin from bovine serum), and 5.9 µg/l sodium selenite to improve yields of glutamate-sensitive cells. Pieces of the skate retina were incubated (70 min) in the unsupplemented medium to which 2.14 mg/ml papain, activated with 1mg/ml L-cysteine, and 0.36 mg/ml protease had been added. Following a thorough rinse in enzyme-free medium, cells were separated by gentle trituration of the retinal tissue through a sterile pipette, plated on plastic culture dishes and stored at 14°C. Prior to patch-clamp recording, the medium was replaced with a skate-modified Ringer solution (Qian et al.1997. J. Neurophysiol. 78:2402). Results: Isolated bipolar cells were identified morphologically and tested with a variety of glutamate receptor agonists. Cells voltage clamped at -25 to -50 mV could be identified as ON-like cells by the outward current induced by glutamate (0.3 - 10 µM), APB (1 - 10 µM), and/or t-ACPD (30 µM) as well as the absence of a response to kainate (100 - 300 µM). Conversely, OFF-like cells were identified with an inward current induced by glutamate and kainate with no response to APB or t-ACPD under these conditions. Currents for each cell type reversed when they were clamped at +30 mV. Testing for kainate sensitivity, the dose-response curve obtained for OFF-like bipolar cells was found to have an EC50 of 23.8 µM kainate and a Hill coefficient of 1.16. Desensitization observed at higher glutamate concentrations (≷10 µM) made it difficult to fit a Hill equation to the ON-like bipolar cell responses. However, by fitting data from lower concentrations (0.3 to 7 µM) an EC50 of 0.49 µM glutamate with a Hill coefficient of 1 was approximated. No morphological characteristics distinguishing isolated ON- from OFF-like bipolar cells were observed. Conclusion: These findings provide direct physiological evidence for the presence of both ON- and OFF-like bipolar cells in the retina of the skate. This supports the notion (Chappell and Rosenstein. 1996. J. Gen. Physiol. 107:535) of a functional OFF-bipolar afferent pathway for the all-rod visual system of the skate. Support: NIH Grant EY00777 and PSC/CUNY Grant 63213-0032
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