Neuronal signaling within the vertebrate retina is highly dependent upon the pH of the extracellular fluid. For example, the modest alteration of external pH from 7.8 to 7.0 has been shown to suppress almost completely the light-induced responses of second order neurons in the retina of the tiger salamander (1-3). Further, stimulation of the retina by light can induce significant changes in extracellular pH (4, 5), raising the possibility that normal light-induced alterations in the extracellular proton concentration might play an important role in modulating the flow of visual information within the retina. Ratiometric imaging techniques using fluorescent pH indicator dyes such as BCECF have begun to expand our knowledge of the control of pH within retinal neurons (cf. 6, 7). However, this method does not readily permit direct examination of fluxes of protons across the membranes of neurons and glia. This limitation, coupled withthe cellular heterogeneity of the vertebrate ret ina,has made it difficult to study the cellular and molecular mechanisms responsible for the maintenance of extracellular pH and its alteration during light stimulation. The purpose of this study was to determine if a different method could be used to examine proton flux directly from isolated retinal neurons, setting the stage for future experiments designed to understand at a molecular level the mechanism that might be responsible for changes in external pH. We used an electrode in the self-referencing format that permits examination of the small ionic gradients expected to be generated at the plasma membrane-saline interface (see Smith, Sanger & Jaffe (8) for review). Isolated retinal neurons from skate (Raja erinacea/R. ocellata) were prepared using the enzymatic dissociation protocol described by Malchow et al. (9). Our results demonstrate that self-referencing pH-selective microelectrode can indeed be used successfully to examine proton flux across the membranes of isolated retinal neurons. We are currently employing specific pharmacological probes known to selectively inhibit various pH transport mechanisms in an effort to clarify the molecular mechanisms responsible for the fluxes we have detected. References 1. Kleinschmidt, J. 1990. Soc. Neurosci. (Abstr.) 16: 465. 2. Kleinschmidt, J. 1991. Ann N.Y. Acad. Sci. 635: 468-470. 3. Barnes, S., Merchant, V. & Mahmud, F. 1993. Proc. Natl. Acad. Sci 90: 10081-10085. 4. Oakley, B. II & Wen, R. 1989. J. Physiol. (Lond.) 419: 353-378. 5. Borgula, G.A., Karwoski, C.J., &Steinberg, R.H. 1989. Vision Res. 29: 1069-1077. 6. Haugh-Scheidt, L. & Ripps, H. 1998. Exp. Eye Research 66: 449-464. 7. Saarikoski, J., Ruusuvuori, E., Koskelainen, A. & Donner, K. 1997. J. Physiol. (Lond.) 498: 61-72. 8. Smith, P.J.S., Sanger, R.H. & Jaffe, L. F. 1994. Meth. Cell Biol. 40: 115-134. 9 .Malchow, R.P., Qian, H., Ripps, H. & Dowling, J.E. 1990. J. Gen. Physiol. 95: 177-198.
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