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Hydrogen Ion Fluxes from Isolated Retinal Horizontal Cells: Modulation by Glutamate.

Peter J. S. Smith [1]

Robert Paul Malchow [2]

Retinal horizontal cells are second order neurons that receive direct input from photoreceptors. These cells are believed to play a crucial role in the formation of the surround aspect of the classic center-surround receptive fields of visual neurons. Debate still persists as to the molecular mechanisms used by horizontal cells to establish the surround portion of these receptive fields. One hypothesis, promulgated recently by Kamermans and colleagues [1], suggests that horizontal cells may exert their lateral inhibitory actions by modulating the calcium flux into the synaptic terminals of photoreceptors, thus altering the release of the photoreceptor neurotransmitter. Hydrogen ions are among several agents that have been proposed to act in this modulatory role [2], and in fact, the responses to light by second order retinal neurons are very sensitive to changes in extracellular pH [3, 4]. In an elegant series of experiments, Barnes and coworkers [5] demonstrated that this pH-dependent modulation of synaptic transmission was due to the marked sensitivity of calcium channels in the photoreceptors to extracellular hydrogen ions. These investigators found that elevated concentrations of [H.sup.+] shifted the voltage-dependence of the calcium current activation curve of the photoreceptors to more depolarized levels and also reduced the calcium conductance. Moreover, small light-induced changes in extracellular pH within the intact retina have been reported by Oakley and Wen [6].

Horizontal cells could thus exert their inhibitory influences by modifying the concentration of hydrogen ions in the external milieu. In the present work, we have used pH-selective microelectrodes to monitor the flux of hydrogen ions surrounding isolated retinal horizontal cells. In particular, we examined whether the amino acid glutamate could alter the flux of hydrogen ions recorded from these cells. We reasoned that, if the release of hydrogen ions from horizontal cells is indeed a key factor in the creation of the surround portion of retinal receptive fields, then such a flux should be modified by glutamate, the neurotransmitter believed to be released by vertebrate photoreceptors [7].

The pH-selective electrodes were used in a self-referencing mode [8], which greatly enhances their signal sensitivity and stability, eliminating much of the electrical noise and drift inherent in such electrodes. In this format, the electrode is first placed just adjacent to the membrane of the cell, and a reading taken; the electrode is then moved a set distance away (typically 30 [micro]m), and a second reading taken. The difference between the voltage readings at the two positions reflects differences in the free hydrogen activity at the two locations. This method allowed us to measure the small hydrogen ion fluxes that would otherwise have been lost in the noise of the recordings.

pH selective electrodes were prepared by pulling thin-walled glass capillary tubing (o.d. 1.5 mm) to a tip diameter of 2-4 [micro]m. The pipettes were silanized and back-filled with 100 mM potassium chloride, and the fluid was forced to the tip of the pipette by air pressure applied to the back of the pipette from a syringe. The pipette tip was then filled with a pH-selective resin (hydrogen ionophore 1-Cocktail B, Fluka Chemica); the tip was placed in contact with a source pipette containing the resin, and about 50 [micro]m of the resin was then drawn up by suction on the back of the pipette. The resin employed here has a particularly high selectivity for hydrogen ions, and is reported to be more than [10.sup.9] times more sensitive to hydrogen ions than to either sodium or potassium ions [9]. Isolated retinal horizontal cells were obtained by enzymatic dissociation of the retina of the skate (Raja erinacea or R. ocellata) as described in Malchow et al. [10]. Briefly, the animals were chilled in ice, cervic ally transected, and double pithed. The eyes were removed, and the retinas were isolated and placed for 45 min under gentle agitation into a skate-modified L-15 culture medium containing 2 mg/ml papain and 1 mg/ml cysteine. The retinas were then rinsed 8 times in media lacking papain and cysteine, and then mechanically agitated through a 5-ml graduated glass pipette. Single drops of this cellular suspension were placed in 35-mm plastic culture dishes that had previously been coated with 1% protamine sulfate and 0.1% concanavalin A. Cells were stored at 14[degrees]C for up to 4 days before use. Recordings were made in a skate Ringer's solution containing 2 mM of the pH buffer HEPES and no added bicarbonate. A 5 mM glutamate stock solution was prepared in skate Ringer and adjusted to pH 7.6 with 1 M NaOH. Glutamate was applied by adding 1 ml of the 5 mM glutamate solution to 4 ml of Ringer already present in the culture dish, resulting in a final concentration of 1 mM glutamate.

Under these conditions, a steady differential signal was obtained from horizontal cells indicative of a higher concentration of hydrogen ions near the membranes of the cells. The size of this signal decreased as the concentration of the pH buffer HEPES was increased, consistent with the hypothesis that the signal detected indeed reflected hydrogen ions. Moreover, as shown in Figure 1, the application of 1 mM glutamate resulted in a marked decrease in the size of the differential signal. A differential signal of approximately 100 [micro]V was initially recorded from this cell. The actual proton flux represented by this differential voltage can be calculated using an equation derived by D. M. Porterfield [in prep.; see also (12)] as follows:

J = - D([delta][[H.sup.+]] + [Buffer] * 0.25[delta][[H.sup.*]] * [[K.sup.-1].sub.a]) * [delta][r.sup.-1]

Where J is the flux, D is the diffusion coefficient for hydrogen ions, [delta][[H.sup.+]] the change in hydrogen ion activity between the two poles of measurement, [Buffer] is the buffer concentration expressed in moles per [cm.sup.-3], [K.sup.a] is the p[K.sub.a] of the buffer expressed in [cm.sup.-3] and [delta]r is the distance in cm between the two measuring positions of the probe. Taking into account a small loss of the signal within the electronics of the amplification system (8), under our experimental conditions the 100 [micro]V signal we observe is then estimated to be indicative of a proton flux of [sim]75 pM [cm.sup.-2] [s.sup.-1]. In 6 cells studied in this fashion, 1 mM glutamate reliably reduced the differential signal by an average of 60%.

We thus conclude that glutamate, the presumed neurotransmitter from vertebrate photoreceptors, can indeed alter the flux of hydrogen ions from horizontal cells. In this context, it is interesting to note that glutamate has previously been reported to promote an acidification of the internal milieu of catfish retinal horizontal cells as measured using the pH-indicator dye BCECF [11]. We hypothesize that glutamate may shut down the transport of hydrogen ions from horizontal cells, thus trapping hydrogen ions in the interior of the cell. This would account for the increased intracellular acidity and the alkalinization of the extracellular milieu that we have observed. The alteration in extracellular pH induced by glutamate may be important in modifying signaling within the outer plexiform layer of the retina. Indeed, extracellular alkalinizations induced by neuronal activity occur in several other regions of the nervous system (reviewed by Chesler [13]), and excitatory amino acid receptors have been implicated in the generation of these phenomena. Thus, modulation of extracellular pH within the CNS by glutamate may be a common means by which synaptic activity is altered. Future experiments are planned in which specific pharmacological agents will be used to determine which transporter or transporters may be involved in the glutamate-induced changes in extracellular hydrogen ion concentrations.

We are grateful to Kasia Hammar for her generous assistance with electrode preparation and cell culture, Naomi Rosenkranz for help preparing isolated cells, and Richard H. Sanger for electronic and computer assistance. This work was supported by grants EY09411 from the National Eye Institute, P41 RR01394 from the National Center for Research Resources, and a grant from the Campus Research Board of the University of Illinois at Chicago.

(1.) BioCurrents Research Center, Marine Biological Laboratory, Woods Hole, MA.

(2.) Departments of Biological Sciences and Ophthalmology, University of Illinois at Chicago, Chicago, IL.

Literature Cited

(1.) Verweij, J., M. Kamermans, and H. Spekreijse. 1996. Vision Res. 36: 3943-3953.

(2.) Kamermans, M., and H. Spekrcijse. 1999. Vision Res. 39: 2449-2468.

(3.) Kleinschmidt, J. 1991. Ann. N.Y. Acad. Sci. 635: 468-470.

(4.) Harsanyi, K., and S. C. Mangel. 1993. Vis. Neurosci. 10: 81-91.

(5.) Barnes, S., V. Merchang, F. Mahmud. 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 10081-10085.

(6.) Oakley, B. and R. Wen. 1989. J. Physiol. (Land) 419: 353-378.

(7.) Copenhagen, D. R., and C. E. Jahr. 1989. Nature 341: 536-539.

(8.) Smith, P. J. S., K. Hammar, D. M. Porterfield, R. H. Sanger, and J. R. Trimarchi. 1999. Microsc. Res. Tech. 46: 398-417.

(9.) Fluka, 1991. Selectophore, Ionophores for Ion-Selective Electrodes and Optrodes Fluxa Chemie A. G. Ronkonkoma, New York.

(10.) Malchow, R. P., H. Qian, H. Ripps, and J. E. Dowling. 1990. J. Gen. Physiol. 95: 177-188.

(11.) Dixon, D. B., K-I. Takahashi, and D. R. Copenhagen. 1993. Neuron 11: 267-277.

(12.) Smith, P. J. S. and J. Trimarchi. 2000. Am. J. Physiol. (in press.)

(13.) Chesler, M. 1990. Progr. Neurobiol. 34: 401-427.
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Author:Molina, Anthony J. A.; Smith, Peter J. S.; Malchow, Robert Paul
Publication:The Biological Bulletin
Geographic Code:1USA
Date:Oct 1, 2000
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