Robert Paul Malchow

Modulation of Extracellular Proton Fluxes from Retinal Horizontal Cells of the Catfish by Depolarization and Glutamate

Self-referencing H+-selective microelectrodes were used to measure extracellular proton fluxes from cone-driven horizontal cells isolated from the retina of the catfish (Ictalurus punctatus). The neurotransmitter glutamate induced an alkalinization of the area adjacent to the external face of the cell membrane. The effect of glutamate occurred regardless of whether the external solution was buffered with 1 mM HEPES, 3 mM phosphate, or 24 mM bicarbonate. The AMPA/kainate receptor agonist kainate and the NMDA receptor agonist N-methyl-d-aspartate both mimicked the effect of glutamate. The effect of kainate on proton flux was inhibited by the AMPA/kainate receptor blocker CNQX, and the effect of NMDA was abolished by the NMDA receptor antagonist DAP-5. Metabotropic glutamate receptor agonists produced no alteration in proton fluxes from horizontal cells. Depolarization of cells either by increasing extracellular potassium or directly by voltage clamp also produced an alkalinization adjacent to the cell membrane. The effects of depolarization on proton flux were blocked by 10 μM nifedipine, an inhibitor of L-type calcium channels. The plasmalemma Ca2+/H+ ATPase (PMCA) blocker 5(6)-carboxyeosin also significantly reduced proton flux modulation by glutamate. Our results are consistent with the hypothesis that glutamate-induced extracellular alkalinizations arise from activation of the PMCA pump following increased intracellular calcium entry into cells. This process might help to relieve suppression of photoreceptor neurotransmitter release that results from exocytosed protons from photoreceptor synaptic terminals. Our findings argue strongly against the hypothesis that protons released by horizontal cells act as the inhibitory feedback neurotransmitter that creates the surround portion of the receptive fields of retinal neurons.

Gap-junctional properties of electrically coupled skate horizontal cells in culture

Whole-cell voltage-clamp recordings were used to examine the unusual pharmacological properties of the electrical coupling between rod-driven horizontal cells in skate retina as revealed previously by receptive-field measurements (Qian & Ripps, 1992). The junctional resistance was measured in electrically coupled cell pairs that had been enzymatically isolated and maintained in culture; the typical value was about 19.92 M omega (n = 45), more than an order of magnitude lower than the nonjunctional membrane resistance. These data and the intercellular spread of the fluorescent dye Lucifer Yellow provide a good indication that skate horizontal cells are well coupled. The junctional conductance between cells was not modulated by the neurotransmitters dopamine (200 microM) or GABA (1 mM), nor was it affected by the membrane-permeable analogues of cAMP or cGMP, or the adenylate cyclase activator, forskolin. Although resistant to agents that have been reported to alter horizontal-cell coupling in cone-driven horizontal cells, the junctional conductance between paired horizontal cells of skate was greatly reduced by the application of 20 mM acetate, which is known to effectively reduce intracellular pH. Together with the results obtained in situ on the receptive-field properties of skate horizontal cells, these findings indicate that the gap-junctional properties of rod-driven horizontal cells of the skate are fundamentally different from those of cone-driven horizontal cells in other species. This raises the possibility that there is more than one class of electrical synapse on vertebrate horizontal cells.

GABA TRANSPORT AND CALCIUM DYNAMICS IN HORIZONTAL CELLS FROM THE SKATE RETINA

1. Changes in intracellular calcium concentration [Ca2+]i in response to extracellularly applied gamma‐aminobutyric acid (GABA) were studied in isolated horizontal cells from the all‐rod skate retina. 2. Calcium measurements were made using fura‐2 AM, both with and without whole‐cell voltage clamp. Superfusion with GABA, in the absence of voltage clamp, resulted in an increase in [Ca2+]i; the threshold for detection was approximately 50 microM GABA, and a maximal response was elicited by 500 microM GABA. 3. The rise in [Ca2+]i was not mimicked by baclofen nor was it blocked by phaclofen, picrotoxin or bicuculline. However, the GABA‐induced [Ca2+]i increase was completely abolished when extracellular sodium was replaced with N‐methyl‐D‐glucamine. 4. With the horizontal cell voltage clamped at ‐70 mV, GABA evoked a large inward current, but there was no concomitant change in [Ca2+]i. Nifedipine, which blocks L‐type voltage‐gated Ca2+ channels, suppressed the GABA‐induced increase in [Ca2+]i. These findings suggest that the calcium response was initiated by GABA activation of sodium dependent electrogenic transport, and that the resultant depolarization led to the opening of voltage‐gated Ca2+ channels, and a rise in [Ca2+]i. 5. The GABA‐induced influx of calcium appears not to have been the sole source of the calcium increase. The GABA‐induced rise in [Ca2+]i was reduced by dantrolene, indicating that internal Ca2+ stores contributed to the GABA‐mediated Ca2+ response. 6. These observations demonstrate that activation of the GABA transporter induces changes in [Ca2+]i which may have important implications for the functional properties of horizontal cells.

Neurotransmitter modulation of extracellular H+ fluxes from isolated retinal horizontal cells of the skate

Self‐referencing H+‐selective microelectrodes were used to measure extracellular H+ fluxes from horizontal cells isolated from the skate retina. A standing H+ flux was detected from quiescent cells, indicating a higher concentration of free hydrogen ions near the extracellular surface of the cell as compared to the surrounding solution. The standing H+ flux was reduced by removal of extracellular sodium or application of 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA), suggesting activity of a Na+–H+ exchanger. Glutamate decreased H+ flux, lowering the concentration of free hydrogen ions around the cell. AMPA/kainate receptor agonists mimicked the response, and the AMPA/kainate receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) eliminated the effects of glutamate and kainate. Metabotropic glutamate agonists were without effect. Glutamate‐induced alterations in H+ flux required extracellular calcium, and were abolished when cells were bathed in an alkaline Ringer solution. Increasing intracellular calcium by photolysis of the caged calcium compound NP‐EGTA also altered extracellular H+ flux. Immunocytochemical localization of the plasmalemma Ca2+–H+‐ATPase (PMCA pump) revealed intense labelling within the outer plexiform layer and on isolated horizontal cells. Our results suggest that glutamate modulation of H+ flux arises from calcium entry into cells with subsequent activation of the plasmalemma Ca2+–H+‐ATPase. These neurotransmitter‐induced changes in extracellular pH have the potential to play a modulatory role in synaptic processing in the outer retina. However, our findings argue against the hypothesis that hydrogen ions released by horizontal cells normally act as the inhibitory feedback neurotransmitter onto photoreceptor synaptic terminals to create the surround portion of the centre‐surround receptive fields of retinal neurones.

Zinc Enhances Ionic Currents Induced in Skate Muller (Glial) Cells by the Inhibitory Neurotransmitter GABA

We describe here a novel effect of zinc on GABA receptors of glial cells in the skate retina. The GABA-induced currents of skate Mϋller cells, the radial glia of the retina, are mediated by activation of GABAA receptors (GABAARs). In other parts of the nervous system, GABAA-mediated currents are inhibited by zinc. However, in isolated, voltage-clamped Mϋller cells, coapplication of zinc (10 μM) and GABA (1 μM) resulted in an enhancement of the GABAAR current. Surprisingly, zinc alone induced a current similar in many respects to that elicited by GABA, i. e. the reversal potential was the same as for the GABA-induced current, the current was blocked by bicuculline and picrotoxin, and the current-voltage relation obtained in the presence of 10 μM zinc was virtually identical to that obtained with 1 μM GABA. Both bicuculline and picrotoxin suppressed a current that was present with cells bathed only in Ringer, suggesting that some of the GABA channels were spontaneously open in the absence of externally applied GABA. This possibility was supported by cell-attached patch recordings. Under conditions in which potassium and calcium currents were suppressed, spontaneous channel activity was observed. Moreover, the frequency of these channel openings was greater when zinc was included in the pipette solution, and reduced when bicuculline was added. These findings suggest that zinc acts directly to enhance the GABAA receptor activity of the Mϋller cells, and raise the possibility that the subunit composition of the GABAARs of skate Mϋller cells differs from that of GABAARs identified previously in other neuronal and glial preparations.

The effects of lowered extracellular sodium onγ-aminobutyric acid (GABA)-induced currents of muller (glial) cells of the skate retina

Summary1.The effects of external sodium on GABA-induced chloride currents were examined with whole-cell voltage-clamp recordings obtained from enzymatically dissociated solitary Muller cells in culture. Our goal was to determine whether a sodium-dependent GABA uptake mechanism influences the GABAa-mediated responses of skate Muller cells.2.At low concentrations of GABA (0.01 to 0.5µM), removal of sodium from the external solution resulted in a marked increase in the ligand-gated currents mediated by activation of GABAa receptors. The enhancement by lowered sodium was greatest at hyperpolarizing potentials and decreased progressively as the cell was depolarized.3.The reversal potential for the GABA-induced response was not significantly altered by the removal of sodium, suggesting that sodium ions did not directly contribute to the GABAa-mediated current.4.Lowering external sodium had no effect on the currents induced by the GABAa-agonist muscimol, consistent with its much lower affinity for the GABA transport carrier.5.Application of the GABA uptake blocker nipecotic acid also abolished the effects of lowered sodium.6.These findings suggest that the effects of lowered external sodium resulted from a decrease in the uptake of GABA into the Muller cells, thus raising the effective concentration of GABA acting upon the GABAa receptors.

Extracellular pH dynamics of retinal horizontal cells examined using electrochemical and fluorometric methods

Extracellular H(+) has been hypothesized to mediate feedback inhibition from horizontal cells onto vertebrate photoreceptors. According to this hypothesis, depolarization of horizontal cells should induce extracellular acidification adjacent to the cell membrane. Experiments testing this hypothesis have produced conflicting results. Studies examining carp and goldfish horizontal cells loaded with the pH-sensitive dye 5-hexadecanoylaminofluorescein (HAF) reported an extracellular acidification on depolarization by glutamate or potassium. However, investigations using H(+)-selective microelectrodes report an extracellular alkalinization on depolarization of skate and catfish horizontal cells. These studies differed in the species and extracellular pH buffer used and the presence or absence of cobalt. We used both techniques to examine H(+) changes from isolated catfish horizontal cells under identical experimental conditions (1 mM HEPES, no cobalt). HAF fluorescence indicated an acidification response to high extracellular potassium or glutamate. However, a clear extracellular alkalinization was found using H(+)-selective microelectrodes under the same conditions. Confocal microscopy revealed that HAF was not localized exclusively to the extracellular surface, but rather was detected throughout the intracellular compartment. A high degree of colocalization between HAF and the mitochondrion-specific dye MitoTracker was observed. When HAF fluorescence was monitored from optical sections from the center of a cell, glutamate produced an intracellular acidification. These results are consistent with a model in which depolarization allows calcium influx, followed by activation of a Ca(2+)/H(+) plasma membrane ATPase. Our results suggest that HAF is reporting intracellular pH changes and that depolarization of horizontal cells induces an extracellular alkalinization, which may relieve H(+)-mediated inhibition of photoreceptor synaptic transmission.

Glutamate modulation of GABA transport in retinal horizontal cells of the skate

Transport of the amino acid GABA into neurons and glia plays a key role in regulating the effects of GABA in the vertebrate retina. We have examined the modulation of GABA‐elicited transport currents of retinal horizontal cells by glutamate, the likely neurotransmitter of vertebrate photoreceptors. Enzymatically isolated external horizontal cells of skate were examined using whole‐cell voltage‐clamp techniques. GABA (1 mm) elicited an inward current that was completely suppressed by the GABA transport inhibitors tiagabine (10 μm) and SKF89976‐A (100 μm), but was unaffected by 100 μm picrotoxin. Prior application of 100 μm glutamate significantly reduced the GABA‐elicited current. Glutamate depressed the GABA dose‐response curve without shifting the curve laterally or altering the voltage dependence of the current. The ionotropic glutamate receptor agonists kainate and AMPA also reduced the GABA‐elicited current, and the effects of glutamate and kainate were abolished by the ionotropic glutamate receptor antagonist 6‐cyano‐7‐nitroquinoxaline. NMDA neither elicited a current nor modified the GABA‐induced current, and metabotropic glutamate analogues were also without effect. Inhibition of the GABA‐elicited current by glutamate and kainate was reduced when extracellular calcium was removed and when recording pipettes contained high concentrations of the calcium chelator BAPTA. Caffeine (5 mm) and thapsigargin (2 nm), agents known to alter intracellular calcium levels, also reduced the GABA‐elicited current, but increases in calcium induced by depolarization alone did not. Our data suggest that glutamate regulates GABA transport in retinal horizontal cells through a calcium‐dependent process, and imply a close physical relationship between calcium‐permeable glutamate receptors and GABA transporters in these cells.

Pharmacological characterization, localization, and regulation of ionotropic glutamate receptors in skate horizontal cells

Glutamate is believed to be the primary excitatory neurotransmitter in the vertebrate retina, and its fast postsynaptic effects are elicited by activating NMDA-, kainate-, or AMPA-type glutamate receptors. We have characterized the ionotropic glutamate receptors present on retinal horizontal cells of the skate, which possess a unique all-rod retina simplifying synaptic circuitry within the outer plexiform layer (OPL). Isolated external horizontal cells were examined using whole-cell voltage-clamp techniques. Glutamate and its analogues kainate and AMPA, but not NMDA, elicited dose-dependent currents. The AMPA receptor antagonist GYKI 52466 at 100 microm abolished glutamate-elicited currents. Desensitization of glutamate currents was removed upon coapplication of cyclothiazide, known to potentiate AMPA receptor responses, but not by concanavalin A, which potentiates kainate receptor responses. The dose-response curve to glutamate was significantly broader in the presence of the desensitization inhibitor cyclothiazide. Polyclonal antibodies directed against AMPA receptor subunits revealed prominent labeling of isolated external horizontal cells with the GluR2/3 and GluR4 antibodies. 1-Naphthylacetyl spermine, known to block calcium-permeable AMPA receptors, significantly reduced glutamate-gated currents of horizontal cells. Downregulation of glutamate responses was induced by increasing extracellular ion concentrations of Zn2+ and H+. The present study suggests that Ca2+-permeable AMPA receptors likely play an important role in shaping the synaptic responses of skate horizontal cells and that alterations in extracellular concentrations of calcium, zinc, and hydrogen ions have the potential to regulate the strength of postsynaptic signals mediated by AMPA receptors within the OPL.

Distinctive patterns of alterations in proton efflux from goldfish retinal horizontal cells monitored with self-referencing H + -selective electrodes

The H+ hypothesis of lateral feedback inhibition in the outer retina predicts that depolarizing agents should increase H+ release from horizontal cells. To test this hypothesis, self‐referencing H+‐selective microelectrodes were used to measure extracellular H+ fluxes from isolated goldfish horizontal cells. We found a more complex pattern of cellular responses than previously observed from horizontal cells of other species examined using this technique. One class of cells had an initial standing signal indicative of high extracellular H+ adjacent to the cell membrane; challenge with glutamate, kainate or high extracellular potassium induced an extracellular alkalinization. This alkalinization was reduced by the calcium channel blockers nifedipine and cobalt. A second class of cells displayed spontaneous oscillations in extracellular H+ that were abolished by cobalt, nifedipine and low extracellular calcium. A strong correlation between changes in intracellular calcium and extracellular proton flux was detected in experiments simultaneously monitoring intracellular calcium and extracellular H+. A third set of cells was characterized by a standing extracellular alkalinization which was turned into an acidic signal by cobalt. In this last set of cells, addition of glutamate or high extracellular potassium did not significantly alter the proton signal. Taken together, the response characteristics of all three sets of neurons are most parsimoniously explained by activation of a plasma membrane Ca2+ ATPase pump, with an extracellular alkalinization resulting from exchange of intracellular calcium for extracellular H+. These findings argue strongly against the hypothesis that H+ release from horizontal cells mediates lateral inhibition in the outer retina.