Responses of small- and large-field bipolar cells to GABA and glycine.

Morphologically distinct subtypes of retinal bipolar cells transmit information along parallel pathways to convey different aspects of the visual scene, but the synaptic mechanisms that regulate signal transmission are largely unknown. The all-rod retina of skate provides a comparatively simple system in which to correlate bipolar cell morphology with responses to the inhibitory neurotransmitters GABA and glycine. Two subtypes of bipolar cells can be identified when isolated in culture: large-field bipolar cells with extensive dendritic arbors, and small-field bipolar cells with one or two dendritic branches. Under voltage-clamp, glycine elicited significant current responses from small-field cells, but not from large-field bipolar cells. Although all bipolar cells displayed GABA-activated chloride currents mediated by both GABA(A) and GABA(C) receptors, the small-field bipolar cells showed a significantly greater contribution from GABA(A) receptors. The results of the present study reveal for the first time that the relative expression of the two classes of GABA receptor on each bipolar cell type correlates with cell morphology and the presence of the glycine receptor.

Excitatory amino acids and serotonin uptake blockers reveal two physiologically distinct serotonin systems in the retina of the skate, Raja erinacea.

The retina of the skate (Raja erinacea) contains at least 2 types of cell (amacrines and bipolars) that can be visualized with an antiserum against serotonin. We have employed serotonin immunocytochemistry in combination with pharmacological manipulation of retinal tissue to analyze physiological properties of serotonergic amacrine cells and serotonin-accumulating bipolar cells. Excitatory amino acids (NMDA, aspartate) had no detectable effects on serotonin-immunoreactivity in bipolar cells but decreased staining in amacrine cells. High K+ Ringer increased staining in bipolar cell somata, however, it depleted the inner plexiform layer of the retina of serotonin. Zimelidine, a serotonin uptake inhibitor, completely blocked serotonin accumulation by bipolar cells but had no effect on amacrine cells, whereas incubation of the retinas in fluoxetine (Prozac), a different inhibitor of serotonin uptake, did not block serotonin accumulation into bipolar cells which was actually enhanced in some cases. We conclude that amacrine and bipolar cells of the skate retina employ two different serotonin uptake carrier systems, thus generating two distinct pharmacological components that are capable of interacting with each other as they compete for extracellular serotonin. Similar mechanisms may exist in the vertebrate CNS and further examination of the interaction of these systems could provide important insights into the action and possible side effects of serotonin-related drugs.

GABA receptors of bipolar cells from the skate retina: actions of zinc on GABA-mediated membrane currents.

GABA receptors of bipolar cells from the skate retina: actions of zinc on GABA-mediated membrane currents. J. Neurophysiol. 78: 2402-2412, 1997. gamma-Aminobutyric acid (GABA)-induced currents were recorded from isolated bipolar cells of the skate retina using perforated patch-clamp methodology. Pharmacological analysis of the responses, using selective agonists and antagonists of the major classes of GABA receptor, revealed the presence of both GABAA and GABAC receptors at both the dendrites and axon terminals of the bipolar cells. The two receptor types showed very different reactions to zinc, a divalent metallic cation that was detected in the synaptic terminal region of skate photoreceptors. Currents mediated by the activation of GABAC receptors were down-regulated by zinc, a feature that is typical of the action of zinc on GABAC receptors. On the other hand, the effects of zinc on GABAA receptor-mediated activity was highly dependent on zinc concentration. Unlike the GABAA receptors on other neurons, responses mediated by activation of the GABAA receptor of skate bipolar cells were significantly enhanced by zinc concentrations in the range of 0. 1-100 mu M; at higher concentrations of zinc (>100 mu M), response amplitudes were suppressed below control levels. The enhancement of GABAA receptor activity on skate bipolar cells showed little voltage dependence, suggesting that zinc is acting on the extracellular domain of the GABAA receptor. In the presence of 10 mu M zinc, the dose-response curve for 4,5,6, 7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP; a GABAA agonist that suppresses GABAC-activated currents) was shifted to the left of the curve obtained in the absence of zinc, but without a significant change in the response maximum. This finding indicates that the enhancing effect of zinc is due primarily to its ability to increase the sensitivity of the GABAA receptor. The novel enhancement of neuronal GABAA receptor activity by zinc, observed previously in the GABAA-mediated responses of skate Müller (glial) cells, may reflect the presence of a unique subtype of GABAA receptor on the bipolar and Müller cells of the skate retina.

Two classes of bipolar cell in the retina of the skate Raja erinacea.

We have used immunoreactions against serotonin and protein kinase C to visualize two distinct classes of bipolar cell in the all-rod retina of the skate, Raja erinacea. To enhance the immunoreaction in serotonin-accumulating bipolar cells, prior to fixation, some retinas were incubated in Ringer's solution containing serotonin and pargyline. We found the somata of serotonin-accumulating bipolar cells to be located slightly distal to the midline of the inner nuclear layer. With increasing eccentricity from the visual streak, the size of the perikarya increases, concomitant with a decline in density of their distribution. Dendrites emanate from stout primary stalks and branch out before reaching the outer plexiform layer. Axons are bistratified within the inner plexiform layer with ramifications at the border of strata 1 and 2 and in stratum 4. The overall morphology of serotonin-accumulating bipolar cells is similar to that of serotonin-accumulating OFF bipolar cells of other non-mammalian vertebrates. Protein kinase C immunoreactive cells display the typical appearance of rod bipolar cells. Somata of protein kinase C immunoreactive bipolar cells are spindle-shaped and located distal to the serotonin-accumulating bipolar cells. Dendrites of these bipolars do not ramify before reaching the outer plexiform layer. Thin axons of protein kinase C immunoreactive bipolar cells end in large, club-shaped terminals in stratum 5 of the inner plexiform layer, bearing a striking similarity to axon terminals of mammalian ON rod bipolar cells. Our findings suggest that the all-rod retina of the skate contains at least two distinct vertical pathways including an OFF bipolar cell pathway in addition to a classical rod ON bipolar pathway.

Zinc enhances ionic currents induced in skate Müller (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 Müller cells, the radial glia of the retina, are mediated by activation of GABA(A) receptors (GABAARS). In other parts of the nervous system, GABz*-)ediated currents are inhibited by zinc. However, in isolated, voltage-clamped Müller cells, coapplication of zinc (10 microM) and GABA (1 microM) resulted in enhancement of the GABA(A)R 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 microM zinc was virtually identical to that obtained with 1 microM 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 GABA(A) receptor activity of the Müller cells, and raise the possibility that the subunit composition of the GABA(A)Rs of skate Müller cells differs from that of GABA(A)Rs identified previously in other neuronal and glial preparations.

Pharmacology of the skate electroretinogram indicates independent ON and OFF bipolar cell pathways.

Organization of afferent information into parallel ON and OFF pathways is a critical feature of the vertebrate visual system. All afferent visual information in the vertebrate retina reaches the inner plexiform layer (IPL) via bipolar cells. It is at the bipolar cell level that separation of ON and OFF information first appears for afferent information from cones. This may also hold true for the rod pathway of cold-blooded vertebrates, but not for mammals. The all-rod retina of the skate presents an opportunity to examine such pathways in a retina having but a single class of photoreceptor. Immunocytochemical evidence suggests that both ON and OFF bipolar cells are present in the skate retina. We examined the pharmacology of the skate electroretinogram (ERG) to test the hypothesis that independent ON and OFF bipolar cell pathways are functional as rod afferent pathways from outer to inner plexiform layer in the skate. 100 microM 2-amino-4-phosphonobutyric acid (APB) reversibly blocked the skate ERG b-wave. A small d-wave-like OFF component of the ERG revealed by DC recording of response to a prolonged (10 s) flash of light was reduced or blocked by 5 mM kynurenic acid (KYN). We found that addition of 200 microM picrotoxin to the Ringer's solution revealed prominent ON and OFF components of the skate ERG while reducing the c-wave. These ON and OFF components were reversibly blocked by 100 microM APB and 5 mM KYN, respectively. Reversible block of the OFF component by KYN was also accomplished in the presence of 500 microM N-methyl-DL-aspartate. From these findings, we conclude that ON and OFF bipolar cells are likely to be functional as parallel afferent interplexiform pathways in the all-rod retina of the skate.

Ultrastructural and electrophysiological changes associated with K(+)-evoked release of neurotransmitter at the synaptic terminals of skate photoreceptors.

Bathing the skate retina in a Ringer solution containing a high concentration (100 mM) of potassium ions depolarized the visual cells, depleted the receptor terminals of synaptic vesicles, and suppressed completely the b-wave of the ERG and the intracellularly recorded response of horizontal cells (the S-potential). The depletion of synaptic vesicles was accompanied by a large increase in the extent of the plasma membrane resulting in distortion of the normal terminal profile, i.e. distension of the basal surface and elaborate infolding of protoplasmic extensions. Morphometric analysis showed that despite the changes in vesicle content and terminal structure, the combined linear extent of the vesicular and plasma membranes was unchanged from control (superfusion with normal Ringer solution); the increase in plasma membrane was equivalent to the observed loss of vesicular membrane. When returned to a normal Ringer solution, the terminals rapidly began to reform, and in about 10 min they were morphologically indistinguishable from receptor terminals seen in control preparations. After 30 min in the normal Ringer solution, the amount of membrane associated with the vesicles and the plasma membrane had reverted to control values, and once again the total membrane estimated morphometrically remained essentially the same. Thus, there is an efficient mechanism at the photoreceptor terminal for the recycling of vesicle membrane following exocytosis. The K(+)-induced depletion of synaptic vesicles was paralleled by a precipitous loss of responsivity in both the b-wave of the ERG and the S-potential of the horizontal cells. However, after 30-min exposure to the high K+ and a return to normal Ringer solution, the recovery of electrophysiological activity followed a much slower time course from that associated with the structural changes; 60 min or longer were required for the potentials to exhibit maximum response amplitudes. It appears that the rate-limiting step in restoring normal synaptic function following massive depletion of vesicular stores is transmitter resynthesis and vesicle loading rather than vesicle recycling.

Sensitivity transformation for vertebrate vision.

The visual response to a flash given in the dark is known to saturate according to the Michaelis-Menten relationship. Nevertheless, the incremental response from increasing levels of mean luminance tends to follow a Weber-Fechner relationship well into the saturation range determined from the Michaelis-Menten results. This sensitivity transformation from Michaelis-Menten to Weber-Fechner is an important characteristic of light adaptation in the vertebrate retina. Recent studies concerning the role of calcium in photoreceptor adaptation have shown that the relaxation from peak to plateau in the response of isolated photoreceptors was absent under conditions in which adaptation was blocked. Comparing the pronounced relaxation from peak to plateau in turtle horizontal cells with the absence of such relaxation in the catfish response, we noted also that turtle incremental sensitivity shows a Weber-Fechner relationship while catfish incremental sensitivity more closely follows the local slope of the Michaelis-Menten relation. Based on these observations, we have obtained an expression to relate the relaxation from peak to plateau with the sensitivity transformation. We assume that adaptation shifts the half-maximum point of the Michaelis-Menten curve so that the light response relaxes to a plateau value equal to a specified fraction phi of the peak response. We show that this manipulation alone results in a transformation from Michaelis-Menten kinetics to Weber-Fechner sensitivity.

Membrane current responses of skate photoreceptors.

Light-evoked membrane currents were recorded with suction electrodes from the outer segments of individual photoreceptors enzymatically dissociated from the skate retina. The intensity-response relation of dark-adapted cells closely followed a Michaelis function for which a half-saturating response was elicited by a flash intensity that produced about 36 photoisomerizations. Dim-light responses, as well as the early rising phase of the responses to a wide range of flash intensities, could be described by a reaction scheme that involved a series of four first-order delay stages. The number of delay stages required to model the rising phase of the photocurrents did not change in light adaptation. However, background illumination that reduced sensitivity by 1.5 log units, or a bleaching exposure that resulted in a nearly equivalent desensitization, shortened significantly the time scale of the responses. In both instances there were two- to threefold increases in the rate constants of the transitional delays, and almost complete suppression of the tail current that characterized the response of the dark-adapted cell. These findings suggest that although light adaptation alters the gain and kinetics of the transduction mechanism, the nature of the intervening processes is the same in dark- and light-adapted photoreceptors. Moreover, the results show clearly that there is no need to postulate the existence of a second class of cone-like rods to account for the remarkable ability of skate photoreceptors to respond to incremental stimuli presented on "saturating" background fields or after exposure to an intense bleaching light.

Dynamics of skate horizontal cells.

The all-rod retina of the skate (Raja erinacea or R. oscellata) is known to have the remarkable capability of responding to incremental flashes superimposed on background intensities that initially block all light-evoked responses and are well above the level at which rods saturate in mixed rod/cone retinas. To examine further the unusual properties of the skate visual system, we have analyzed responses of their horizontal cells to intensity-modulated step, sinusoidal, and white-noise stimuli. We found that during exposures to mean intensities bright enough to block responses to incremental stimuli, decremental stimuli were also initially blocked. Thereafter, the horizontal cells underwent a slow recovery phase during which there was marked nonlinearity in their response properties. The cell first (within 2-3 min) responded to decrements in intensity and later (after greater than 10 min) became responsive to incremental stimuli. After adaptation to a steady state, however, the responses to intensity modulation were nearly linear over a broad range of modulation depths even at the brightest mean levels of illumination. Indeed, examination of the steady-state responses over a 5-log-unit range of mean intensities revealed that the amplitude of the white noise-evoked responses depended solely on contrast, and was independent of the retinal irradiance as the latter was increased from 0.02 to 20 muW/cm2; i.e., contrast sensitivity remained unchanged over this 1,000-fold increase in mean irradiance. A decrement from the mean as brief as 2 s, however, disturbed the steady state. Another unexpected finding in this all-rod retina concerns surround-enhancement, a phenomenon observed previously for cone-mediated responses of horizontal cells in the retinas of turtle and catfish. While exposure to annular illumination induced response compression and a pronounced sensitivity loss in response to incremental light flashes delivered to the dark central region, the cell's sensitivity showed a significant increase when tested with a white noise or sinusoidally modulated central spot. Unlike horizontal cells in other retinas studied thus far, however, response dynamics remained unchanged. Responses evoked either by a small spot (0.25-mm diam) or by a large field light covering the entire retina were almost identical in time course. This is in contrast with past findings from cone-driven horizontal cells whose response waveform (dynamics) was dependent upon the size of the retinal area stimulated.

Dynamics of turtle cones.

The response dynamics of turtle photoreceptors (cones) were studied by the cross-correlation method using a white-noise-modulated light stimulus. Incremental responses were characterized by the kernels. White-noise-evoked responses with a peak-to-peak excursion of greater than 5 mV were linear, with mean square errors of approximately 8%, a degree of linearity comparable to the horizontal cell responses. Both a spot (0.17 mm diam) and a large field of light produced almost identical kernels. The amplitudes of receptor kernels obtained at various mean irradiances fitted approximately the Weber-Fechner relationship and the mean levels controlled both the amplitude and the response dynamics; kernels were slow and monophasic at low mean irradiance and were fast and biphasic at high mean irradiance. This is a parametric change and is a piecewise linearization. Horizontal cell kernels evoked by the small spot of light were monophasic and slower than the receptor kernels produced by the same stimulus. Larger spots of light or a steady annular illumination transformed the slow horizontal cell kernel into a fast kernel similar to those of the receptors. The slowing down of the kernel waveform was modeled by a simple low-pass circuit and the presumed feedback from horizontal cells onto cones did not appear to play a major role.