GABA(C) receptors control adaptive changes in a glycinergic inhibitory pathway in salamander retina.

We studied the role of GABA in adaptive changes in a lateral inhibitory system in the tiger salamander retina. In dark-adapted retinal slice preparations picrotoxin caused a slow enhancement of glycine-mediated IPSCs in ganglion cells. The enhancement of glycinergic IPSCs developed slowly over the course of 5-20 min, even though picrotoxin blocked both GABA(A) and GABA(C) receptors within a few seconds. The slow enhancement of glycinergic IPSCs by picrotoxin was much weaker in light-adapted preparations. The slow enhancement of glycinergic inhibitory inputs was not produced by bicuculline, indicating that it involved GABA(C) receptors. The responses of ganglion cells to direct application of glycine were not enhanced by picrotoxin, indicating that the enhancement was not caused by an action on glycine receptors. In dark-adapted eyecup preparations picrotoxin caused a slow enhancement of glycinergic IPSPs and transient lateral inhibition produced by a rotating windmill pattern, similar to the effect of light adaptation. The results suggest that the glycinergic inhibitory inputs are modulated by an unknown substance whose synthesis and/or release is inhibited in dark-adapted retinas by GABA acting at GABA(C) receptors.

Action potentials are required for the lateral transmission of glycinergic transient inhibition in the amphibian retina.

Transient lateral inhibition (TLI), the suppression of responses of a ganglion cell to light stimuli in the receptive field center by changes in illumination in the receptive field surround, was studied in light-adapted mud puppy and tiger salamander retinas using both eyecup and retinal slice preparations. In the eyecup, TLI was measured in on-off ganglion cells as the ability of rotating, concentric windmill patterns of 500-1200 micron inner diameter to suppress the response to a small spot stimulus in the receptive field center. Both the suppression of the spot response and the hyperpolarization produced in ganglion cells by rotation of the windmill were blocked in the presence of 2 microM strychnine or 500 nM tetrodotoxin (TTX), but not by 150 microM picrotoxin. In the slice preparation in which GABA-mediated currents were blocked with picrotoxin, IPSCs elicited by diffuse illumination were blocked by strychnine and strongly reduced by TTX. The TTX-resistant component was probably attributable to illumination of the receptive field center. TTX had a much greater effect in reducing the glycinergic inhibition elicited by laterally displaced stimulation versus nearby focal electrical stimulation. Strychnine enhanced light-evoked excitatory currents in ganglion cells, but this was not mimicked by TTX. The results suggest that local glycinergic transient inhibition does not require action potentials and is mediated by synapses onto both ganglion cell dendrites and bipolar cell terminals. In contrast, the lateral spread of this inhibition (at least over distances >250 micron) requires action potentials and is mainly onto ganglion cell dendrites.

Localization of gap junctions and tracer coupling in retinal Müller cells.

Physiological studies have demonstrated the existence of direct intercellular communication, presumably mediated by gap junctions, both between neurons and between glial cells in the vertebrate retina. We localized gap junctions in the retinas of rat, goldfish, and mudpuppy by using antisera directed against proteins that make up the connexon channels in two tissues from which connexins have been isolated: liver (connexin 32; CX32) and heart (connexin 43; CX43). Although the antiserum against CX32 stained liver gap junctions, it did not reveal any staining in rat or goldfish retina. The antiserum against CX43 stained gap junctions associated with the intercalated disk in rat heart and also stained gap junctions between pigment epithelium cells in rat, goldfish, and mudpuppy retina. Anti-CX43 also stained gap junctions between Müller cells in goldfish and mudpuppy retina but not in rat retina. Intracellular injections of the tracer Neurobiotin into Müller cells in the mudpuppy retina revealed that these glial cells are extensively tracer coupled. Staining with the tracer formed a syncytium of thin processes surrounding every neuron from the outer limiting membrane to the inner limiting membrane. Confocal microscopy demonstrated that the Müller cells were in close apposition with one another at every level of the retina. However, CX43 immunoreactivity was heaviest at the outer limiting membrane, where the apical processes of Müller cells are located. Some anti-CX43 staining was observed at the level of the outer nuclear layer and the inner plexiform layer but not in the ganglion cell layer or at the Müller cell end feet forming the inner limiting membrane.

Modulation of sustained and transient lateral inhibitory mechanisms in the mudpuppy retina during light adaptation.

Two functionally and anatomically distinct types of lateral inhibition contribute to the receptive field organization of ganglion cells in the vertebrate retina: sustained lateral inhibition (SLI), which is present during steady illumination and transient lateral inhibition (TLI), evoked by changes in illumination. We studied adaptive changes in these two lateral inhibitory mechanisms in the mudpuppy retina by measuring the responses of ON-OFF ganglion cells to spots of light in the receptive field center, in the absence and presence of a concentric broken annulus (windmill) pattern, which was either stationary or rotating. SLI was measured as the percent suppression of the centered spot response by the stationary windmill and TLI was measured as the additional suppression produced when the windmill was rotating. In dark-adapted retinas SLI was elicited by windmills of 600 or 1,200 micron ID, but TLI could not be elicited by windmills of any size, over a wide range of windmill intensities and rotation rates. Exposure of dark-adapted retinas to diffuse adapting light caused an immediate decrease in the response to the spot alone, followed by slowly developing changes in both SLI and TLI: SLI produced by 1,200 micron ID windmills became weaker, whereas SLI produced by 600 micron ID windmills became stronger. After several minutes strong TLI could be elicited by both 600 and 1,200 micron ID windmills. The changes in SLI and TLI were usually complete within 5 and 15 min, respectively, and recovered to dark-adapted levels slightly more slowly after the adapting light was turned off. However the changes in sensitivity of the spot response were complete within one minute after onset and termination of the adapting light. The adaptive changes in SLI and TLI did not depend on the presence of the adapting light; after a brief (1 min) exposure to the adapting light, the changes in SLI and TLI slowly developed and then decayed back to the dark-adapted level. The effects of the adapting light on SLI were mimicked by dopamine and blocked by D1 dopamine receptor antagonists. However dopamine did not enable TLI in dark-adapted retinas and dopamine antagonists did not prevent enablement of TLI when dark-adapted retinas were exposed to light or disable TLI when applied to light-adapted retinas. The results suggest that light-adaptive changes in SLI are mediated by dopamine and are consistent with a reduction in electrical coupling between neurons that conduct the SLI signal laterally in the retina. In contrast, TLI appears to be switched off or suppressed in the dark-adapted retina and enabled in light-adapted retinas, by a relatively slow modulatory mechanism that does not involve dopamine.

Lateral inhibition in the inner retina is important for spatial tuning of ganglion cells.

The center-surround receptive-field organization in retinal ganglion cells is widely believed to result mainly from lateral inhibition at the first synaptic level (in the outer retina). Inhibition at the second synaptic level (in the inner retina) is thought to mediate more complex response properties. Here we show that much of the sustained surround antagonism in certain on-center ganglion cells results from lateral inhibition in the inner retina, via GABAergic amacrine cells, and that the lateral conduction of this signal requires voltage-gated sodium currents. Blocking lateral inhibition in the inner retina eliminates the preference of small-center ganglion cells for small stimuli but has little effect on ganglion cells with large receptive-field centers. These results illustrate how lateral inhibition at successive synaptic stages can selectively control the size of neural receptive-field centers.

Cholinergic modulation of dopamine release and horizontal cell coupling in mudpuppy retina.

The effects of cholinergic agonists and antagonists on electrical coupling between horizontal cells were studied in dark-adapted mudpuppy retinas. Carbachol and the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) uncoupled horizontal cells, but the muscarinic agonist oxotremorine did not. The uncoupling effects of carbachol and DMPP were blocked by the nicotinic antagonist D-tubocurarine and by the dopamine antagonist fluphenazine, indicating that carbachol uncoupled horizontal cells by stimulating dopamine release via nicotinic receptors. Carbachol also caused an increase in release of [3H]dopamine from retinas. D-Tubocurarine increased horizontal cell coupling, indicating that tonic cholinergic input was present in dark-adapted retinas. D-Tubocurarine did not reduce light-evoked uncoupling of horizontal cells, suggesting that cholinergic neurons are not an essential part of the direct pathway by which light causes an immediate increase in dopamine release.

Cones contribute to light-evoked, dopamine-mediated uncoupling of horizontal cells in the mudpuppy retina.

1. The relative effectiveness of adapting lights of different wave-lengths on uncoupling of horizontal cells was measured in dark-adapted mudpuppy retinas. Diffuse blue (470 nm) or red (620 nm) adapting stimuli were adjusted in intensity to be equally effective for rods or for cones. Uncoupling of horizontal cells was measured by intracellular recording of changes in their responses to spot and annulus stimuli. The intensities of the adapting light pairs were varied over 3 log units. The responses of the horizontal cells indicated that both rods and cones were stimulated by the adapting lights. 2. Relatively dim adapting lights did not produce detectable changes in horizontal cell coupling. Brighter adapting lights caused uncoupling of horizontal cells. When the brighter adapting lights were rod matched, the uncoupling effect of the 620-nm light was significantly greater than that of the 470 nm light, indicating that cones contribute to the uncoupling effect. 3. When the adapting lights were cone matched, the effects of the two wavelengths were not significantly different, but this did not rule out a rod contribution because the effective adapting lights probably produced maximal or nearly maximal, and hence equal or nearly equal, responses in rods. 4. The results indicate that cones contribute to the light-evoked uncoupling of horizontal cells in mudpuppy, although a contribution from rods could not be ruled out. Because it was shown previously that light-evoked uncoupling of horizontal cells in mudpuppy is mediated by dopamine, the results also suggest that cones contribute to the light-evoked release of dopamine.

An intensity-dependent biphasic neuron in mudpuppy retina.

Intracellular recordings in dark-adapted mudpuppy retinas have revealed a type of infrequently encountered cell with unusual response properties. These cells may be a subclass of horizontal cell since they are encountered at the same depth as horizontal cells and have large receptive fields and response amplitudes. However, they differ from typical horizontal cells in that they are depolarized by low intensity illumination and hyperpolarized by higher intensity illumination at all wavelengths. Both types of responses appear to be driven mainly by 572 nm cones. Both the depolarizing and hyperpolarizing responses were unaffected by APB, indicating that they are not mediated by on-center bipolar cells.

Comparison of the effects of flickering and steady light on dopamine release and horizontal cell coupling in the mudpuppy retina.

1. The effects of flickering adapting illumination (repetitive flashes) on horizontal cell responses to illumination of the center and surround portions of the receptive field were compared with those of steady adapting illumination in dark-adapted mudpuppy retinas. 2. Exposure to flickering adapting light caused an increase in amplitude of responses to small spots in the receptive-field center and a decrease in the response to a concentric annulus. This is interpreted as due to an increase in coupling resistance between horizontal cells. 3. The uncoupling effect of flickering adapting light was no greater than that of the same quantity of steady adapting light at the same intensity, even when the rate of flickering was varied by a factor of 10. 4. The uncoupling effect of flickering light was blocked by the dopamine antagonists fluphenazine and SCH23390, indicating that it is mediated by dopamine release. 5. The uncoupling effect of flickering light was also blocked in the presence of 2-amino-4-phosphonobutyrate (APB), which prevents light responses of on-center but not off-center bipolar cells, suggesting that flickering light increases dopamine release via the on-pathway. 6. The gamma-aminobutyric acid (GABA) antagonist bicuculline had an uncoupling effect similar to that of adapting illumination. This effect was blocked by dopamine antagonists, indicating that there is tonic GABA-mediated inhibition of dopamine release in mudpuppy retina similar to that previously reported by others in fish and turtle retinas. 7. The uncoupling effect of bicuculline was not reversed by APB. However, APB alone caused an increase in coupling that was rapidly reversed by bicuculline.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between light, dopamine release and horizontal cell coupling in the mudpuppy retina.

1. The effect of different experimental conditions on electrical coupling between horizontal cells in the mudpuppy retina was studied by comparing the changes in responses to illumination of the central and peripheral portions of the receptive field, using centred spot and annulus stimuli. An increase in the amplitude of the response to a centred spot stimulus and a decrease in the amplitude of the response to a concentric annulus indicated a decrease in coupling, and vice versa. 2. Dopamine (10-250 microM) caused a decrease in coupling between horizontal cells. The uncoupling effect of dopamine was much greater in dark-adapted than in light-adapted retinas. The effect of the D1-receptor agonist SKF38393 was similar to that of dopamine. The effect of the D2-receptor agonist LY171555 on coupling was opposite to that of dopamine; this was attributed to a reduction in endogenous dopamine release. 3. The D1 antagonist SCH23390 (15 microM) caused an increase in coupling between horizontal cells. This effect was much greater in light-adapted than in dark-adapted retinas. 4. The glutamate analogue 2-amino-4-phosphonobutyrate (APB), which hyperpolarizes on-centre bipolar cells and blocks their responses to light, caused an increase in coupling between horizontal cells. This effect of APB was greater in light-adapted retinas than in dark-adapted retinas. The effect of APB on coupling could be reversed by the addition of dopamine, but the effect of dopamine on coupling could not be reversed by the addition of APB. These results suggest that APB increases horizontal cell coupling by causing a decrease in dopamine release. 5. In dark-adapted retinas, 2.5 min exposure to an adapting light caused a decrease in coupling between horizontal cells; the uncoupling effect of the adapting light was blocked in the presence of either SCH23390 or APB. 6. The results suggest that coupling between horizontal cells in the mudpuppy retina is decreased by dopamine acting at D1 receptors, that the release of dopamine affecting horizontal cells is greater under light-adapted conditions, and that the pathway by which exposure to light increases this dopamine release is mainly via on-centre bipolar cells.

Time-dependent differential effects of cobalt ions on rod- and cone-driven responses in the isolated frog retina.

The effects of cobalt ions on 502-nm rod- and 575-nm cone-driven components of the b-wave of the electroretinogram were studied in the isolated frog retina. Addition of 100-150 microM cobalt initially caused a suppression of rod-driven responses and an enhancement of cone-driven responses. In the continued presence of cobalt, however, the rod-driven responses gradually recovered and the cone-driven responses became suppressed. These concentrations of cobalt had no effect on the rod- and cone-driven mass receptor potentials which were isolated in the presence of 4 mM glutamate. At higher concentrations of cobalt (1 mM or greater), both rod- and cone-driven b-wave responses were eliminated and there was no recovery in the continued presence of cobalt. The results suggest that cobalt has markedly different, time-dependent effects on signal transmission from rods and cones to second-order cells.

APB increases apparent coupling between horizontal cells in mudpuppy retina.

2-Amino-4-phosphonobutyrate (APB), an agonist at a unique type of glutamate receptor on depolarizing bipolar cells, caused an apparent increase in coupling between horizontal cells as evidenced by a decrease in amplitude of responses to illumination of the receptive field center and an increase in responses to illumination of the peripheral part of the receptive field. APB also caused a hyperpolarization of horizontal cells in darkness and increased the amplitude of responses to full-field illumination, which cannot be explained by an increase in electrical coupling between horizontal cells. Possible mechanisms for these actions are discussed.

Suppression of cone-driven responses by rods in the isolated frog retina.

The sensitivity of rod- and cone-driven responses was studied in the isolated frog retina during the period of rapid dark adaptation following a conditioning flash which bleached a negligible amount of visual pigment. Following a conditioning flash, cone-driven b-wave responses were first enhanced and then depressed. The time courses of the enhancement and subsequent depression of cone-drive responses varies greatly with the intensity and wavelength of the conditioning flash, but were identical when the conditioning flashes were matched for equal excitation of 502 nm rods. These changes in cone-driven response sensitivity were correlated with the desensitization and recovery of rods following the conditioning flash. When signal transmission from rods to second-order cells was interrupted by the addition of L-glutamate, the conditioning flash did not produce the above-described enhancement and subsequent depression of long-wavelength receptor potential responses. The suppression of cone-driven response therefore appears to be due to a synaptically mediated influence from 502 nm rods which is maximal when the rods are in the dark-adapted state, with little or no contribution from 433 nm rods, and no involvement of the pigment epithelium.

Push-pull effect of surround illumination on excitatory and inhibitory inputs to mudpuppy retinal ganglion cells.

1. Changes in membrane potential and conductance were measured in on-centre and off-centre ganglion cells during the responses to illumination of different portions of the receptive field. 2. In on-centre ganglion cells the sustained depolarizing response to steady illumination of the receptive field centre was associated with a net increase in conductance. In the presence of centre illumination, stimulation of the surround with an annulus of light caused a hyperpolarization and a net decrease in conductance, and the reversal potential of the light-evoked response was shifted in a negative direction. In the absence of centre illumination the same annular stimulus caused a hyperpolarization and a net increase in conductance. 3. In off-centre ganglion cells the sustained hyperpolarizing response to centre illumination was associated with a net increase in conductance. In the presence of centre illumination, stimulation of the surround with an annulus caused a depolarization and a net decrease in conductance, and the reversal potential of the light-evoked response was shifted in a positive direction. In the absence of centre illumination the same annulus caused a depolarization and a net increase in conductance. 4. The results indicate that illumination of the receptive field surround can affect both the excitatory and inhibitory sustained inputs to a given ganglion cell in a 'push-pull' manner, by decreasing the synaptic input that was increased by centre illumination and increasing the synaptic input of opposite sign. The relative effect of a given surround illumination on these two inputs, and hence the sign and magnitude of the net conductance change, varied with the amount of centre illumination.

Excitatory amino acids have different effects on horizontal cells in eyecup and isolated retina.

Horizontal cells in the mudpuppy eyecup responded to continuous superfusion with L-glutamate, L-aspartate, kainate and quisqualate with a transient depolarization and reduction of the light evoked responses. However, in isolated retina preparations, in which these substances were applied to the photoreceptor side of the retina, the effects were sustained as long as the agonists were present. These results suggest that the transient action of these agonists in eyecup preparations was due to the rapid development of an intraretinal diffusion barrier, and are consistent with the hypothesis that photoreceptors release an excitatory amino acid transmitter.

Synaptic transmission at N-methyl-D-aspartate receptors in the proximal retina of the mudpuppy.

The effects of excitatory amino acid analogues and antagonists on retinal ganglion cells were studied using intracellular recording in the superfused mudpuppy eyecup preparation. Aspartate, glutamate, quisqualate (QA), kainate (KA) and N-methylaspartate (NMA) caused depolarization and decreased input resistance in all classes of ganglion cells. The order of sensitivity was QA greater than or equal to KA greater than NMA greater than aspartate greater than or equal to glutamate. All of these agonists were effective when transmitter release was blocked with 4 mM-Co2+ or Mn2+, indicating that they acted at receptor sites on the ganglion cells. At a concentration of 250 microM, 2-amino-5-phosphonovalerate (APV) blocked the responses of all ganglion cells to NMA, but not to QA or KA, indicating that NMA acts at different receptor sites from QA or KA. Responses to bath-applied aspartate and glutamate were reduced slightly or not at all in the presence of APV, indicating that they were acting mainly at non-NMDA (N-methyl-D-aspartate) receptors. In all ganglion cells 250 microM-APV strongly suppressed the sustained responses driven by the 'on'-pathway but not those driven by the 'off'-pathway. In most on-off ganglion cells the transient excitatory responses at 'light on' and 'light off' were not reduced by 500 microM-APV. APV-resistant transient excitatory responses were also present in some on-centre ganglion cells. APV did not block the transient inhibitory responses in any class of ganglion cells. At concentrations which blocked the sustained responses of ganglion cells, APV did not affect the sustained responses of bipolar cells, indicating that it acted at sites which were post-synaptic to bipolar cells. The simplest interpretation of these results is that the transmitter released by depolarizing bipolar cells acts at NMDA receptors on sustained depolarizing amacrine and ganglion cells. It may act at non-NMDA receptors at synapses which produce transient excitatory responses, but this could not be proved. The transmitter released by hyperpolarizing bipolar cells does not appear to act at NMDA receptors on any post-synaptic cells.

Strychnine blocks transient but not sustained inhibition in mudpuppy retinal ganglion cells.

Transient and sustained inhibitory synaptic inputs to on-centre, off-centre, and on-off ganglion cells in the mudpuppy retina were studied using intracellular recording in the superfused eye-cup preparation. When chemical transmission was blocked with 4 mM-Co2+, application of either glycine or gamma-aminobutyric acid (GABA) caused a hyperpolarization and conductance increase in all ganglion cells. For both amino acids, the responses were dose dependent in the range 0.05-10 mM, with a half-maximal response at about 0.7 mM. Glycine and GABA sensitivities were very similar in all three types of ganglion cells. The response to applied glycine was selectively antagonized by 10(-5) M-strychnine and the response to applied GABA was selectively antagonized by 10(-5) M-picrotoxin. In all ganglion cells, 10(-5) M-strychnine eliminated the transient inhibitory events which occur at the onset and termination of a light stimulus. The block of transient inhibition was associated with a relative depolarization of membrane potential and decrease in conductance at these times. Strychnine had no effect on membrane potential or conductance in darkness or during sustained inhibitory responses to light. Picrotoxin (10(-5) M) did not block transient inhibitory events in any ganglion cells, but did affect other components of their responses. The results suggest that in all three classes of ganglion cells transient inhibition, but not sustained inhibition, may be mediated by glycine or a closely related substance.

Autoradiographic studies of [3H]-glycine, [3H]-GABA, and [3H]-muscimol uptake in the mudpuppy retina.

Autoradiographic studies showed selective accumulation of [3H]-glycine, [3H]-GABA, and the GABA agonist [3H]-muscimol by neurons of the mudpuppy retina. [3H]-Glycine was taken up by bipolar cells, amacrine cells, and displaced amacrine or ganglion cells. Both [3H]-GABA and [3H]-muscimol were also accumulated by bipolar cells, amacrine cells and ganglion layer cells. However, the [3H]-GABA uptake pattern differed from that of [3H]-muscimol in showing labeling of horizontal cells, an increased percentage of cells in the ganglion cell layer, and a band in the most proximal portion of the inner plexiform layer. Variations in grain density suggested the presence of multiple subpopulations of [3H]-glycine- and [3H]-GABA-labeled amacrine cells. The labeled cells may play a role in inhibitory pathways in the inner retina.

Sustained and transient synaptic inputs to on-off ganglion cells in the mudpuppy retina.

Synaptic inputs to on-off ganglion cells in mudpuppy retina were studied by measuring current-voltage relations in darkness, during different phases of the response to light, and in the presence of 4 mM-Co2+. The addition of Co2+ to the bathing medium usually caused a hyperpolarization of the membrane potential in darkness and an increase in input resistance, indicating that on-off ganglion cells receive tonic excitatory synaptic input in darkness. Other results suggest that an additional synaptic input, with a reversal potential near the dark potential, may also be active in darkness. At the onset of a light stimulus in the receptive field centre all on-off ganglion cells responded with transient excitatory and inhibitory synaptic events, both of which were due to increases in conductance. Similar transient excitatory and inhibitory events occurred at the termination of the light stimulus. In about one-half of the on-off ganglion cells studied the synaptic activity during steady illumination was the same as in darkness. In the remaining cells steady illumination caused an increase in sustained inhibition.