Localization of voltage-sensitive L-type calcium channels in the chicken retina.

L-type calcium channels have been associated with synaptic transmission in the retina, and are a potential site for modulation of the release of neurotransmitters. The present study documents the immunohistochemical localization of neuronal alpha1 subunits of L-type calcium channels in chicken retina, using antibodies to the alpha1c, alpha1d and alpha1f subunits of L-type calcium channels. The alpha1c-like subunits were localized to Müller cells, with predominantly radial processes, and a prominent band of horizontal processes in the outer plexiform layer. The antibody to alpha1d subunits labelled most, if not all, cell bodies. The antibody to a human alpha1f subunit strongly labelled photoreceptor terminals. Fainter immunoreactivity was detected in the inner segments of the photoreceptors, a subset of amacrine cells, two bands of labelling in the inner plexiform layer and many ganglion cells. The differential cellular distributons of these alpha1-subunits suggests subtle functional differences in their roles at different cellular locations.

Enkephalin, neurotensin and somatostatin increase cAMP levels in the chicken retina.

PURPOSE: Enkephalin, neurotensin and somatostatin are released at high rates in the dark and at low rates in the light n the chicken retina. The present study examines the effects of these peptide transmitters on retinal cAMP METHODS: Chicken retinas were incubated in vitro with various drugs for 10min. Cyclic AMP was extracted with acidified ethanol and retinal levels of cAMP were measured using a radioassay kit. RESULTS/CONCLUSIONS: These peptides increased cAMP levels in the chicken retina in vitro, which is surprising as their receptors are generally thought to be negatively coupled to adenylate cyclase. The paradoxical increase in retinal cAMP may be due to unique types of peptide receptors that are positively coupled to adenylate cyclase. A more plausible explanation is that these peptides act indirectly and change the rate of release of another transmitter, whose receptor is coupled to adenylate cyclase.

Light-stimulated release of dopamine from the primate retina is blocked by 1-2-amino-4-phosphonobutyric acid (APB).

Macaca mulatta retinas were superfused, in vitro, to measure the efflux of dopamine. Steady light, in the low photopic range, stimulated dopamine release slightly. Flashing light (3 Hz) superimposed over the steady background increased dopamine efflux significantly. This increase was completely blocked by the addition of d,1-2-amino-4-phosphonobutyric acid (d,l-APB, 10-100 microM) to the superfusion medium, but not by the addition of the inactive enantiomer d-APB (10 microM). The results suggest that ON bipolar cells provide the excitatory drive to dopaminergic amacrine cells in primates, as in other species.

Development of the enkephalin-, neurotensin- and somatostatin-like (ENSLI) amacrine cells in the chicken retina.

The development of the enkephalin-, neurotensin- and somatostatin-like immunoreactive (ENSLI) amacrine cells in the chicken retina has been investigated by radioimmunoassay (RIA) and immunocytochemistry (ICC). By RIA, enkephalin-like immunoreactivity (ENK-LI) was detected at embryonic day (E) 5 at only very low levels, which gradually increased until E17. From E18 to E21, there was a relatively rapid increase in ENK-LI levels, and just after hatching, there was a very steep rise. By ICC, the cell bodies of the ENSLI amacrine cells were first detected in the inner nuclear layer on E18, with no immunostaining in the inner plexiform layer (IPL). On E21, more cells were detected and processes in the IPL were visible, but detailed arborisations were not clear. On postnatal day (P) 1, the ENSLI amacrine cells showed a morphology similar to that in mature retina in both the density of cell bodies and the ramification pattern of processes. Antibodies to neurotensin and somatostatin revealed a similar developmental pattern. Thus, the three peptides appear to follow a similar developmental pattern in the ENSLI amacrine cells, suggesting that the three peptides respond similarly to developmental stimuli, just as they are released in parallel in response to physiological stimulation from mature ENSLI amacrine cells. After hatching, higher levels of ENK-LI were detected by RIA and more ENSLI amacrine cell bodies and processes were detected by ICC in animals kept in the light than in those kept in the dark. In retinas kept in the light for 12 h, it was found that immunoreactive processes in the IPL formed strongly stained patches, but this was not observed in retinas kept in the dark for 12 h.

Localization of D1 dopamine receptors in the chicken retina.

PURPOSE: The localization of dopamine D1 receptors (DIR) in the chicken retina was examined using an anti-human DIR monoclonal antibody and PAP techniques. RESULTS: A clear band of staining was seen in the outer plexiform layer, as well as cellular staining in the outer-most part of the inner nuclear layer, probably in a subset of horizontal cells. Many different amacrine cell bodies were labelled in the inner one-third of the inner nuclear layer. There was also extensive staining in the inner plexiform layer, which showed some striation. Occasional labelled ganglion cells were also detected. CONCLUSION: Localization of D1-dopamine receptors has been shown in the chicken retina.

A fundamental step-transition in retinal function at low light intensities.

There appears to be a fundamental step-transition in retinal function at low light intensities, close to the scotopic-mesopic transition. This step-transition is observed for elements of the retinal dark-light switch, which has been described in the chicken retina. Over the same range of light intensities, there is a step-transition in photoreceptor retinomotor movements and in the coupling of horizontal and All amacrine cells, which suggests a switch in retinal circuitry from rod-processing to cone-processing regimes. A similar step-transition in pineal function suggests that the retinal step-transition signals to the central circadian systems. Finally, this step-transition may also inhibit eye growth, and thus be responsible for the reported diurnal rhythm in eye growth. Disturbances to this step-transition may be the initial cause of disordered eye growth in the form-deprivation myopia paradigm.

Dopaminergic behaviour in chicken retina and the effect of form deprivation.

PURPOSE: Dopamine (DA) is considered to be a neurotransmitter involved in light-adaptive responses in the retina and has been implicated in the control of the eye growth induced by form deprivation. Vitreal DOPAC was shown to be a good indicator of retinal dopaminergic activity. METHODS/RESULTS: Dopaminergic activity was highest during the light; with vitreal DOPAC levels rising within 3 h of light exposure. Form deprivation attenuated dopaminergic activity, as the rise in vitreal DOPAC levels on light exposure was reduced in form-deprived eyes, compared with control eyes. CONCLUSION: The lack of sustained activation of DA release may explain the role of DA in the control of eye-growth.

Deprivation of form vision suppresses diurnal cycling of retinal levels of leu-enkephalin.

Deprivation of form vision by the fitting of translucent occluders suppressed the diurnal cycling of enkephalinergic amacrine cells (the ENSLI amacrine cells), in the chicken. Daily periods of normal vision or enforcing temporal contrast using strobe lighting appeared to restore normal functioning of the ENSLI cells. These results suggest that the ENSLI cells are involved in retinal circuits that assess the quality of the visual image and control eye growth.

A retinal dark-light switch: a review of the evidence.

We propose that there exists within the avian, and perhaps more generally in the vertebrate retina, a two-state nonadapting flip-flop circuit, based on reciprocal inhibitory interactions between the photoreceptors, releasing melatonin, the dopaminergic amacrine cells, and amacrine cells which contain enkephalin-, neurotensin-, and somatostatin-like immunoreactivity (the ENSLI amacrine cells). This circuit consists of two loops, one based on the photoreceptors and dopaminergic amacrine cells, and the other on the dopaminergic and ENSLI amacrine cells. In the dark, the photoreceptors and ENSLI amacrine cells are active, with the dopaminergic amacrine cells inactive. In the light, the dopaminergic amacrine cells are active, with the photoreceptors and ENSLI amacrine cells inactive. The transition from dark to light state occurs over a narrow (< 1 log unit) range of low light intensities, and we postulate that this transition is driven by a graded, adapting pathway from photoreceptors, releasing glutamate, to ON-bipolar cells to dopaminergic amacrine cells. The properties of this pathway suggest that, once released from the reciprocal inhibitory controls of the dark state, dopamine release will show graded, adapting characteristics. Thus, we postulate that retinal function will be divided into two phases: a dopamine-independent phase at low light intensities, and a dopamine-dependent phase at higher light intensities. Dopamine-dependent functions may show two-state properties, or two-state properties on which are superimposed graded, adapting characteristics. Functions dependent upon melatonin, the enkephalins, neurotensin, and somatostatin may tend to show simpler two-state properties. We propose that the dark-light switch may have a role in a range of light-adaptive phenomena, in signalling night-day transitions to the suprachiasmatic nucleus and the pineal, and in the control of eye growth during development.

Parallel suppression of retinal and pineal melatonin synthesis by retinally mediated light.

We have recently shown that light, over a narrow range of low intensities suppresses the activity of the enkephalin-immunoreactive amacrine cells of the chicken retina. In this paper, we show that over the same range of low light intensities the rate of melatonin synthesis in both the retina and the pineal of the chicken is suppressed. We further show that the effects of light on the pineal at these low intensities are mediated by the retina and not by direct actions on the pineal. Combined with our evidence that dopaminergic pathways within the retina are involved in controlling the state of activity of the pineal, these results suggest, but do not prove, that the change in state of a microcircuit within the retina involving the photoreceptors, dopaminergic amacrine cells and enkephalin-immunoreactive amacrine cells may be causally related to changes in the state of the pineal.

Pineal activity is under the control of retinal D1-dopaminergic pathways.

The role of dopaminergic pathways in the retina in controlling the functional state of the pineal was investigated. Dopaminergic agents were injected into the eyes of dark-adapted chickens which were maintained in the dark. Changes in the activity of N-acetyltransferase (NAT) in the retina and pineal were then monitored. Injection of the non-specific dopamine agonist 6,7-ADTN depressed retinal and pineal NAT. The D1-specific agonist SKF38393 did not affect retinal NAT but depressed pineal NAT. In contrast, quinpirole, a D2-specific agonist, depressed retinal NAT, but did not depress pineal NAT. Thus, D1- rather than D2-dopaminergic pathways in the retina are involved in the retinal circuit which control pineal function.

A role for the enkephalin-immunoreactive amacrine cells of the chicken retina in adaptation to light and dark.

The functional state of the amacrine cells which contain enkephalin-, neurotensin- and somatostatin-like immunoreactivity of the chicken retina was monitored by measuring the rate of change in the levels of [Leu]enkephalin-like immunoreactivity in the retina. Dark-adapted birds were exposed to lights of different intensities for 12 h. At light levels of < or = 0.03 microW/cm2, the ENSLI amacrine cells were highly active but, by 0.08 microW/cm2, they reached a state of maximum inactivation. Thus, the ENSLI amacrine cells act as flip-flop devices, inactivated by critical levels of light, which correspond to those which inactivate pineal melatonin synthesis. They may, therefore, be involved in retinal pathways which signal the difference between day and night.

Endogenous dopamine inhibits the release of enkephalin-like immunoreactivity from amacrine cells of the chicken retina in the light.

The activity of the enkephalin-immunoreactive (ENSLI) amacrine cells of the chicken retina is low in the light and high in the dark, resulting in parallel increases and decreases in the levels of the enkephalins. In vivo, the selective dopaminergic D1 antagonist SCH23390 increased the activity of the ENSLI amacrine cells in the light (ED50; 20 pmol), but had a much lesser effect in the dark, whereas the selective dopaminergic D2 antagonist sulpiride had effects only at very high concentrations (ED50; 39 nmol). In contrast, the non-selective dopamine agonist ADTN hardly affected the activity of the ENSLI amacrine cells in the light, but markedly reduced their activity in the dark. This pattern of effects suggests that dopamine actively inhibits the ENSLI amacrine cells in the light, but exerts much less inhibitory activity in the dark, consistent with the idea that dopamine is released during the exposure of the retina to light. Thus dopaminergic controls over the ENSLI amacrine cells appear to contribute to the light:dark differences in activity of the ENSLI amacrine cells. Results obtained on the dopaminergic control of enkephalin release in vitro were generally consistent with this model, except that ADTN appeared to stimulate the ENSLI amacrine cells in the dark.

Somatostatin-14 and somatostatin-28 levels are light-driven and vary during development in the chicken retina.

The relative levels of somatostatin-14 and somatostatin-28 were determined during both perinatal development and variations in lighting conditions in the chicken retina. During perinatal development of the retina, somatostatin-14 predominated in recently hatched chickens, whereas somatostatin-28 predominated in the retinas of older chickens. In mature chickens, the levels of both somatostatin-14 and somatostatin-28 increased during the light and decreased during the dark. Our results suggest that these two forms of somatostatin are released proportionally and in parallel.

[Leu5]enkephalin-like immunoreactive amacrine cells are under nicotinic excitatory control during darkness in chicken retina.

Based on the principle that retinal levels of [Leu5]enkephalin-like immunoreactivity (LELI) are set by the rate of release and thus reflect neural activity, we partially defined the dark-associated increase in excitatory control of LELI amacrine cells in chicken. Retinal levels of LELI were measured by radioimmunoassay (RIA). Intravitreal injection of cholinergic antagonists decreased the rate of depletion of LELI during the dark phase, suggesting the presence of cholinergic excitatory control of the LELI neurons. This cholinergic control involves nicotinic rather than muscarinic receptors, as tubocurarine appeared over 100 times more effective than atropine in inhibiting the decrease in retinal levels of LELI in the dark. (The ED50s were estimated at 3.2 and 450 nmol, respectively.) The lack of effect of the antagonists when applied during the light phase, suggest that there is little cholinergic input to the LELI amacrine cells in the light. Superfusing isolated retinas with buffer containing tubocurarine (10 microM) decreased the efflux of LELI by 35%, compared to the spontaneous release during the dark. Atropine (10 microM) had no effect on the release of LELI, and pilocarpine (100 microM) increased the release of LELI from retinas superfused in the light by 20%. We conclude that, in addition to previously reported glycinergic and dopaminergic inhibition, the LELI amacrine cells receive cholinergic excitatory input. A shift in balance between glycinergic and dopaminergic inhibitory, and cholinergic excitatory control may underly the light-driven variation in activity of the LELI neurons in chicken retina.

Glycinergic control of [Leu5]enkephalin levels in chicken retina.

Retinal levels of [Leu5]enkephalin-like immunoreactivity (LE-LI) increase during the light and decrease during darkness, in vivo15. Intravitreal injection of the GABA antagonist picrotoxin had no effect on the accumulation of LE-LI during the light, suggesting the absence of significant GABAergic control over LE-LI cells. However, injection of the glycine antagonist strychnine, prevented the light-induced increase of retinal levels of LE-LI during 6 h exposure to light, indicating the presence of glycinergic control over the LE-LI neurons. When applied during the dark, strychnine increased the depletion of LE-LI by 34% compared to vehicle-injected eyes, suggesting that the LE-LI neurons receive some glycinergic input during the dark as well. The release of LE-LI from retinas superfused in vitro is depressed by exposing the preparation to light. Superfusing isolated retinas with physiological buffer containing picrotoxin (100 microM), GABA (50 mM), or the GABA agonists muscimol (100 microM), (+)-baclofen (200 microM), or 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) (100 mM), had no effect on the efflux of LE-LI. Strychnine (100 mM) however increased the efflux of LE-LI by 64%, compared to the spontaneous efflux during the light. Glycine (15 and 50 mM) decreased the spontaneous efflux of LE-LI from retinas superfused in darkness by 44-48% and by 31% at 5 mM. These data are consistent with the results from pharmacological manipulations in vivo. We conclude that the LE-LI amacrine cells are under inhibitory control from glycinergic but not from GABAergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Light inhibits the release of both [Met5]enkephalin and [Met5]enkephalin-containing peptides in chicken retina, but not their syntheses.

The levels of native and cryptic [Met5]enkephalin in the chicken retina were found to vary during a 12:12 h light-dark cycle, both rising in the light and falling during the dark. Such variations could conceivably arise from (a) changes in the rate of release and subsequent degradation of native and/or cryptic [Met5]enkephalin, (b) changes in the rate of proenkephalin A synthesis, or (c) changes in the rate of proenkephalin A processing. Measurement of the rate of release of native and cryptic [Met5]enkephalin in vitro indicated that the increased rate of release of both of these forms of [Met5]enkephalin during the dark quantitatively accounted for the fall in their retinal levels during the dark. This indicated that the biosynthesis of proenkephalin A was not activated during the light-dark cycle. Molecular weight fractionation of retinal extracts also supported this idea, since the pool of high molecular weight precursors did not vary in size, suggesting that processing was not modulated during the light-dark cycle. Instead, the fall in both cryptic and native [Met5]enkephalin during the dark was due to their increased rate of release together with a rate-limiting conversion of high molecular weight [Met5]enkephalin-containing peptides to low molecular weight [Met5]enkephalin-containing peptides. The enkephalinergic cells of the retina seem to cope with physiological variations in demand by accumulating a large pool of peptide during periods of low stimulation (light), so that when stimulation and release is high (dark), the decrease in pool levels does not compromise the function of the cells and their postsynaptic targets.

The release of Leu5-enkephalin-like immunoreactivity from chicken retina is reduced by light in vitro.

A superfusion system was established to examine the efflux of endogenous Leu5-enkephalin-like immunoreactivity (LE-LI) from isolated chicken retinas. Superfusion with buffer containing high concentration of K+ (60 mM KCl) increased the efflux of LE-LI by 96%. This effect was not observed when Co2+ (4 mM CoCl2) was present. Exposing the retinas to light decreased the efflux of LE-LI by 59% compared to that observed during superfusion in the dark. No effect of ambient light could be detected in the presence of Co2+. Upon reverse-phase high-performance liquid chromatography the material released by the retina comigrated with synthetic Leu5-enkephalin. These results demonstrate that the release of LE-LI from retinal neurons is increased during the dark, and it is concluded that the lighting conditions exert their effects by modifying the state of polarization of the LE-LI amacrine cells and hence the release of LE-LI from these neurons.