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Electrophysiological recordings on retinal rod cells, horizontal cells and on‐bipolar cells indicate that exogenous nitric oxide (NO) has neuromodulatory effects in the vertebrate retina. We report here endogenous NO formation in mammalian photoreceptor cells. Photoreceptor NO synthase resembled the neuronal NOS type I from mammalian brain. NOS activity utilized the substrate L‐arginine (Km = 4 microM) and the cofactors NADPH, FAD, FMN and tetrahydrobiopterin. The activity showed a complete dependence on the free calcium concentration ([Ca2+]) and was mediated by calmodulin. NO synthase activity was sufficient to activate an endogenous soluble guanylyl cyclase that copurified in photoreceptor preparations. This functional coupling was strictly controlled by the free [Ca2+] (EC50 = 0.84 microM). Activation of the soluble guanylyl cyclase by endogenous NO was up to 100% of the maximal activation of this enzyme observed with the exogenous NO donor compound sodium nitroprusside. This NO/cGMP pathway was predominantly localized in inner and not in outer segments of photoreceptors. Immunocytochemically, we localized NO synthase type I mainly in the ellipsoid region of the inner segments and a soluble guanylyl cyclase in cell bodies of cone photoreceptor cells. We conclude that in photoreceptors endogenous NO is functionally coupled to a soluble guanylyl cyclase and suggest that it has a neuromodulatory role in visual transduction and in synaptic transmission in the outer retina.

Using the in vivo rabbit eyecup, we have studied the light-evoked release of acetylcholine (ACh) which is presumed to indicate the activity of cholinergic amacrine cells. Gamma-Aminobutyric acid (GABA) inhibited the light-evoked release of ACh (IC50 congruent to 1 mM), but the GABA antagonists bicuculline (5 micro M) and picrotoxin (20 micro M) potentiated the light-evoked release and markedly increased the resting release of ACh. This bicuculline/picrotoxin-evoked release was calcium dependent and the effects of bicuculline, but not picrotoxin, were blocked by muscimol, a potent GABA agonist. Muscimol also inhibited the light-evoked release of ACh (IC50 less than 1 micro M) and was at least 1000 times more potent than GABA. Nipecotic acid (1 mM), a GABA transport blocker, also inhibited the light-evoked release of ACh, but the effect was slow in onset and recovery was prompt. We conclude that the cholinergic amacrine cells of rabbit retina are inhibited by GABA. The relatively weak action of GABA, compared to muscimol, may be due to the presence of avid GABA transport systems. We ascribe the excitatory effects of bicuculline and picrotoxin to the antagonism of endogenous GABA, suggesting that the cholinergic cells are influenced by a tonic release of GABA. This is consistent with the effects of nipecotic acid. Although we are unable to specify the synaptic arrangements involved, we suggest that the most likely interaction is directly between GABA amacrine cells and the cholinergic amacrine cells and/or their presumed bipolar cell inputs.

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Light and electron microscopic autoradiography demonstrates that 3H-GABA is accumulated by horizontal cells in neonatal rabbit retina but not in the adult. A specific population of horizontal cells appears to be mature at birth and they avidly accumulate 3H-GABA during a 15-minute incubation period in vitro. Uptake into horizontal cells is not observed after the fifth postnatal day; 3H-GABA-accumulating horizontal cell bodies and their processes are the first identifiable components that clearly mark the future location of the outer plexiform layer at birth and as such, may be considered pioneering elements. Our observations raise the interesting possibility that the pioneering horizontal cell may provide structural and/or chemical factors necessary for the subsequent development of the outer plexiform layer of the retina. Labeling patterns of other retinal cells also show varying degrees of change during development. A population of amacrine cells accumulate 3H-GABA at birth. These cells show little change in their morphological or 3H-GABA uptake properties from birth to adulthood. Müller cells show weak accumulation of 3H-GABA at birth. Subsequent to this time, labeling of Müller cells is significantly more robust, resulting in Müller cell domination of retinal autoradiographic patterns in more mature retinas. Every cell body in the ganglion cell layer accumulates 3H-GABA at birth. The number of labeled cells declines during postnatal development, resulting in a very limited adult population. We conclude that the ability of retinal cells to accumulate 3H-GABA does not remain constant during postnatal development; rather each cell population displays a unique maturation sequence that results in a dramatic developmental shift in the number and types of GABA-accumulating cells present in the retina.

Horizontal cells are among the first to mature in the neonatal mammalian retina and they are the first to establish the position of the outer synaptic layer which is subsequently formed by invading terminals of both rod and cone photoreceptors. During the period of cone synaptogenesis, horizontal cells transiently express the full complement of GABAergic properties (uptake, release, synthesis and storage of GABA); later during development of rod terminals, these properties are down-regulated. Given the reports of GABA's role in other developing neuronal systems (for review: 10), we have examined the effect that GABA, produced from horizontal cells, might have on photoreceptor maturation in rabbit retina. Results from our previous studies show that lesioning the horizontal cell with kainic acid in vivo leads to a displacement of cone photoreceptor cells and a disappearance of their synaptic terminals, while rod cells maintain their normal position and produce an overabundance of terminals. Similar effects are seen with the GABA-A receptor antagonists, picrotoxin and bicuculline. New evidence from 3H-thymidine studies suggests that the effects of kainic acid are specific and that cell division, migration and differentiation in other cell types do not appear to be affected. This body of work is summarized and possible mechanisms of action are suggested which could account for the apparent ability of GABA to help maintain the normal position of cone cell bodies and regulate cone synaptogenesis.

No abstract

[3H]Serotonin is accumulated by a specific set of amacrine cells in the rabbit retina. These cells also accumulate the neurotoxin, 5,7-dihydroxytryptamine, and show signs of necrosis within 4 h of in vivo exposure to the drug. Biochemical analysis of [3H]serotonin uptake reveal a sodium-and temperature-dependent, high affinity uptake system with a Km of 0.94 #M and Vmax of 1.08 pmol/mg protein/min. [3H]Tryptophan is also accumulated in rabbit retinal homogenates by a high affinity process. Accumulated [3H]serotonin is released in response to potassium-induced depolarization of intact, isolated retinas. In vitro binding studies of rabbit retinal homogenate membranes demonstrate specific sets of binding sites with characteristics of the postsynaptic serotonin receptor. These data strongly suggest that rabbit retina contains virtually all of the molecular components required for a functional serotonergic neurotransmitter system. The only significant difference between the serotonin system in rabbit retina and that in the well-established serotonin transmitter systems in nonmammalin retinas and in brains of most species is the relatively low concentration of endogenous serotonin in rabbit retinas, as demonstrated by high-performance liquid chromatography, histofluorescence, or immunocytochemistry.Serotonin has been identified as a transmitter in nervous tissue from the brain and retina of many vertebrates. Retinas from frogs and goldfish contain relatively high levels of endogenous serotonin (14,22), while bird and lizard retinas contain somewhat lower levels (7,15). In these species, neurons that possess endogenous stores of serotonin (demonstrated by histofluorescence or immunocytochemical techniques) and/or serotonin uptake systems (demonstrated by histofluorescence, autoradiography, or neurotoxic reactions) have neurites that are limited to the inner plexiform layer. The cell bodies of these neurons are found primarily in the amacrine cell layer of the inner nuclear layer, although some are found in the ganglion cell layer and are thought to be displaced amacrine cell bodies. One exception is the report by Tornquist et al. (22) that describes accumulation of [3H]-serotonin by cells in the outer plexiform layer of pigeon and chick retinas, tentatively designated interplexiform cells.Although serotonin is considered a strong transmitter candidate in the retinas of the nonmammalian species mentioned above, its role in mammalian retinas has been questioned because of the relatively low concentration of endogenous serotonin. Consistent with this finding, attempts to demonstrate endogenous serotonin by histofluorescence or immunocytochemistry have failed. Thus, Ehinger et al. (4) (see also reference 7) have proposed that the true indoleamine transmitter is not serotonin but some other closely related compound. By using a microdansylation procedure, Osborne et al. (10,12) observed that the low concentrations of serotonin in the bovine retina were localized in the inner nuclear and inner plexiform layers that ...