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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.