N-methyl-D-aspartate-mediated glutamate toxicity in the developing rabbit retina.

Extracellular levels of endogenous glutamate are relatively high in the developing rabbit retina but nonetheless appear to promote cell survival and developmental processes at concentrations considered toxic in the adult. We wished to examine the development of retinal susceptibility to glutamate toxicity as well as the protective effects of two N-methyl-D-aspartate (NMDA) antagonists, 2-amino-5-phosphono-5-valeric acid (APV) and dextromethorphan (Dex), and the nitric oxide synthase (NOS) inhibitor, NG-methyl-L-arginine (metARG). One day in vitro retinal explants of adult and neonatal rabbits were incubated with various agonists and antagonists, and stained with trypan blue to visualize necrotic cells. The density of the necrotic cells was analyzed using the Zeiss Videoplan 2. Immature neurons were approximately 10-fold less sensitive to NMDA toxicity compared to the adult. Although both NMDA antagonists and metARG provided marked protection for adult retinal neurons against glutamate toxicity, the modest susceptibility of the immature neuron was blocked only by Dex and not APV or metARG. At least two factors may contribute to the ability of the neonatal retina to survive in the presence of high levels of endogenous extracellular glutamate. First, the 10-fold developmental increase in NMDA toxicity occurs simultaneously with a 12-15-fold downregulation of extracellular glutamate, probably through the actions of maturing Muller cells. Second, the NMDA/NO excitotoxic pathway may not be active at birth since an NOS inhibitor had little effect at this stage and our previous morphological data demonstrate that NOS-containing cells are not present in their mature configuration until the second postnatal week.

GABA and GABA-A receptors are maximally expressed in association with cone synaptogenesis in neonatal rabbit retina.

Previous studies have shown the cone photoreceptors form reciprocal synapses with horizontal cells during the first week after birth in rabbits. These synapses constitute pioneering elements of the developing outer plexiform layer. We now report that antibodies against the alpha-1 and against the beta-2/3 subunits of the GABA-A receptor label a highly restricted sublamina in the developing outer plexiform layer known to contain nascent cone photoreceptor terminals. Staining is relatively weak at birth, increases to maximal levels between postnatal days 5 and 7, and is significantly reduced in the adult. These results support recent calcium imaging studies which have shown that the activation of GABA-A receptors causes an increase in intracellular free calcium in cones, an effect which is observed only at 3-9 days after birth. The transient expression of GABA-A receptors in this region coincides with the period of peak expression of GABA immunoreactivity in horizontal cells. A direct functional link between GABAergic transmission and cone synaptogenesis is suggested by previous reports that GABA-A receptor antagonists cause disruption of cone synaptogenesis. Together these findings support the notion that GABA functions as a developmental neurotransmitter which is produced by horizontal cells and interacts with developing cone axons in order to facilitate synaptic linkage between these two cells types.

GABA-induced increases in [Ca2+]i in retinal neurons of postnatal rabbits.

Previous studies have indicated that gamma-aminobutyric acid (GABA) plays an important trophic role in the synapse formation between horizontal cells and photoreceptors in postnatal rabbit retina. However, the mechanism of the GABA effect has not been identified. Using fluo-3 Ca2+ imaging and confocal laser scanning microscopy we examined the effect of GABA on [Ca2+]i during postnatal retinal development. GABA (100 microM) evoked a fast and transient increase of [Ca2+]i in selected populations of freshly dissociated retinal cells from postnatal rabbits. This increase was apparent on postnatal day 1 and reached a maximum on day 5. Little increase in [Ca2+]i was observed in retinal cells isolated from adult rabbits. GABA receptor antagonists, picrotoxin and bicuculline, significantly reduced the response. The GABAB agonist, baclofen, did not evoke any [Ca2+]i changes. The GABA-induced increase in [Ca2+]i was observed in all retinal layers in neonatal retinal whole-mount explants. In the outer retina, the increase was seen in cone photoreceptors which were specifically labeled with peanut agglutinin (PNA). The GABA-induced increase in [Ca2+]i may provide an important mechanism for regulating cone synaptogenesis in the outer plexiform layer of the postnatal retina.

Expression and downregulation of the GABAergic phenotype in explants of cultured rabbit retina.

PURPOSE: To study the morphologic and neurochemical development of the rabbit retina in explant culture. METHODS: Explants of retina from newborn rabbits were cultured in defined medium in the absence of serum or soluble growth factors. The morphology of the explant and the neurochemical development of the GABAergic system were examined by light microscopy, autoradiography, and immunohistochemistry for 7 days and compared to those of the postnatal rabbit retina in vivo. RESULTS: Cultured explants from newborn rabbit retina develop and maintain well-defined plexiform and cellular layers up to 7 days. Exogenous 3H-gamma-aminobutyric acid (GABA) and antibodies against GABA labeled a population of horizontal, amacrine and displaced amacrine cells in the ganglion cell layer during the first 3 days in culture. After 4 days in culture, the extent of uptake and immunolabeling was diminished among all three cell types, but labeled horizontal cells were markedly rare. At 7 days in culture, uptake and GABA-like immunoreactivity could not be detected in horizontal cells, but antibodies to calbindin-D reacted with horizontal and amacrine cells in the appropriate retinal layers. Peanut agglutinin lectin binding studies revealed a mosaic of cone photoreceptor inner segments indistinguishable from that of neonatal retina in vivo. CONCLUSIONS: The experiments show that the maturation of cellular layers and the developmental expression of the GABAergic phenotype can be observed in retinal explants cultured under chemically defined conditions. Histochemical evidence is presented that indicates cultured explants of newborn rabbit retinas express markers of the GABAergic phenotype in a manner consistent with that observed in vivo. The authors show that horizontal cells continue to survive in culture after the diminution in GABA immunoreactivity.

High levels of extracellular glutamate are present in retina during neonatal development.

The three major classes of neurons which comprise the primary visual pathway in retina are glutamatergic. These cells are generated in two separate developmental stages, with one subclass of photoreceptors (cones) and ganglion cells generated before birth; and the other subclass of photoreceptors (rods) and bipolar cells generated during the first week after birth. Gas chromatography/mass spectroscopy analysis coupled with a new method for collecting small samples of extracellular fluids from retina were used to determine the levels of endogenous glutamate present during differentiation and synaptogenesis of these different cell types. As expected the total retinal content of glutamate increased during the postnatal period in synchrony with the generation and maturation of glutamatergic cells. However, a significant proportion of the endogenous pool was found extracellularly at birth. Intracellular glutamate is localized within cell bodies and growing processes of cones and ganglion cells at this time but few glutamatergic synapses are present. The extracellular concentration of glutamate actually declined during the most active period of synaptogenesis, reaching very low levels in the adult. The high concentrations of extracellular glutamate in neonatal retina could play an important role in a variety of developmental events such as dendritic pruning, programmed cell death and neurite sprouting.

Calbindin immunoreactivity of horizontal cells in the developing rabbit retina.

Horizontal cells are retinal interneurons that establish inhibitory feedback loops within the outer plexiform layer of the primary visual pathway. Most mammalian retinas contain two types of horizontal cells. A-type horizontal cells have neuritic branches that contact cone photoreceptors exclusively, while the B-type horizontal cells have dendritic branches that contact cones, in addition to axons that form synapses with rod photoreceptors. Immunoreactivity for calbindin, a calcium binding protein involved in calcium transport, was used as a marker for horizontal cells during post-natal development of the rabbit retina. On post-natal days 1, 3 and 5, calbindin immunoreactivity is limited to a single population of A-type horizontal cells. They appear as a monolayer of cells with broad tapering processes, establishing the proximal border of the nascent outer plexiform layer and forming a target for ingrowing cone photoreceptor terminals. The size and density of the cell bodies and the length of neuritic processes are essentially unchanged during this period, which corresponds to the time of peak expression of GABAergic markers in horizontal cells. Coincident with a decrease in GABAergic markers and the completion of cone-to-horizontal cell synaptogenesis by day 7, changes within the horizontal cell mosaic are detected morphometrically. A delayed phase of overall cell growth results in a 70% increase in average somal diameter (representing a 3.7-fold increase in spherical volume), a six-fold increase in mean neurite length and a decrease in cell density to one-third of that found in the newborn. We conclude that the process of terminal differentiation of horizontal cells is not complete until some time after the second post-natal week. Furthermore, the expression of GABAergic markers is associated primarily with early maturational events, whereas expression of calbindin is sustained throughout post-natal development, suggesting a prominent role for calcium dependent mechanisms at all development stages.

Modulatory actions of gamma aminobutyric acid (GABA) on GABA type A receptor subunit expression and function.

Gamma aminobutyric acid (GABA) is present in the central nervous system (CNS) during very early embryogenesis. It is therefore likely to play a role not only as a neurotransmitter but also as a signal molecule for neuronal differentiation, growth, and development. It has been firmly established that formation of synapses is strengthened by GABA, and the expression of certain subunits of the GABA type A (GABAA) receptor complex is clearly promoted by GABA. This latter effect of GABA may have profound implications for the functional activity of GABAergic synapses since the pharmacological properties of GABAA receptors are governed by the subunit composition of the receptor complex. Dynamic changes in GABAA receptor expression and diversity during development and differentiation may therefore play important roles for the inhibitory potential of the CNS during mature stages.

The role of GABA during development of the outer retina in the rabbit.

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.

gamma-Aminobutyric acid immunoreactivity in multiple cell types of the developing rabbit retina.

We have previously demonstrated that the neonatal rabbit retina contains a larger complement of cells that accumulate [3H]-GABA than does the adult. In order for these neurons to be classified as GABAergic, they must also contain endogenous GABA. We now report that these same neonatal cell populations are also immunoreactive to GABA antisera. In frozen sections from rabbit retina, treated with GABA antisera, immunoreactive processes in both synaptic layers were observed at postnatal day 1. The appearance of immunofluorescent fibers precedes that of photoreceptor and bipolar cell terminals in the outer plexiform layer and is diminished by postnatal day 5. Also noted, was a 50% decrease in the density of GABA-immunoreactive cell bodies in the inner nuclear and ganglion cell layers, accompanied by an increase in cell volume and a shift toward a more spherical cell shape of the remaining cells. At postnatal day 1 and 3, we also observed immunoreactive cells having the characteristic morphology of interplexiform cells. This cell type sends branches to both the outer and inner plexiform layers, thus a morphological basis for communication between the two developing plexiform layers is present as early as postnatal day 1. Thus, retinas from neonatal rabbits have a larger complement of cells that stain for endogenous GABA than does the adult. These results coupled with our previous studies suggest that GABAergic properties are expressed by a larger number of cell types in the neonate than in the adult and are consistent with the general hypothesis that GABA functions as a trophic agent during development.

Development of the glutamate system in rabbit retina.

We have investigated two characteristics of the glutamate system in the developing rabbit retina. 1) Glutamate immunoreactivity was observed at birth within developing processes of four cell types; two of which, photoreceptors and ganglion cells, are known to be glutamatergic in the adult. Two other cell types, type A horizontal cells and amacrine cells, are immunoreactive to both glutamate and GABA at birth, suggesting that endogenous pools of glutamate in GABAergic neurons serve as precursor for GABA synthesis. Thus it appears that endogenous glutamate pools are present within neurons prior to synaptogenesis as part of the early expression of either the glutamate or GABA transmitter phenotype. 2) Analysis of 3H-glutamate metabolism during retinal development showed that rapid conversion of glutamate to glutamine does not occur until the second postnatal week, coincident with the expression of Muller (glial) cell activity. In the absence of glial metabolism in the neonate, extracellular concentrations of glutamate remain relatively high and are likely to have major effects on neuronal maturation.

Melatonin inhibits ACh release from rabbit retina.

Previous studies have suggested that melatonin, released from photoreceptors, may modulate retinal dark-adaptive responses by inhibition of dopamine release from retinal interneurons. We have broadened these studies to examine the effect of melatonin on release of another retinal neurotransmitter, acetylcholine (ACh). The ACh system in rabbit retina has been localized to starburst amacrine cells, which release ACh in response to a variety of experimental stimuli, including direct potassium depolarization, flashing light, and glutamatergic as well as GABAergic inputs. The effect of melatonin on release of endogenously synthesized [3H]-ACh was measured in perfusates from retinas or retinal synaptosomes preloaded with [3H]-choline chloride. Melatonin significantly inhibited ACh release stimulated by potassium in intact retina but not in synaptosomes. Stimulation of intact retina by flashing light or by the glutamate receptor agonist, kainic acid, was also inhibited by melatonin. In contrast, there was no significant effect of melatonin on picrotoxin-induced release. These findings suggest that melatonin does have an inhibitory effect on ACh release, either by direct interaction with the cholinergic amacrine cell, or indirectly via GABAergic but not glutamatergic neurons.

Acetylcholine release from the rabbit retina mediated by kainate receptors.

The cholinergic amacrine cells of the rabbit retina may be labeled with 3H-choline (3H-Ch), and the activity of the cholinergic population may be monitored by following the release of 3H-ACh. Glutamate analogs caused massive ACh release, up to 50 times the basal efflux, with the following rank order of potency: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) greater than quisqualate (QQ) = kainate (KA) much greater than NMDA (in magnesium-free medium) much greater than glutamate greater than aspartate. In contrast, the release of 3H-Ch was unchanged. Submaximal doses of each agonist were used to establish the specifity of glutamate antagonists. Kynurenic acid was selective for KA much greater than QQ, and 6,7-dinitroquinoxaline-2,3-dione (DNQX) was selective for KA greater than QQ much greater than NMDA. At low doses, which selectively blocked the response to KA, both antagonists blocked the light-evoked release of ACh. These results suggest that ACh release may be produced via several glutamate receptors, but the physiological input to the cholinergic amacrine cells is mediated by KA receptors. Because these cells receive direct input from cone bipolar cells, this work supports previous evidence that the bipolar cell transmitter is glutamate.

Evidence for functionally distinct subclasses of gamma-aminobutyric acid receptors in rabbit retina.

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the mammalian retina, where it serves many roles in establishing complex response characteristics of ganglion cells. We now provide biochemical and physiological evidence that at least three subclasses of GABA receptors (A1, A2, and B) contribute to different types of synaptic integration. Receptor binding studies indicate that approximately three-fourths of the total number of [3H]GABA binding sites in retina are displaced by the GABAA receptor antagonist, bicuculline, whereas one-fourth are displaced by the GABA-B receptor agonist, baclofen. GABAA receptors can be described by a three-site binding model with KD values of 19 nM, 122 nM, and 5.7 microM. Benzodiazepines and barbiturates potentiate binding to the GABAA site, which suggests that significant numbers of GABAA receptors are coupled to regulatory sites for these compounds and thus are classified as GABAA1 receptors. The response to pentobarbital appears to involve a conversion of low-affinity sites to higher-affinity sites, and is reflected in changes in the densities of sites at different affinities. Functional studies were used to establish which of the different receptor subclasses regulate release from cholinergic amacrine cells. Our results show that GABA suppresses light-evoked [3H]acetylcholine release via GABAA2 receptors not coupled to a benzodiazepine or barbiturate regulatory site, and enhances release via GABAB receptors. GABAA1 sites do not appear to control acetylcholine release in rabbit retina.

Kainic acid lesioning alters development of the outer plexiform layer in neonatal rabbit retina.

The first synaptic relay in the primary visual pathway occurs between terminals of photoreceptors and second-order neurons within the outer plexiform layer of the retina. During development, one of these types of second-order neurons, the type A horizontal cell, differentiates and assumes mature characteristics several days before any other cells ramifying in that synaptic layer. In neonates, horizontal cells appear to be GABAergic during the first 5 days of postnatal life and in addition they also are responsive to kainic acid. We have previously suggested that they may play a pioneering role in the postnatal development of the outer plexiform layer, perhaps providing structural guidance or trophic substances such as GABA, for synaptic development. To test this hypothesis, we first demonstrated that a single intraocular injection of kainic acid within 24 hr of birth results in a permanent and selective loss of type A horizontal cells in the outer retina. Retinas from animals maintained for 5 days postinjection were harvested for analysis of postnatal development of the outer plexiform layer in the absence of horizontal cells. One of the major findings was that kainic acid treatment caused a reversal of the normal complement of photoreceptor cell types, resulting in an abnormally high rod/cone ratio. The distribution of cell processes within the outer plexiform layer was also altered and normal synaptic connections were not made. In spite of these changes in the constituents of the outer plexiform layer, the normal position of the synaptic layer was not affected by the loss of horizontal cells. These results rule out the possibility that horizontal cells provide a structural barrier which is an absolute requirement for establishing the location of the outer plexiform layer. Rather, these cells may be more involved in cell differentiation and synaptogenesis.

Darkness stimulates rapid synthesis and release of melatonin in rat retina.

The presence of melatonin in retina has been widely reported for over two decades although studies of its functional importance within the retina have only recently been emphasized. We have analyzed the biochemical characteristics of melatonin synthesis and release, focusing on rapid changes in response to light/dark conditions. Our major findings are consistent with the following conclusions: (1) melatonin synthesis is stimulated within minutes after exposure to darkness, and may reflect an increase in N-acetyl transferase activity; (2) melatonin is not stored, but rather it diffuses freely throughout the retina immediately after it is synthesized; and (3) the dark-induced increase in retinal melatonin release is a synthesis-coupled response and does not involve separate secretion mechanisms. The characteristics of melatonin synthesis and release described herein would be consistent with the proposed role of melatonin as a local paracrine effector of dark-adaptive responses in retina.

AP4 inhibits chloride-dependent binding and uptake of [3H]glutamate in rabbit retina.

Glutamate is one of the major neurotransmitters used by primary and secondary neurons of the visual pathway in retina. AP4(2-amino-4-phosphonobutyric acid) preferentially blocks the activity of one functional subclass of retinal neurons, ON bipolar cells, apparently by acting as an agonist at a hyperpolarizing glutamate receptor. We have used in vitro binding assays to examine different subclasses of presumptive glutamate receptors in retinal membrane fractions. One subclass consists of AP4-sensitive binding sites which require calcium and chloride for maximal binding and which are inhibited by freeze-thaw procedures. In addition, AP4 inhibits chloride-dependent [3H]glutamate uptake into retinal synaptosomes and intact retina. [3H]glutamate which is accumulated via the AP4-sensitive mechanism can be subsequently released by depolarizing levels of potassium. The pharmacological selectivity of AP4-sensitive glutamate receptors on ON bipolar cells measured electrophysiologically is very similar to that of AP4-sensitive, [3H]glutamate binding and uptake, measured biochemically in subcellular fractions. These results raise the possibility that AP4-sensitive glutamate recognition sites in retina may be linked to two separate effectors, one which gates ion channels and leads to hyperpolarization, and another which acts as a glutamate transporter.

Neurotransmitter systems in the outer plexiform layer of mammalian retina.

Melatonin represents a second type of chemical signal released from photoreceptors in response to increased darkness, one with characteristics which are significantly different from those of glutamate. Concise spatial and temporal aspects of the photoreceptor signal are conserved through discrete glutamatergic synapses. Different classes of post-synaptic neurons each have appropriate subclasses of glutamate receptors which transmit sign conserving or sign inverting images of the visual mosaic. In contrast, melatonin, because of its highly lipophilic nature is not released by stimulus-coupled secretion mechanisms, but rather by simple diffusion. Thus control of melatonin "release" may be less concise than glutamate. In addition, melatonin may diffuse beyond the confines of the synaptic area to target cells throughout the retina. Effects of melatonin in retina are not well understood; however, current hypotheses suggest that, perhaps via its control of dopamine systems in the inner retina, melatonin plays an important role in dark adaptation and in various retinal processes which exhibit a circadian rhythm. Melatonin and glutamate may represent "co-transmitters" which provide the visual pathway with two types of signals, with melatonin providing widespread modulatory influences on the discrete visual information conveyed via glutamatergic circuits.

Synaptosomal neurotransmitter uptake systems in the retina and brain nuclei of light- and dark-adapted rabbits.

High affinity uptake rate for [14C]aspartate and [3H]dopamine by retinal homogenate (H), Pl (outer plexiform layer, OPL), and P2 (inner plexiform layer, IPL) retinal synaptosomal fractions were not significantly different between light- and dark-adapted rabbits. However, there were significant increases in the dark in [3H]gamma-aminobutyric acid high affinity uptake rate by retinal H and P2 but not that of Pl. There was a significantly higher [3H]choline uptake rate by retinal H, Pl and P2 in the dark-adapted compared to light-adapted rabbits, but there was no significant change in this rate for synaptosomal fractions from the lateral geniculate body, superior colliculus, visual cortex (VA I + II), caudate nucleus (CN) and hippocampus (HP). Data obtained in this study, along with reports of others, indicate that the change in retinal neurotransmission functions may not always be parallel with the change in high affinity uptake rates of neurotransmitters by retinal synaptosomal fractions. Data obtained indicate an increase in retinal cholinergic neuronal activities in the dark and indicate that optic nerves are not cholinergic and cholinergic neurons in brain nuclei, such as VA, CN and HP, are not significantly influenced by optic nerve inputs in light and dark conditions.