Membrane morphogenesis in retinal rod outer segments: inhibition by tunicamycin.

Isolated Xenopus laevis retinas were incubated with 3H-labeled mannose or leucine in the presence or absence of tunicamycin (TM), a selective inhibitor of dolichyl phosphate-dependent protein glycosylation. At a TM concentration of 20 micrograms/ml, the incorporation of [3H]mannose and [3H]leucine into retinal macromolecules was inhibited by approximately 66 and 12-16%, respectively, relative to controls. Cellular uptake of the radiolabeled substrates was not inhibited at this TM concentration. Polyacrylamide gel electrophoresis revealed that TM had little effect on the incorporation of [3H]leucine into the proteins of whole retinas and that labeling of proteins (especially opsin) in isolated rod outer segment (ROS) membranes was negligible. The incorporation of [3H]mannose into proteins of whole retinas and ROS membranes was nearly abolished in the presence of TM. Autoradiograms of control retinas incubated with either [3H]mannose or [3H]leucine exhibited a discrete concentration of silver grains over ROS basal disc membranes. In TM-treated retinas, the extracellular space between rod inner and outer segments was dilated and filled with numerous heterogeneously size vesicles, which were labeled with [3H]leucine but not with [3H]mannose. ROS disc membranes per se were not labeled in the TM-treated retinas. Quantitative light microscopic autoradiography of retinas pulse-labeled with [3H]leucine showed no differences in labeling of rod cellular compartments in the presence or absence of TM as a function of increasing chase time. These results demonstrate that TM can block retinal protein glycosylation and normal disc membrane assembly under conditions where synthesis and intracellular transport of rod cell proteins (e.g., opsin) are not inhibited.

Turnover of rod photoreceptor outer segments. II. Membrane addition and loss in relationship to light.

The rate of disk addition to rod outer segments (ROS) varies widely in Xenopus laevis tadpoles kept in cyclic light (12L:12D). When measured as radioactive band (3H-band) displacement during the 2nd day after injection of [3H]leucine, 75% of the daily increment of displacement occurred during the first 8 h of light. During the same interval, the number of open disks at the ROS base increased more than threefold. During the last 8 h of darkness, 3H-band displacement was undetectable and the number of open disks was reduced. These observations suggest the possibility that disk addition may occur discontinuously. During the 3rd and 4th days after injection of [3H]leucine, maximal displacement of the 3H-band occurred later in the day than on the 2nd day, its movement no longer corresponding to the increase in open disks. This delay in 3H-band displacement may reflect a time delay as a result of propagation of compressive stress in an elastic ROS system. Maximal disk loss from ROS as reflected in counts of phagosomes in the pigment epithelium occurred within 1 h of light exposure, and phagosome counts remained high for 4 h before declining to a low level in darkness. Modified lighting regimes affected the daily rhythms of shedding and disk addition differently, suggesting that control mechanisms for the two processes are not directly coupled. During 3 days in darkness, disk addition was reduced 50% compared to controls (12L:12D), whereas shedding was reduced by about 40%. Although reduced in level, shedding occurred as a free-running circadian rhythm. There was no evidence of rhythmicity of disk addition in darkness. In constant light, the rate of disk addition was not different from controls, but shedding was reduced by about 80% after the 1st day. This resulted in a 21% increase in ROS length. Among animals kept on a 2.5L:21.5D cycle, the rate of disk addition was reduced by 40% while shedding was maintained near control levels, resulting in a slight decrease in ROS length. These observations indicate that normal shedding requires alternating light and darkness, and that the daily rhythm of disk addition is due primarily to daily stimulation by light.

Turnover of rod photoreceptor outer segments. I. Membrane addition and loss in relationship to temperature.

Membrane turnover in outer segments of Rana pipiens red rods (ROS) was studied in tadpoles maintained under cyclic lighting (12L:12D) at 23 degrees, 28 degrees, and 33 degrees C. Large fragments (greater than 2 microns in diameter or length) were shed from the ROS tips shortly after the onset of light. These were phagocytized by the pigment epithelium (PE) which caused an increase in the number of phagosomes greater than 2 microns in size (large phagosomes). Large phagosomes were present in highest numbers 2-4 h after light exposure and were degraded by 8-12 h. The proportion of ROS that shed each day after the onset of the light cycle increased with increment increases in temperatures (23 degrees C-18%, 28 degrees C-33%, 33 degrees C-42% per day), resulting, in a reduction in the average interval of time between repeated sheddings (23 degrees C-5.6 days, 28 degrees C-3 days, 33 degrees C-2.4 days) though the average numbers of disks shed from ROS at the various temperatures were not significantly different (23 degrees C-139.5 +/- 5.7, 28 degrees C-129.4 +/- 7.6, 33 degrees C-129.9 +/- 4.8 disks/shed packet). Phagosomes in the PE that were less than 2 microns in diameter (small phagosomes) were present in relatively constant numbers throughout the day, and their numbers increased at higher temperatures. The absence of a concomitant increase in small phagosomes as large phagosomes were degraded indicates that large phagosomes were not the major source of small phagosomes. When the PE was isolated to culture in the absence of the retina, these small phagosomes were degraded. The rate of disk addition to the ROS base was determined by autoradiography after [3H]leucine injection. The number of disks added per day increased with elevations of temperature (23 degrees C-32.4; 28 degrees C-55.9; 33 degrees C-65.5). The average number of disks added to the ROS between repeated sheddings (23 degrees C-181.4; 28 degrees C-167.7; 33 degrees C-157.2) was greater than the number of disks shed after light exposure. Inasmuch as the ROS show no net increase in length during the tadpole stages utilized, the remaining disks must be lost at some other time. Electron microscope analysis revealed the presence of small groups of disks in curled configurations at the tips of ROS, suggesting possible stages of detachment.(ABSTRACT TRUNCATED AT 400 WORDS)

Endocytosis and degradation of interstitial retinol-binding protein: differential capabilities of cells that border the interphotoreceptor matrix.

Between the pigment epithelium and the outer limiting membrane of the retina is an extracellular compartment filled with the interphotoreceptor matrix (IPM). A prominent component of the IPM is a glycoprotein known as interstitial retinol-binding protein (IRBP). Using in vitro techniques, we compared the ability of the cells that border this compartment to internalize colloidal gold (CG) coated with either IRBP or ovalbumin, a glycoprotein not found in the IPM. Neither IRBP-CG nor ovalbumin-CG was internalized by the Muller's cells. Both rod and cone photoreceptors take up IRBP-CG, which is observed in small vesicles and multivesicular bodies. Neither photoreceptor type takes up ovalbumin-CG. Acid phosphatase cytochemistry indicates that acid phosphatase reaction product in the multivesicular bodies co-localizes with IRBP-CG, which suggests that this molecule is degraded by rod and cone photoreceptors and is not recycled. The pigment epithelium internalizes IRBP-CG and ovalbumin-CG, both of which remain in small cytoplasmic vesicles near the apical plasma membrane. There is no indication that vesicles that contain either IRBP-CG or ovalbumin-CG fuse with the lysosomal system in the pigment epithelial cells during the incubation.

Photoreceptor outer segments: accelerated membrane renewal in rods after exposure to light.

The rate of rod outer segment renewal in Rana pipiens tadpoles under constant light and under diurnal conditions of 12 or 2 hours light per day is significantly increased compared to that in animals in darkness. Furthermore, during 24 hours in light after 6 days in darkness the rate of renewal is three to four times that in darkness. In Xenopus laevis tadpoles the rate of renewal is more than five times greater during the first 8 hours of a normal diurnal cycle than during the following 16 hours. These observations demonstrate that bursts of renewal activity occur as a response to light, and suggest that a normal pattern of light alternating with darness plays a fundamental role in the regulation of rod outer segment turnover.

Cyclic GMP accumulation causes degeneration of photoreceptor cells: simulation of an inherited disease.

Guanosine 3',5'-monophosphate (cyclic GMP) metabolism in developing eye rudiments of Xenopus laevis embryos in culture is disrupted by the phosphodiesterase inhibitor isobutylmethylxanthine. At low concentrations of inhibitor the rudiments develop normally, but at higher concentrations of the inhibitor, cyclic GMP accumulates in the rudiments and the retinal photoreceptor cells degenerate selectively. The isobutylmethylxanthine-induced photoreceptor degeneration is associated with an accumulation of cyclic GMP and, in this respect, it stimulates an early biochemical defect in the inherited degenerative disease of rd mice.

Histopathology of the Retina from a Three Year-Old Suspected to Have Joubert Syndrome.

PURPOSE: To define the retinal pathology in a 3 year-old eye donor who died from complications of an undiagnosed genetic syndrome. METHODS: Eyes were fixed and analyzed using macroscopic fundus photography (MF), confocal scanning laser ophthalmoscopy (cSLO) and spectral-domain optical coherence tomography (SD-OCT). Small areas from the perifovea and periphery were processed for histology and indirect immunofluorescence, using antibodies specific to retinal proteins such as rhodopsin, cone arrestin, RPE65 and others. Available medical records were also reviewed. RESULTS: With all three imaging modalities, the affected donor's eyes lacked the distinct morphological detail typically observed with these techniques in postmortem control eyes. MF images showed a "photonegative effect" due to a hypopigmented macula relative to a hyperpigmented retinal background. cSLO imaging demonstrated a weak autofuorescence signal that was largely devoid of the usual retinal structures compared to the control. SD-OCT suggested disorganization of the affected retina, absence of a photoreceptor layer, and degeneration of the choroid in the macular area. Histologic findings indicated a highly disorganized photoreceptor layer in the macula and periphery. The RPE layer displayed thinning in some regions of the periphery and decreased pigmentation in most areas. Rods and cones were significantly reduced in the affected retina but a few cones were detected in the perifovea. Centrin-2 labeling was mostly absent from the connecting cilium of the photoreceptor cells. Medical record review pointed to a possible clinical diagnosis of Joubert syndrome. CONCLUSIONS: The retinal degenerative findings, and absence of centrin-2 labeling are compatible with the expected retinal phenotype in patients with Joubert syndrome.

Serotoninergic neurons in the retina of Xenopus laevis: selective staining, identification, development, and content.

Uptake of 3H-serotonin followed by autoradiography, and uptake of the serotonin analog 5,7-dihydroxytryptamine (5,7-DHT), with subsequent staining, were each used to define a unique set of neurons in the retina of the African clawed frog, Xenopus laevis. Both techniques demonstrated the same population of neurons, on the basis of perikaryal size, shape, and position within the retina. Two classes of amacrine cells accumulated 5,7-DHT at the proximal (vitread) margin of the inner nuclear layer; the two classes were distinguished by the size of their perikarya. Two similar populations of cells, observed in the ganglion cell layer with lower frequency, may represent "displaced" counterparts of these two amacrine cell types. A class of bipolar cells whose perikarya were located in middle-to-distal regions of the inner nuclear layer also accumulated 5,7-DHT and 3H-serotonin. Processes of these cells contributed to a dense plexus of fine fibers that appeared evenly distributed throughout the inner plexiform layer. 3H-Serotonin-accumulating cells first appeared in the developing retina at stage 35/36, a time immediately after retinal stratification but before elaboration of either plexiform layer. Electron microscopic analysis permitted an identification of 3H-serotonin-accumulating terminals in the inner plexiform layer. Serotonin-labeled terminals containing conventional contacts, suggestive of amacrine cells, were presynaptic to unidentified processes and postsynaptic to bipolar cells. Labeled terminals containing ribbon contacts, indicative of bipolar cells, were postsynaptic to amacrine cells. The amount of serotonin contained in isolated retinas was 15 pmol/mg protein as measured by HPLC with electrochemical detection. We attempted to stimulate the release of accumulated 3H-serotonin from mature retinas by increasing the K+-concentration in the bathing medium. Although preloaded glycine is readily released from 14C-glycine-accumulating neurons, from the same retinas there was no calcium-dependent, K+-stimulated release of 3H-serotonin. This finding suggests that serotonin and glycine are processed differently by retinal neurons, the consequence of which results in differing responses to 40 mM K+.

Interphotoreceptor matrix in the human retina: cone-like domains surround a small population of rod photoreceptors.

The interphotoreceptor matrix (IPM) in mammalian retinas is subdivided into rod and cone specific compartments: peanut agglutinin (PNA) binding glycoconjugates are associated with cones, whereas wheat germ agglutinin (WGA) binding glycoconjugates are associated with rods. To establish the identity of a photoreceptor cell type in the human retina with rod dimensions but with a matrix domain which stains with PNA, double label studies, using PNA-ferritin to decorate the extracellular domains and immunocytochemical techniques using a rod specific anti-opsin antibody were conducted. The PNA-binding domains were observed in the cone-associated IPM as well as in the IPM surrounding a small population of rod-shaped photoreceptors. The outer segments of these rod-shaped photoreceptors showed intense labeling with a rod specific anti-opsin antibody as did all other rods which were free of PNA-labeling. A quantitative analysis of all retinal quadrants indicates that this novel rod represents approximately 0.3% of the total rod population in the human retina.

Glycinergic neurons in the human retina.

Neurotransmitter-specific properties of glycinergic neurons in the human retina were studied using 11 pairs of eyes from donors ranging from 2 1/2 to 54 years in age. A mean endogenous level of 10.3 nmoles glycine per mg protein was measured by amino acid analysis in retinas isolated within 1 hour postmortem. When retinas were incubated with 3H-glycine (2 microM) and processed for autoradiography, label was found associated with neurons whose somata reside within the inner nuclear layer. Some heavily labeled neurons located at the vitread border of the inner nuclear layer were identified as amacrine cells based on ultrastructural verification of the conventional synaptic contacts made by their processes in distal regions of the inner plexiform layer. In proximal regions of the inner plexiform layer, dendrites of glycine-accumulating amacrine cells were postsynaptic to both ribbon and conventional synaptic contacts, suggesting input from bipolar and other, nonglycinergic amacrine cells. Their density (30 +/- 11 S.D. cells/mm linear retinal expanse) tended to be greater toward the central fundus. A second population of lightly labeled, probable bipolar cells was present in the middle of the inner nuclear layer; the density of this second set of glycine-accumulating cells approximated that of the heavily labeled population from the fovea, centrally, to the ora serrata, peripherally. Release of either accumulated or endogenous glycine was elicited by K+-depolarization in a Ca2+-dependent manner. Tissue fragments exposed for 6 minutes to normal medium, 40 mM K+-substituted medium, or K+-substituted medium with Co2+, release endogenous glycine into each bathing solution in average amounts of 0.6, 2.6, and 0.7 nmoles per mg protein, respectively. Together these data strongly implicate glycine as a neurotransmitter in the human retina.

The emergence, localization, and maturation of neurotransmitter systems during development of the retina in Xenopus laevis: II. Glycine.

The high-affinity uptake and release of glycine was studied in retinas of Xenopus laevis. In the toad and tadpole retina, 3H-glycine was accumulated by a population of cells located predominantly in the inner nuclear layer. When retinas preloaded with 3H-glycine were subjected to high K+-concentrations, these retinas released large amounts of 3H-glycine by a Ca++-dependent mechanism. The appearance and maturation of these putative glycinergic properties was followed during retinal development. Our results indicate that the high-affinity uptake of glycine first appears around stage 33/34 whereas K+-stimulated glycine release cannot be detected until stage 42.

The emergence, localization, and maturation of neurotransmitter systems during development of the retina in Xenopus laevis. III. Dopamine.

The uptake, synthesis, and release of dopamine was studied in retinas of Xenopus laevis. In the tadpole and adult retina, 3H-dopamine is accumulated by cells located in the inner nuclear layer. Retinas preloaded with 3H-dopamine release this compound in response to high K+ concentrations in the medium. This release is probably Ca++-dependent as it is inhibited by Co++ in the medium. Adult retinas are also capable of synthesizing 3H-dopamine from 3H-tyrosine. The appearance and maturation of these dopaminergic properties were followed during retinal development. Our data indicate that synthesis of dopamine can first be detected as early as stage 35/36 whereas uptake of dopamine first occurs at stage 43. K+-stimulated release of preloaded 3H-dopamine from putative dopaminergic neurons is, however, not evident until stage 46. These results show that similar to the development of GABA-ergic and glycinergic properties, the uptake, synthesis, and release mechanisms for dopamine emerge at different stages during retinal differentiation in Xenopus Laevis.

Dopaminergic neurons in the human retina.

The utilization of dopamine in the adult human retina was examined by using high-affinity uptake, localization, synthesis, and release as neurotransmitter-specific physiological probes. Autoradiographic and histochemical studies have shown that dopamine-accumulating and dopamine-containing cells of the human retina belong to a population of neurons whose somata are located in the proximal regional of the inner nuclear layer. Some of these are amacrine cells which are pre- and postsynaptic to other amacrine cells exclusively in the inner plexiform layer. However, evidence is presented which indicates the existence of interplexiform dopaminergic neurons which send processes to both plexiform layers of the retina. These neurons contain a high concentration of dopamine, take up 3H-dopamine by a hig-affinity mechanism, and release endogenous or accumulated dopamine by a Ca2+-dependent mechanism upon depolarization with high extracellular K+. An endogeneous level of about 20 pmoles dopamine per mg protein was measured in freshly isolated retina using high-pressure liquid chromatography with electrochemical detection. These results demonstrate that mechanisms for dopaminergic neurotransmission are present in the human retina.

The emergence, localization and maturation of neurotransmitter systems during development of the retina in Xenopus laevis. I. Gamma aminobutyric acid.

The high-affinity uptake, biosynthesis and release of GABA have been studied in the retina of Xenopus laevis. In the mature retina, [3H]-GABA is accumulated predominantly by horizontal cells. A second population of cells located in the inner nuclear layer (possibly a type of amacrine cell) also showed a specific GABA uptake. In addition, this retina contains significant activities of L-glutamic acid decarboxylase and also releases [3H]-GABA in response to increasing K+ concentrations in the medium. We have followed the appearance and maturation of these GABA-ergic properties during embryonic development of this retina. Our results indicate that these properties emerge in a precise temporal pattern during retinal differentiation: the specific neuronal uptake of GABA precedes GABA synthesis which is followed by K+-stimulated GABA release.

SPACRCAN in the developing retina and pineal gland of the rat: spatial and temporal pattern of gene expression and protein synthesis.

SPACRCAN is a hyaluronan-binding proteoglycan that is present in the pineal gland and interphotoreceptor matrix of the retina. Here, we evaluate the pattern of SPACRCAN gene expression and protein appearance during retinal and pineal gland development in the rat. In situ hybridization histochemistry with SPACRCAN riboprobes indicates that hybridization signals are first evident in the retina over developing photoreceptor cells at embryonic day 16 (E16) and in the pineal gland at E21. Immunocytochemistry using a SPACRCAN antibody shows localization of SPACRCAN protein in the developing interphotoreceptor matrix by Postnatal day 5 (P5) and in the pineal gland by P6. These studies suggest that SPACRCAN mRNA expression may occur substantially earlier than the time when SPACRCAN protein is detectable in both the retina and the pineal gland. The period of retinal histogenesis when SPACRCAN is detected first is coincident with the time photoreceptors begin to extend from the outer retinal surface, suggesting that SPACRCAN may participate in the maturation and maintenance of the light-sensitive photoreceptor outer segment.

Photoreceptor outer segment development: light and dark regulate the rate of membrane addition and loss.

The rate of membrane addition during outer segment development and the onset of membrane loss through shedding was evaluated for Xenopus laevis embryos reared at 23 degrees C in constant light (LL), constant darkness (DD), and cyclic light (12 hr L/12 hr D; LD). Embryos were placed under these lighting conditions 1 day following fertilization at open neural plate stages and were sampled each day for 9 days. Rod and cone outer segments were present in each treatment group on day 3 of development. At the end of 10 days of development, rod outer segment volumes were 708 +/- 195, 380 +/- 40, and 297 +/- 33 micrometer3 (mean +/- S.D.) for LL, LD, and DD treatment groups, respectively, whereas mean cone outer segment volumes were 57 +/- 8, 48 +/- 8, and 35 +/- 9 micrometer3 for these same treatment groups. The loss of outer segment material through shedding was assessed by monitoring the phagosome content of the pigment epithelium. Phagosomes were present in the pigment epithelium in DD embryos as early as 6 days of development, were present at day 5 only after the onset of the light cycle in LD embryos, and were rarely observed in LL embryos. The rate of outer segment membrane elaboration in rods (determined with 3H-leucine autoradiography) was virtually identical in LL and LD (100 and 98 micrometer3/day) but was greatly reduced in DD (70 micrometer3/day). These findings indicate that the relatively rapid rate of rod outer segment volume accumulation in LL embryos is due to a reduction in membrane loss through shedding and not to a higher rate of membrane addition as compared to animals reared in LD. In contrast, the reduced rate of rod outer segment development in DD embryos is the result of both a slower rate of membrane elaboration accompanied by the early onset and enhanced rate of membrane loss through shedding in darkness.

Neurotransmitter properties of the newborn human retina.

Human retinal tissue from a newborn was examined autoradiographically for the presence of high-affinity uptake and localization of the following putative neurotransmitters: dopamine, glycine, GABA, aspartate, and glutamate. In addition, the dopamine content of this newborn retina was measured by high pressure liquid chromatography. Our study reveals that specific uptake mechanisms for 3H-glycine, 3H-dopamine, and 3H-GABA are present at birth. However, the number and distribution of cells labeled with each of these 3H-transmitters are not identical to those observed in adult human retinas. Furthermore, the amount of endogenous dopamine in the newborn retina is approximately 1/20 the adult level. Photoreceptor-specific uptake of 3H-glutamate and 3H-aspartate are not observed. These findings indicate that, while some neurotransmitter-specific properties are present at birth, significant maturation of neurotransmitter systems occurs postnatally.

Membrane addition to rod photoreceptor outer segments: light stimulates membrane assembly in the absence of increased membrane biosynthesis.

The effect of light on the biosynthesis and the assembly of rod photoreceptor outer segment membranes was analyzed in vitro using retinas from Xenopus laevis. The number of open discs at the base of the outer segment was used as a morphologic index to evaluate relative differences in rates of membrane assembly. Assembly was stimulated in vitro by light, as evidenced by a greater number of open discs that accumulated in light-exposed retinas than that in retinas maintained under the same conditions but in darkness. Quantitative autoradiography indicated, however, that unlike membrane assembly, the incorporation of 3H-leucine or 3H-mannose into proteins by rods was nearly identical in light-stimulated and dark-maintained retinas. Likewise, the synthesis of opsin, a protein destined for the rod outer segment, was not affected by light. We conclude from these studies that the enhanced rate of outer segment membrane assembly that occurs during the early light phase of the diurnal cycle takes place in the absence of an increase in the rate of biosynthesis of opsin, the major outer segment membrane protein.

Synthesis and secretion of interstitial retinol-binding protein by the human retina.

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein present between the retina and pigmented epithelium, which may function to shuttle vitamin A derivatives between these tissues. While previous studies have shown that the retina is solely responsible for IRBP synthesis, the specific retinal cell(s) in which this occurs has not been established. Since the carbohydrate moiety of IRBP contains fucose, the authors have analyzed the sites of incorporation of 3H-fucose in the human retina in vitro, using autoradiography. Following a 30-min pulse incubation, all retinal layers exhibited incorporation of label; however, the rod photoreceptor inner segments contained one- to two-fold more radioactivity than was present in any other retinal compartment. In autoradiographs of retinas recovered following a 4 hr chase incubation, all retinal layers retained similar levels of radioactivity with the exception of the rod photoreceptors, cone photoreceptors and cells in the inner nuclear layer, which lost 75, 11, and 14 percent, respectively of the radioactivity present immediately following the 30-min pulse. Proteins present in the chase incubation medium were analyzed by polyacrylamide gel electrophoresis and fluorography. The principal labeled component in the chase medium was identified as IRBP by immunoprecipitation with antibovine-IRBP immunoglobulins. Thus, the major loss of 3H-fucose radioactivity from rod photoreceptors coupled with the appearance of 3H-labeled IRBP in the incubation media suggests that the rod photoreceptors are primarily responsible for the synthesis and secretion of IRBP.

Retinal development: Time and order of appearance of specific neuronal properties.

The high affinity uptake, biosynthesis and K(+)-stimulated release of certain neurotransmitter candidates was studied in adult and developing retinas of Xenopus laevis. In the adult retina, (3)H-GABA was accumulated predominantly by horizontal cells while (3)H-glycine and (3)H-dopamine were accumulated by cells located deeper in the inner nuclear layer (possibly a type of amacrine or interplexiform cells). This retina also synthesized GABA, dopamine and acetylcholine from their precursors supplied exogenously. Furthermore, adult retinas preloaded with (3)H-GABA, (3)H-glycine and (3)H-dopamine, released these transmitters in response to increased K(+)-concentration in the medium. We have determined the time of appearance and maturation of these properties during embryonic development. With GABA, the appearance of the high affinity uptake system appeared first, preceding GABA synthesis which was followed by the development of K(+)-stimulated transmitter release mechanism. Similary, (3)H-glycine uptake appeared several stages before its release. In the case of dopamine, however, its biosynthesis occurred first, followed by the development of the high affinity uptake system and finally the release mechanism appeared. The initiation of this sequence of events for the three transmitter systems studied occurred at different developmental stages: the GABA-ergic properties appeared first, followed shortly by the glycinergic properties, which preceded the dopaminergic properties.