Journal ArticleHuman retinal tissue from a newborn was examined autoradiographically for the presence of highaffinity  uptake and localization of the following putative neurotransmitters: dopamine, glycine, GAB A,  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 3Hglycine,  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  V20 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

Frederick, Jeanne M.

Hollyfield, Joe g.

English

The University of Utah: J. Willard Marriott Digital Library

Neurotransmitter Properties of the Newborn Human Retina Joe G. Hollyfield, Jeanne M. Frederick, and Mary E. Rayborn 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 Iho 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. Invest Ophthalmol Vis Sci 24:893-897, 1983 Several amino acids, such as gamma aminobutyric acid (GABA), aspartic acid, glutamic acid, and gly-cine, as well as the catecholamine, dopamine, have been implicated as possible neurotransmitters in the vertebrate retina. We have recently extended neuro-transmitter studies from animal retinas to the adult human retina by documenting the uptake, localiza-tion, synthesis, content, and release properties of these compounds. I - 3 Integral to these studies is the ability of neurons to take up their specific neurotrans-mitter from the extracellular compartment via high-affinity uptake systems. By incubating isolated retinas for short intervals in 3H-transmitter candidates, neu-rons that use a specific transmitter accumulate that 3H-transmitter and can later be visualized in standard histologic sections following autoradiography. Here we present new data on the state of differentiation of the high-affinity uptake systems of several neurotrans-mitter candidates in the newborn human retina, as well as an analysis of dopamine content. Methods and Results The pair of eyes used in these studies was obtained through the cooperation of the Lions Eyes of Texas From the Cullen Eye Institute, Baylor College of Medicine, Houston, Texas. Supported by the Retina Research Foundation, Houston, Texas; Fight for Sight, Inc., New York City, New York; and The National Institutes of Health, Bethesda, Maryland. JGH is the recipient of an Olga K. Wiess Scholars award from Research to Prevent Blind-ness, New York City, New York. JMF is the recipient of a" National Research Service Award from the National Institutes of Health. Submitted for publication: August 23, 1982. Reprint requests: Joe G. Hollyfield, PhD, Cullen Eye Institute, Baylor College of Medicine, Houston, TX 77030. Eye Bank in the Cullen Eye Institute (Houston). The eyes measured 1.47 cm in diameter and were ob-tained from a full-term hydrocephalic infant that died shortly after parturition. They were placed on ice fol-lowing enucleation and reached the laboratory within 20 min after death. The anterior segment was re-moved and the retina isolated from the vitreous hu-mor and posterior segment. The retinas were cut into small strips along the central to peripheral axis, and these radial strips were further divided so that rep-resentative retinal expanses from the center, mid-, and far-periphery could be identified. Retinal fragments were incubated for 10 min in oxygenated mammalian Ringer's solution, supple-mented with 5% fetal calf serum and 5 mM Tris-HCI (pH 7.35), and containing 20 uCi per 400 ~l of either L-3H-aspartic acid (10.7 Ci/mmol), L-3H-glutamic acid (18.8 Ci/mmol), D-3H-aspartic acid (16.0 Ci/ mmol), 3H-muscimol (9.3 Ci/mmol), gamma-3H-aminobutyric acid (25.0 Ci/mmol), 3H-glycine (15.0 Ci/mmol), or 3H-dopamine (33.5 Ci/mmol), all sup-plied by New England Nuclear. After a brief wash in fresh media, the retinal fragments were fixed in 1 % formaldehyde and 2% glutaraldehyde in 58 mM So-renson's phosphate buffer (pH 7.4). Following de-hydration and embedding in Epon, the tissues were prepared for light microscope autoradiography by methods previously described. I Autoradiographs were exposed for 4-12 days prior to developing. The retina showed distinct lamination of outer nuclear, inner nuclear, and ganglion cell layers, sep-arated by interposed neuropile throughout the entire retinal expanse. The fovea contained few cells and was virtually identical in morphology to the newborn 0146-0404/83/0700/893/$1.05 © Association for Research in Vision and Ophthalmology 893 894 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE I July 198J Vol. 24 Figs. 1-2. Newborn retina from central (Fig. I) and from peripheral (Fig. 2) regions incubated with lH-dopamine. Note the dense band of radioactivity at the border of the inner nuclear and plexiform layers (arrows). Compare photoreceptor development in Figures 1 and 2. Figures 3-4. Newborn retina incubated with 3H-glycine. Five labeled cell bodies (arrowheads) in the inner nuclear layer are present in the central retina (Fig. 3) whereas over 20 labeled cells are present in the peripheral region (Fig. 4). Bar in lower right representS 50 ",m. fovea recently described.4 In extrafoveal regions, the photoreceptor layer contained large numbers of cells that approached adult density. Although retinal strat-ification was complete throughout the retina, pho-toreceptor differentiation was more advanced in the central retina than in the periphery (Figs. 1-8). Retinal fragments incubated with 3H-dopamine showed a distinct band of radioactivity over the most distal region of the inner plexiform layer (sublamina I). Localization of label to this position (Figs. 1-2), is identical to the location of 3H-dopamine uptake in the inner plexiform layer in the adult. Only one la-beled cell body was observed in over 11 mm of retinal expanse examined. Labeled processes of interplexi-form cells were not observed traversing the inner nu-clear layer nor were punctate tenninals observed in No. 7 NEUP.OTP.AN5MITIEP. PP.OPEP. TIES OF NEW[)OP.N ~ETINA / Hollyfield er 01. 895 ® Figs. 5-6. Newborn retina from the central (Fig. 5) and peripheral (Fig. 6) regions incubated with 3H-GABA. Note extensive labeling of glial cell processes with grain distributions at both the inner and outer limiting membranes. Numerous perikarya are labeled in the inner nuclear layer, and are predominantly situated adjacent to the inner plexiform layer. Arrow points 10 a labeled perikaryon in the ganglion cell layer. Figs. 7-8. Retinas incubated in the presence of 3H·muscimol. Mid-peripheral (Fig. 7) and far-peripheral (Fig. 8) regions show labeled cells in the inner nuclear layer (arrowheads) and in the ganglion cell layer (arrows). Bars in lower right represents 50 ~m. Figures 5-8 photographed and printed to identical magnifications. the outer plexifonn layer. The absence of interplexi-form cell labeling with 3H-dopamine in the newborn differs from our findings in the adult.3 Small fragments of the newborn retina were also extracted and analyzed by HPLC-EC as previously descritJedJ to determine endogenous dopamine con-tent. The level measured was 0.93 pmol/mg protein, a value significantly lower than the mean value mea-sured in mature retinas (19.0 ± 8.6 pmol/mg protein). Retinas incubated in the presence of 3H-glycine 896 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1983 Vol. 24 showed uptake of the label by cells located in the inner region of the inner nucle~r layer in positions consistent with the location of 3H -glycine labeled cells in the adult (Figs. 3-4). However, the density of these cells varied from center to periphery. The number of cells in the central retina was 20 ± 1.4 cells per mm linear retinal expanse (mean ± SO), which is ap-proximately half the number present in the adult,) whereas in the peripheral retina, the number of gly-cine labeling cells was 73 ± 12, approximately twice the density present in the adult. Retinal tissues incubated with 3H-GABA showed extensive uptake in glial processes in all regions, ie, extensive labeling was present from inner to outer limiting membranes (Figs. 5-6). Additionally, large numbers oflabeled cells were also present in the inner nuclear layer, and occasionally, labeled cells in the ganglion cell layer were observed (Fig. 7). This pattern of glial uptake of 3H-GABA has been described in a variety of mammalian species. We had previously incubated human retinas with 3H-muscimol and found that this analog of GABA was selectively ac-cumulated by GABAergic neurons, but not by glia. Accordingly, we incubated the newborn human ret-ina with 3H-muscimol and observed the uptake of this GABA analog which is presumed to be exclu-sively neuronal (Figs. 7-8). 3H-muscimol-Iabeled cells were present at the border of the inner nuclear and inner plexiform layer as well as in the ganglion cell layer. The density of the muscimol-Iabeled cells was 23 ± 9 cells per mm linear retinal expanse and was similar throughout all regions of the retina examined. This density of 3H-muscimol-labeling in the newborn retina was virtually identical to that seen in adult human retina (22.1 cells per mm linear retinal ex-panse).) Retinas labeled with either 0- or L-3H-aspartic acid or L-3H-glutamic acid showed extensive glial cell up-take in each preparation. Although we have reported that, among photoreceptors, rods selectively accu-mulate aspartic acid while both rod and cone pho-toreceptors take up glutamic acid,2 extensive photo-receptor labeling was not observed with either 3H_ aspartate or 3H-glutamate in the newborn retina. Discussion These observations indicate that while a number of neurotransmitter specific properties are present at birth, some characteristics of the adult retina have not developed by this time. Our observations with 3H-dopamine clearly demonstrate the same inner plexiform layer labeling pattern present in adult ret-ina, but fail to demonstrate the interplexiform cell terminals in the outer plexiform layer. In the adult human retina, processes of interplexiform cells ex-tending to the outer retina take up 3H-dopamine. Our analysis does not allow us to distinguish whether the interplexiform processes are entirely absent at birth or whether the interplexiform cell uptake properties are only immature at this time. It is also noteworthy that the dopamine content of the newborn retil1a is only 1/20 that present in the adult.3 A factor to be considered in a comparison of dopamine content between newborn and adult human retinas stems from an observation that tyrosine hydroxylase (TH) is activated by light in the neonatal rat. If TH in the human retina is also light sensitive, the low dopamine content at birth could in part be related to the absence of light stimulation in utero. Another apparent difference from the adult is the variation in the number of 3H-glycine-accumulating cells from central to peripheral retina. In the central retina of the newborn, the number of 3H-glycine-Ia-beling cells is approximately one-half that found in the adult whereas in the periphery the number of these cells is approximately twice the adult density. Postnatal maturation of the human retina may there-fore involve redistribution of these glycinergic neu-rons. If mitotic activity has stopped in the central retina, it" might be predicted that the large population of glycinergic neurons in the peripheral retina may contribute to the central glycinergic amacrine cell population. This could be accomplished by lateral migration from peripheral to central locations. Lat-eral migration of cells from the peripheral germina-tive zone through the inner nuclear layer has been demonstrated during maturation in the frog retina.6 The density of 3H-muscimol-Iabeled neurons (GABAergic neurons) is similar to the number pres-ent in the adult retina in all regions examined. It should be emphasized that a normal density of GA-BAergic cells coexist in the newborn retina with pop;.. ulations of glycinergic cells that are below adult levels in the central retina, and above adult levels in the peripheral retina. Because of these differences it is likely that specific translocation of individual cells within the retina must occur in order to achieve the appropriate distribution of cells relative to each other. It is unlikely that simple stretching of the tissue at the periphery to reduce the number of cells per linear expanse would be sufficient to achieve the appropriate cell densities. On the other hand, a combination of stretching of the peripheral retina to reduce the den-sity of glycinergic cells, coupled with the additional proliferation of GABAergic cells to increase their number in the periphery, might be a mechanism by which)this could be accomplished. Additional studies that combine an analysis of the mitotic capabilities of the newborn retina along with uptake studies will be required to answer this question. A recent study reports that the density of foveal No. 7 NEUROTRANSMITIER PROPERTIES OF NEWOORN RETINA / Hollyfield er 01. 897 photoreceptors at birth is below the number present in the adult.4 Our studies on 3H-aspartate and 3H_ glutamate uptake indicate that photoreceptors, foveal and extrafoveal, are immature at birth in that they have not yet developed specific uptake properties. Unlike the adult, photoreceptors in the newborn were relatively free of label, whereas heavy labeling over . glial cells was comparable to that previously observed in mature retina. 1,2 The exact time during development when the up-take properties for glycine, dopamine, and GABA first emerge must await the analyses of retinas ob-tained from prenatal stages. We have studied the up-take of these three putative transmitters in a single retina from a 9-week aborted fetus. While diffuse la-beling was present throughout this retina, no specific neuronal labeling was evident suggesting the absence of high-affinity uptake properties. On the other hand, studies using material from later (postnatal) times will be required in order to determine when the glutamate and aspartate uptake properties in photoreceptors first appear, as well as to determine the time of mat-uration of the dopaminergic interplexiform cells. Key words: retinal development, human retina, newborn retina, autoradiography, neurotransmitters, glycine, dopa-mine, GABA, aspartate, glutamate Acknowledgments The authors thank the Lions Eyes of Texas Eye Bank (Houston) for retrieval of these tissues and the Life Flight Helicoptor Ambulance service for transportation of the tis-sues to the laboratory. Special thanks go to the mother of the infant from which this tissue was taken for her generos-ity in making this very special gift. References 1. Lam DMK and Hollyfield JG: Localization of putative amino acid neurotransmitters in the human retina. Exp Eye Res 31:729, 1980. 2. Lam DMK, Frederick JM, and Hollyfield, JG: Identification of putative neurotransmitters in the human retina. In The Structure of the Eye (Proceedings of the 4th International Sym-posium), Hollyfield JG, editor. New York, Elsevier Biomedi-cal, 1982, pp. 205-213. 3. Frederick JM, Rayborn ME, Laties AM, Lam DMK, and Hollyfield JG: Dopaminergic neurons in the human retina. J Comp Neurol 210:65, 1982. 4. Abramov I, Gordon J, Hendrickson A, Hainline L, Dobson V, and LaBossiere E: The retina of the newborn human infant. Science 217:265, 1982. 5. Cohen J, and Neff, NH: Retinal amacrine cell system tyrosine hydroxylase: the development of responsiveness to light and neuroleptic drugs. Brain Res 255: 160, 1982. 6. Hollyfield JG: Differential addition of cells to the retina in Rana pipiens tadpoles. Develop BioI 18: 163, 1968. 

Neurotransmitter properties of the newborn human retina.

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Neurotransmitter properties of the newborn human retina.

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