Quantitative distribution of six amino acids in rat retinal layers

Vision Res. Vol. 29, No. 9, pp. 1079-1084,1989 Printed in Great Britain. All rights reserved

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RESEARCH NOTE QUANTITATIVE

DISTRIBUTION IN RAT RETINAL

OF SIX AMINO ACIDS LAYERS

C. DAVID Ross, JUDY A. PARLI and DONALD A. GODFREY* Department of Physiology, Oral Roberts University, Tulsa, OK 74171, U.S.A. (Received 17 August 1988; in revisedform 26 January 1989)

Abstract-Concentrations of glutamate, aspartate, glutamine, glycine, GABA, and taurine were determined in samples microdissected from rat retinal layers and assayed by HPLC. Glutamate and glutamine were relatively high in the inner nuclear (INL) and ganglion cell (GCL) layers; aspartate was relatively high in the outer nuclear layer (ONL), outer plexiform layer, and INL. Distributions of glutamate and aspartate did not correlate well with those of enzymes involved in their metabolism. Glycine and GABA were highest in the inner plexiform layer, with increasing concentrations through the INL, and were relatively high in the GCL. Taurine was highest in the ONL. Glutamate Aspartate Quantitative histochemistry

Glutamine Glycine Rat Amino acids HPLC

INTRODUCTION

Results from studies involving many different techniques give support for the involvement of amino acids in retinal neurotransmission. Evidence is strongest for excitatory roles of glutamate and aspartate and for inhibitory roles of GABA and glycine (Massey & Redburn, 1987; Miller & Slaughter, 1985; Yazulla, 1986), although a transmitter function for taurine has also been suggested (Lin, Song & Win, 1985). One type of evidence favoring a transmitter function for these amino acids in the retina is their relatively high concentrations in this structure. Quantitative assays of amino acids in samples microdissected from individual retinal layers have been performed in several species, including glycine and GABA in rabbit and mudpuppy (Dick & Lowry, 1984), glutamate, aspartate, GABA and glycine in monkey (Berger, McDaniel, Carter & Lowry, 1977; Lowry, Roberts & Lewis, 1956), GABA in rat and frog (Graham, 1974), and taurine in rabbit, monkey, cat, chick and frog (Orr, Cohen & Lowry, 1976). Some measurements of amino acids in rat retina have been made, but the resolutions of the approaches were less than that which can be achieved using the Lowry *Present address: Dept. of Otolaryngology, Medical College of Ohio, Toledo, OH 43699, U.S.A.

GABA

Taurine

Retina

microdissection method (Lowry, 1953) with serial sections of retina (Ross, Dunning, Juengel & Godfrey, 1985) and the results have not been in complete agreement (Graham, 1974; Kennedy, Neal & Lolley, 1977; Morjaria & Voaden, 1979; Yates & Keen, 1976). Assays of free amino acids employing high performance liquid chromatography (HPLC) have achieved sufficient sensitivity to enable measurements of amino acid concentrations in submicrogram samples, such as those obtained from discrete retinal layers. This study reports the distributions of glutamate, aspartate, glutamine, GABA, glycine, and taurine concentrations in samples microdissected from layers and sublayers of rat retina, as well as from choroid, pigmented epithelium, and optic nerve. Eyes from 8 pigmented rats (Long-Evans Hooded) were removed within 2 min of decapitation, immediately frozen in Freon cooled to - 130°C in liquid nitrogen, mounted onto aluminium dowels with brain paste, and sectioned tangentially at 7.5 pm thickness (Lowry, 1953; Ross & McDougal, 1976). After freeze-drying, samples were dissected from the centers of sections (Ross et al., 1985) through one eye of each rat, weighed on a “fishpole” quartz fiber balance (Lowry & Passonneau, 1972), and loaded into poly-propylene tubes (Waters limited volume inserts No. 72030) to which were subsequently added 13 ~1 of 0.01 M NaOH to extract 1079

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x 10; nlnutem Fig. 1.Chromatograms of a calibrated standard (upper) and of a retinal homogenate (lower). The amounts of amino acid standards analyzed in 5~1 (upper trace) were 0.70pmoles for asparagine, glutamine, taurine and GABA, and 0.75 pmoles for the other amino acids. In the retinal homogenate (lower trace), 0.059 pg (proton) was analyzed, which is equivalent to about 0. I pg dry weight. Taurine peaks at 0.190 V in the lower trace, which is off-scale at the magnification shown. Chromatograms were stored and plotted using a Maxima chromatographic workstation.

the free amino acids. For analysis of amino acid concentrations in a retinal homogenate (Fig. I), an aliquot of the homogenate (both retinae from 1 rat, weighed and homogenized in 9 volumes of 50mM potassium phosphate buffer, pH 7.0) was diluted in 0.01 M NaOH. Amino acids were detected by the fluorescence of the derivatized product of the reaction between primary amino acids and o-phthalaldehyde (OPA) (Hill,

Walters, Wilson & Stuart, 1979). All chromatography equipment used was from Waters (Milford, MA), unless otherwise specified. Briefly, 5 ~1 of OPA, immediately followed by 5 ~1 of dissolved sample, were withdrawn by a WISP autosampler and injected onto a reverse phase column (5 pm particle size, Altex Ultrasphere Octyl or DuPont Zorbax CS), heated at 30°C. The mobile phase, a two-solvent system

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composed of (A) buffer (9.8 mM sodium hydrogen phosphate/24.8 mM sodium acetate in 2.0% methanol/2.2% THF/95.8O/ H20, adjusted to pH 6.45 & 0.05) and (B) methanol, flowed at a

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rate of 1.5 ml/min, with the non-linear gradient changing from 78% solvent A and 22% solvent B to 0% A and 100% B. Fluorescence was quantified using either a Kratos Spectroflow 980

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Fig. 2. Concentrations of 6 amino acids in rat retinal layers. Abbreviations are Ch (choroid), PE (pigmented epithelium), OS (outer segments of photoreceptors), IS (inner segments of photoreceptors), ONL (outer nuclear layer, containing nuclei of photoreceptors), OPL (outer plexiform layer, containing processes of photoreceptors, horizontal’cells and bipolar cells), INL (inner nuclear layer, containing somata of horizontal cells predominantly in outer part, bipolar cells predominantly in middle part, and amacrine cells predominantly in inner part), IPL (inner plexiform layer, containing processes of bipolar, amacrine and ganglion cells) and GCL (ganglion cell layer, containing somata of ganglion cells and displaced amacrine cells). All layers from GCL through ONL contain some cytoplasm of Miiller glial cells. Averages of amio acid concentrations in all retinal layers (not including choroid, PE, or ON) are 40 (glutamate), 11 (aspartate), 14 (glutamine), 21 (glycine), 20 (GABA), and 220 (taurine) mmol/kg dry weight.

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detector, equipped with a 5 ~1 flow cell and set at 389 nm emission and 325 nm excitation (Fig. 2) or with a Spectrovision FD-100 detector, with an 8 ~1 flow cell and 400 nm emission and 330 nm excitation filters (Fig. 1). Data were recorded and stored using a data module or a computer with Maxima chromatographic software, and amino acid concentrations were calculated by comparing peak heights obtained from samples with a standard curve of peak heights of calibrated standards, run at 3 different concentrations at the beginning and end of each set of samples. An internal standard (/I-Zthienyl-DL-serine) was included with each sample and amino acid standard. Representative chromatograms of a calibrated standard, containing less than 1 pmole of each amino acid, and a retinal homogenate are given in Fig. 1. Amino acids in retinal layers (Fig. 2) Glutamate and aspartate. Glutamate concentration did not differ greatly among samples between the proximal portion of the inner segment (IS) region and the ganglion cell layer (GCL), although slight peaks were found in the middle of the inner nuclear layer (INL) and the innermost part of the GCL. Glutamate concentration dropped between the basal and apical portions of the IS and was significantly lower in the outer segments (OS) of the photoreceptors. Aspartate concentration did not vary significantly across the retinal layers, but was relatively higher in the outer plexiform layer (OPL) and outer nuclear layer (ONL) and lower in the OS and IS. Neither glutamate nor aspartate concentrations in the rat GCL were as proportionately high as has been reported in monkey, where concentrations of both amino acids were much higher in GCL than in other retinal layers, being about twice those in ONL (Berger, et al., 1977). Glutamate-like and aspartate-like immunoreactive staining in rat retina has been localized primarily in the IPL, GCL, and in some somata of the INL (Madl, Larsen, Beitz & Johnson, 1985). Although this distribution corresponds somewhat to the locations of small peaks of glutamate and aspartate concentrations (Fig. 2), the lack of significant differences in glutamate and aspartate concentrations in the inner layers makes difficult any rigorous comparison with this staining result. Glutamine. Glutamine concentration was highest in the INL and GCL, relatively low in the OPL, ONL and IS, and lowest in the OS.

Some of the glutamine distribution may be related to Mtiller cell cytoplasm, which, in rat, constitutes a relatively higher proportion of the volume of the INL and GCL and does not extend past the ONL (Rasmussen, 1972). Glutaminase, the enzyme which catalyzes synthesis of glutamate from glutamine, has been suggested as a marker for neurons using glutamate as a neurotransmitter (Wenthold & Altschuler, 1983), and aspartate aminotransferase (AAT), catalyzing the interconversion between glutamate + oxalacetate and aspartate + a-ketoglutarate, has been suggested as a marker for glutamatergic and/or aspartatergic neurons (Altschuler, Mosinger, Ha&son, Parakkal & Wenthold, 1982). Unfortunately, the distributions of activities of these enzymes do not correlate well with the quantitative distributions of their related amino acids in retinal layers (Ross & Godfrey, 1985; Ross, Bowers & Godfrey, 1987a,b). Activities of both glutaminase and AAT are highest in the IS, where glutamate concentration is not especially high and where aspartate concentration is relatively low. Perhaps the high glutaminase activity in IS correlates better with the ratio of glutamate to glutamine concentrations, which is also highest in that layer. Glycine. Glycine concentration was low in the photoreceptor layers and OPL, increased steeply from the outer to inner parts of the inner nuclear layer (INL) to a peak in the inner plexiform layer (IPL). The pattern of high glycine concentration in the IPL, INL and ganglion cell layer (GCL) is consistent with glycinergic amacrine cells, with somata conventionally located in the INL and displaced in the GCL, and with processes predominantly in the more superficial part of the IPL. In cat INL, conventionally placed amacrine cells that accumulate glycine and stain for glycine-like immunoreactivity have been reported (Pourcho & Goebel, 1985, 1987b). In addition, staining was more prominent in the outer part of the IPL than in the inner (Pourcho & Goebel, 1987b). The relatively high glycine concentration in rat GCL suggests that somata of displaced amacrine cells may also be glycinergic. The relatively high glycine concentration in the outer and middle INL could be associated with bipolar cells. Glycine immunoreactivity has been associated with populations of cone bipolar cells in cat (Pourcho & Goebel, 1987b), and glycine uptake by bipolar cells has also been reported in several species (Pourcho, Goebel &

Research Note

McReynolds, 1984; Pourcho & Goebel, 1987a; Voaden, 1986). GABA. GABA concentration was extremely low in photoreceptor layers and low in the OPL, but increased significantly through the INL, to a peak near the border between the INL and IPL. GABA concentration was highest in the IPL, particularly near the GCL, and was only slightly lower in the GCL. The gradient of GABA in the INL is consistent with GABAergic amacrine cells, with somata located in the INL near the border with the IPL. The high GABA concentration in the GCL would suggest that many displaced amacrine cells are GABAergic. The peak of GABA in the deep IPL would indicate a considerable arborization of amacrine processes in sublamina b or in the deepest 20% of the IPL. The low GABA concentration in the photoreceptor layes and the OPL does not correlate well with a suggestion that 25% on monkey photoreceptors utilize GABA as a transmitter because their terminals contain GABA immunoreactivity (Nishimura, Schwartz & Rakic, 1986). GABA concentration in OPL of monkey is also very low and is primarily in the vitreal half of that layer (Berger et al., 1977), suggesting more of an association with horizontal cells than with photoreceptor terminals. The distributions of both glycine and GABA in rat retina are similar to those found in monkey (Berger et al., 1977) and rabbit (Dick & Lowry, 1984), except that GABA is relatively higher in the outer parts of the INL in monkey and relatively lower in the GCL of both species. This relative difference in the GABA content of the GCL suggests that rat may have more GABAergic amacrine cells displaced to the GCL than monkey or rabbit. In rabbit, as in rat, the highest retinal GABA concentration is in the IPL next to the GCL (Dick & Lowry, 1984). Taurine. Taurine concentration was extremely high in the photoreceptors, particularly in the ONL. The concentration progressively fell in deeper layers, leveling off in the IPL and GCL. Taurine concentrations of up to 400 mmol/kg dry wt in rat ONL have been reported in previous studies (Morjaria & Voaden, 1979; Orr et al., 1976).

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tamine in the PE. The aspartate and glutamine peaks in the PE correspond in location to a small peak in activity of the mitochondrial isoenzyme of aspartate aminotransferase (mAAT, ROSS et al., 1987a), although glutaminase activity is low in the PE (Ross et al., 1987b). Aspartate concentration is also relatively high in PE of monkey (Berger et al., 1977). Amino acids in optic nerve (ON)

Lipids contribute much more significantly to the dry weight of optic nerve samples than to retinal samples, comprising about 50% of the dry weight in optic nerve and about 20% of the dry weight in GCL (Berger et al., 1977). Therefore, on a lipid-free dry weight basis, the amino acid concentrations in optic nerve (Fig. 2) would be about twice their dry weight values, while the GCL values would be only about 1.25 times higher. Using such a basis, glutamate, aspartate, glutamine, glycine, GABA, and taurine concentrations in ON are about 40, 30, 108, 23, 0.4 and 44% of the values in GCL, respectively. The relatively high concentrations of glutamate, aspartate, and glutamine in GCL and in ON could support an association between these amino acids and the optic projection. It has been suggested that glutamate is involved with the retinotectal projection in pigeon (Fonnum & Henke, 1982) and in goldfish (Langdon & Freeman, 1987). GABA-like immunoreactivity has been associated with ganglion cells in the GCL and with fibers in the optic nerve of rabbit, leading to the suggestion that some ganglion cells are GABAergic (Yu, Watt, Lam & Fry, 1988). It has been reported that a considerable number of rat ganglion cells that project to the superior colliculus also display GABAlike immunoreactivity (Caruso, Owczarzak, Hazelett & Pourcho, 1988). However, the low GABA content of the optic nerve (Fig. 2) implies that the number of such cells should be extremely small. This discrepancy between GABA-like immunoreactivity in ganglion cells and the low GABA content in the optic nerve, containing cell axons, needs to be resolved. Acknowledgements~The

authors thank John Schemenaur and Marc Bowers for excellent technical assistance. Supported by N.I.H. grant EY-03838.

Amino acids in choroid and pigmented epithelium (Pfi)

Concentrations of all amino acids in the choroid and PE were low compared to those in retinal layers, except for aspartate and glu-

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