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[3H]-d -aspartate accumulation in the retina of pigeon, guinea-pig and rabbit
Exp. Eye Res. (1981) 33, 381-391
[3H]=D-Aspartate A c c u m u l a t i o n in the Retina of Pigeon, Guinea-Pig and Rabbit B. EHINGER
Department of Ophthalmology, University of Lund, Lund, Sweden (Received 16 October 1980 and accepted 24 February 1981, London) Retinas from pigeons, guinea-pigs and rabbits were exposed to [aH]-D-aspartate either by intraocular injections or by incubating undetached or detached retinas in it. In all species about 5-10% of the cells in the ganglion cell layer became radioactive. In the pigeon most of the photoreeeptors became radioactive when the substance reached them (i.e. when the retina was detached ) and, in guinea-pigs and rabbits, about 10 % of them also became radioactive. In the latter case the radioactive photoreceptors were characterized by long myoids protruding out among the surrounding outer segments and by having their perikarya in the outermost cell row of the outer nuclear layer. The frequency distribution and morphology of the radioactive photoreceptors suggest they are cones. Since [3H]-D-aspartate can be presumed to label neurons using aspartate or glutamate as transmitter, the work suggests that cones and about 5-10 % of M1ganglion cells use either of the two substances as transmitter. 1. I n t r o d u c t i o n U p t a k e mechanisms exist in m a n y types of neurons as a m e t h o d for inactivating and conserving their neurotransmitters. The presence of such an uptake mechanism can be shown autoradiographically and this has now become one of the s t a n d a r d methods for demonstrating aminergic, glycinergic or GABA-ergic neurons. However, the presumed natural transmitters of m a n y excitatory neurons, L-glutamic acid and L-aspartic acid, have n o t yielded the same results because of a strong glial u p t a k e (an d metabolism) disguising w h a t neuronal u p t a k e there might be. However, the isomer, [3H]-D-aspartic acid, has been shown to be accumulated by brain neurons likely to use glutamic acid as their neurotransmitter (Balcar and Johnston, 1972; Davies and Johnston, 1976; L u n d Karlsen and F o n n u m , 1978; Storm-Mathisen and W o x e n Opsahl, 1978; Takagaki, 1978; Malthe-Sorensen, Skrede and F o n n u m , 1979, 1980; F o n n u m , L u n d Karlsen, Malthe-Sorensen, Skrede and Walaas, 1979) and should be useful as a morphological autoradiographic marker for neurons using aspartate or glutamate as transmitter. There is much evidence suggesting either glutamic acid a n d / o r aspartic acid as possible neurotransmitters in the outer plexiform layer (see section 4) and it has also been suggested t h a t g l u t a m a t e could be the transmitter of ganglion cells (Henke, Schenker and Cu@nod, 1976). We have therefore performed autoradiographic studies on the cellular localization of [aH]-D-aspartate uptake in retinas from pigeon, guinea-pig and rabbit. The results are compatible with glutamate or aspartate as transmitter(s) in cones (or cone-like cells) and in a small population of the ganglion cells (about 5-10 % ). A preliminary report has appeared (Ehinger, 1980). Glutamate u p t a k e into ganglion cells has previously been observed (Bruun and Ehinger, 1974) but could not be clearly distinguished from non-specific uptake.
0014-4835/81/100381 + 11 $01.00/0
9 1981 Academic Press Inc. {London) Limited 381
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2. M a t e r i a l s a n d M e t h o d s The eyes of wild, adult pigeons, pigmented adult guinea-pigs and pigmented adult rabbits were used. Five or 20 #Ci of [3H]-D-aspartic acid was injected intravitreally in three light-adapted eyes of each species. Only topical anaesthesia (0"4% oxibuprocaine) was used. The eyes were obtained after 30 rain or 4 hr and pieces of the posterior pole were processed as described below. The same injections were given (under infrared illumination and with the aid of an infrared converting viewing system) in two eyes from each species, dark adapted for 12-16 hr. Doses and times were as for light adapted eyes. The dark adapted animals were left in the dark after the injections but were killed and dissected in ambient laboratory light (about 200 lux). Posterior segments were incubated in a balanced salt solution (Ames, 1965). After 10 rain, [3H]-D-aspartate was added to a final concentration of 1, 3, 5 or 20/tCi/ml and the incubations were carried on for 15, 30 or 60 min at 37~ The tissue pieces were then washed twice for 5 min in the salt solution at 0~ and processed for autoradiography. In a second series, retinas were incubated as above (5/tCi/ml) for 15 rain and then incubated at 37~ for another 15, 30 or 60 rain with the salt solution without [3H]-D-aspartate, washed as described above and processed for autoradiography. Each group consisted of six pieces of retinas from two animals. Guinea-pigs and rabbits were dark adapted for 12-16 hr and incubated as above in the dark. The salt solution was always carefully aerated with a mixture of 5 Yo CO2 in oxygen. The incubation flasks were gently rocked during the incubation. In about half of the specimens, the retina detached during the experiment but often only partially, allowing the analysis of both detached and undetached retina in the same specimen. Tissue pieces were always taken from the posterior pole. The pecten was avoided in the birds as was the area with medullated nerve fibres in rabbits but no other attempts were made to analyse any specific region. The various tissue pieces were freeze-dried and fixed in gaseous formaldehyde at 80~ for 1 hr. The formaldehyde was generated from about 5 g paraformaldehyde placed together with the dry tissue pieces in a 1 l jar, which was then heated in an oven. The tissue pieces were then embedded in plastic (Durcopan| FLUKA) in vacuo without any intermediate steps in any solvent and cut (4 #m) with a glass knife. They were then covered with autoradiographic stripping film (Kodak AR 10), exposed for 1-12 weeks, developed and fixed. If needed, they were stained with 1 Yo toluidine blue in 40 % ethanol.
3. R e s u l t s
Photoreceptors Certain photoreceptors became radioactive in all a n i m a l species. However, their radioactivity was very low after intraocular injections, both after 30 a n d after 240 min. I n fact, it was so low as to be judged insignificant before the results from the i n c u b a t i o n s were available (see Fig. 7). I n these, strong radioactivity appeared in some photoreceptors in detached retinas. The r a d i o a c t i v i t y was much less e v i d e n t in retinas which were still attached. There was no obvious difference between light- a n d d a r k - a d a p t e d retinas. However at times there were for no a p p a r e n t reason differences in the photoreceptor r a d i o a c t i v i t y in different sectors of one a n d the same retina. I n the pigeons, the radioactive photoreceptors were numerous. The r a d i o a c t i v i t y was concentrated in two b a n d s in the inner segments (arrows in Fig. 1), one corresponding to the outermost layer of perikarya in the outer nuclear layer a n d one near the i n n e r limit of the i n n e r segments at the level of the synaptic terminals of the photoreceptors (Fig. 1). The outer segments were usually n o t very radioactive; however, moderate r a d i o a c t i v i t y was seen in some structures e x t e n d i n g out a m o n g
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FIG. 1. Pigeon retina, incubated 30 rain in [3H]-D-aspartate, 1 #Ci/ml. There is radioactivity in the photoreceptor inner segments in one band corresponding to the outermost cell bodies (large arrowheads) and in another band corresponding to the synaptic terminals (small arrowheads). Ph, Photoreceptors; ONL, outer nuclear layer; OPL, outer plexi.formlayer; INL,: inner nuclear layer; IPL, inner plexiform layer; G, ganglion cell layer. Phase contrast mierographs of autoradiograph. Left, focus on the silver grains. Right, focus on the section, x 224.
t h e i n n e r m o s t p a r t o f the o u t e r segments: R a d i o a c t i v e p e r i k a r y a were f r e q u e n t t h r o u g h o u t the inner n u c l e a r layer, a l t h o u g h t h e y were clearly m o r e n u m e r o u s in its o u t e r p a r t s as i n d i c a t e d b y t h e b a n d of r a d i o a c t i v i t y seen i n 'the o u t e r m o s t p a r t of t h e o u t e r n u c l e a r layer, I n r a b b i t s a n d guinea-pigs certain p h o t o r e c e p t o r s also b e c a m e r a d i o a c t i v e when i n c u b a t i n g d e t a c h e d retinas, less when i n c u b a t i n g r e t i n a s still a t t a c h e d to t h e p i g m e n t e p i t h e l i u m a n d only little a f t e r t h e i n t r a o e u l a r injections. The r a d i o a c t i v i t y was seen in s t r u c t u r e s a m o n g the p h o t o r e c e p t o r o u t e r segments. A b o u t 10 % o f the p h o t o r e c e p t o r s showed this d i s t r i b u t i o n of r a d i o a c t i v i t y . A t high m a g n i f i c a t i o n (Fig. 2) it was seen t h a t it was t h e myoid, e x t e n d i n g o u t a m o n g the a d j o i n i n g rods, t h a t was
FIG. 2. Autoradiograph of dark-adapted rabbit retina incubated 30 min in [3H]-D-aspartate, 1 #Ci/ml. The arrow points to a radioactive cone, Note its short outer segment and long, radioactive myoid. Phase contrast micrograph. Left, focus on the grains. Right, focus on the section, x 910.
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FIo. 3. Guinea-pig retina (light-adapted), incubated 15 rain in [3H]-n-aspartate, 20 #Ci/ml. Note the radioactivit~ of the photoreceptors (cone-like cells), in their perikarya in the outermost part of the outer nuclear layer (ONL) and in their synaptie terminals in the outer plexiform layer (OPL). INL, Inner nuclear layer; IPL, part of the inner plexiform layer. Phase contrast micrographs of autoradiograph. Left, focus on the grains. Right, focus on the section. • 420.
FIG. 4. Pigeon retina, incubated 30 rain in [aH]-D-aspartate, 1 #Ci/ml. There is one ganglion cell (arrow) which is radioactive in its cytoplasm (but less in the nucleus). Another ganglion cell (arrow and D) is doubtfully positive. IPL, inner plexiform layer; G, ganglion cell layer. Phase contrast micrographs of autoradiograph. Left, focus on the grains. Right, focus on the section, x 500. radioactive. The outer segment of t h e cell was less radioactive. A n u m b e r of r a d i o a c t i v e cell bodies were seen in t h e o u t e r m o s t p a r t of t h e o u t e r nuclear l a y e r (Fig. 3), being a p p r o x i m a t e l y equal in n u m b e r with the r a d i o a c t i v e p h o t o r e c e p t o r segment. R a d i o a c t i v i t y was also present in a position corresponding to the p h o t o r e c e p t o r terminals, again with a frequency a p p r o x i m a t i n g t h a t of the outer segments.
Ganglion cells Most ganglion cells were clearly n o t a c c u m u l a t i n g r a d i o a c t i v i t y either in vivo or in vitro, or in light Or darkness. However, in all species there was a p o p u l a t i o n of cells in the ganglion cell layer which was r a d i o a c t i v e (Figs 4-6). T h e y were seen b o t h after i n t r a o c u l a r injections of [3H]-D-aspartate a n d incubations in it, b u t the m o r p h o l o g y was much b e t t e r after the i n t r a o c u l a r injections. The degree of labelling was variable, m a k i n g a b o u t 30 % of t h e m only d o u b t f u l l y positive. E v e n if there was positive labelling of a ganglion cell, the surrounding tissue was often seen to be more r a d i o a c t i v e (Fig. 5). The r a d i o a c t i v e cells were often smaller and more d a r k l y stained
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FIG. 5. Autoradiograph of guinea-pig retina, 4 hr after the intraoeular injection of 10 #Ci [3H]D-aspartate. There is a gradient of radioactivity through the retina and highest in the innermost part. One of the ganglion cells is radioactive (arrow), although less than the surrounding tissue. Most of the radioactivity is arranged in 'vertical' strands typical of glial localization. Phase contrast micrographs. Above, focus on the grains. Below, focus on the section. Designation of layers as in Fig. 1. • 560.
Fio. 6. Guinea-pig retina, 4 hr after the intraoeular injection of 25 #Ci [3H]-D-aspartate. A large ganglion cell (arrow) is labelled. Phase contrast micrograph. Left, focus on the grains. Right, focus on the section. Designation of layers as in Fig. 1. • 735. b y the toluidine blue (Fig. 4) b u t large labelled ganglion cells (Fig. 6) were also e n c o u n t e r e d (see below for frequency figures). The nucleus was always m u c h less radioactive t h a n the cytoplasm. All ganglion cells (negative, d o u b t f u l l y positive, or positive) were i n f r e q u e n t l y seen to be s u r r o u n d e d b y a t h i n sheath of radioactivity. W h e n the cell was small (less t h a n a b o u t 8 #m), it was n o t always possible to decide whether this r a d i o a c t i v i t y a c t u a l l y was in the cell cytoplasm or in the s u r r o u n d i n g structure. 14
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About 400 ganglion cells were counted in each species. The proportion of positive plus doubtfully positiva ganglion cells was 11% in pigeons, 15 % in guinea-pigs and 12 % in rabbits. For reasons which are given below, these figures are likely to have a bias upwards. I f only large (more than about 8 #m) and clearly positive cells were counted, the figures were 3 %, 5 % and 5 %, respectively. Roughly one-third of all cells in the ganglion cell layer were seen to be of the small (less t h a n about 8 #m) variety. More precise figures on the distribution of ganglion cells have recently been published for the rabbit and they agree very well (Hughes and Vaney, 1980). Other neurons
There was no definite accumulation of radioactivity in either of the plexiform layers (apart from t h a t of the photoreceptor terminals in the outer plexiform layer) t h a t could signal any uptake of [aH]-D-aspartate in significant populations of horizontal cells, bipolar cells, amacrine cells, or interplexiform cells. However, it is thereby not excluded t h a t small subpopulations of either cell type might exist, disguised by the glial radioactivity. Radioactive cell bodies were occasionally seen which could have represented such cells (particularly among horizontal cells in dark-adapted rabbits) but it was at the same time not possible to exclude t h a t they represented glial cells with an unusual position of the perikaryon.
FIG. 7. Autoradiograph of guinea-pig retina, 4 hr after the intraocular injection of 10#Ci [3H]-D-aspartate close to the retina. There is a gradient of radioactivity through the retina, highest in the innermost part. The radioactivity is arranged in radially directed strands which expand near the vitreous surface of the retina, in the middle of the inner nuclear layer (where the glial cell bodies are) and (slightly) at the level of the outer limiting membrane. The distribution is characteristic of glial cell radioactivity. Note that there are several non-radioactive ganglion cells (G). Phase contrast micrograph. Designation of layers as in Fig. 1. • 300. Glial cells
There was always a considerable radioactivity in glial cells, most pronounced in experiments with intraocular injections (Fig. 7) but present to a significant extent also in all incubations. This radioactivity was seen as radial strands through several retinal layers. After intraocular injections and in incubations with retinas attached to the pigment epithelium there was a clear gradient, decreasing from the vitreal side outwards. The perikarya of glial cells are usually in the middle of the inner nuclear layer, where radioactivity was always found. Most of this radioactivity is likely to be in the glial cells but, as noted above, it cannot be excluded t h a t it also represents radioactivity in small numbers of neurons with perikarya in the inner nuclear layer.
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Pigment epithelium The pigment epithelium became significantly radioactive only in the incubation experiments and then only when the retina had detached.
4. D i s c u s s i o n
The present series of experiments show that at least two of the neuron types in the retina accumulate [3H]-D-aspartate more than the others. Glia does also accumulate the substance, although not enough to hide the neuronal uptake nearly as much as was previously found with [~H]-L-aspartate or [ZH]-L-glutamate (Ehinger and Falck, 1971; Bruun and Ehinger, t974; Kennedy, Voaden and Marshall, 1974; White and Neal, 1976; Voaden, 1976, 1978). The identifiable neurons becoming radioactive are subpopulations of ganglion cells and photoreceptors. The counts of radioactive ganglion cells are likely to have systematic errors for several reasons. I f all cells in the ganglion cell layer are counted, glial cells are likely to be included. The proportion of glia among ganglion cells was reported as 5-12 % in pigeons (Binggeli and Panle, 1969) and 5-8 % in central and 15-19 % in peripheral rabbit retina (Vaney, 1980; Hughes and Vaney, 1980). Further, about 40 % of the positive cells were noted as doubtfully so and an unknown but presumably significant number of these doubtful cells must be regarded as falsely scored. There may also have been false negatives and it is not possible to assess accurately if they balance the false positive ones. Given that [aH]-D-aspartate accumulating cells are a new observation in the retina, it would seem that observer bias would favour the falsely positive cells, making the total figure falsely high and adding to the error caused by glial cells in the ganglion cell layer. The figures around 11-15 % positive cells are thus likely to be maximum figures. Counting only big (larger than 8 #m) cells (which definitely were neurons) gave figures between 3 and 5 %. However, some of the small cells in the ganglion cell layer are undoubtedly true nerve cells (Vaney, 1980; Hughes and Vaney, 1980) and this type of count therefore gives too low figures. The true figure is likely to be in between the two extremes but at present the technique does not allow any better accuracy. The observations thus show that there is a population of ce!ls in the ganglion cell layer that accumulate [aH]-D-aspartate and the size of this population is likely to be between 5 and 10 % of the total number of ganglion cells; the definite extreme limits being 3 and 15%. Both large and small (smaller than 8/~m) cells accumulate [~H]-D-aspartate. Cones occur in rabbits (see l~ohen, 1964) and were recently described in detail (Bunt, 1978). They represent about 8 % of all photoreceptors and have their nucleus in the outermost part of the outer nuclear layer. Both these characteristics fit precisely with the [SH]-D-aspartate accumulating photoreceptors, which thus are likely to be cones. The marked and easily recognizable radioactivity of the cones that appears among the rod outer segments in rabbits does not reside in cone outer segments but rather in the very long myoid (Fig. 2) which extends almost all the way out to the pigment epithelium in these cells (Bunt, 1978). The short outer segment accumulates radioactivity less readily. Cones or at least cone-like cells are also known in guinea-pigs (Kolmer, 1936; SjSstrand, I969) and the similarities with the radioactive photoreceptors in rabbits makes it likely that cones are the cells accumulating [aH]D-aspartate also in guinea-pigs. In pigeons, finally, cones are the dominating types 14~2
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ofphotoreceptors and since most photoreceptors accumulated [aH]-D-aspartate in this species it again seems that the uptake was in cones. Summarizing the morphological evidence it seems that in the three species investigated, cones accumulate [3H]-D-aspartate. Most of the radioactivity appears in the ellipsoid, the myoid, the perikaryon and the synaptic structures. As might be expected, comparatively little radioactivity is accumulated by the outer segments (but in rabbits and presumably also in guinea-pig the myoid of the cones or cone-like cells extends out among the rod outer segments so that in low-power micrographs it seems as if outer segments were heavily labelled). The results discussed above raise two important questions. First, to what extent can the neuronal uptake be regarded as a marker for neurons operating with glutamate or aspartate as their transmitter and, second, do the results fit with what is previously known about glutamate and aspartate in the retina ? 4.1. [3H]-I)-aspartate uptake as marker for neurons using glutamate or aspartate as transmitter Balcar and Johnston (1972) described strong competitive inhibition of L-glutamate and L-aspartate uptake by D-aspartate. The metabolism of D-aspartate was found to be small (Davies and Johnston, 1976): after 1 hr of incubation in [14C]-D-aspartate, 97 % of the tissue radioactivity in rat brain slices appeared as aspartate and similar figures were found in 1 hr perfusion experiments. Takagaki (1978) obtained similar figures in rat brain synaptosomal preparations. Metabolites are therefore not likely to influence the results. High-affinity, sodium-dependent uptake of [aH]-D-aspartate into neurons presumed to use glutamate as their transmitter has been shown in a number of brain preparations (Davies and Johnston, 1976; Land Karlsen and Fonnum, 1978; Storm-Mathisen and Woxen Opsahl, 1978; Takagaki, 1978; MaltheSorensen, Skrede and Fonnum, 1979, 1980; Fonnum, Lund Karlsen, Malthe-S~rensen, Skrede and Walaas, 1979), and the accumulated [3H]-D-aspartate can be released by a Ca2+-dependent mechanism (Davies and Johnston, 1976; Fonnum, Lund Karlsen, Malthe-Sorensen, Skrede and Walaas, 1979; Malthe-Sorensen, Skrede and Fonnum, 1980). There is thus little doubt that [3H]-D-aspartate will label glutamate neurons. Since the uptake of [~H]-D-aspartate was equally inhibited by aspartate and glutamate (Balcar and Johnston, 1972 ; Takagaki, 1978), [3H]-D-aspartate must be suspected to label also aspartate neurons but there seems to be no direct previous evidence for this in the brain. 4.2. Comparison between the uptake of [aH]-D-aspartate and other results in the retina Pigeon ganglion cells were proposed to use glutamate as neurotransmitter (Henke, Schenker and Cu6nod, 1976; Cudnod and Henke, 1978) because retinal ablation caused a decrease in high-affinity uptake of glutamate (by a decrease in t h e Vmax) in a synaptosome-rich fraction from the optic rectum. Similarly, Yates and Roberts (1974) found a decrease in the frog tectum glutamate content after enucleation. In rabbits, however, Margolis, Heller and Moore (1968) were unable to find any such effect in the dorsal lateral geniculate body. In the monkey retina, the glutamate and aspartate concentration shows an increase in the innermost layer (Berger, McDaniel, Carter and Lowry, 1977) and both substances are present in significant concentrations in the ganglion cell layer in rats and frogs (see Voaden, 1978; Voaden, Morjaria and Oraedu, 1980). The evidence from the literature together with the present experiments thus suggest that a proportion of ganglion cells (about 5-10%) use either glutamate or
[3H]-D-ASPARTATE UPTAKE IN RETINA
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aspartate as their transmitter. A considerable fraction of the cells in the ganglion cell layer are not true ganglion cells but 'displaced amacrine cells' (renamed 'coronate cells'; Hughes and Vaney, 1980; Vaney, 1980), particularly in the pigeon (Binggeli and Paule, 1969). However, it seems unlikely that the [aH]-D-aspartate accumulating cells could be such displaced amacrine cells, because neither is any significant number of other amacrine cells labelled (see above) nor would it be possible for displaced amacrine cells to give the results described by Yates and Roberts (1974) or Henke, Schenker and Cudnod (1976). The weight of evidence thus suggests that most or all of the labelled cells in the ganglion cell layer are conventional ganglion cells, sending axons to the optic nerve. The fact that neither the glutamate nor the aspartate concentration is very high in the optic nerve (Johnson and Aprison, 1971 ; Berger et al., 1977) is no contradiction because preterminal axons normally do not have any high transmitter content and only at most 15 % of the axons operate with glutamate or aspartate. The comparatively low percentage can also explain why it has not always been possible to detect any decrease in glutamate content in the brain region with the nerve endings of the ganglion cells after the eye had been removed (e.g. Margolis, Heller and Moore, 1968). Aspartate and glutamate have been of interest as possible photoreceptor neurotransmitters since it was shown that horizontal cells and bipolar cells are very sensitive to these agents (Dowling and Ripps, 1972; Mnrakami, Ohtsu and Ohtsuka, 1972; Cervetto and MacNiehol, 1972; Murakai, Ohtsuka and Shimazaki, 1975; see also Negishi and Drujan, 1979 or Voaden, Mor]aria and Oraedu, 1980 for further references and recent results). More recently it was shown in the carp retina that cone horizontal cells are fifty times more sensitive to aspartate than glutamate, that the effect of both the natural photoreceptor transmitter and exogenously applied aspartate are blocked by alpha-aminoadipic acid, an aspartate antagonist; and that exogenously applied aspartate has much greater effects of horizontal cells when the retina is treated with Co 2+, an agent which depresses the spontaneous dark release of transmitter from the photoreceptors (Wu and Dowling, 1978). These findings suggest that aspartate is the transmitter of cones in the carp retina. Aspartate and glutamate are present in significant concentrations (about 2080 nmol/mg protein) in the photoreceptors in a number of species (Berger et al., 1977; Voaden, 1978) but other parts of the retina contain more glutamate and the concentration differences for aspartate are small. Considering the present results, the aspartate (or glutamate) concentration in the photoreceptor layer could be expected to be more impressive if analyzed in cone-rich retinas (from, for example, pigeons) but no such figures are available. Further, Thomas and Redburn (1978) reported highaffinity uptake of glutamate and aspartate in a photoreceptor cell preparation from rabbits. Finally, Neal, Collins and Massey (1979) and Neal and Massey (1980) reported a presumably significant (P < 0-05) fall in the endogenous aspartate release upon light stimulation; precisely the expected effect if aspartate is transmitter in photoreceptors. Glutamate showed no such decrease. Since aspartate seems to be present presynaptically, to be released by the appropriate stimulus (darkness) and has striking postsynaptic effects, indistinguishable from the effect of the physiological stimulation, it is a strong candidate as a photoreceptor neurotransmitter. The experiments reported in this paper agree, and further suggest that it is a transmitter in cones. Further work will show whether it is the transmitter of all cones and in all species.
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B. EHINGER ACKNOWLEDGMENTS
This work was supported by grants from the Swedish Medical Research Council (project 14X-2321), by the Thorsten and Elsa Segcrfalks Stiftelse, by the Golje Foundation and by the Faculty of Medicine, University of Lund. REFERENCES Ames, A. (1965). Studies of morphology, chemistry and function in isolated retina. In Biochemistry of the Retina (Ed. Graymore, C. N.). Pp. 22-30. Academic Press, London & New York. Balcar, V. J. and Johnston, G. A. (1972). Glutamate uptake by brain slices and its relation to the depolarization of neurones by acidic amino acids. J. Neurobiol. 3, 295-301. Berger, S. J., McDaniel, M. L., Carter, J. G. and Lowry, 0. H. (1977). Distribution of four potential transmitter amino acids in monkey retina. J. Neurochem. 28, 159-63. Binggeli, R. L. and Paule, W. J. (1969). The Pigeon Retina. Quantitative aspects of the optic nerve and ganglion cell layer. J. Comp. Neurol. 137, 1-18. Bruun, A. and Ehinger, B. {1974). Uptake of certain putative neurotransmitters into retinal neurons of some mammals. Exp. Eye Res. 19, 435-47. Bunt, A. H. (1978). Fine structure and autoradiography of rabbit photoreeeptor cells. Invest. Ophthalmol. 17, 90-104. Cervetto, L. and MacNichol, E. F. (1972). Inactivation of horizontal cells in turtle retina by glutamate and aspartate. Science 178, 767-8. Cudnod, M. and Henke, H. (1978). Neurotransmittcrs in the avian visual system. In Amino Acids as Neurotransmitters (Ed. Fonnum, F.). Pp. 221-40. NATO Advanced Study Institute Series A. Davies, L. P. and Johnston, G. A. R. (1976). Uptake and release of D-and L-aspartate by rat brain slices. J. Neurochem. 25, 1007-14. Dowling, J. E. and Ripps, H. (1972). Adaptation in skate photoreceptors. J. Gen. Physiol. 60, 698-719. Ehinger, B. (1980). Photoreceptor accumulation of [3H]-D-aspartate. Association for Eye Research, 14-17 September, Parma. Ehinger, B. and Falck, B. (1971). Autoradiography of some suspected neurotransmitter substances: GABA, glycine, aspartic acid, glutamic acid, histamine, dopamine, and L-dopa. Brain Res. 33, 157-72. Fonnum, F., Lund Karlsen, R., Malthe-SSrenssen, D., Skrede, K. K. and Walaas, I. (1979). Localization of neurotransmitters, particularly glutamate, in hippocampus, septum, nucleus accumbeus and superior colliculus. Prog. Brain Res. 51, 167-91. Henke, H., Schenker, T. M. and Cu6nod, M. (1976). Uptake of neurotransmitter candidates by pigeon optic rectum. J. Neurochem. 26, 125-30. Hughes, A. and Vaney, D. I. (1980). Coronate cells: displaced amaerines of the rabbit retina ? J. Comp. Neurol. 189, 169-89. Johnson, J. L. and Aprison, M. H. (1971). The distribution of glutamate and total free amino acids in thirteen specific regions of the cat central nervous system. Brain Res. 26, 141-8. Kennedy, A. J., Voaden, M. J. and Marshall, J. (1974). Glutamate metabolism in the frog retina. Nature 252, 50-2. Kolmer, W. (1936). Die Netzhaut. i n Handbuch der Mikroskopischen Anatomic des Menschen, Vol. IIL 2 (Ed. v. MSllendorff, W.). Pp. 295-466. Springer-Verlag, Berlin. Lund Karlsen, R. and Fonnum, F. (1978). Evidence for glutamate as a neurotransmitter in the corticofugal fibres to the dorsal lateral geniculate body and the superior colliculus in rat. Brain Res. 151,457-67. Malthe-SSrenssen, D., Skrede, K. K. and Fonnum, F. (1979). Calcium-dependent release of D-[aH]-aspartate evoked by selective electrical stimulation of excitatory afferent fibres to hippocampal pyramidal cells in vitro. Neuroscienee 4, 1255-63. Malthe-SSrensen, D., Skrede, K. K. and Fonnum, F. (1980). Release of D-[3H]-aspartate from the dorsolateral system after electrical stimulation of the fimbria in vitro. Neuroscience 5, 127-33.
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Margolis, R. K., Heller, A. and Moore, R. Y. (1968). Effects of changes in cellular composition following neuronal degeneration on amino acids in brain. Brain Res. 11, 19-31. Murakami, M., Ohtsu, K. and Ohtsuka, T. (1972). Effects of chemicals on receptors and horizontal cells in the retina. J. Physiol. 227, 899-913. Murakami, M., Ohtsuka, T. and Shimazaki, H. (1975). Effects of aspartate and glutamate on the bipolar cells in the carp retina. Vision Res. 15, 456-8. Neal, M. J., Collins, G. G. and Massey, S. G. (1979). Inhibition of aspartate release from the retina of the anaesthetised rabbit by stimulation with light flashes. Neurosci. Letters 14, 241-5. Neal, M. J. and Massey, S. G. (1980). The release of acetyleholine and amino acids from the rabbit retina in vivo. Neurochem. Int. 1, 191-208. Negishi, K. and Drujan, B. D. (1979). Effects of some amino acids on horizontal cells in the fish retina. J. Neurosci. Res. 4, 351-63. Robert, J. (1964). Das Auge und seine Hilfsorgane. In Handbuch der Mikroskopischen Anatomie des Mensechen, Vol. IH, 4 (Eds v. MLllendorff, W. and Bargmann, W.). Springer-Verlag, Berlin. SjLstrand, F. (1969). The outer plexiform layer and the neural organization of the retina. In The Retina. Morphology, Function and Clinical Characteristics (Eds Straatsma, B. R., Hall, M. 0., Allen, R. A. and Crescitelli, F.). Pp. 63-100. University of California Press, Berkeley and Los Angeles. Storm-Mathisen, J. and Woxen Opsahl, M. (1978). Aspartate and/or glutamate may be transmitters in hippocampal efferents to septum and hypothalamus. Neurosci. Letters 9, 65-70. Takagaki, G. (1978). Sodium and potassium ions and accumulation of labelled D-aspartate and GABA in crude synaptosomal fraction from rat cerebral cortex. J. Neurochem. 30, 47-56. Thomas, T. N. and Redburn, D. A. (1978). Uptake of [14C]-aspartic acid and [14C]-glutamie acid by retinal synaptosomal fractions. J. Neurochem. 31, 63-8. Vaney, D. J. (1980). A quantitative comparison between the ganglion cell populations and axonal outflows of the visual streak and periphery of the rabbit retina. J. Comp. Neurol. 189, 215-33. Voaden, M. J. (1976). Gamma aminobutyric acid and glycine as retinal neurotransmitters. In Transmitters in the Visual Process (Ed. Bonting, S. L.). Pp. 107-26. Pergamon Press, Oxford, New York, Toronto, Sydney, Frankfurt. Voaden, M. J. (1978). The localization and metabolism of neuroactive amino acids in the retina. In Amino Acids as Chemical Transmitters (Ed. Fonnum, F.). Pp. 257-74. Plenum Press, New York. Voaden, M. J., Morjaria, B. and Oraedu, A. C. I. (1980). The localization and metabolism of glutamate, aspartate and GABA in the rat retina. Neurochem. Int. 1, 151-66. White, R. O. and Neal, M. J. (1976). The uptake of L-glutamate by the retina. Brain Res. 111, 79-93. Wu, S. M. and Dowling, J. E. (1978). L-Aspartate: evidence for a role in cone photoreceptor synaptic transmission in the carp retina. Proc. Natl. Aead. Sci. U.S.A. 75, 5205-9. Yates, R. A. and Roberts, P. J. (1974). Effects of enucleation and intraocular colchicine on the amino acids of frog optic tectum. J. Neurochem. 23, 391-3.
[3H]=D-Aspartate A c c u m u l a t i o n in the Retina of Pigeon, Guinea-Pig and Rabbit B. EHINGER
Department of Ophthalmology, University of Lund, Lund, Sweden (Received 16 October 1980 and accepted 24 February 1981, London) Retinas from pigeons, guinea-pigs and rabbits were exposed to [aH]-D-aspartate either by intraocular injections or by incubating undetached or detached retinas in it. In all species about 5-10% of the cells in the ganglion cell layer became radioactive. In the pigeon most of the photoreeeptors became radioactive when the substance reached them (i.e. when the retina was detached ) and, in guinea-pigs and rabbits, about 10 % of them also became radioactive. In the latter case the radioactive photoreceptors were characterized by long myoids protruding out among the surrounding outer segments and by having their perikarya in the outermost cell row of the outer nuclear layer. The frequency distribution and morphology of the radioactive photoreceptors suggest they are cones. Since [3H]-D-aspartate can be presumed to label neurons using aspartate or glutamate as transmitter, the work suggests that cones and about 5-10 % of M1ganglion cells use either of the two substances as transmitter. 1. I n t r o d u c t i o n U p t a k e mechanisms exist in m a n y types of neurons as a m e t h o d for inactivating and conserving their neurotransmitters. The presence of such an uptake mechanism can be shown autoradiographically and this has now become one of the s t a n d a r d methods for demonstrating aminergic, glycinergic or GABA-ergic neurons. However, the presumed natural transmitters of m a n y excitatory neurons, L-glutamic acid and L-aspartic acid, have n o t yielded the same results because of a strong glial u p t a k e (an d metabolism) disguising w h a t neuronal u p t a k e there might be. However, the isomer, [3H]-D-aspartic acid, has been shown to be accumulated by brain neurons likely to use glutamic acid as their neurotransmitter (Balcar and Johnston, 1972; Davies and Johnston, 1976; L u n d Karlsen and F o n n u m , 1978; Storm-Mathisen and W o x e n Opsahl, 1978; Takagaki, 1978; Malthe-Sorensen, Skrede and F o n n u m , 1979, 1980; F o n n u m , L u n d Karlsen, Malthe-Sorensen, Skrede and Walaas, 1979) and should be useful as a morphological autoradiographic marker for neurons using aspartate or glutamate as transmitter. There is much evidence suggesting either glutamic acid a n d / o r aspartic acid as possible neurotransmitters in the outer plexiform layer (see section 4) and it has also been suggested t h a t g l u t a m a t e could be the transmitter of ganglion cells (Henke, Schenker and Cu@nod, 1976). We have therefore performed autoradiographic studies on the cellular localization of [aH]-D-aspartate uptake in retinas from pigeon, guinea-pig and rabbit. The results are compatible with glutamate or aspartate as transmitter(s) in cones (or cone-like cells) and in a small population of the ganglion cells (about 5-10 % ). A preliminary report has appeared (Ehinger, 1980). Glutamate u p t a k e into ganglion cells has previously been observed (Bruun and Ehinger, 1974) but could not be clearly distinguished from non-specific uptake.
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2. M a t e r i a l s a n d M e t h o d s The eyes of wild, adult pigeons, pigmented adult guinea-pigs and pigmented adult rabbits were used. Five or 20 #Ci of [3H]-D-aspartic acid was injected intravitreally in three light-adapted eyes of each species. Only topical anaesthesia (0"4% oxibuprocaine) was used. The eyes were obtained after 30 rain or 4 hr and pieces of the posterior pole were processed as described below. The same injections were given (under infrared illumination and with the aid of an infrared converting viewing system) in two eyes from each species, dark adapted for 12-16 hr. Doses and times were as for light adapted eyes. The dark adapted animals were left in the dark after the injections but were killed and dissected in ambient laboratory light (about 200 lux). Posterior segments were incubated in a balanced salt solution (Ames, 1965). After 10 rain, [3H]-D-aspartate was added to a final concentration of 1, 3, 5 or 20/tCi/ml and the incubations were carried on for 15, 30 or 60 min at 37~ The tissue pieces were then washed twice for 5 min in the salt solution at 0~ and processed for autoradiography. In a second series, retinas were incubated as above (5/tCi/ml) for 15 rain and then incubated at 37~ for another 15, 30 or 60 rain with the salt solution without [3H]-D-aspartate, washed as described above and processed for autoradiography. Each group consisted of six pieces of retinas from two animals. Guinea-pigs and rabbits were dark adapted for 12-16 hr and incubated as above in the dark. The salt solution was always carefully aerated with a mixture of 5 Yo CO2 in oxygen. The incubation flasks were gently rocked during the incubation. In about half of the specimens, the retina detached during the experiment but often only partially, allowing the analysis of both detached and undetached retina in the same specimen. Tissue pieces were always taken from the posterior pole. The pecten was avoided in the birds as was the area with medullated nerve fibres in rabbits but no other attempts were made to analyse any specific region. The various tissue pieces were freeze-dried and fixed in gaseous formaldehyde at 80~ for 1 hr. The formaldehyde was generated from about 5 g paraformaldehyde placed together with the dry tissue pieces in a 1 l jar, which was then heated in an oven. The tissue pieces were then embedded in plastic (Durcopan| FLUKA) in vacuo without any intermediate steps in any solvent and cut (4 #m) with a glass knife. They were then covered with autoradiographic stripping film (Kodak AR 10), exposed for 1-12 weeks, developed and fixed. If needed, they were stained with 1 Yo toluidine blue in 40 % ethanol.
3. R e s u l t s
Photoreceptors Certain photoreceptors became radioactive in all a n i m a l species. However, their radioactivity was very low after intraocular injections, both after 30 a n d after 240 min. I n fact, it was so low as to be judged insignificant before the results from the i n c u b a t i o n s were available (see Fig. 7). I n these, strong radioactivity appeared in some photoreceptors in detached retinas. The r a d i o a c t i v i t y was much less e v i d e n t in retinas which were still attached. There was no obvious difference between light- a n d d a r k - a d a p t e d retinas. However at times there were for no a p p a r e n t reason differences in the photoreceptor r a d i o a c t i v i t y in different sectors of one a n d the same retina. I n the pigeons, the radioactive photoreceptors were numerous. The r a d i o a c t i v i t y was concentrated in two b a n d s in the inner segments (arrows in Fig. 1), one corresponding to the outermost layer of perikarya in the outer nuclear layer a n d one near the i n n e r limit of the i n n e r segments at the level of the synaptic terminals of the photoreceptors (Fig. 1). The outer segments were usually n o t very radioactive; however, moderate r a d i o a c t i v i t y was seen in some structures e x t e n d i n g out a m o n g
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FIG. 1. Pigeon retina, incubated 30 rain in [3H]-D-aspartate, 1 #Ci/ml. There is radioactivity in the photoreceptor inner segments in one band corresponding to the outermost cell bodies (large arrowheads) and in another band corresponding to the synaptic terminals (small arrowheads). Ph, Photoreceptors; ONL, outer nuclear layer; OPL, outer plexi.formlayer; INL,: inner nuclear layer; IPL, inner plexiform layer; G, ganglion cell layer. Phase contrast mierographs of autoradiograph. Left, focus on the silver grains. Right, focus on the section, x 224.
t h e i n n e r m o s t p a r t o f the o u t e r segments: R a d i o a c t i v e p e r i k a r y a were f r e q u e n t t h r o u g h o u t the inner n u c l e a r layer, a l t h o u g h t h e y were clearly m o r e n u m e r o u s in its o u t e r p a r t s as i n d i c a t e d b y t h e b a n d of r a d i o a c t i v i t y seen i n 'the o u t e r m o s t p a r t of t h e o u t e r n u c l e a r layer, I n r a b b i t s a n d guinea-pigs certain p h o t o r e c e p t o r s also b e c a m e r a d i o a c t i v e when i n c u b a t i n g d e t a c h e d retinas, less when i n c u b a t i n g r e t i n a s still a t t a c h e d to t h e p i g m e n t e p i t h e l i u m a n d only little a f t e r t h e i n t r a o e u l a r injections. The r a d i o a c t i v i t y was seen in s t r u c t u r e s a m o n g the p h o t o r e c e p t o r o u t e r segments. A b o u t 10 % o f the p h o t o r e c e p t o r s showed this d i s t r i b u t i o n of r a d i o a c t i v i t y . A t high m a g n i f i c a t i o n (Fig. 2) it was seen t h a t it was t h e myoid, e x t e n d i n g o u t a m o n g the a d j o i n i n g rods, t h a t was
FIG. 2. Autoradiograph of dark-adapted rabbit retina incubated 30 min in [3H]-D-aspartate, 1 #Ci/ml. The arrow points to a radioactive cone, Note its short outer segment and long, radioactive myoid. Phase contrast micrograph. Left, focus on the grains. Right, focus on the section, x 910.
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FIo. 3. Guinea-pig retina (light-adapted), incubated 15 rain in [3H]-n-aspartate, 20 #Ci/ml. Note the radioactivit~ of the photoreceptors (cone-like cells), in their perikarya in the outermost part of the outer nuclear layer (ONL) and in their synaptie terminals in the outer plexiform layer (OPL). INL, Inner nuclear layer; IPL, part of the inner plexiform layer. Phase contrast micrographs of autoradiograph. Left, focus on the grains. Right, focus on the section. • 420.
FIG. 4. Pigeon retina, incubated 30 rain in [aH]-D-aspartate, 1 #Ci/ml. There is one ganglion cell (arrow) which is radioactive in its cytoplasm (but less in the nucleus). Another ganglion cell (arrow and D) is doubtfully positive. IPL, inner plexiform layer; G, ganglion cell layer. Phase contrast micrographs of autoradiograph. Left, focus on the grains. Right, focus on the section, x 500. radioactive. The outer segment of t h e cell was less radioactive. A n u m b e r of r a d i o a c t i v e cell bodies were seen in t h e o u t e r m o s t p a r t of t h e o u t e r nuclear l a y e r (Fig. 3), being a p p r o x i m a t e l y equal in n u m b e r with the r a d i o a c t i v e p h o t o r e c e p t o r segment. R a d i o a c t i v i t y was also present in a position corresponding to the p h o t o r e c e p t o r terminals, again with a frequency a p p r o x i m a t i n g t h a t of the outer segments.
Ganglion cells Most ganglion cells were clearly n o t a c c u m u l a t i n g r a d i o a c t i v i t y either in vivo or in vitro, or in light Or darkness. However, in all species there was a p o p u l a t i o n of cells in the ganglion cell layer which was r a d i o a c t i v e (Figs 4-6). T h e y were seen b o t h after i n t r a o c u l a r injections of [3H]-D-aspartate a n d incubations in it, b u t the m o r p h o l o g y was much b e t t e r after the i n t r a o c u l a r injections. The degree of labelling was variable, m a k i n g a b o u t 30 % of t h e m only d o u b t f u l l y positive. E v e n if there was positive labelling of a ganglion cell, the surrounding tissue was often seen to be more r a d i o a c t i v e (Fig. 5). The r a d i o a c t i v e cells were often smaller and more d a r k l y stained
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FIG. 5. Autoradiograph of guinea-pig retina, 4 hr after the intraoeular injection of 10 #Ci [3H]D-aspartate. There is a gradient of radioactivity through the retina and highest in the innermost part. One of the ganglion cells is radioactive (arrow), although less than the surrounding tissue. Most of the radioactivity is arranged in 'vertical' strands typical of glial localization. Phase contrast micrographs. Above, focus on the grains. Below, focus on the section. Designation of layers as in Fig. 1. • 560.
Fio. 6. Guinea-pig retina, 4 hr after the intraoeular injection of 25 #Ci [3H]-D-aspartate. A large ganglion cell (arrow) is labelled. Phase contrast micrograph. Left, focus on the grains. Right, focus on the section. Designation of layers as in Fig. 1. • 735. b y the toluidine blue (Fig. 4) b u t large labelled ganglion cells (Fig. 6) were also e n c o u n t e r e d (see below for frequency figures). The nucleus was always m u c h less radioactive t h a n the cytoplasm. All ganglion cells (negative, d o u b t f u l l y positive, or positive) were i n f r e q u e n t l y seen to be s u r r o u n d e d b y a t h i n sheath of radioactivity. W h e n the cell was small (less t h a n a b o u t 8 #m), it was n o t always possible to decide whether this r a d i o a c t i v i t y a c t u a l l y was in the cell cytoplasm or in the s u r r o u n d i n g structure. 14
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About 400 ganglion cells were counted in each species. The proportion of positive plus doubtfully positiva ganglion cells was 11% in pigeons, 15 % in guinea-pigs and 12 % in rabbits. For reasons which are given below, these figures are likely to have a bias upwards. I f only large (more than about 8 #m) and clearly positive cells were counted, the figures were 3 %, 5 % and 5 %, respectively. Roughly one-third of all cells in the ganglion cell layer were seen to be of the small (less t h a n about 8 #m) variety. More precise figures on the distribution of ganglion cells have recently been published for the rabbit and they agree very well (Hughes and Vaney, 1980). Other neurons
There was no definite accumulation of radioactivity in either of the plexiform layers (apart from t h a t of the photoreceptor terminals in the outer plexiform layer) t h a t could signal any uptake of [aH]-D-aspartate in significant populations of horizontal cells, bipolar cells, amacrine cells, or interplexiform cells. However, it is thereby not excluded t h a t small subpopulations of either cell type might exist, disguised by the glial radioactivity. Radioactive cell bodies were occasionally seen which could have represented such cells (particularly among horizontal cells in dark-adapted rabbits) but it was at the same time not possible to exclude t h a t they represented glial cells with an unusual position of the perikaryon.
FIG. 7. Autoradiograph of guinea-pig retina, 4 hr after the intraocular injection of 10#Ci [3H]-D-aspartate close to the retina. There is a gradient of radioactivity through the retina, highest in the innermost part. The radioactivity is arranged in radially directed strands which expand near the vitreous surface of the retina, in the middle of the inner nuclear layer (where the glial cell bodies are) and (slightly) at the level of the outer limiting membrane. The distribution is characteristic of glial cell radioactivity. Note that there are several non-radioactive ganglion cells (G). Phase contrast micrograph. Designation of layers as in Fig. 1. • 300. Glial cells
There was always a considerable radioactivity in glial cells, most pronounced in experiments with intraocular injections (Fig. 7) but present to a significant extent also in all incubations. This radioactivity was seen as radial strands through several retinal layers. After intraocular injections and in incubations with retinas attached to the pigment epithelium there was a clear gradient, decreasing from the vitreal side outwards. The perikarya of glial cells are usually in the middle of the inner nuclear layer, where radioactivity was always found. Most of this radioactivity is likely to be in the glial cells but, as noted above, it cannot be excluded t h a t it also represents radioactivity in small numbers of neurons with perikarya in the inner nuclear layer.
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Pigment epithelium The pigment epithelium became significantly radioactive only in the incubation experiments and then only when the retina had detached.
4. D i s c u s s i o n
The present series of experiments show that at least two of the neuron types in the retina accumulate [3H]-D-aspartate more than the others. Glia does also accumulate the substance, although not enough to hide the neuronal uptake nearly as much as was previously found with [~H]-L-aspartate or [ZH]-L-glutamate (Ehinger and Falck, 1971; Bruun and Ehinger, t974; Kennedy, Voaden and Marshall, 1974; White and Neal, 1976; Voaden, 1976, 1978). The identifiable neurons becoming radioactive are subpopulations of ganglion cells and photoreceptors. The counts of radioactive ganglion cells are likely to have systematic errors for several reasons. I f all cells in the ganglion cell layer are counted, glial cells are likely to be included. The proportion of glia among ganglion cells was reported as 5-12 % in pigeons (Binggeli and Panle, 1969) and 5-8 % in central and 15-19 % in peripheral rabbit retina (Vaney, 1980; Hughes and Vaney, 1980). Further, about 40 % of the positive cells were noted as doubtfully so and an unknown but presumably significant number of these doubtful cells must be regarded as falsely scored. There may also have been false negatives and it is not possible to assess accurately if they balance the false positive ones. Given that [aH]-D-aspartate accumulating cells are a new observation in the retina, it would seem that observer bias would favour the falsely positive cells, making the total figure falsely high and adding to the error caused by glial cells in the ganglion cell layer. The figures around 11-15 % positive cells are thus likely to be maximum figures. Counting only big (larger than 8 #m) cells (which definitely were neurons) gave figures between 3 and 5 %. However, some of the small cells in the ganglion cell layer are undoubtedly true nerve cells (Vaney, 1980; Hughes and Vaney, 1980) and this type of count therefore gives too low figures. The true figure is likely to be in between the two extremes but at present the technique does not allow any better accuracy. The observations thus show that there is a population of ce!ls in the ganglion cell layer that accumulate [aH]-D-aspartate and the size of this population is likely to be between 5 and 10 % of the total number of ganglion cells; the definite extreme limits being 3 and 15%. Both large and small (smaller than 8/~m) cells accumulate [~H]-D-aspartate. Cones occur in rabbits (see l~ohen, 1964) and were recently described in detail (Bunt, 1978). They represent about 8 % of all photoreceptors and have their nucleus in the outermost part of the outer nuclear layer. Both these characteristics fit precisely with the [SH]-D-aspartate accumulating photoreceptors, which thus are likely to be cones. The marked and easily recognizable radioactivity of the cones that appears among the rod outer segments in rabbits does not reside in cone outer segments but rather in the very long myoid (Fig. 2) which extends almost all the way out to the pigment epithelium in these cells (Bunt, 1978). The short outer segment accumulates radioactivity less readily. Cones or at least cone-like cells are also known in guinea-pigs (Kolmer, 1936; SjSstrand, I969) and the similarities with the radioactive photoreceptors in rabbits makes it likely that cones are the cells accumulating [aH]D-aspartate also in guinea-pigs. In pigeons, finally, cones are the dominating types 14~2
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ofphotoreceptors and since most photoreceptors accumulated [aH]-D-aspartate in this species it again seems that the uptake was in cones. Summarizing the morphological evidence it seems that in the three species investigated, cones accumulate [3H]-D-aspartate. Most of the radioactivity appears in the ellipsoid, the myoid, the perikaryon and the synaptic structures. As might be expected, comparatively little radioactivity is accumulated by the outer segments (but in rabbits and presumably also in guinea-pig the myoid of the cones or cone-like cells extends out among the rod outer segments so that in low-power micrographs it seems as if outer segments were heavily labelled). The results discussed above raise two important questions. First, to what extent can the neuronal uptake be regarded as a marker for neurons operating with glutamate or aspartate as their transmitter and, second, do the results fit with what is previously known about glutamate and aspartate in the retina ? 4.1. [3H]-I)-aspartate uptake as marker for neurons using glutamate or aspartate as transmitter Balcar and Johnston (1972) described strong competitive inhibition of L-glutamate and L-aspartate uptake by D-aspartate. The metabolism of D-aspartate was found to be small (Davies and Johnston, 1976): after 1 hr of incubation in [14C]-D-aspartate, 97 % of the tissue radioactivity in rat brain slices appeared as aspartate and similar figures were found in 1 hr perfusion experiments. Takagaki (1978) obtained similar figures in rat brain synaptosomal preparations. Metabolites are therefore not likely to influence the results. High-affinity, sodium-dependent uptake of [aH]-D-aspartate into neurons presumed to use glutamate as their transmitter has been shown in a number of brain preparations (Davies and Johnston, 1976; Land Karlsen and Fonnum, 1978; Storm-Mathisen and Woxen Opsahl, 1978; Takagaki, 1978; MaltheSorensen, Skrede and Fonnum, 1979, 1980; Fonnum, Lund Karlsen, Malthe-S~rensen, Skrede and Walaas, 1979), and the accumulated [3H]-D-aspartate can be released by a Ca2+-dependent mechanism (Davies and Johnston, 1976; Fonnum, Lund Karlsen, Malthe-Sorensen, Skrede and Walaas, 1979; Malthe-Sorensen, Skrede and Fonnum, 1980). There is thus little doubt that [3H]-D-aspartate will label glutamate neurons. Since the uptake of [~H]-D-aspartate was equally inhibited by aspartate and glutamate (Balcar and Johnston, 1972 ; Takagaki, 1978), [3H]-D-aspartate must be suspected to label also aspartate neurons but there seems to be no direct previous evidence for this in the brain. 4.2. Comparison between the uptake of [aH]-D-aspartate and other results in the retina Pigeon ganglion cells were proposed to use glutamate as neurotransmitter (Henke, Schenker and Cu6nod, 1976; Cudnod and Henke, 1978) because retinal ablation caused a decrease in high-affinity uptake of glutamate (by a decrease in t h e Vmax) in a synaptosome-rich fraction from the optic rectum. Similarly, Yates and Roberts (1974) found a decrease in the frog tectum glutamate content after enucleation. In rabbits, however, Margolis, Heller and Moore (1968) were unable to find any such effect in the dorsal lateral geniculate body. In the monkey retina, the glutamate and aspartate concentration shows an increase in the innermost layer (Berger, McDaniel, Carter and Lowry, 1977) and both substances are present in significant concentrations in the ganglion cell layer in rats and frogs (see Voaden, 1978; Voaden, Morjaria and Oraedu, 1980). The evidence from the literature together with the present experiments thus suggest that a proportion of ganglion cells (about 5-10%) use either glutamate or
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aspartate as their transmitter. A considerable fraction of the cells in the ganglion cell layer are not true ganglion cells but 'displaced amacrine cells' (renamed 'coronate cells'; Hughes and Vaney, 1980; Vaney, 1980), particularly in the pigeon (Binggeli and Paule, 1969). However, it seems unlikely that the [aH]-D-aspartate accumulating cells could be such displaced amacrine cells, because neither is any significant number of other amacrine cells labelled (see above) nor would it be possible for displaced amacrine cells to give the results described by Yates and Roberts (1974) or Henke, Schenker and Cudnod (1976). The weight of evidence thus suggests that most or all of the labelled cells in the ganglion cell layer are conventional ganglion cells, sending axons to the optic nerve. The fact that neither the glutamate nor the aspartate concentration is very high in the optic nerve (Johnson and Aprison, 1971 ; Berger et al., 1977) is no contradiction because preterminal axons normally do not have any high transmitter content and only at most 15 % of the axons operate with glutamate or aspartate. The comparatively low percentage can also explain why it has not always been possible to detect any decrease in glutamate content in the brain region with the nerve endings of the ganglion cells after the eye had been removed (e.g. Margolis, Heller and Moore, 1968). Aspartate and glutamate have been of interest as possible photoreceptor neurotransmitters since it was shown that horizontal cells and bipolar cells are very sensitive to these agents (Dowling and Ripps, 1972; Mnrakami, Ohtsu and Ohtsuka, 1972; Cervetto and MacNiehol, 1972; Murakai, Ohtsuka and Shimazaki, 1975; see also Negishi and Drujan, 1979 or Voaden, Mor]aria and Oraedu, 1980 for further references and recent results). More recently it was shown in the carp retina that cone horizontal cells are fifty times more sensitive to aspartate than glutamate, that the effect of both the natural photoreceptor transmitter and exogenously applied aspartate are blocked by alpha-aminoadipic acid, an aspartate antagonist; and that exogenously applied aspartate has much greater effects of horizontal cells when the retina is treated with Co 2+, an agent which depresses the spontaneous dark release of transmitter from the photoreceptors (Wu and Dowling, 1978). These findings suggest that aspartate is the transmitter of cones in the carp retina. Aspartate and glutamate are present in significant concentrations (about 2080 nmol/mg protein) in the photoreceptors in a number of species (Berger et al., 1977; Voaden, 1978) but other parts of the retina contain more glutamate and the concentration differences for aspartate are small. Considering the present results, the aspartate (or glutamate) concentration in the photoreceptor layer could be expected to be more impressive if analyzed in cone-rich retinas (from, for example, pigeons) but no such figures are available. Further, Thomas and Redburn (1978) reported highaffinity uptake of glutamate and aspartate in a photoreceptor cell preparation from rabbits. Finally, Neal, Collins and Massey (1979) and Neal and Massey (1980) reported a presumably significant (P < 0-05) fall in the endogenous aspartate release upon light stimulation; precisely the expected effect if aspartate is transmitter in photoreceptors. Glutamate showed no such decrease. Since aspartate seems to be present presynaptically, to be released by the appropriate stimulus (darkness) and has striking postsynaptic effects, indistinguishable from the effect of the physiological stimulation, it is a strong candidate as a photoreceptor neurotransmitter. The experiments reported in this paper agree, and further suggest that it is a transmitter in cones. Further work will show whether it is the transmitter of all cones and in all species.
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