Glutamate in some retinal neurons is derived solely from glia

Vol. 60, No. 2, pp. 355 366, 1994 Elsevier ScienceLtd Copyright © 1994 IBRO Printed in Great Britain. All rights reserved 0306-4522/94 $7.00 + 0.00

Neuroscience

Pergamon

0306-4522(94) E0024-X

G L U T A M A T E IN SOME RETINAL N E U R O N S IS DERIVED SOLELY FROM GLIA D. V. P o w and S. R. ROBINSON Vision, Touch and Hearing Research Centre, Department of Physiology and Pharmacology, The University of Queensland, Brisbane 4072, Australia Al~traet--Glutamate is the most abundant excitatory neurotransmitter in the vertebrate central nervous system. It is widely assumed that neurons using this transmitter derive it from several sources: (i) synthesizing it themselves from ~t-ketoglutarate or aspartate, (ii) synthesize it from glial-derived glutamine, or (iii) take up glutamate from the extracellular space. By use of immunocytochemistry we show that glutamate is abundant in the retinal ganglion and bipolar cells of the rabbit, but that immunoreactivity for glutamate in these neurons is reduced below immunocytochemical detection limits after the specific inhibition of glutamine synthesis in glial cells by D,L-methionine O,L-sulphoximine. GABA immunoreactivity in retinal amacrine cells was also reduced after inhibition of glutamine synthetase but the patterns and densities of immunoreactivity for taurine and glycine were unaffected. Therefore, this experimental paradigm does not induce generalized metabolic changes in neurons or glia. This study demonstrates that some glutamatergic neurons are dependent on the synthetic processes in glia for their neurotransmitter content.

Glutamate is the predominant excitatory transmitter in the mammalian CNS. 36 Glutamate does not readily traverse the blood-brain barrier, unless the barrier is first perturbed, 27,3° so glutamate must be synthesized within the CNS. The cellular site(s) of glutamate synthesis within the C N S have not been determined, but it is widely assumed 2~ that neurons synthesize glutamate themselves using aspartate, or precursors derived from the Krebs cycle. Glutamate can also be recycled: after its release from neurons, glutamate is quickly taken up by glial cells and amidated to form the non-neuroactive compound, glutamine/3,~4.27.57 This amidation is catalysed by the enzyme glutamine synthetase, which is present only in glia, 22 including the radial glial cells (Miiller cells) in the retina. 49 Glutamine is released by the glial cells and taken up by neurons for conversion back to glutamate. 1°,13,14,23,27,2s,52,56.57 It is generally considered that this glutamine "cycle" primarily recycles " u s e d " glutamate and does not contribute to the de n o v o formation of " n e w " glutamate in the CNS. 25 In this study we have sought to examine the relative contributions that de n o v o synthesis of glutamate, and the glutamine cycle, make to the maintenance of glutamate content in neurons. Our null hypothesis was that if neurons synthesize significant quantities of glutamate themselves, using Krebs cycle precursors or aspartate, then inhibition of glutamine synthetase should have little effect on the neuronal content of glutamate. To evaluate this hypothesis, we have compared the patterns of glutamate and glutamine 355

immunoreactivity in normal retinae, and in retinae where glutamine biosynthesis in glial cells had been inhibited with methionine sulphoximine (a compound which does not affect high affinity uptake of glutamate or glutamine, or the uptake and metabolism of other transmitters)/6,37,44 In addition, G A B A immunoreactivity was examined, since G A B A is synthesized in neurons from glutamate.17 Glycine and taurine- immunoreactivities were also examined since they are derived from biosynthetic pathways distinct from those which form glutamate or glutamine. 39,58 EXPERIMENTAL PROCEDURES

Five male pigmented rabbits aged three to four months were obtained from the Central Animal Breeding House, University of Queensland. They were killed with sodium pentobarbitone administered intravenously (60mg/kg), enucleated, and the retinae quickly dissected from their globes in oxygenated Ames' medium (Sigma)? Retinae were bisected along the vertical midline; two hemisections were immediately placed into fixative whilst the remainder were placed into warm (36+ I°C) oxygenated Ames' medium (controls), or an identical medium containing 2 mM D,L-methionine D,L-sulphoximine (Sigma) (experimental group). At this concentration, D,L-methionine D,L-sulphoximine specifically inhibits glutamine synthetase but does not affect high affinity uptake of glutamate or glutamine, or the uptake and metabolism of other transmitters. 4'5'6'37''~ Ames' media 2 contains glutamate (7~M) and glutamine (0.5mM), but no ct-ketoglutarate; to examine whether these exogenous molecules could gain entry to the retina in immunocytochemically detectable quantities, a further six retinae were incubated in media containing 2 mM methionine sulpboximine, but where the

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concentrations of either glutamate or glutamine had been raised five-fold, or where :~-ketoglutaratae was present at 5 mM. Retinae were maintained in these media for 90 min, and then placed into 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 6.5) for 2h, followed by fixation overnight at pH 10.1. One additional rabbit was anaesthetized with halothane and then injected intraocularly in one eye with 0.36mg D,L-methionine l),L-sulphoximine in 25/tl of Ames' media (which, assuming the eye has a volume of [ ml, gives a final methionine sulphoximine concentration of 2 mM within the eye), Alter 90 min the rabbit was killed as described above, the retinae removed and fixed as above. Samples from the medullary ray regions of control and experimental retinae were embedded together in Durcupan resin and sectioned transversely at 0.5~m. Sections were mounted on glass multiwell microscope slides (Roboz, Rockville, MD, U.S.A.) and dried down on to the slides using a hotplate (40C. The sections were then etched with 11% sodium ethoxide (5 min), washed with distilled water, and immunoperoxidase-labeled using well characterized, highly specific and sensitive polyclonal antisera for glutamate, glutamine, GABA, taurine and glycine at dilutions of 1: 600,000, 1: 350,000, l : 600,000, l : 900,000 and 1: 300,000, respectively, as described previously:~-~ The peroxidase reaction product was subsequently intensified with silver) s In some cases, serial 0.5-/tm-thick sections were cut and labelled alternately for glutamate and glutamine. Micrographs derived from such serial sections were scanned into a computer, assigned pseudocolours, and the images superimposed using Abobe photoshop. By this means the relationship between glutamate and glutamine-containing elements could be readily illustrated. Additional pieces of retinae were processed for electron microscopy as previously described:~' To illustrate the specificity and sensitivity of the antisera used in this study we have generated test sections similar to those used by Ottersen and co-workers43 consisting of pieces of gelatin, containing known concentrations of various free amino acids, which have been fixed with glutaraldehyde and embedded in resin. The rationale for this approach is based on the tenets expounded by Marc et al. 34 who suggested that relative glutamate levels in tissues can be estimated provided three conditions are met: "'(1) Under identical assay conditions the stoichiometry of antibody binding in semithin sections is the same as that observed with model antigens. (2) The intracellular cross-linking environment is similar across cell types, rendering the observed immunoreactivities primarily dependent on hapten concentration. (3) The measured immunoreactivity is due to glutamate." These tenets require that the test system be subject to similar processing conditions as tissue sections, and that the amino acid be cross-linked to a matrix of protein(s) similar to insoluble structural proteins of the brain. Porcine gelatin (BDH Australia, 150 bloom, derived from pig skin) was used as a matrix protein (to which to fix the amino acids. Briefly, a warm 10% solution of gelatin was prepared in 0.1 M phosphate buffer, pH 7.2. To aliquots of this solution, an equal volume of free amino acid solution was added. For comparisons of the specificity of each antiserum, each amino acid was made up as a 2 mM solution in distilled water (thus when added to the gelatin its final concentration would be l mM), whilst for sensitivity testing, a range of concentrations were used. When the warm gelatin amino acid solutions had cooled and gelled, they were fixed overnight with 2.5% glutaraldehyde in 0.l M phosphate buffer. pH 7.2. The gelatin blocks were then treated as if they were pieces of tissue; they were dehydrated and embedded in durcupan resin as described above. After the blocks had polymerized overnight they were sectioned on a sledge microtome, yielding 25-g~m-thick slices. These slices were re-embedded in Durcupan resin with slices from other

blocks, in the form of a "stack" of sections, containing a variety of gelatin-amino acid slices. These stacks were re-polymerized and then 0.5-~m-thick sections were cut and processed for immunocytochemistry as if they were tissue sections. RESULTS

Sensitivity and spee!fieity :~/ antisera Immunocytochemistry o f the test sections (Fig. 1A) indicated that each antiserum was highly specific for the appropriate antigen, with no evidence of detectable cross-reactivity under these conditions. The sensitivity tests (Fig. 1B, C) indicated that the lower concentration limit for detection o f amino acids was approximately 4 0 # M under the conditions employed here.

Examination o f retinae Control retinae which had been incubated in normal Ames' media for 9 0 m i n were o f good morphology as assessed electron-microscopically and were, in all respects, including their patterns o f immunocytochemical labelling, indistinguishable from tissues which had been fixed immediately after enucleation. No detectable differences were observed between retinae which had been exposed to methionine sulphoximine by way o f intraocular injection, or by exposure in vitro. Subsequent descriptions and illustrations as to the actions o f methionine sulphoximine apply equally to retinae exposed to methionine sulphoximine in vitro or in vit,o.

Glutamate immunoreaetivity In control retinae, ganglion cell somata and their axons were strongly immunoreactive for glutamate. The somata of bipolar cells were also strongly immunoreactive, and the inner segments o f rod and cone photoreceptors showed moderate immunoreactivity (Fig. 2A). There was a striking difference in the pattern o f glutamate immunoreactivity after inhibition of glutamine synthetase. Glutamate immunoreactivity was no longer observed within somata or axons of ganglion cells, nor in the somata of bipolar cells, even if the concentration of glutamate or glutamine in the bathing medium were increased five-fold or the media supplemented with 5 m M ~-ketoglutarate (not illustrated). By contrast, glutamate immunoreactivity was intense in the somata and processes o f Mfiller cells. Weak labelling was still present in the inner segments of photoreceptors (but the intensity of labelling was always less than that in control specimens) (Fig. 2C).

Glutamine immunoreactiviO' The present study is the first to describe the distribution o f glutamine in the retina. In normal retinae, glutamine immunoreactivity was present in glial (Mfiller) cells, being most a b u n d a n t in their vitread portions (Fig. 2B). However, the most

Glia make new glutamate

A

357

C

B

!i¸ i:~ii~

1

2

3

4

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¸

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Fig. 1. (A) Stack of six gelatin test sections, containing taurine, glycine, glutamate, glutamine, GABA or aspartate. The stack in row I was stained with Toluidine Blue, whilst those in rows 2 ~ have been immunocytochemically labelled for GABA, glutamine, glutamate, glycine and taurine, respectively. For each antiserum, specific labelling is restricted to the test section containing the appropriate amino acid. B and C are stacks of gelatin test sections which contain a series of concentrations (0-160/tM) of glutamate (B) or glutamine (C). B was labelled with antiserum to glutamate, C with antiserum to glutamine. Both amino acids are clearly detectable at concentrations as low as 40/~M.

intensely labelled elements were the somata of ganglion cells and bipolar cells (Fig. 2B). Glutamine immunoreactivity was absent from ganglion cell axons. After inhibition of glutamine synthetase, no glutamine immunoreactivity was observed in the retina (Fig. 2D) even when the concentration of glutamine in the bathing media was raised five-fold (not shown).

Relationship between glutamate containing elements

and glutamine-

Labelling of serial sections for glutamate or glutamine permits the graphic illustration of the relationship between glutamine and glutamatecontaining elements (Fig. 3). Somata of ganglion cells show clear co-localization of both glutamate and glutamine; axons of ganglion cells contain only glutamate, whilst Miiller cells contain only glutamine.

GABA immunoreactivity G A B A was a b u n d a n t in many amacrine cells and displaced amacrine cells (Fig. 4A); no label was seen in photoreceptors, M/iller cells, bipolar or horizontal cells. The majority of ganglion cells did not appear to contain GABA. After inhibition of

glutamine synthetase a similar distribution of GABAimmunoreactive cells was observed, but the cells were only weakly labelled (Fig. 4B).

Glycine immunoreact&ity Glycine immunoreactivity was restricted to amacrine cells, a few displaced amacrine cells and occasional ganglion cells (Fig. 4C, D); light labelling was also observed in the somata of some bipolar cells. No changes in the patterns or intensities of labelling were observed after inhibition of glutamine synthetase.

Taurine immunoreactivity In normal retinae, taurine immunoreactivity was a b u n d a n t in M/iller cells, particularly their vitread portions, and in photoreceptors, whilst labelling was absent from ganglion, amacrine, bipolar and horizontal cells (Fig. 4E). No change in the pattern or intensity of labelling was observed after inhibition of glutamine synthetase (Fig. 4F).

Electron -microscopy Examination of transversely sectioned retinae revealed that glial (M/iller) cell processes completely wrapped most synapses, including the glutamatergic

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Fig. 2. Light micrographs of radial sections through the medullary ray regions of rabbit retinae. (A) Control retina that has been immunolabelled for glutamate. The radial processes of M/iller cells are conspicuous by their lack of immunoreactivity, while ganglion cell somata and axons are densely labelled. (B) Control retina that has been immunolabelled for glutamine. Both M/iller cells and ganglion cell somata are intensely labelled. Ganglion cell axons are unlabelled. (C) Glutamine synthetase-inhibited retina, that has been immunolabelled for glutamate. Glutamate-labelling is prominent in Mfiller cells but it is absent from neurons. (D) Glutamine synthetase-inhibited retina that has been immunolabelled for glutamine. Neither neuronal nor glial elements are labelled. M, M/iller cell; Ax, axon bundles in the nerve fibre layer; arrows indicate the somata of ganglion cells. Scale bar = 50 ~m.

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Fig. 3. Pseudocolour image showing two serial 0.5-p m-thick sections of retina labelled for glutamate and glutamine, respectively (see Fig. la and b, for comparison). Structures which contain only glutamine (M/iller cells) are coloured green; structures which contain only glutamate, such as ganglion cell axon bundles, are colour-coded red. Where glutamate and glutamine are co-localized such as in the somata of ganglion cells, the structures are coloured yellow. On the basis of this type of illustration we suggest that the somata of ganglion cells are the sites of glutamine uptake from the Miiller cells; the glutamine is then converted into glutamate and then exported down the axons.

p h o t o r e c e p t o r synapses (identified by the presence o f synaptic ribbons) (Fig. 5). All n e u r o n a l s o m a t a were completely encapsulated by the M/iller cells, as were o t h e r n e u r o n a l c o m p a r t m e n t s such as dendrites a n d axons (Fig. 6).

DISCUSSION

The antisera used in this study were d e m o n s t r a t e d to be highly specific as assessed by o u r test system; m o r e o v e r the sensitivity tests indicated that the lower

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Fig. 4. Normal retina (A, C, E) and glutamine synthetase-inhibited retina (B, D, F) immunolabelled for GABA, glycine and taurine. GABA immunocytochemistry (A, B) reveals similar populations of amaerine cells in both preparations, but after inhibition of glutamine biosynthesis (B) there is a noticeable decrease in the intensity of GABA immunoreactivity, lmmunolabelling for glycine in normal (C) and glutamine synthetase-inhibited retina (D) reveals the presence of glycine in some amacrine cells. There is no apparent difference in the pattern of the immunolabelling in the two preparations. Taurine-immunoreactivity is identical in normal (E) and inhibited (F) retinae: label is present in Mfiller cells and photoreceptors. Scale bar 501~m.

Glia make new glutamate

Fig. 5. Electron-micrograph of tissue from a retina, which had been incubated for 90 min in control media, showing the intimate association between Miiller cell processes (m) and the glutamate-containing photoreceptor terminals (p) in the outer plexiform layer, which are characterized by the presence of ribbon synapses. Scale bar = 0.5/~m. Fig. 6. Electron micrograph of tissue from a retina which had been incubated for 90 min in control media, showing M/iller cell processes (m) surrounding somata (S) and dendrites (D) of retinal neurons. Scale bar = 0.5 ~m.

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concentration limit for detection of amino acids in our test system was approximately 4 0 # M , under the conditions employed here. Assuming that the detection sensitivity of the test system, and the detection sensitivity in tissue sections is comparable, then cells containing concentrations of glutamate or glutamine below 4 0 # M would be classified as immunonegative. Synthesis of glutamate

This study shows, for the first time, that the synthesis of glutamine from glutamate by glial cells is essential for the maintenance of a content of glutamate in at least some neurons, such as ganglion cells. Moreover the lack of any immunocytochemically detectable glutamate in such neurons after inhibition of glutamine synthesis indicates that these neurons do not themselves make significant amounts of glutamate from aspartate, or substrates derived from the Krebs cycle. The inhibitor of glutamine biosynthesis used in this study (methionine sulphoximine) did not perturb the distribution of other metabolically unrelated amino acids such as glycine, in neuronal or glial elements. Glutamate is a precursor for GABA; L7 hence it was not surprising that depletion of neuronal glutamate also led to a partial depletion of GABA in GABAergic neurons. Co-localization of glutamate and glutamine in neuronal somata

Since ganglion cells lack glutamine synthetase, they cannot synthesize their own glutamine. 33'42 Therefore the presence of glutamine in somata of these neurons suggests that they must import it, and, moreover, that the somata are probably the sites of such importation. This accords with previous suggestions that high-affinity uptake sites for glutamine are restricted to neuronal somata. 65°56 The lack of detectable glutamine in the axons of ganglion cells and the presence of glutamate in both the somata and axons of these cells suggests that glutamine is converted into glutamate in the somata before being transported down the axons, to be used by their synaptic terminals, several centimetres away. The depletion of the glutamate reserves in ganglion cells after methionine sulphoximine treatment is presumably a consequence both of this transport process, and of the failure of glia to re-supply the neurons with new glutamine. Source ~[ glutamine in neuronal somata

Glutamine in the extracelullar spaces surrounding the somata of ganglion cells and bipolar cells must originate either from MiJller cells, or from extra-retinal sources, such as blood or vitreous humour. The latter sources can be ruled out because immunoreactivity for glutamine and glutamate was absent from ganglion and bipolar cells after inhibition of glutamine synthetase, despite the presence of significant amounts of glutamate and

glutamine in the culture media. 2 Similarly, after D,Lmethionine O,L-sulphoximine had been administered intraocularly in a living rabbit, glutamate was also absent from ganglion cells and bipolar cells, but present in Miiller cells (not illustrated). In this latter situation, retinal cells have normal access to any molecular species transferred from blood or vitreous humour. Taken together, these observations indicate that ganglion cells and bipolar cells in the retina obtain their content of glutamine exclusively from Miiller cells, and that such glutamine is the sole precursor for conversion back to glutamate in these neurons. Structural features contributing to metabolic relationships between neurons and gila

We suggest that in the retina, the complete ensheathment of neuronal elements by glial cells forms a barrier which prevents exogenous glutamate or glutamine from reaching neuronal uptake sites. Consequently, only glial cells can directly supply neurons with molecules such as glutamate or glutamine. This concept of a permeability barrier is consistent with the observations of other researchers who have shown that threshold doses for neuronal stimulation by exogenous glutamate are 2 3 log units higher in intact tissue than in isolated neurons.~,8.9.19.51 Glutamate in glial cells

In control retinae, glial cells lack a detectable content of glutamate. This concurs with the observations of Marc et al., 34 with respect to the goldfish retina where it has been suggested that the concentration of glutamate in Mfiller cells is probably two orders of magnitude lower than that in bipolar cells, being about 50 #M (or less), which places it at, or below, the limit of immunocytochemical detection. The Miiller cells do, however, contain an abundance of glutamine. This apparent anomaly arises because of the presence of glutamine synthetase in glial cells, which catalyses the rapid conversion of glutamate into glutamine. 7~5'3~ 3~,49 Consequently, when glutamine synthetase is inhibited, abundant glutamate immunoreactivity is detectable in Mfiller cells (Fig. 2C). What are the roles ~)/ glia and neurons in de novo glutamate synthesis?

The carboxylation of pyruvate (derived from glucose) to oxaloacetate is the principal means for ultimately generating a net excess of ~-ketoglutarate. This carboxytation reaction is catalysed by pyruvate carboxylase, an enzyme present only in glia. This localization, which has been extensively documented, 27 implicates glia in one of the steps leading to the de novo synthesis of glutamate. It is not generally appreciated, however, that several other essential enzymes leading to to formation of glutamate also appear to be preferentially localized within

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Glia make new glutamate

GLUCOSE

glutamate, it is probable that in the CNS, much of the d e n o v o synthesis of glutamate occurs principally in glia. This conclusion is supported by our new PYRUVATE observations that inhibition of the glial enzyme glutamine synthetase causes an accumulation of glutamate in glial cells, rather than in neurons, OXALOACETATE and moreover, to a total lack of glutamate immunoreactivity in two classes of glutamate-using CITRATE neurons. A schemata of the proposed relationship is illustrated in Fig. 8. However, other synthetic pathways may be employed in the synthesis of glutamate. Aspartate amino transferase catalyses the interconversion of aspartate and glutamate, whilst glutamate dehydrogenase, converts ~-ketoglutarate (present in blood) to glutamate. However, neither of these enzymes are present in ganglion cells or bipolar cells, 1'2°'55 even though they are present in retinal glial cells. 2°'53 I r "6"~'] ASPARTATE Furthermore, when retinae are exposed to methionine sulphoximine in Ames' media which has been GLUTAMINE supplemented with 5 m M ~-ketoglutarate, glutamate Fig. 7. Schema(based on Ref. 26), outlining the major was still absent from the ganglion cells and bipolar metabolicpathwaysinvolvedin the synthesis,and recycling cells. By contrast photoreceptors contain both asparof glutamate and the cellular localization of the enzymes, tate amino transferase and glutamate dehydrowhere known. Glutamate is converted into pyruvate by genase, L2°'55indicating that they may have a capacity glycolysis. Pyruvate is converted into oxaloacetate using the enzyme pyruvate carboxylase (PC) which, in the brain, is to use aspartate or ct-ketoglutarate as substrates for located only in glial cells. This oxaloacetate enters the the d e n o v o synthesis of glutamate, as an adjunct to Krebs cycle. Within the Krebs cycle, citrate synthase the use of glutamine. Photoreceptors also have the (CS) converts oxaloacetate into citrate, which is then ability to take up glutamate by an electrogenic transconverted into isocitrate by aconitase (A). Isocitrate dehyporter. ~6This uptake is likely to occur via their outer drogenase (ID) converts isocitrate into ct-ketoglutarate. -Ketoglutarate leaves the Krebs cycle and is converted by segments which extend into the sub-retinal space glutamate dehydrogenase (GD; which is, in cerebellum (where they are not enveloped by glial processes), and retina, predominantly within glia), 2°m thereby forming whilst their synaptic terminals in the outer plexiform glutamate. Aspartyl amino transferase (AAT) catalyses layer are thought to take up and recycle some of the the reversible interconversion of aspartate and glutamate. Glutamate may also be used to form glutamine using the glial enzyme glutamine synthetase (GS). This enzyme is inhibited by methionine sulphoximine (MS). Glutamine can be converted back into glutamate by the enzyme phosphateactivated glutaminase (PAG). Glutamate may serve as a precursor for formation of GABA, using the enzyme glutamate decarboxylase (GAD). In turn GABA may reenter the citric acid cycle as succinate by means of the GABA shunt.

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glia (Fig. 7). Thus: (1) The metabolic toxin, fluorocitrate, acts on the Krebs cycle at the level of the enzyme aconitase (which converts citrate to isocitrate). Fluorocitrate impairs metabolism in glial cells, but spares neurons. 24 This specificity suggests either that glia, and not neurons contain aconitase, or that fluorocitrate is selectively accumulated by glia. (2) Isocitrate dehydrogenase which catalyses the conversion of isocitrate to ~t-ketoglutarate, is present at high levels in astrocytes relative to average brain content? 4 (3) Glutamate dehydrogenase which converts ~t-ketoglutarate to glutamate is present predominantly in glia in cerebellum, hippocampus and retina.2°' 53 Given that glia possess many of the key enzymes required for the conversion of pyruvate into NSC 60/2 D

O

n0, on

/ aLNX.._ Fig. 8. Schema of the deduced flux of glutamine between glia and neurons in the retina. Glucose taken up by Mfiller cells is converted into pyruvate, which is then converted, via the Krebs cycle to form ~-ketoglutarate and then glutamate (GLU). This glutamate is converted into glutamine (GLN) and then provided to ganglion cells and bipolar cells. These neurons convert the glutamine back into glutamate in their somata using phosphate-activated glutaminase. Ganglion cells transport the glutamate from their somata into their axons. The dashed lines represent the inner and outer limiting membranes of the retina.

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glutamate which they have released. ~ Nevertheless, uptake or re-uptake of glutamate, or synthesis of glutamate from aspartate or c~-ketoglutarate does not supply the entire glutamate requirements of the photoreceptors because the intensity of glutamateimmunoreactivity in photoreceptors is always diminished after methionine sulphoximine treatment. Thus, some glial-derived glutamine is p r o b a b l y required to m a i n t a i n the n o r m a l glutamate content of photoreceptors. Functional significance

Since ganglion cells utilize glutamate as their principal neurotransmitter, TM~-''1829'4°41 their dependence on Mfiller cells provides the latter with the potential to influence signal t r a n s d u c t i o n in the retinofugal pathway. It is not yet k n o w n whether glutamatergic neurons in o t h e r parts of the central nervous system have a similar dependency on glia, but this possibility deserves consideration because it has implications that extend well beyond the m o d u l a t i o n of neuronal signals by glia. Neurons that are dependent on glia for g l u t a m a t e (or G A B A , which is derived within neurons from glutamate), will be extremely sensitive

to any changes in the ability of glia to synthesise or release glutamine. CONCLUSION This study shows that: (i) some retinal glutamatergic neurons are d e p e n d e n t upon an exogenous source of glutamine for their content of glutamate; (ii) glial cells are the only source of this glutamine; and (iii) glutamine is not synthesized exclusively from glutamate that has been released from neurons. These findings, c o m b i n e d with the fact that glia, rather than neurons, possess m a n y of the critical enzymes involved in de novo synthesis of glutamate, lead us to suggest that, in the retina at least, it is glia, rather than neurons, that are the source of glutamate. Preliminary reports on this work have been made in abstract form. 47"4~This work was supported by an NH&MRC project grant to Dr D. I. Vaney. We thank Dr V. Balcar for insightful discussions, Drs Vaney, Wong and Prof. Pettigrew for helpful comments on the manuscript, and D. Crook and D. Noone for technical assistance. D.V.P. was supported by an NH&MRC project grant to Dr David Vaney, and S.R.R. by an ARC Research Fellowship. Acknowledgements

REFERENCES 1. Altschuler R. A., Mossinger J. L., Harmison G. G., Parakkal M. H. and Wenthold R. J. (1982) Aspartate aminotransferase-like immunoreactivity as a marker for aspartate/glutamate in guinea pig photoreceptors. Nature 298, 657 659. 2. Ames A. and Nesbett F. B. (1981) In zitro retina as an experimental model of the central nervous system. J. Neurochem. 37, 867 877. 3. Ariel M., Laseter E. M., Mangel S. C. and DoMing J. E. (1984) On the sensitivity of HI horizontal cells of the carp retina to glutamate, aspartate and their agonists. Brain Res. 295, 179 183. 4. Balcar V. J. and Johnston G. A. R. (1972) The structural specificity of the high affinity uptake of L-glutamate and L-aspartate by rat brain slices. J. Neurochem. 19, 2657 2666. 5. Batcar V. J. and Johnston G. A. R. (1973) High affinity uptake of transmitters: studies on the uptake of L-aspartate, GABA, L-glutamate and glycine in cat spinal cord. J. Neurochem. 20, 529 539. 6. Balcar V. J. and Johnston G. A. R. (1975) High affinity uptake of L-glutamine in rat brain slices. J. Neurochem. 24, 875-879. 7. Barbour B., Brew H. and Attwell D. (1990) Properties of electrogenic glutamate uptake in retinal gila. In Sensory Transduetion (eds Borsellino A., Cervetto L. and Torre V.), pp. 247 256. Plenum Press, New York. 8. Bloomfield S. A. and Dowling J. E. (1985) Roles of aspartate and glutamate in synaptic transmission in rabbit retina. 1. Outer plexiform layer. J. Neurophysiol. 53, 699 713. 9. Bloomfield S. A. and DoMing J. E. (1986) Roles of aspartate and glutamate in synaptic transmission in rabbit retina. II. Inner plexiform layer. J. Neurophysiol. 53, 714 725. 10. Bradford H. F., Ward H. K. and Thomas A. J. (1978) Glutamate as a substrate for nerve endings. J. Neurochem. 30, 1453 1459. 11. Canzek V., Wolfensberger M., Amsler U. and Cu~nod M. (1981) In vivo release of glutamate and aspartate following optic nerve stimulation. Nature 293, 572 574. 12. Castel M., Belenky M., Cohen S., Ottersen O. P. and Storm-Mathisen J. (1993) Glutamate-like immunoreactivity in retinal terminals of the mouse suprachiasmatic nucleus. Eur. J. Neurosci. 5, 368 381. 13. Curtis D. R. and Watkins J. C. (1960) The excitation and depression of spinal neurones by structurally related amino acids. J. Neurochem. 6, 117 141. 14. Curtis D. R. and Watkins J. C. (1965) The pharmacology of amino acids related to gamma-aminobutyric acid. Pharmac. Rev. 17, 347-391. 15. Ehinger B., Ottersen O. P., Storm-Mathisen J. and DoMing J. E. (1988) Bipolar cells in the turtle retina are strongly immunoreactive for glutamate. Proc. natn. Acad. Sci. U.S.A. 85, 8321 8325. 16. Eliasof S. and Werblin F. (1993) Characterization of the glutamate transporter in retinal cones of the tiger salamander. J. Neurosci. 13, 402M11. 17. Erecinska M. and Silver 1. A. (1990) Metabolism and role of glutamate in mammalian brain. Prog. NeurobioL 35, 245 296. 18. Fonnum F. and Henke H. (1982) The topographical distribution of alanine, aspartate, ?,-amino butyric acid, glutamate, glutamine, and glycine in the pigeon optic rectum and the effect of retinal ablation. J. Neurochem. 38, 1130 1134. 19. Garthwaite J. (1985) Cellular uptake disguises action of L-glutamate on N-methyl-D-aspartate receptors. Br. J. Pharmae. 85, 297-307.

Glia make new glutamate

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20. Gebhard R. (1992) Histochemical demonstration of glutamate dehyrogenase and phosphate-activated glutaminase activities in semithin sections of the rat retina. Histochemistry 97, 101-103. 21. Hall Z. W. (1992) An Introduction to Molecular Neurobiology. Sinauer, Sunderland MA. 22. Hamberger A., Cotman C. W., Sellstr6m /~. and Weiler C. T. (1978) In Dynamic Properties o f Glial Cells (eds Schoffeneils E., Frank G., Hertz L. and Tower D. B.), pp. 163-172. Pergamon Press, Oxford. 23. Hamberger A. C., Chiang G. H., Nylen E. S., Scheff S. W. and Cotman C. W. (1979) Glutamate as a CNS transmitter. I. Evaluation of glucose and glutamine as precursors for the synthesis of preferentially released glutamate. Brain Res. 168, 513-530. 24. Hassel B., Paulsen R. E., Johnsen A. and Fonnum F. (1992) Selective inhibition of glial cell metabolism in vivo by fluorocitrate. Brain Res. 576, 120-124. 25. Hertz L. (1989) Is Alzheimer's disease an anterograde degeneration, originating in the brainstem, and disrupting metabolic and functional interactions between neurons and glial cells? Brain Res. Rev. 14, 335-353. 26. Hertz L. and Peng L. (1992) Energy metabolism at the cellular level of the CNS. Can. J. Physiol. Pharmac. 70, Suppl. 145 157. 27. Hertz L., Peng L., Westergaard N., Yudkoff M. and Schousboe A. (1992) Neuronal-astrocyte interactions in metabolism of transmitter amino acids of the glutamate family. In Drug Research Related to Neuroactive Amino Acids (eds Schousboe A., Diemer N. H. and Kofod H.), pp. 3048. Munksgaard, Copenhagen. 28. Kaneko T. and Mizuno N. (1992) Mosaic distribution of phosphate-activated glutaminase-like immunoreactivity in the rat striatum. Neuroscience 49, 329-345. 29. Kemp J. A. and Sillito A. M. (1982) The nature of the excitatory transmitter mediating X and Y cell inputs to the cat dorsal lateral geniculate nucleus. J. Physiol. 323, 377 391. 30. Kempski O., von Andrian U., Schurer L. and Baethmann A. (1990) Intravenous glutamate enhances edema formation after a freezing lesion. Adv. Neurol. 52, 219-223. 31. Linser P. and Moscona A. A. (1979) Induction of glutamine synthetase in embryonic neural retina: localisation in M/Jller fibres and dependence on cell interactions. Proc. natn. Acad. Sci. U.S.A. 76, 647~6480. 32. Linser P. J. (1991) Comparative immunochemistry of elasmobranch retina Muller cells and horizontal cells. J. exp. Zool., Suppl. 5, 88-96. 33. Linser P. J., Sorrentino M. and Moscona A. A. (1984) Cellular compartmentalization of carbonic anhydrase-C and glutamine synthetase in developing and mature mouse neural retina. Devl Brain Res. 13, 65-73. 34. Marc R. E., Liu W.-L. S., Kalloniatis M., Raiguel S. F. and Van Haesendonck E. (1990) Patterns of glutamate immunoreactivity in the goldfish retina. J. Neurosci. 10, 4006-4034. 35. Massey S. C. (1990) Cell types using glutamate as a neurotransmitter in the vertebrate retina. Prog. Ret. Res. 9, 399-425. 36. Mayer M. L. and Westbrook G. L. (1987) The physiology of excitatory amino acids in the vertebrate central nervous system. Prog. Neurobiol. 28, 197-276. 37. Meister A. (1974) Glutamine synthetase of mammals. In The Enzymes (ed. Boyer P. D.), vol. 10, pp. 699-754. Academic Press, New York. 38. Merchenthaler I., Gallyas F. and Liposits Z. (1989) Silver intensification in immunocytochemistry. In Techniques in lmmunocytochemistry (eds Bullock G. R. and Petrusz P.), Vol. 4, pp. 217-252. Academic Press, New York. 39. Metzler D. E. (1977) Biochemistry: The Chemical Reactions o f Living Cells, p. 847. Academic Press, New York. 40. Montero V. M. and Wenthold R. J. (1989) Quantitative immunogold analysis reveals high glutamate levels in retinal and cortical synaptic terminals in the lateral geniculate nucleus of the macaque. Neuroscience 31, 639~i47. 41. Morino P., Bahro M., Cu6nod M. and Streit P. (1991) Glutamate-like immunoreactivity in the pigeon optic tectum and effects of retinal ablation. Eur. J. Neurosci. 3, 366-378. 42. Moscona A. A. (1983) On glutamine synthetase, carbonic anhydrase and M/iller glia in the retina. Prog. Ret. Res. 2, 111 135. 43. Ottersen O. P., Zhang N. and Walberg F. (1992) Metabolic compartmentation of glutamate and glutamine: morphological evidence obtained by quantitative immunocytochemistry in rat cerebellum. Neuroscience 46, 519-534. 44. Pace J. and McDermott E. E. (1952) Methionine sulfoximine and some enzyme systems involving glutamine. Nature 169, 415-416. 45. Pow D. V. (1993) Immunocytochemistry of amino acids in the rodent pituitary using extremely specific, very high titre antisera. J. Neuroendocr. 5, 349 356. 46. Pow D. V. and Crook D. K. (1993) Extremely high titre antisera against small neurotransmitter molecules: rapid production, characterisation and use in light- and electron-microscopic immunocytochemistry. J. Neurosci, Meth. 48, 51~63. 47. Pow D. V. and Robinson S. R. (1993) Retinal glutamatergic neurones are totally dependent on glia for their neurotransmitter content. Proc. int. Union Physiol. Socs 244, 12P. 48. Pow D. V., Robinson S. R. and Noone D. (1993) Compartmentation of amino acids in the retina: interrelationships between glia and neurones. Proc. Aust. Neurosci. Soc. 4, 118. 49. Riepe R. E. and Norenberg M. D. (1977) M/iller cell localisation of glutamine synthetase in rat retina. Nature 268, 654~555. 50. Roberts P. J. and Keen P. (1974) High-affinity uptake system for glutamine in rat dorsal roots but not in nerve-endings. Brain Res. 67, 352-357. 51. Rosenberg P. A. and Aizenman E. (1989) Hundred-fold increase in neuronal vulnerability to glutamate toxicity in astrocyte-poor cultures of rat cerebral cortex. Neurosci. Lett. 103, 162 168. 52. Ross C. D., Bowers M. and Godfrey D. A. (1987) Distribution of glutaminase activity in retinal layers of rat and guinea pig. Brain Res. 401, 168-172. 53. Rothe F., Wolf G. and Schunzel G. (1990) Immunohistochemical demonstration of glutamate dehydrogenase in the postnatally developing rat hippocampal formation and cerebellar cortex: comparison to activity staining. Neuroscience 39, 419-429. 54. Rust R. S. Jr, Carter J. G., Martin D., Nerbonne J. M., Lampe P. A., Pusateri M. E. and Lowry O. H. (1991) Enzyme levels in cultured astrocytes, oligodendrocytes and Schwann cells, and neurons from the cerebral cortex and superior cervical ganglia of the rat. Neurochem. Res. 16, 991499.

366

D.V. Pow and S. R. ROBINSON

55. Sarthy P. V., Hendrickson A. E. and Wu J.-Y. (1986) L-glutamate: a neurotransmitter candidate for cone photoreceptors in the monkey retina. J. Neurosci. 6, 637~43. 56. Schousboe A., Hertz L., Svenneby G. and Kvamme E. (1979) Phosphate activated glutaminase activity and glutamine uptake in primary cultures of astrocytes. J. Neurochem. 32, 943 950. 57. Shank R. P. and Aprison M. H. (1981) Present status and significance of the glutamine cycle in neural tissues. Lil~' Sci. 28, 837-842. 58. Stryer L. (1975) Biochemisto,, p. 491. W. H. Freeman, San Francisco.

(Accepted 5 January 1994)