Gen. Pharmac., 1976, Vol. 7, pp. 321 to 332. Pergamon Press. Printed in Great Britain

MINIREVIEW AMINO ACID TRANSMITTER SUBSTANCES IN THE VERTEBRATE RETINA MIO-U~FI J. NF~d. Department of Pharmacology, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N lAX, England

(Received 23 March 1976)

INTRODUCTION

The retina originates embryologically as an outgrowth of the diencephalon, and apart from the photoreceptor layer, it can be regarded as grey matter. It is reasonable to assume that the retina utilizes the same transmitter substances as the rest of the central nervous system and so study of retinal transmitters, in addition to providing information on visual physiology, may also provide dues to understanding central synaptic mechanisms in general The retina possesses several experimental advantages for the study of transmitter substances. In particular, it is the only area of the CNS which can be isolated in vitro, intact, without any preliminary slicing or chopping procedures. In addition, it can be stimulated physiologically with flashes of light and the response of the tissue can be easily monitored by recording the electroretinogram (e.r.g.). The organization of the retina is relatively well understood and its well-defined layered structure facilitates neurochemical studies. In spite of these apparent advantages, the retina until recently has been relatively ignored in the investigation of central transmitters. However, the increasing interest in amino acid transmitters in the retina is shown by the fact that in the present brief review most of the papers quoted have appeared in the last five years. ORGANIZATION OF THE VERTEBRATE RETINA

Cellular organization Before discussing retinal transmitters it may be helpful to briefly outline the organization of the retina [see Dowling (1970) for review]. As illustrated in Fig. 1, the retina consists of essentially five types of neurones and one type of glial cell (Mtiller fibres). The cell bodies are arranged in three nuclear layers (the outer nuclear, inner nuclear, and ganglion cell layer) and the synapses are arranged in two plexiform layers. In each plexiform layer the processes of three cell types interact. The photoreceptor cell bodies are located in the outer nuclear layer:

in the outer plexiform layer these cells synapse with bipolar and horizontal cells whose perikarya are located in the outer half of the inner nuclear layer. The amacrine cells occur in the inner half of the inner nuclear layer. In the inner plexiform layer, the bipolar and amacrine cells synapse with one another and with ganglion cells, whose perikarya are located along the inner margin of the retina. The neuroglial Miiller cells extend radially across the retinal layers, i.e. parallel to the photoreceptor and bipolar cells.

Outer plexiform layer In the outer plexiform layer, the terminals of the photoreceptors synapse with horizontal and bipolar cells. The precise anatomical arrangements differ with species and according to whether the receptors are cones or rods. The organization of receptor terminals with second order retinal neurones is illustrated in Fig. 1 and Fig. 2. The photoreceptor terminal contains an electron dense "ribbon" surrounded by a "halo" of synaptic vesicles and an invagination, into which are inserted two horizontal cell processes and one bipolar process (triad). In addition to these ribbon synapses, the outer plexiforrn layer also possesses "conventional" synapses. These are made between processes of horizontal cells with other horizontal ceils and with bipolar cells. Flat bipolar cells synapse with photoreceptor pedicles by superficial contacts and not by ribbon synapses (Fig. 1).

Inner plexiform layer This layer is much thicker than the outer synaptic layer and differs greatly between species. The bipolar terminals contact amacrine and ganglion cell processes at ribbon synapses. The ribbon terminal of the bipolar cell nearly always synapses with two postsynaptic processes (dyad) (Figs. l and 2), i.e. ganglion cell dendrite and amacrine cell process; two amacrine processes; or rarely, two ganglion cell dendrites. The frequency of the particular pairings depends on the species. Amacrine cell processes form conventional synapses on bipolar terminals, ganglion cell dendrites, and other amacrine cell processes. At many dyads, 321

MICHAELJ. NEAL

322

OUTER LIMBS

"--- O.L.M. OUTER OUTER

INNER

NUCLEAR PLEXIFORM

NUCLEAR

INNER PLEXIFORM

GANGLION CELL NERVE FfBRE I.L.M.

Fig. 1. Diagram of vertebrate retina. In the outer plexiform layer, photoreceptor (R) cell pedicles synapse with processes from bipolar (B) and horizontal (H) cells. Horizontal cells make conventional synaptic contacts on to bipolar dendrites and other horizontal cell process (not shown). In the inner plexiform layer, bipolar terminals may contact one ganglion cell dendrite (G) and one amaerine cell process (A) at ribbon synapses or two amacrine cell processes. When the latter arrangement predominates in a retina, numerous conventional synapses between amacrine processes (serial synapses) are observed and ganglion cells are contacted mainly by amacrine processes (right side of diagram). Amacrine processes in all retinae made synapses of the conventional type back on to bipolar terminals (reciprocal synapses). The glial Mtiller cells (M) run parallel to the photoreeeptor and bipolar cells and extend from the outer limiting membrane (O.LM.) to the inner limiting membrane (I.L.M.) [Modified from Dowling (1970)], an amacrine cell process synapses back on to the bipolar terminal (reciprocal synapse) (Fig. 2) suggesting that feedback interaction may occur between the amacrine process and the bipolar cell presynaptic terminal. TRANSMITrERS IN THE RETINA In addition to the putative amino acid transmitters which are the subject of this review, the reader should be aware that there is much evidence to suggest that ACh and dopamine are retinal transmitter substances. Both ACh and dopamine are probably transmitters released from different populations of amacrine cells, although in some species and particularly in invertebrates, ACh may be a photoreceptor transmitter [for reviews see GRAHAM (1974); N ~ L (1976), ACh; EHINGER (1976), dopamine]. Glutamate and/or aspartate may be the transmitters released from photoreceptor and possibly bipolar cell terminals. The inhibitory amino acids, 7-aminobutyric acid (GABA), glycine, and taurine have all been proposed as retinal transmitters that might be released from the terminals of horizontal and amacrine cells, since these neurones are known to be inhibitory.

7-AMINOBUTYRICACID (GABA) Content and distribution GABA is present in the retinae of mammals, amphibia, birds and fish (Kojima et al., 1958; Kubic'ek & Dol6nek, 1958; Graham et al., 1970; Kuriyama et al., 1968; Lain, 1972; Macaione, 1972; Pasantes-Morales et al., 1972a; Starr, 1973; Cohen et al., 1973). The concentration of GABA in the whole retina of most species is comparable with that found in whole brain and is in the range of about 1 3 ~mole/g wet weight. The higher concentration of GABA in frog and goldfish retinae may be related to their more complex organization. The retinal levels of "transmitter" amino acids in some species are given in Table 1. The intraretinal localization of the GABA system has been studied in varying detail by the use of selective degeneration and microdissection techniques. In rat retinae where the photoreceptors were destroyed by injecting the animals with iodoacetate and malate and in mice retinae with congenital absence of photoreceptors, the GABA levels were almost the same as normal control retinae. In contrast, destruction of the inner layers with glutamate was associated with a

Amino acid transmitter substances

ri

bbon~

I ~m

,

A

Fig. 2. Synaptic contacts in the vertebrate retina. (A) Synapse of rod photoreceptor pedicle with one bipolar and two horizontal cell H process (triad). (B) Cone terminal forming two triads. In cones, three bipolar and two horizontal processes may synapse in an invagination. S indicates superficial contact. (C) Synapse in inner plexiform layer between a bipolar terminal B and amacrine A and ganglion cell G processes. Note reciprocal synapse (arrows show direction of transmission). (D) Serial type of synapse found in inner plexiform layer of complex retinae. large decrease in retinal GABA (Macaione, 1972; Cohen et al., 1973). A finer localization of GABA was reported by Kuriyama et al. (1968) who dissected the rabbit retina into receptor, bipolar, ganglion cell and nerve fibre layers and found that the ganglion cell layer, which also included the inner synaptic layer, Table 1. Levels of some free amino acids in light- and dark-adapted retinae from different species Amino

acid c o n c e n t r a t i o n

(~moles/g

w e t weight)

Species Taurine Chicken

Rat

Goldfish

Frog

Glutamate

Aspartate

GABA

Glycine

L

i0.2

4.6

0.3

3.7

2.0

D

9.8

4.2

0.3

3.4

2.0

L

12.7

.4.7

1.4

1.9

2.3

D

13.3

4.4

1.5

1.9

2.5

L

14.0

1.7

0.3

2.0

0.6

D

13.0

1.7

0.3

1.4"

0.7

L

12.2

3.9

0.7

2.7

1.6

D

12.7

4.0

0.7

2.2*

1.7

contained the highest GABA and GAD activity. In a similar type of study, Graham (1972, 1974) dissected the frog and rat retina into six layers and showed that there were large regional differences in G A D activity (Table 2). In both rat and frog retinae the highest GAD activity was in the inner synaptic layer, followed by the amacrine-rich bipolar and ganglion cell layers, both of which had about half the activity of the inner synaptic layer. The horizontal cell layer had about 25~o of the activity of the inner synaptic layer, and in agreement with degeneration experiments, the photoreceptors had little or no GAD activity. There was much less difference in the actual levels of GABA in the different layers possibly due to Miiller cell accumulation of GABA released into the extracellular space. In this respect it is interesting that histochemical studies indicate that in the retina, the glial cells appear to be the main site of GABA catabolism (Hyde & Robinson, 1974; Moore & Gruberg, 1974). On the assumption that GABA is a retinal transmitter, it has been reasoned from these distribution studies that since the optic nerve does not contain GABA, the ganglion cells themselves are unlikely to contain GABA. Also, the bipolar cells are unlikely to be GABAergic since they are excitatory. Thus, the most likely neuronal source of the retinal GABA is the amacrine cells which are inhibitory in function. The smaller amounts of GABA in the outer synaptic layer may be associated with the horizontal cells. This would be consistent with the inhibitory function of these cells, and Lam (1975) has recently shown that isolated fusiform axons from goldfish horizontal cells are capable of synthesizing GABA but not ACh or dopamine. The adaptational state of the retina might be expected to influence transmitter systems since the neuronal activity of the light-adapted retina is greater than that of the completely dark-adapted retina. The influence of the adaptational state on the retinal GABA system has been studied but unfortunately the results vary considerably in different species and have proved difficult to interpret in functional terms. In the rat and chicken retina, GAD, GABA-T and GABA levels were unaltered by the adaptational state (Starr, 1973) but the dark-adapted frog retina possessed significantly less G A D activity and GABA than the light-adapted retina (Table 1) (Graham et al., 1970; Starr, 1973). Dark-adapted goldfish retinae were Table 2. GABA content and GAD activity in layers of dark-adapted frog retina (from Graham, 1972) Retinal

.....

L = light-adapted. D = dark-adapted. * Significant difference between light- and dark-adapted retinae. (From Starr, 1973.) Note the strikingly high concentration of taurine and the relatively low concentrations of glutamate and aspartate in the retina compared with whole brain, G.P. 7 / 5 - - s

323

Receptor Receptor Horizontal o....................... seg~nts bodies bipolar

AmacrJne ich sI~e~ic Gang .... bipolar Y p cell+fibre

GABA

~.~o~e~/g~

1.82

dry weight

G AD activity ,~le,/h/g d~y

weight

5.84

14.8

21.6

30.1

26.4

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MICHAEL J. NEAL

also found to have lower GABA levels than lightadapted retinae, although in this species, no change in G A D activity was observed (Lam, 1972; Starr, 19731. Pasantes-Morales et al. (1973b) found that retinae of chicks reared in continuous light conditions for 6 days had significantly higher GABA levels than those from animals reared in continuous darkness, a result consistent with those obtained from the frog and goldfish retina, but different from Starr's (1973) findings using adult chicken retinae. In an attempt to obtain further information on the effects of adaptation on the GABA system, Starr (1975) studied the effect of light stimulation on the incorporation of label from [14C]glucose or [3H]acetare into GABA, glutamate, aspartate and glutamine, both in vitro and in vivo, in dark-adapted frog and rat retinae. It was found in the frog that with [14C]glucose, but not with [3H]acetate, the in vivo synthesis of GABA and glutamine was stimulated by light at the expense of glutamate. This light evoked increase in GABA turnover probably occurred in neurones, since glucose is incorporated into amino acids in both neurones and glia, but acetate is thought to label mainly glial pools of amino acids. No increase in GABA synthesis was seen in the rat retina, either in vivo or in vitro, or in the frog retina in vitro. The increased synthesis of GABA seen in the retina of certain species in response to light is consistent with GABA being a transmitter substance, since it can be argued that in the light more GABA is required for release from inhibitory nerve terminals. On the other hand, had a fall in GABA levels been observed in light-stimulated retinae, it could have been argued that this was due to the release of GABA from nerve terminals followed by its destruction by GABA-T. It is difficult at present to believe that any of the neurochemical changes sometimes seen in the GABA system in response to light or dark adaptation have much significance. The changes are only seen in some species and in the frog, only in vivo. Furthermore, important seasonal variations are observed (Graham et al., 1970). For example, at certain times of the year, the fall in GABA levels seen in dark-adapted frog retinae do not occur (Graham, personal communication). Similarly, in the winter, the uptake of [3H]GABA by the light-adapted rat retina was about twice as great as that by dark-adapted retinae. This difference could not be observed in the summer, although it reappeared the following winter (Neal, unpublished results). Release

In spite of much inferential evidence which suggests that GABA might be a retinal transmitter, there has been no report of GABA release from the retina being increased by photic stimulation. The release of labelled GABA from the rat and frog retina was not increased by photic stimulation although a calciumdependent release of GABA was produced by high

concentrations of potassium (Voaden & Starr, 1972; Kennedy & Voaden, 1974a). The failure of light to evoke a release of labelled GABA from the rat retina is hardly surprising, since uptake of GABA in this species is into glial cells. However, this explanation cannot account for the similar failure to see a light evoked release from the frog retina, which accumulates GABA in neurones. The release of endogenous GABA from the retina has been reported by Pasantes-Morales et al. (1974) who found that light stimulation of the fowl retina did not significantly increase the efflux of GABA, glutamate, glycine, alanine or lysine. The effiux of 3H-GABA from the fowl retina was also unaffected by light although electrical field stimulation produced a six-fold increase in the efflux of labelled amino acid. Uptake

Following their release from presynaptic nerve terminals, amino acid transmitters are believed to be inactivated by active transport systems present in neurones and/or glial cells. Thus, the demonstration of such uptake processes in the retina is important in establishing transmitter roles for amino acids, although it should be remembered that high affinity uptake processes, for example, for GABA, also occur in tissues where the amino acid has no transmitter role. The uptake of labelled GABA by the retina has been characterized by Goodchild & Neal (1970, 1973) and by Starr & Voaden (1972a). The uptake process for GABA in the rat retina was remarkably similar to that described for rat cerebral cortex (Iversen & Neal, 1968), and was temperature sensitive, sodium dependent, inhibited by metabolic inhibitors and capable of achieving a large net uptake of GABA. The process is specific, being inhibited only by close analogues of GABA. Although [3H]GABA is accumulated by glia in the rat retina, the pattern of specificity of the uptake process resembles that reported for uptake into neurones rather than glia, i.e. the uptake is inhibited by L-2,4-DABA and is relatively unaffected by/~-alanine. Again, in contrast to other areas of the CNS, the accumulation of [~H]GABA by the rat retina was unaffected or increased by aminooxyacetic acid (Neal & Starr, 1973). Kinetic studies indicated that GABA transport in the retina is saturable and possesses both a high affinity (apparent K,, = 40/~M, Goodchild & Neal, 1970; 1973; Start & Voaden, 1972a) and a low affinity component (Kin = 0.5 mM, Neal et al., 1973). The uptake of [3H]GABA was not influenced by the adaptational state of the rat retina (Starr & Voaden, 1972a) although the dark-adapted goldfish retina accumulated less [3H]GABA than retinae stimulated with light (Lam & Steinman, 1971). The sites of uptake of GABA in the retina have been extensively studied by autoradiography. The results of these studies have been somewhat confusing and the sites of GABA uptake in the retina seem to vary considerably with different species and also with

Amino acid transmitter substances the technique used to label the tissue. In general, the evidence available at present suggests that non-mammalian retinae may take up labelled GABA predominantly into neurones, whilst mammalian retinae tend to accumulate GABA in the neuroglial MOiler cells. However, the picture in mammalian retinae is not quite clear-cut, and in some species, neuronal uptake as well as glial uptake is seen if the labelling is accomplished by intravitreal injection in vivo. In one of the earliest autoradiographical studies of GABA localization, Ehinger & Falck (1971) found that in the rabbit retina [3H]GABA was accumulated in the inner synaptic layer, in some ganglion cells and in some cells situated in the inner part of the inner nuclear layer which they tentatively identified as amacrines. This pattern of labelling was essentially the same when the retina was labelled by intravitreal injection or by incubation of the isolated retina, therefore the polar distribution of the [3H]GABA could not be due merely to a concentration gradient. The number of cells in the inner nuclear layer taking up GABA was smaller than the total number of amacrines, indicating that the cells taking up GABA must be a subgroup of amacrines. The uptake of [3H]GABA by ganglion ceils was variable but could not have been due to intraretinal cells since after chronic sectioning of the optic nerve no ganglion cells were seen to take up GABA. A neuronal uptake of [3H]GABA was also reported in the goldfish retina both in vitro and following intravitreal injections (Lain & Steinman, 1971). The GABA was accumulated predominantly in horizontal cells and in cells with the position of amacrines. A much greater uptake of GABA was observed in horizontal cells of light-stimulated retina. A predominantly neuronal uptake of [3H]GABA has also been reported in the frog (Voaden et al., 1974), chicken and pigeon (Marshall & Voaden, 1974a). A different pattern of labelling was found in the isolated rat retina by Neal & Iversen (1972). In this species the retinal uptake of [3H]GABA appeared to be predominantly into the neuroglial MOiler cells. The failure to demonstrate neuronal uptake in this retina was not due to a rapid metabolism of GABA in neurones with a subsequent loss of labelled metabolites, since in the presence of AOAA, the same pattern of uptake was observed. Glial uptake by the isolated rat retina was confirmed by Bruun & Ehinger (1974) and Marshall & Voaden (1974b). The factors influencing the sites of uptake of GABA have been somewhat clarified by Bruun & Ehinger (1974), who found that both the species of animals and the method of labelling were important. Thus, [3H]GABA applied by intravitreal injection into eyes of rats, guinea-pigs, cats and monkeys was preferentially accumulated by amacrines although glial cell uptake also occurred in the monkey and rat. In vitro labelling resulted in predominantly glial uptake except in cats and guinea-pigs, where labelled amacrines were still observed. In rats and monkeys, glial

325

uptake of [3H]GABA often disguised amacrine uptake, even in vitro. In a recent species comparison of GABA uptake in mammalian retinae labelled in vitro, Marshall & Voaden (1975) found a predominantly glial uptake in the cat, baboon, guinea-pig, goat and rabbit retina, although in the rabbit and cat retina, amacrine cell uptake was also observed. In contrast to GABA, glycine always seems to be taken up by retinal neurones whatever the species, and both in vivo and in vitro. Similarly, glutamate is consistently taken up by glial cells in the retina. It is not known why the pattern of GABA uptake shows such wide species variation. The subcellular distribution of [3H]GABA accumulated by the isolated rabbit retina has been studied by Neal & Atterwill (1974), and Atterwill & Neal (1976). The [3H]GABA was accumulated by osmotically sensitive particles which could be partially separated on sucrose density gradients from particles which accumulated labelled dopamine. The particles accumulating [3H]GABA were tentatively identified as synaptosomes since the fractions rich in these particles also possessed most of the GAD activity on the gradient. An interesting discovery in these studies was that the large photoreceptor terminals sedimented at much lower centrifugal forces than synaptosomes from conventional synapses. The preparation of pure photoreceptor terminals may prove valuable in the identification of photoreceptor transmitter substances. Effect on cells

Like most central neurones, retinal cells are inhibited by GABA. This was first shown by Noell (1959) who reported that the iontophoretic application of GABA on to ganglion cells of the rabbit retina rapidly and reversibly decreased the response to flashing light. Kishida & Naka (1968) showed that GABA inhibited spike discharges in ganglion cells of the isolated bullfrog retina in vitro and Straschill (1968) showed that intracarotid injections of GABA depressed both the spontaneous and light-driven activity of ganglion cells of the cat retina. Straschill & Perwein (1969) subsequently showed that GABA, applied iontophoretically to cat retinal ganglion cells, inhibited spontaneous and light-evoked activity. Ames & Pollen (1969) using the isolated superfused rabbit retina found that GABA could always depress the spontaneous and evoked activity of ganglion cells. However, because of the rather high concentrations required, they considered that the effect might not be synaptic. Murakami et al. (1972) recording intracelhdarly from the isolated carp retina, showed that both GABA and glycine, when applied as an atomized spray, hyperpolarized L and C types of horizontal cells and abolished their responses to flashes of light. The same types of cell were depolarized by aspartate and glutamate but were unaffected by ACh. It is clear that GABA consistently inhibits retinal neurones; however, the mechanisms underlying this

M~CHAELJ. NEAL

326

inhibition are unknown. The one study using intracellular recording indicated that GABA hyperpolarizes horizontal cells, but so far there has been no report concerning the membrane conductance changes underlying this effect. The presence of receptors for GABA is not proof that GABA is a retinal transmitter and what is needed is the demonstration of physiological and pharmacological identity between synaptic inhibition and iontophoretically applied GABA. Effect on electro-retinogram (e.r.9.)

GABA inhibits the b-wave of the e.r.g., which is the result of neuronal activity proximal to the photoreceptors, but does not effect the a-wave, the leading edge of which is due to photoreceptor activity, or the c-wave, which originates from the pigment epithelium but depends on photoreceptor activity. The depressant effect of intravitreal injections of GABA on the b-wave of the anaesthetized chicken e.r.g, was antagonized by picrotoxin but not by strychnine (Bonaventure et al., 1974) and the presence of GABA receptors on retinal neurones was supported by the results of Graham (1974), who found that intravitreal injections of the GABA antagonists, picrotoxin and bicuculline, produced rhythmic potentials in the rat retina. These potentials were not seen after the intravitreal injection of the glycine antagonist, strychnine, and the authors (Graham & Pong, quoted by Graham, 1974) suggested that GABA might be involved in a feed-back "damping circuit". In further experiments, Graham & Pong used the b-wave threshold of the e.r.g, as a measure of retinal sensitivity and found that GABA decreased the sensitivity of the retina to light, whilst picrotoxin and bicuculline had the opposite effect. These results were taken as support for the concept of GABA acting as an inhibitory transmitter mediating a negative feed-back control process. Burkhardt (1972) studied the effects of picrotoxin and strychnine on the proximal negative response (PNR) of the isolated frog retina. This response, which is believed to originate from the amacrine cells, was increased in amplitude by both picrotoxin and strychnine. However, whilst picrotoxin also prolonged the response, strychnine only affected the amplitude. These results indicate that there are at least two classes of inhibition in the retina, and Burkhardt suggested that picrotoxin might be blocking a presynaptic inhibitory effect of GABA released from amacrine nerve terminals on to bipolar cells. Burkhardt made no comment regarding the action of strychnine on the PNR, but clearly the effects of this drug could be due to the antagonism of glycine, or possible taurine, released from the nerve terminals of a different population of amacrine cells. TAUR1NE Content amt distribution

Taurine is by far the most abundant free amino acid in the retina (Kubic'ek & Dol6nek, 1958; Broth-

erton, 1962; Pasantes-Morales et al., 1972a; Cohen et al., 1973; Starr, 1973) representing in the rat, frog, chicken and goldfish retinae between 40~ and 50% of the free amino acid pool (Table 1). The concentration of taurine in the whole retina (10-15 pmoles/g wet weight) is much higher than in the rest of the central nervous system, where in most areas, the concentration is around 1-2 pmoles/g. Kennedy & Voaden (1974b) found that taurine was more concentrated in the outer retinal layers of frog, and this was confirmed by Orr et al. (1976) who showed that in five species all the retinal layers contained substantial levels of taurine which was more localized in the outer layers. This study indicated that a large proportion of the taurine must be located in the photoreceptors and is consistent with the 60-70% loss of taurine observed in mice and cat retinae with absence of photoreceptors (Cohen et al., 1973; Hayes et al., 1975). However, there is no evidence at present to suggest that taurine is concentrated in the photoreceptor terminals, and Cohen et al. (1973) reported that taurine levels in newborn, 9-, and 90-day old mice were very similar, although in newborn mice, receptor terminals have not developed. The levels of taurine in the rat, frog, chicken and goldfish retina were unaffected by their state of adaptation (Starr, 1973; Table l). There has been no report, as far as I am aware, on the intraretinal distribution or subcellular distribution of cysteinesulphinate decarboxylase, the enzyme responsible for the formation of taurine. Release

Start & Voaden (1972b) found that the turnover of [14C]taurine was not more than 1~o of the amino acid accumulated by the rat retina in 1 hr, a finding consistent with their report that [14C]taurine was released extremely slowly from the retina. Light stimulation did not cause an increase in the efflux of labelled taurine from the rat retina (Voaden, personal communication). In contrast, Pasantes-Morales et al. (1973c; 1974) have reported large increases in the release of both [35S]taurine and endogenous taurine from isolated chicken retinae stimulated by very high intensity flashes of light. A single flash of light apparently increased the release of [35S]taurine fivefold and the endogenous taurine two-fold. The release of [35S]taurin e was calcium dependent and no change was observed in the efflux of endogenous glutamate, glycine, alanine, GABA or lysine. The origin of the labelled taurine released in these experiments is not clear since it is not known which cells in the chicken accumulate exogenous taurine. Also, the cellular origin of the endogenous taurine released from the retina after light stimulation is unknown but it should be possible to determine whether the taurine is released from photoreceptor terminals by exposing the superfused retina to aspartate, thus blocking neuronal activity beyond the photoreceptor cells, which are themselves resistant to the depolarizing effect of aspartate.

Amino acid transmitter substances Uptake

The uptake of taurine has been demonstrated in the rat (Start & Voaden, 1972b; Neal et al., 1973), rabbit (Ehinger, 1973) and chick retina (PasantesMorales et al., 1972b). These transport systems for taurine appear to be sodium dependent, temperature sensitive and are inhibited by metabolic inhibitors. Although the above authors agree that taurine uptake in the retina is saturable, there are considerable discrepancies in the quoted kinetic parameters. Neal et al. (1973) found that the uptake process for taurine could be resolved into two components with apparent K,, values of 27 pM and 0.54 raM. Starr & Voaden (1972b), also using rat retina, reported only a single transport system for taurine with an apparent K m value (1 raM) which corresponded roughly with the low affinity uptake reported by Neal et al. (1973). The narrow concentration range used by Starr & Voaden explains why they only found a one component transport system for taurine. Pasantes-Morales et al. (1972b) also quoted only a single K,, value for taurine uptake (1.53 mM) although according to their published data their Km value should be about 16/A/I. Ehinger (1973) using the rabbit retina also claimed a single Km value of 43 #M, but again, examination of the reported data seems to indicate a K,, value of about 2 #M. The accumulation of [14C]taurine by the rat, frog and chicken retina is the same in light- and darkadapted retinae, although the uptake of taurine was slightly reduced in dark-adapted goldfish retinae (Starr, 1973). Somewhat in contrast, Pasantes-Morales et al. (1972b) found that taurine uptake in retina from chicks reared in the dark was slightly greater than in retinae from chicks reared in light. Autoradiographic studies indicate that in the rabbit retina [aH]taurine is taken up mainly into glial cells (Ehinger, 1973). Effect on cells and the e.r.g.

There has been no report on the action of taurine on single cells in the retina but the amino acid has been shown to affect the e.r.g. Taurine depressed the b-wave amplitude, without affecting the a-wave when injected into the vitreous body of the anaesthetized chicken (Pasantes-Morales et al., 1973a) or added to the superfusion fluid of the isolated frog retina. The depressant effect of taurine on the b-wave of the chicken e.r.g, in vivo, but not that of GABA, was antagonized by strychnine. The effect of GABA, but not that of taurine, was antagonized by picrotoxin (Bonaventure et al., 1974). These results imply that separate receptors for taurine and GABA may be present on retinal neurones but the mechanisms underlying the depression of the b-wave are unknown. In the spinal cord, taurine has been classified as a "glycine-like" agonist, i.e. it inhibits cells that are inhibited by glycine, and these effects are antagonized by strychnine. Since glycine may be a retinal transmitter substance,

327

one must be very cautious in deducing a transmitter role for taurine on the basis of its effects on the e.r.g. GLYCINE Content and distribution

There is good evidence tl~t this amino acid is an inhibitory transmitter substance in the spinal cord and there is some evidence that it may be a transmitter in the retina. Glycine has been demonstrated in the rat, frog, chicken and goldfish retina (Pasantes-Momles et al., 1972a; Starr, 1973; Table 1). The concentration of glycine in the retina is similar to that in other areas of the CNS, the reported values being from 0.6 pmoles/g (goldfish retina) to 2.3 #moles/g (rat retina) (Starr, 1973). The distribution of glycine within the retina has not been studied in detail but it does not seem to be associated with the photoreceptors (Kennedy & Voaden, 1974b) and the glycine content of mice retinae with destroyed inner layers was about half that of controls (Cohen et al., 1973). There was no difference in the levels of glycine in light- or darkadapted rat, frog, chicken or goldfish retinae (Starr, 1973) or in chicken retinae reared in continuous dark or light conditions (Pasantes-Morales et al., 1973b). Release

The release of [3H]glycine from the superfused rabbit retina in vitro and from the cat retina in vivo following light stimulation has been reported (Ehinger & Lindberg, 1974). The increased release from the rabbit retina after photic stimulation was small (1.3 times the prestimulation release) but was highly significant. The experiments on anaesthetized cats were only successful in about 5 out of 20 attempts but in the successful experiments the increase in glycine efflux was 2.9 times the resting spontaneous rate. Since it was shown by autoradiography that the [3H]glycine was present predominantly in amacrine cells and that only a very small proportion was metabolized (less than 5%), it is reasonable to believe that the glycine released by light stimulation probably originated from amacrine cell nerve endings. Voaden (1974) studied the release of [3H]glycine from the isolated frog eye cup preparation. No difference in the spontaneous efflux of [3H]glycine from dark-adapted retinae and retinae exposed to flashing light was observed but fully bleached (non-functional) retinae released [aH]glycine faster than those subjected to flashing light. The reason for this difference is not clear, but may be due to the fact that bleached, but not light-stimulated retinae, accumulate radioactivity in ganglion cells as well as amacrines. In 6 out of 16 experiments a transient increase in [3H]glycine was observed at the start of light stimulation. Uptake

Bruun & Ehinger (1972) have shown that the rabbit retina possesses an uptake system for glycine. This

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process is apparently similar to that described in the spinal cord by Neal (1971), i.e. it is specific, saturable, temperature sensitive and inhibited by metabolic inhibitors. It is of particular interest that in addition to a low affinity uptake process (K,, = l mM), the retina also possesses a high affinity uptake process with a K,, of about 7 pM (Neal et al., 1973) since high affinity uptake processes for glycine are absent in other areas of the CNS where glycine is not thought to be a transmitter. Autoradiographic studies of the localization of glycine accumulated by human, rabbit, pigeon, chicken, monkey, cat, guinea-pig, frog and rat retinae after either intravitreal injections, or incubation in vitro, all suggest that the amino acid is taken up into a population of amacrine cells and some ganglion cells (Ehinger & Falck, 1971 ; Ehinger, 1972; Voaden et al., 1974; Marshall & Voaden, 1974b; Bruun & Ehinger, 1974). Effect on cells

There have been few reports on the effects of glycine on retinal neurones, but Ames & Pollen (1969) found that glycine consistently depressed the spontaneous and evoked activity of all cell types in the isolated rabbit retina, and these effects were indistinguishable from those of GABA. Glycine applied as an atomized spray on to gecko and carp retinae was shown by intracellular recording to hyperpolarize the horizontal cells and to reduce the S-potentials, but had no appreciable effect on receptor potentials (Murakami et al., 1972). These authors also showed that glycine inhibited the b-wave of the e.r.g, and reasonably ascribed this effect to the hyperpolarization of the horizontal cells. They did not report whether these effects of glycine were antagonized by strychnine. Effect o f strychnine on the e.r.g.

The effects of strychnine on the e.r.g, are not clear. Wohlzogen (1956) found that strychnine had no effect on the e.r.g, of cats although ganglion cell activity was increased. However, V6rkel & Hanitzsch (1971) showed that "low" concentrations (3.5 x 10-s M) of strychnine clearly increased the amplitude of the b-wave in the isolated rabbit and frog retina, whilst high concentrations (7 x 10-4M) abolished the b-wave. These authors suggested that the failure of Wohlzogen (1956) to observe an effect of strychnine on the e.r.g, might have been due to the barbiturate anaesthetic used. Strychnine has also been shown to increase the amplitude, but not the duration, of the PNR (Burkhardt, 1972). If it is assumed that the actions of strychnine on the retina are due to antagonism of glycine released from nerve terminals, it would seem that glycine may only play a relatively minor role since Bornschein & Heiss (1966), in experiments where activity in optic nerve fibres of the cat was measured by placing a microelectrode on the optic chiasm, found that convulsant doses of strych-

nine did not affect the inhibitory phases of "'off' and "off' effects. GLUTAMATE AND ASPARTATE Though direct evidence is lacking, it is widely believed that these amino acids may be important excitatory synaptie transmitter substances in the brain and there have been suggestions that they may also be transmitters released at certain retinal synapses, particularly by the photoreceptor terminals (DoMing & Ripps, 1973). Photoreceptor cells and horizontal cells have low resting membrane potentials in the dark and most respond to light by hyperpolarizing. It has been suggested that in the dark there is a continuous release of an excitatory transmitter from the photoreceptor terminals which maintains the bipolar and horizontal cells in a depolarized state. When the retina is exposed to light the photorcceptors hyperpolarize, the release of transmitter is reduced, and the second-order neurones hyperpolarize. This scheme is supported by several pieces of evidence: (1) the hyperpolarization of horizontal cells produced by light stimulation is often accompanied by an increase in membrane resistance, (2) the application of magnesium ions or cobalt to the retina, which would be expected to reduce transmitter release, produces hyperpolarization of the horizontal cells (Dowling & Ripps, 1973; Cervetto & Piccolino, 1974), (3) peroxidase, which is believed to be accumulated only by 'active' nerve-endings, is taken up by rod terminals, only in the dark-adapted retina (Schacher et al. 1974). The identity of this presumed excitatory transmitter is unknown but in vertebrates it is unlikely to be ACh since the photoreceptor layer contains little or no cholineacetyltransferase (ChAc) activity and ACh has no effect on horizontal cells (Murakami et al., 1972). Similarly, the photoreceptor transmitter is unlikely to be dopamine (or any other catecholamine), since fluorescence histochemical studies indicate that dopaminergic neurones in the retina are located only in the inner nuclear layer. It is possible that glutamate and/or aspartate may be the elusive photoreceptor transmitter although as in other areas of the CNS this suggestion has proved hard to confirm. Content and distribution

Glutamate is present in the retina in relatively high concentrations. In the rat, frog and chicken retina, the glutamate concentration is about 4 #moles/g, but is rather lower in the goldfish (1.7prnoles/g) (Starr, 1973; Table 1). Glutamate represents about 10-15~o of the free amino acid pool in the retina, a value considerably lower than that of brain (40~5~) (Pasantes-Morales et al., 1972a). Similarly, aspartate levels in the retina (0.2 1.5 pmoles/g, Starr, 1973) are generally lower than those in brain, representing about 2% and 10% of the free amino acid pool, respectively (Pasantes-Morales et al., 1972a).

Amino acid transmitter substances Graham et al. (1970) found that glutamate was rather evenly distributed across the frog retina, but Kennedy & Voaden (1974b) who compared the glutamate and aspartate concentrations in sections 70 #m thick from the inner and outer surface of the frog retina found that both amino acids were relatively concentrated in the outer layers. In contrast, Lowry et al. (1956) showed that in the monkey retina, glutamate is relatively concentrated in the ganglion cell layer, although the distribution in other layers appeared to be fairly even. A striking finding was the high glutamic-aspartic transaminase (and malic dehydrogenase) activity in the inner segments of the photoreceptors. The high activity of these enzymes in the inner segments may account for the relatively high concentration of aspartate found in crude nuclear pellets prepared by centrifugation of homogenates of rabbit retinae, since these fractions contain many pinched-off photoreceptor terminals and inner segments (Neal & Atterwill, 1974; Atterwill & Neal, 1976). Lowry et al. (1956) suggested that these enzymes may cooperate in the conversion of glutamate to aspartate and provide an emergency mechanism for the use of glutamate when other metabolites are lacking. However, an alternative possibility is that they could be concerned with producing a rapidly available transmitter pool of aspartate. Glutamate and aspartate levels were not influenced by the state of adaptation of the retina (Graham et al., 1970; Starr, 1973)(Table 1) or by rearing the animals in continuous light or dark (Pasantes-Morales et al., 1973b). Release

The spontaneous release of endogenous glutamate from the chicken retina in vitro has been reported, this release was not enhanced by stimulation of the retina with light (Pasantes-Morales et al., 1974). Neal & Collins (unpublished observations) also found that photic stimulation of the rat retina in vitro did not increase the etitux of endogenous glutamate but did cause a reduction in the release of aspartate, a result that is consistent with aspartate being a photoreceptor transmitter. The release of labelled glutamate and metabolites from retinae previously loaded with [3H]-glutamate has been reported by van Harreveld & Fifkova (1970, 1972). However, since exogenous glutamate is accumulated predominantly by glial cells, these studies are presumably not concerned with transmitter pools of glutamate. Uptake

Glutamate is accumulated in the retina by a temperature-sensitive, sodium-dependent uptake process which possesses a high and low affinity component with apparent Km values of 21 #M and 0.63 mM, respectively (Neal & White, 1971 ; White & Neal, 1976). The glutamate transport system in the retina is not specific, being competitively inhibited by other acidic amino acids, such as aspartate and cysteate. Aspar-

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tate is also taken up by the retina by a saturable process with a high and low affinity component (K,, = 26 pM and 0.68 mM, respectively) and kinetic studies suggest that the retina accumulates glutamate and aspartate by a common mechanism (White & Neal, 1976). Autoradiographic examination of rat retinae incubated in vitro with [all]glutamate indicated that the amino acid was accumulated predominantly in the glial cells (White & Neal, 1976). Labelled glutamate and aspartate are also taken up by the glial cells in the rabbit retina following intravitreal injections (Ehinger & Falck, 1971). The demonstration of glial uptake of acidic amino acids in the retina is consistent with results obtained in cerebetlar slices where glutamate uptake is confined to glia (H/Skfelt & Ljungdahl, 1972). Effect on cells

Kishida & Naka (1968) showed that glutamate and aspartate, when applied through a pipette on to ganglion cells of the frog retina always increased their spike discharge rate. These effects could be prevented by the simultaneous application of inhibitory amino acids such as GABA or alanine. The authors suggested that the excitability of the CNS might be controlled by changes of the proportion of excitatory to inhibitory amino acids in the extracellular fluid. Ames & Pollen (1969) using the superfused rabbit retina in vitro and recording extracellularly from ganglion cells also found that glutamate increased the spontaneous activity of on-cells and on-off cells without much change in their evoked response. High concentrations tended to cause a fall in spike amplitude. The authors considered that the high concentrations of glutamate required to modify ganglion cell activity made it an unlikely transmitter candidate. A similar view was expressed by Trubatch et al. (1973) because they found that in the chicken retina, glutamate superfusion abolished tectal responses to light, but these soon returned in spite of the continued presence of glutamate. Glutamate and aspartate have been shown to depolarize horizontal cells in the skate, carp, and turtle retina (Cervetto & MacNichol, 1972; Dowling & Ripps, 1972; Murakami et al., 1972; Sugawam & Negishi, 1973), and suppress responses of the proximal elements. These amino acids abolished the hyperpolarizing S-potentials but left relatively unaltered the photoreceptor responses themselves. Thus, the a-wave and c-wave of the e.r.g, were almost unaltered but the b-wave which is the result of neuronal activity proximal to the receptors was abolished. Recently, Murakami et al. (1975) have shown that aspartate and glutamate also have potent depolarizing effects on bipolar cells. SUMMARY AND C O N C L U S I O N S

The two most important criteria for establishing transmitter substances are (1) release of the substance

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following suitable physiological stimulation and (2) identity of action, i.e. demonstration that the exogenous substance when applied to a neurone has the same physiological effects, and is influenced by pharmacological agents in the same way, as transmitter released by stimulation of presynaptic nerve terminals. None of the amino acids considered in this review satisfies these criteria and the evidence for them being retinal transmitters remains entirely inferential. A problem in establishing identity of action is related to the extraordinary thinness of the retina. The neural part of the retina is less than 200/~m, which is less than the thickness of a single layer in most other regions of the brain. The retinal nerve cells are consequently small and it is therefore difficult to record from them intracellularly and to perform the iontophoretic type of experiments which have proved so valuable in establishing GABA and glycine as central transmitters in other areas of the brain. Furthermore, extracellular studies involving iontophoresis are difficult because action potentials do not appear in the retina except in the amacrine and ganglion cells. The thinness of the retina might be expected to facilitate release studies, but as yet, the release of only taurine and glycine has been claimed and these results have yet to be confirmed by other workers. The failure to show a release of GABA is particularly disappointing since there is much secondary evidence supporting its claim to be a retinal transmitter substance. The failure to demonstrate GABA release may be due to rapid reuptake but this argument is weakened by the fact that both taurine and glycine have reuptake systems with similar kinetic constants to GABA. The role of taurine in the retina remains to be established. This amino acid is concentrated in the photoreceptors, but it is unlikely to be released from their terminals since the photoreceptor transmitter is believed to be an excitatory substance. The significance of the light-evoked release of taurine will remain unknown until the cellular sites of origin of the released taurine can be determined. The evidence that glutamate or aspartate is a photoreceptor transmitter is meagre and totally inferential. The most promising approach to identifying this transmitter is probably to study the release of amino acids from purified fractions of photoreceptor terminals prepared from retinal homogenates. The evidence for glycine being a retinal transmitter is suggestive, but depends mainly on a single report of the release of labelled glycine from the retina following photic stimulation. It must be concluded that a great deal more work is required to establish amino acids as retinal transmitters. A systematic extracellular study of the pharmacology of amino acids in the retina would be extremely valuable as would be more thorough release studies both from the retina itself and from retinal synaptosomes. Further evidence for the role of amino

acids might come from studying the distribution of "receptors" in the retina.

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