Neurochem. Int. Vol. 20, No. 2, pp. 139-191, 1992

0197-0186/9255.00+0.00 Copyright © 1992Pergamon Press plc

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INVITED REVIEW LOCALIZATION A N D F U N C T I O N OF DOPAMINE IN THE A D U L T VERTEBRATE RETINA M. B. A. DJAMGOZ 1 a n d H.-J. WAGNER 2 qmperial College of Science, Technology and Medicine, Department of Biology, Neurobiology Group, London SW7 2BB, U.K. 2philipps University of Marburg, Institute of Anatomy and Cell Biology, 3550 Marburg, Fed. Rep. Germany CRITIQUE b y : J . C. BESHARSE a n d P. M. IUVONE Abstract--Dopamine (DA) has satisfied many of the criteria for being a major neurochemical in vertebrate retinae. It is synthesized in amacrine and/or interplexiform cells (depending on species) and released upon membrane depolarization in a calcium-dependent way. Strong evidence suggests that it is normally released within the retina during light adaptation, although flickering and not so much steady light stimuli have been found to be most effective in inducing endogenous dopamine release. DA action is not restricted to those neurones which appear to be in "direct" contact with pre-synaptic dopaminergic terminals. Neurones that are several microns away from such terminals can also be affected, presumably by short diffusion of the chemical. DA thus affects the activity of many cell types in the retina. In photoreceptors, it induces retinomotor movements, but inhibits disc shedding acting via D2 receptors, without significantly altering their electrophysiological responses. DA has two main effects upon horizontal cells : it uncouples their gap junctions and, independently, enhances the efficacy of their photoreceptor inputs, both effects involving D1 receptors. In the amphibian retina, where horizontal cells receive mixed rod and cone inputs, DA alters their balance in favour of the cone input, thus mimicking light adaptation. Light-evoked DA release also appears to be responsible for potentiating the horizontal cell ~ cone negative feed-back pathway responsible for generation of multi-phasic, chromatic S-potentials. However, there is little information concerning action of DA upon bipolar and amacrine cells. DA effects upon ganglion cells have been investigated in mammalian (cat and rabbit) retinae. The results suggest that there are both synaptic and non-synaptic Dl and D2 receptors on all physiological types of ganglion cell tested. Although the available data cannot readily be integrated, the balance of evidence suggests that dopaminergic neurones are involved in the light/dark adaptation process in the mammalian retina. Studies of the DA system in vertebrate retinae have contributed greatly to our understanding of its role in vision as well as DA neurobiology generally in the central nervous system. For example, the effect of DA in uncoupling horizontal cells is one of the earliest demonstrations of the uncoupling of electrotonic junctions by a neurally released chemical. The many other, diverse actions of DA in the retina reviewed here are also likely to become model modes of neurochemical action in the nervous system. As regards the retinal part of the visual system specifically, it would follow that the dopaminergic pathways are likely to be involved in a variety of functions including sensitivity control, chromaticity, spatial resolution/acuity and temporal signalling.

Abbreviations :

AC, amacrine cell ; ADTN, aminodihydroxytetra-hydronaphthalene; BC, bipolar cell ; CNS, central nervous system; DA, dopamine; DBH, dopamine-fl-hydroxylase ; DOPA, dihydroxyphenyl alanine; DOPAC, dihydroxyphenyl acetic acid; HC, horizontal cell; IBMX, isobutylmethylxanthine ; INL, inner nuclear layer ; IPC, interplexiform cell ; IPL, inner plexiform layer; MEL, melatonin; NAT, N-acetyltransferase ; 6-OHDA, 6-hydroxydopamine; OPL, outer plexiform layer; PNMT, phenylethanolamine-N-methyltransferase ; SP, substance P; TH, tyrosine hydroxylase.

The vertebrate retina is a n e m b r y o n i c projection of the forebrain, representing a model o f n e u r o n a l organization in the central nervous system (CNS). Retinal tissue is some 200-300/~m thick a n d comprises six different types o f neurone, as well as glial cells (mainly Muller cells a n d astrocytes). T h e c o n s t i t u e n t n e u r o n e s are regularly a r r a n g e d a n d m o s t o f their synaptic connections are m a d e in either o f two plexiform layers. In the outer plexiform layer (OPL), pho139

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toreceptor (rod and cone) signals are transmitted to second-order neurones (horizontal and bipolar cells). In the inner plexiform layer (IPL), ganglion cells (output neurones with axons forming the optic nerve) receive synaptic inputs from bipolar and amacrine cells (Fig. 1). Interplexiform cells (IPCs) are "centrifugal" in their organization, conveying signals from the IPL back into the OPL. IPCs also appear to be a target for efferent fibres entering the retina through the optic nerve, at least in lower vertebrates (Stell e t al.~ 1984; Zucker and Dowling, 1987). Thus, two

"radial" and two "lateral" sets of pathways are found. The "radial" circuits involve : (1) the "'through" pathway for transmission of signals in the chain: photoreceptor-* bipolar -~ ganglion cell (GC) ; and (2) the centrifugal feed-back mediated by the IPCs. Horizontal cells (HCs) and amacrine cells (ACs) form the two lateral systems in the OPL and 1PL, respectively (Fig. 1).

PE

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OPL

INL

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Fig. 1. Summary diagram of cell types and synaptic interactions occurring in the vertebrate retina. Excitatory synapses are indicated by O, inhibitory junctions by O, and reciprocal junctions by ,~. PE, pigment epithelium; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer ; GCL, ganglion cell layer ; C, cones ; R, rods ; H, horizontal cell ; BE),depolarizing bipolar cell ; BH, hyperpolarizing bipolar cell ; As, sustained amacrine cell ; Av, transient amacrine cell ; IP, interplexiform cell; G, ganglion cell. From Dowling (1987).

Invited Review Each of the six main types of retinal neurone has several sub-types and these collectively use most, if not all, of the neurotransmitter substances known to exist in the CNS generally (Brecha, 1983 ; Massey and Redburn, 1987). However, the strength of the evidence supporting the candidacy of the different transmitters varies considerably from chemical to chemical and amongst species. Beginning mainly in the mid-70s, a series of papers have been published with steadily increasing momentum, highlighting IPCs as a diversely important cellular component of retinal functioning (Boycott et al., 1975; Dowling and Ehinger, 1975, 1978a; Dowling et al., 1976; Kolb and West, 1977 ; DoMing, 1979 ; Oyster et al., 1985 ; Linberg and Fisher, 1986; Ryan and Hendrickson, 1987). These studies were aided greatly by the finding that a major sub-type of IPC uses dopamine (DA) as its chemical messenger (Ehinger and Falck, 1971 ; Dowling and Ehinger, 1975, 1978a; Dowling et al., 1980; Nguyen-Legros et al., 1982; Yazulla and Zucker, 1988). Other studies have shown that a subpopulation of ACs in some species also are dopaminergic (Dowling and Ehinger, 1978b ; Sarthy et al., 1981 ; Frederick et al., 1982 ; Holmgren-Taylor, 1982 ; Pourcho, 1982; Mariani et al., 1984; Nguyen-Legros et al., 1984, 1985; Witkovsky et al., 1984, 1987; Versaux-Botteri et al., 1986; Chino and Hashimoto, 1986; Keyser et al., 1987; Voigt and W/issle, 1987; Dacey, 1988 ; Mariani and Hokoc, 1988). A wealth of information has accumulated concerning localization, synaptic connectivity and functional role of dopaminergic IPCs and ACs. This article reviews and evaluates the progress made in our understanding of the dopaminergic systems in the adult vertebrate retina with an emphasis on their role in visual processing, where possible. TECHNIQUES FOR IDENTIFICATION A N D LOCALIZATION OF DOPAMINE

Identification and characterization of dopaminergic cells critically depend on the method of labelling since reliable identification and full evaluation of the connectivity only are possible after complete visualization of the entire cell. DA is one of several catecholamines that are putative chemical messengers in the retina. The presence of epinephrine (Cohen et al., 1982; Osborne and Nesselhut, 1983) and norepinephrine (Ehinger and Steinbusch, 1985; Osborne and Patel, 1985) is not a general feature among vertebrates and their physiological importance in the retina is also questionable. In contrast, many of the neurotransmitter criteria for DA have now firmly

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been established. The following methods have mostly been used in identification and localization of DA in vertebrate retinae. Biochemical identification

The presence of catecholamines in the eye, and in the retina in particular, was first noted by Duner et al. (1954). For a specific determination of DA content, spectro-fluorometric methods (Nichols et al., 1967) were originally used. More recently, radioenzyme quantitation of DA contents revealed values ranging from 0.8 ng/mg protein in chicken to 6.2 ng/mg protein for the frog (DaPrada, 1977). High performance liquid chromatography (HPLC) with electrochemical detection (ED) has further improved the precision of DA determination, to a point where differences between light- and dark-adapted specimens were revealed (Osborne, 1981; Nesselhut and Osborne, 1982; Morgan and Kamp, 1980; Proll et al., 1982; Wulle et al., 1990). HPLC-ED has, therefore, become the method of choice for identifying the different kinds of catecholamines, including reliably distinguishing DA from epinephrine and norepinephrine. Histofluorescence

Malmfors (1963) was the first to apply the FalckHillarp method, then newly discovered, for the fluorescence-microscopic demonstration of endogenous catecholamines in the rat retina. This method uses formaldehyde vapour to form a fluorescent product with catecholamines in tissue sections, the specificity of DA being determined by spectrophotometric measurements and/or by previous pharmacological treatment. Comprehensive accounts of catecholaminergic cells in vertebrate retinae have subsequently been presented by Ehinger (1966, 1976, 1983) and colleagues (Ehinger and Dowling, 1987; Ehinger and Falck, 1969; Dowling and Ehinger, 1975). Thus, all dopaminergic cells were found to have perikarya near the vitread border of the inner nuclear layer (INL), an extensive fibre plexus in the IPL and, in certain species, another level of ramification in the OPL. The accuracy of this method of visualization could be enhanced by preloading the retina with DA, other catecholamines or indoleamines, which are absorbed by rapid and specific uptake mechanisms, and could thus help reveal very delicate processes (Dowling and Ehinger, 1975, 1978a, b). Negishi's group [reviewed by Negishi et al. (1990)] subsequently adopted and extensively used a modified ("Faglu") method (Furness et al., 1977; Nakamura, 1979). Although this rather simplified procedure reveals mainly the perikarya of catecholaminergic cells, den-

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dritic trees could also be studied after intracellular filling with Lucifer Yellow (Teranishi and Negishi, 1986, 1988). A utoradiography

In this approach the endogenous rapid uptake mechanism for catecholamines is exploited to introduce radioactively labelled analogues into the specific cell types. Mostly tritiated agonists have been used, as well as antagonists (e.g. 2-aminodihydroxytetrahydronaphthalene, ADTN) or precursor molecules (dihydroxyphenyl alanine, DOPA) (Ehinger and Falck, 1971 ; Ehinger, 1981). Due to the short range of tritium radiation, autoradiography is restricted to the study of thin or ultrathin sections, which makes the technique amenable to high resolution studies including direct identification of synapses (HolmgrenTaylor, 1982). On the other hand, this technique precludes the visualization of single cell details including dendritic processes in whole-mounts or thick sections. Irnmunocytochemistry

This is a powerful and versatile technique capable of combining the advantages of the Falck-Hillarp method and of autoradiography in allowing both high resolution and low-power observation of entire cells. Basically, antibodies against DA itself can be raised [after preparation of conjugates using glutaraldehyde and a protein ; Geffard et al. (1984)] or against the rate limiting enzyme of catecholamine synthesis, tyrosine hydroxylase (TH). The former is highly specific but has to rely on a preparation that binds the antigen rapidly and tightly by the fixative. In contrast, TH can be used as a marker for DA only if parallel biochemical analyses have shown that no other catecholamine is present in the retina being studied, or by also using antibodies for enzymes involved in the synthesis of epinephrine or norepinephrine (dopamine-/~-hydroxylase, DBH ; and phenylethanolamineN-methyltransferase, PNMT). Antibodies can be applied to sections as well as to whole isolated retinae, although when incubating pieces of tissue thicker than about 50/~m (e.g. entire retinae) care must be taken to ensure that the antibodies penetrate throughout the specimen. Otherwise, incomplete staining of the cells may occur thus making it difficult to correctly identify the cell types and establish their synaptology. Neurotoxic destruction

Intraocularly injected neurotoxins that can specifically destroy catecholaminergic cells have been used as markers in order to establish cellular synaptology in the electron microscope. In some of the earliest

studies [e.g. Dowling and Ehinger (1975, 1978a, b)] 5,6-dihydroxytryptamine was used. This drug accumulates in both catecholamine- and indoleaminecontaining cells, thus lacking the specificity of 6-hydroxydopamine (6-OHDA) introduced by Negishi et al. (1981). Short term (4 h) application induces the accumulation of ring-like electron dense material inside large and small synaptic vesicles, while leaving the overall morphology intact. However, after repeated injection and long-term (about 7-10 days) exposure, catecholaminergic cells are destroyed up to the point where only degenerated processes are observed. More recently, ultrastructural studies employing neurotoxins have been superceded by autoradiographic (Holmgren-Taylor, 1982) or immunocytochemical techniques (Yazulla and Zucker, 1988; Wagner and Wulle, 1990). DOPAMINERGIC CELL TYPES ACROSS SPECIES Unlike the brain, where dopaminergic cells are projection neurones that extend their axons over considerable distances (nigro-striatal, meso-limbic and meso-cortical systems on the one hand, and the interhypothalamic group of neurones on the other) DAcontaining cells of the retina are true "local circuit" (intrinsic) neurones which may best be compared to the periglomerular cells of the olfactory bulb. Characteristically, intrinsic neurones only have very short axons, or no axons at all. In the retina, dopaminergic cell bodies are situated mostly in the INL, and the bulk of their processes extends into the IPL. This pattern of ramification corresponds exactly to the classical picture of the amacrine cell (Cajal, 1893). However, in a number of species, dopaminergic cells consistently display processes traversing the INL and terminating in the OPL or at its vitread border. These axon-like, radial branches may originate from the perikaryon or from the fibre plexus in the IPL, carrying information from the inner to the outer retina. The latter group of ceils was first noted in Golgi stained retinae (Cajal, 1893) and have come to be known as #zterplexiform cells (Gallego, 1971). Fishes

In fishes, IPCs are the main representatives of dopaminergic neurones. These cells are not only restricted to teleosts but can be found in such primitive species as lampreys (Negishi et al., 1985a; de Miguel and Wagner, 1990). In adult specimens of these cyclostomes (jawless fishes), immunocytochemical visualization of TH revealed a diffuse plexus o,~ stained processes in the IPL with a tendency of densification

Fig. 2. Immunocytochemical demonstration of tyrosine hydroxylase-like immunoreactivity in 25/tm radial sections (a, h) and 1 #m tangential sections after pre-embedding staining and PAP visualization in a cichlid teleost (a)-(g) and a turtle (h). Because o f the section thickness, unspecific staining of photoreceptor layers becomes apparent in the radial views and must not be confounded with the specific tyrosine hydroxylase label. In the fish, the reaction product indicating the presence of dopamine is contained in an IPC. (a) Radial section showing an immunopositive perikaryon in the INL and three layers of fibres in the IPL as well as a dense plexus o f stained fibres surrounding horizontal cell perikarya. (b)-(g) Tangential views of the same area at various levels of the retina from sclerad to vitread. (b) Photoreceptor layer. (c) External horizontal cell layer. (d) INL. (e)-(g) Three different levels of the IPL. Magnification x 280. In the turtle (h), DA is found in ACs and there is no label in the horizontal cell layer. Magnification x 304.

invited Review

144

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Fig. 3. Semi-schematical synopsis of the types of dopaminergic cells found in different vertebrate retinae. Animal classes with amacrines as dopaminergic cells are given in the upper row, whilst animals with IPCs as dopaminergic neurones are illustrated in the lower row. It is obvious from this diagram, that there is no simple evolutionary trend towards either amacrine or IPC as dopaminergic retinal neurones. It is noteworthy, however, that amongst fishes and mammals, the most advanced species have IPC as dopaminergic units.

at the border with the INL [Figs 2(a)-(g) and 3]. In addition, the HCs are surrounded by many delicate fibres which also spread into the OPL. Interestingly, numerous branches extend even further and appear to "ensheath" the photoreceptor somata; most of these processes stop short of the outer limiting membrane, thus appearing to form a sharp boundary. Occasional processes, however, have been seen to break through this membrane and terminate in the sub-retinal space (de Miguel and Wagner, 1990). Compared with other vertebrates, this would appear to be the most extensive dopaminergic innervation pattern found in the outer retina. Elasmobranchs differ from the other fishes in that they have been found to have only ACs as dopaminergic neurones. According to the observations of Brunken et al. (1986) there are species differences in the number of sublayers occupied by the stained

processes; sharks have dopaminergic fibres in IPL sublayers sl, s3, and s5, whilst rays lack such processes in the vicinity of ganglion cell somata. In teleost fishes, the dopaminergic fibres in the IPL are most often segregated into 2 or 3 discrete sublayers [Figs 2(a)-(g), 3 and 5(a), (c), (e)]. These are most often located at the inner and outer borders as well as in an intermediate sublayer (Dowling and Ehinger, 1978a; Negishi et al., 1980; Teranishi and Negishi, 1986; Wagner and Wulle, 1990). By comparing different species of teleost, it appears that this pattern is prevalent in visually-active fish (e.g. salmonids, perciforms and cyprinids) with a well developed IPL. On the other hand, in species with simpler retinae (e.g. catfish), the branching pattern of IPCs in the IPL is more diffuse [Figs 5(a), (c), (e)]. The density of dopaminergic cell bodies in the INK has been assessed in whole-mount preparations. In

Invited Review carp, a homogenous distribution was found in the fundus of the eye, with a density of about 100 cells/mm2 in young adult specimens. With increasing age and size of the eyes, this cell density decreases (along with the densities of the other retinal cells) to about a third of this value (Negishi et al., 1981). There seems to be not much difference in dopaminergic cell density amongst the teleost fish species studied ; adult catfish retinae, for example, contain 60-128 cells/mm2, with no difference that could be interpreted in terms of a naso-temporal or a ventro-dorsal gradient (Wagner and Wulle, 1990). As for the processes directed towards the outer retina, it is interesting to note that in the roach the sclerad branches originate directly from the soma only in 20% of the IPCs. In the other cases, these processes emerge from lateral branches in the IPL; up to five such processes have been observed belonging to a single cell and running through the INL (Wagner and Wulle, 1990). Since these sclerad fibres are mostly very delicate and, as a rule, lack varicosities, they are often difficult to identify in peroxidase-based immunocytochemical stainings or in micro-injected specimens. Instead, they have been visualized most convincingly in fluorescence micrographs, where they stand out against the dark background. In the Japanese cyprinid, dace, dopaminergic ACs have been described using an intracellular dye injection technique (Chino and Hashimoto, 1986). Although true IPCs were also found, the overall morphology in the IPL of both cell types did not appear to significantly differ in the whole-mounts shown. It would be very important to substantiate this finding because, for teleosts, this would be the only indication of the expression of D A by both ACs and IPCs. Amphibia

Species of amphibia which have been most extensively studied are the mudpuppy, Xenopus, and the larval tiger salamander. In all cases, ACs appear to be the only type of neurone to contain DA, the main level of ramification in the IPL being at the junction with the INL [Figs 3 and 5(g)] [Xenopus: Sarthy et al. (1981); mudpuppy: Adolph et al. (1980); tiger salamander: Watt et al. (1987)]. These studies involved fluorescence microscopic and autoradiographic preparations. Although occasional processes were observed to reach into the I N L in the mudpuppy, no plexus of processes surrounding HCs was detected. It was concluded, therefore, that IPCs do not occur in significant numbers in the mudpuppy retina (Adolph et al., 1980).

145

Reptiles

Among reptiles, the turtle retina has been studied most extensively. As in the case of the amphibians, ACs are the only type of dopaminergic cell found (Witkovsky et al., 1984 ; Nguyen-Legros et al., 1985 ; Kolb et al., 1987). TH immunocytochemistry with various methods of visualization has consistently failed to demonstrate any processes linking the perikarya in the inner part of the INL with the outer retina [Figs 2(h) and 4(a)]. Extensively ramifying plexus of varicose fibres were concentrated at the marginal sublayers of the IPL and in the intermediate layer, which in the usual terminology for IPL sublayering correspond to sl, lower s2 and the s4/s5 border (Kolb et al., 1987). These cells could thus be identified as type A28 of the Golgi classification scheme of Kolb et al. (1986). As for the density of TH-positive perikarya, it seems similar to that found in adult carp: about 60 cells/mm 2 at the centre of the retina, decreasing to about 10 cells/mm2 in the periphery. Since dendritic field size was inversely related to cell density, a constant coverage of the IPL by dopaminergic fibres was maintained (Kolb et al., 1987). In another reptilian species, the more advanced lizard, TH staining revealed a type of AC which lacked the fibre plexus in the middle of the IPL (Engbretson and Batelle, 1987).

Birds

ACs appear to be the only type of dopaminergic neurone found in the chick retina which has been studied mostly in this class [Figs 3 and 4(o)]. Typically, TH-immunoreactive neurones had pyriform perikarya and a dense plexus of delicate, varicose fibres in sl. In addition, thicker processes extended through the IPL to ramify in s4; finally, there were faintly stained fibres and occasional varicosities in s3 (Brecha, 1983; Kato et al., 1984; Su and Watt, 1987). In the extreme periphery of the retina, a second system of dopaminergic fibres (called "marginal zone system") has been detected which has connections to ganglion cells on the one hand and reaches into the ciliary body on the other (Stoeckel et al., 1976).

Mammals

The localization of DA has been investigated in several mammalian species, including primates (Fig. 3). The picture emerging from these studies is not as clear as in other vertebrates and both IPCs and ACs have been identified as dopaminergic neurones [see

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Fig. 4. Light micrographs of semithin (0.5 #m) sections of retinae of some amniote (i.e. higher) vertebrates, arranged in pairs of consecutive sections after postembedding/immuno-staining with antisera to tyrosine hydroxylase (a, c, e) or GABA (b, d, f). Arrows in the anti-GABA stained figures indicate the positions of the corresponding tyrosine hydroxylase-like immuno-positive somata. In these species co-localization of the two markers is observed. Magnification, × 440. Scale bar, 20 #m (valid for all figures). (a) and (b) Radial sections of the turtle retina. Note the offset in the horizontal plane of the two micrographs, resulting in the fact that the labelled cell indicated by the arrow seems to be displaced. (c) and (d) Radial sections of the chick retina. (e) and (f) Tangential sections through the inner nuclear layer of the mouse retina. From Wulle and Wagner (1990).

Invited Review recent review by Nguyen-Legros (1988)]. Based on soma size, two types of ACs could be distinguished and these also seemed to differ in their pattern of ramification in the IPL. "Large" cells (perikaryal diameter about 15 #m) were multipolar with several primary dendrites leaving the perikaryon. By contrast, "small" cells (perikaryal diameter, 8-10 #m) had a single primary dendrite giving rise to radiating branches in the IPL; in the adult, these cells were predominantly PNMT-positive. The large cells could be interstitial or had somata displaced into the GC layer. As regards the stratification levels of these ACs in the IPL, there was a clear preference for the sublayers s 1, s3 and s5. While in rat, rabbit, mouse, guinea pig, cat and squirrel monkey, occasional branches were observed to cross the INL and to terminate at the level of HC somata (Ehinger, 1966 ; Ehinger and Falck, 1969 ; Nyugen-Legros et al., 1981), this finding was not consistent enough to lead the authors to doubt the basic amacrine nature of these cells. In the rhesus monkey retina, two types of TH-positive AC types have been described (Mariani and Hokoc, 1988) differing not only in soma diameter, but also in their cell density and IPL ramification pattern. While the large CAI type arborized almost exclusively at the junction of the I N L and IPL and also sent radial processes into the INL, the smaller CA2 cell stratified in the centre of the IPL. Dopaminergic IPCs, by contrast, had originally only been seen in retinae of Cebus and other New World monkeys (Haggendahl and Malmfors, 1963, 1965 ; Ehinger and Falck, 1971 ; Ehinger and Steinbusch, 1985). Using TH-immunocytochemistry, it has been shown more recently that retinae of rodents, cats and all primates also have IPCs (Ballesta et al., 1984; Frederick et al., 1982; Hendrickson et al., 1981; Negishi et al., 1985b; Oyster et al., 1985). It would appear, therefore, that IPCs, in fact, could be a general feature of mammalian retinae. Conflicting results obtained earlier may be explained by assuming a nonuniform distribution of ACs and IPCs, or by methodological problems restricting the visualization of either the very delicate ascending processes and/or the plexus of terminals in the OPL, some surrounding HCs. As for the density of the large dopaminergic cells, their distribution essentially paralleled that of ganglion cells ; in the rabbit, their density decreased from the visual streak towards the periphery (NguyenLegros, 1988). In primates, on the other hand, THpositive cells had their highest density in a concentric ring around the fovea thus following the distribution of rods (Mariani et al., 1984).

147

Colocalization o f dopamine and G A B A

Using double label immunocytochemistry, it has been found that D A may not be the only neurotransmitter present in TH-positive cells of mammals (Kosaka et al., 1987 ; W~ssle and Chun, 1988). In rats and cats, G A B A and/or glutamate decarboxylase was also found in the DA neurones. In a comparative anatomical approach, Wulle and Wagner (1990) demonstrated that this type of colocalization is not a common feature among all vertebrates. Interestingly, colocalization occurs in retinae of higher i.e. amniote vertebrates (such as reptiles, birds and mammals ; Fig. 4), whilst dopaminergic cells of fish and amphibians (i.e. anamniotes ; Fig. 5) do not contain GABA. Such a pattern of colocalization is clearly not linked to the type of cell (AC and/or IPC) containing TH, and its functional significance remains unknown at present.

C O N T R O L OF D O P A M I N E RELEASE

Since DA has been recognized as a putative chemical messenger in the retina, efforts have been made to characterize and specify the conditions regulating its release. Before endogenous D A could reliably be measured in superfusion experiments, either the total amount of retinal DA was assessed (Drujan et al., 1965; Nichols et al., 1967; Drujan and Diaz-Borges, 1968), or the release of [3H]DA from retinae preloaded with radiolabelled DA or tyrosine was studied [e.g. Kramer (1971); Thomas et al. (1978); Sarthy and Lam (1979); Bauer et al. (1980, 1981); Dubocovich and Weiner (1981 )]. Some of the early studies (Druj an et al., 1965; Nichols et al., 1967; Drujan and DiazBorges, 1968) yielded conflicting results, when the DA content was correlated with different lighting conditions. Thus, in one set of experiments with rabbits, frogs and toads, light adaptation reduced the DA content (Drujan et al., 1965 ; Drujan and Diaz-Borges, 1968), while in other studies with rabbits, rats and guinea pigs, the DA content was apparently increased after 1 h of light stimulation (Nichols et al., 1967). These inconsistencies may in part be due to the different animals used, but also to the difficulty in interpreting the total DA content as the result of synthesis, storage, release, uptake and/or degradation. Release experiments on cat retinae preloaded with [3H]DA using flickering light stimulus identified this paradigm as adequate for eliciting DA release (Kramer, 1971). Superimposing this stimulus on an additional bright background enhanced the rate of release even further. The coupling of flickering stimu-

Fig. 5. Light micrographs of semithin (0.5 Itm) sections of the retinae of anamniote (i.e. lower) vertebrates, arranged in pairs of consecutive sections after postembedding/immunostaining with antisera to tyrosine hydroxylase (a, c, e, g, i) or GABA (b, d, f, h, k). Arrows in the anti-GABA stained figures indicate the positions of the corresponding tyrosine hydroxylase-like immuno-positive somata. In these species, no colocalization is observed. Magnification, × 440. Scale bar, 20 itm (valid for all figures). (a) and (b) Tangential sections of the unction of the INLS and IPLS of the roach retina. (c) and (d) Radial sections of the roach retina. (e) and (f) Radial sections of the glass catfish retina. (g) and (h) Radial sections of the Xenopua" retina. (i) and (k) Tangential sections of the Xenopus retina at the junctions of the INLS and IPLS. From Wulle and Wagner (1990).

Invited Review lus and release was further substantiated by the demonstration that release was proportional to stimulation frequency. Numerous subsequent studies corroborated this finding in other species [for a review see Kamp (1985)]. Furthermore, the role of DA as a transmitter in the retina was substantiated by showing that release occurred upon membrane depolarization (Sarthy and Lam, 1979; Bauer et al., 1980, 1981), could be elicited by electrical stimulation (Dubocovich and Weiner, 1981) and in all cases was Ca2+-depen dent. However, no conclusive evidence was presented to explain whether the " O N " and/or the " O F F " component of the flicker stimulus was the trigger of DA release. On the whole, the apparent ineffectiveness of steady light on D A release would be in good agreement with the complex stimulation patterns preferred by many ACs, which are thought to contribute heavily to the activity of dopaminergic cells. On the other hand, in the Xenopus retina steady (and not flickering) light stimuli were found to be effective in releasing endogenous DA (Boatright et al., 1989). The light stimulus optimal for eliciting DA release from a given retina is probably related to the light-evoked response pattern(s) of the DA-containing neurones therein. However, very little is known about electrophysiological characteristics of dopaminergic neurones in vertebrate retinae [but see Djamgoz et al. (1991)]. The development of the accurate HPLC-ED technique made the retinal D A content and release of endogenous DA amenable to analysis, thus also avoiding possible artifacts involved in preloading with radiolabelled DA (Herdon et al., 1985). The following sections review the available data from various vertebrates, obtained mainly using HPLC. Teleosts Since IPCs are the main dopaminergic cell type in teleosts, their retinae have been a most favourable subject for the study of DA release. It may thus be assumed that the data obtained may not be contaminated significantly by other cell types, which may not necessarily behave identically. Sarthy and Lam (1979) originally showed that [3H]DA taken up by the goldfish retina is released upon stimulation with high K ÷ in a Ca2+-dependent way, indicating strongly that this release is neuronal, vesicular and normally elicited by membrane depolarization. It was already known (Iuvone et aL, 1978) that light stimulation activates TH, the key enzyme in the DA synthesizing pathway. Therefore, it was proposed that light stimulation would also lead to an increase in DA release. This idea was supported by the

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findings of Dearry and Burnside (1986a, b) showing a clear dopaminergic control of light-adaptive cone contraction (see also Photoreeeptors: retinomotor movements section). Nanomolar DA was found to be capable of eliciting cone myoid shortening in the dark. Furthermore, maintenance of light-adapted cone position was also found to be DA-dependent. Taken Logether, these results suggested that DA release is high throughout the light phase. However, this conclusion was contradicted by Mangel and Dowling (1985) who found in the carp retina that prolonged (longer than 90 min) dark adaptation produced electrophysiological effects on HC coupling and sensitivity similar to those observed after exogenous DA application. This led them to propose that D A release is high in the dark. Measuring release of endogenous DA from crucian carp (Carassius earassius) retinae, rather than looking at dopaminergic effects, Kirsch and Wagner (1987, 1989) observed a low-level basal release of DA in the dark, which was enhanced 3 4 fold by stimulation with flickering, but not by steady light [Fig. 6(a), (b)]. The light-induced release of DA from isolated retinae was not maintained at constant high levels throughout the light stimulation period ; instead, it levelled off to near dark values after 15-30 rain. This effect was slower in retinae where DA re-uptake had been blocked by benztropine, a common DA re-uptake blocker. Therefore, it was speculated that uptake was also stimulated by either light stimulation or D A release, implying that the retinal DA uptake mechanism does not operate near saturation conditions as has been proposed for other systems, but can be stimulated further by light. Thus, light would not only stimulate synthesis and release but also re-uptake of released DA from the synaptic cleft. In another set of experiments, levels of retinal dihydroxyphenylacetic acid (DOPAC), one of the main metabolites of DA, were assessed during a 24 h period (see also Cyclic Aspects of Dopamine Function section), in addition to the total DA content (Wulle et al., 1990). A prominent peak of DOPAC accompanied the change from dark to light, providing further evidence that light stimulation in teleosts leads to increased release of DA (Fig. 31). Pharmacological aspects of DA release in fish have also been examined in an attempt to identify the cell types and transmitter systems that interact with the dopaminergic IPCs and control their activity (O'Connor et aL, 1986, 1987; Kirsch and Wagner, 1987, 1989). A GABA-ergic inhibitory influence, long known for mammals and which had previously been postulated for fish on the basis of indirect electro-

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Invited Review

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Localization and function of dopamine in the adult vertebrate retina.

Dopamine (DA) has satisfied many of the criteria for being a major neurochemical in vertebrate retinae. It is synthesized in amacrine and/or interplex...
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