Chapter 1 Neurotransmitter systems in the turtle retina

CHAPTER 1

Neurotransmitter Systems in the Turtle Retina R E T O W E I L E R * , A L E X A N D E R K. B A L L t and J O S E F A M M E R M U L L E R *

*Department of Neurobiology, University of Oldenburg, Oldenburg, FRG tDepartment of Anatomy, McMaster University, Hamilton, Canada

CONTENTS 1. Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Glutamate and Aspartate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. GABA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 4

2. Biogenic Amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Serotonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Dopamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5 5 9

1

3. Neuropeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Corticotropin-Releasing Factor (CRF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Neuropeptide Y (NPY) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Glucagon (GLU) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Methionine Enkephalin (M-ENK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Neurotensin (NT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. LANT-6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Somatostatin (SRIF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Substance P (SP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12 13 15 16 17 18 20 21 21 22

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23

1. AMINO ACIDS All of the amino acids which have been implicated as potential neurotransmitter candidates in the vertebrate retina (for review see Massey and Redburn, 1987) have been localized, or have been shown to have effects on other neurotransmitter pathways in the turtle retina. These amino acids include glutamate, aspartate, glycine, and GABA. 1.1. Glutamate and Aspartate

In the outer retina, studies have shown that glutamate and aspartate are most likely trans-

mitter candidates for photoreceptors and that GABA is a likely transmitter candidate for one type of horizontal cell. Turtle retinas have been homogenized and endogenous levels of amino acids measured in photoreceptor-rich fractions (Lam et al., 1982). Only the sulphur-containing amino acid, taurine, was found to be significantly higher than elsewhere in the retina. Furthermore, immunocytochemical studies have shown that although turtle photoreceptors are glutamate-immunoreactive, the intensity of the label is only half that of putative glutamatergic bipolar cells (Ehinger et al., 1988). Stronger evidence for L-glutamate or

R. WEILER et al.

L-aspartate being a photoreceptor transmitter comes from studies using electrophysioiogical recording from horizontal cells which have been incubated or superfused with suspect neurotransmitter candidates. Application of aspartate or glutamate to the retina cells produces depolarizations in horizontal cells and the elimination of the light response. Since these transmitter candidates mimic the expected effect of the endogenous photoreceptor transmitter, it suggests that aspartate and glutamate may be the transmitters used by these cells. However, the results have not been easy to interpret since the effects of the amino acids vary under different conditions. Prolonged perfusion of the everted turtle eyecup with high concentrations of aspartate or glutamate has little effect on horizontal cell responses (Normann et al., 1986). Nevertheless, a transient depolarization in horizontal cells can be achieved after initial superfusion, presumably after the high affinity uptake mechanisms for these amino acids have been saturated. It has been suggested, however, that some of these effects which are associated with pathological conditions (Normann et aL, 1986), are due to acidic amino acid-induced increase in extracellular potassium (Perlman et aL, 1987) or are due to receptor desensitization. Sustained responses to aspartate, kainate, quisqualate, or N-methyl-o-aspartate (NMDA), without desensitization, can be obtained from horizontal cells in the isolated turtle retina, suggesting that there may be a diffusion barrier to acidic amino acids in the proximal retina which confounds the experiments when the eyecup preparation was used in these studies (Miyachi et al., 1987). Superfusion of the everted turtle eyecup with the analogues of aspartate and glutamate, kainate and NMDA, demonstrated that these agonists were more potent but that their effects differed, suggesting that two types of acidic amino acids may be present on horizontal cell dendrites (Perlman et al., 1987). The existence of discrete aspartate and glutamate receptors on horizontal cells is consistent with the localization of a highaffinity uptake mechanism for [3H]-aspartate in cones and for [~H]-glutamate in rods in goldfish (Marc and Lam, 1981) and mudpuppy (Lam et al., 1982) retina; however similar uptake experiments

have not yet been performed in turtle. Some studies imply that acetylcholine plays a role as a neurotransmitter in the distal turtle retina. Turtle cones have been shown to be immunoreactive for the synthesizing enzyme for acetylcholine, choline acetyltransferase (Criswell and Brandon, 1987). Alpha-bungarotoxin binding sites are located in the outer synaptic layer of the turtle retina and have been shown by EM-autoradiography to be situated on bipolar cell dendritic processes and not horizontal cells (Lam et al., 1982). But while nicotinic acetylcholine receptors have been associated with bipolar cells (James and Klein, 1985), it is muscarinic antagonists that have been shown to block cone to horizontal cell transmission (Gerschenfeld and Piccolino, 1977). Furthermore, turtle photoreceptor cells have acetylcholine and choline concentrations which are similar to that of the whole retina and only 25°70 of the activity of choline acetyltransferase and acetylcholinesterase found in the entire retina (Lam et al., 1982). The transmitters used by turtle photoreceptors, like other vertebrates, is still not known. It is also not clear whether the substances implicated as possible transmitters co-exist in the same photoreceptors or if they are located in different photoreceptor subtypes.

1.2. GABA

GABA has been implicated as a neurotransmitter of one class of turtle horizontal cell, as it has in most other vertebrate retinas. There are four types of horizontal cells which have been suggested to receive chromatic input from cones: H1 cells receive input from rods and red cones (O and R types); H2 cells receive input from red and green cones (Y and N types); H3 cells receive input from blue cones (C type); and H4 cells presumably receive input from red cones (A type) (Lipetz, 1985). The colour-opponent, biphasic spectral sensitivities observed in H2 and H3 cells have been suggested to arise from a circuit involving inhibitory feedback from H1 and H2 horizontal cells, respectively (Lipetz, 1985; Ohtsuka and Kouyama, 1986).

NEUROTRANSMITTER SYSTEMS IN THE TURTLE RETINA

In most vertebrate retinas centre-surround receptive field properties are first detected in bipolar cells and trigger features such as directional selectivity first detected in ganglion cells. These properties have been attributed to synaptic interactions occurring in the outer and inner synaptic layers, respectively. However, many of these properties have been demonstrated in turtle horizontal cells (Normann and Kolb, 1981; Criswell and DeVoe, 1986; Adolph, 1988), but the cellular mechanisms underlying them remain unclear. The H1 horizontal cell has a soma and an axon terminal (L1 and L2, respectively) which have different electrophysiological properties, suggesting that they may make different synaptic connections in the outer synaptic layer (Lipetz, 1985; Ohtsuka and Kouyama, 1986a,b). As in other vertebrate retinas, this monophasic horizontal cell may use GABA as a neurotransmitter. HI horizontal cells are immunoreactive for GABA and accumulate [3H]-GABA in the light (Ball et al., 1988). The feedback from horizontal cells to cones has been shown to be mediated by GABA acting on GABA-A receptors (Tachibana and Kaneko, 1984). While this GABA feedback could account for the receptive field properties of cones, the receptive field properties of horizontal cells may also involve the coupling interactions between the soma and axon terminal through gap junctions, or synaptic interactions between the H1 soma and H1 axon terminals (Piccolino et al., 1982; Kolb and Jones, 1984; Adolph, 1988). The putative GABAergic H1 cell has also been shown to be presynaptic to bipolar cell dendrites as well (Kolb and Jones, 1984). Such feedforward synaptic interactions suggest that direction selective properties of bipolar cells could originate from directionally selective H1 horizontal cells (Adolph, 1988). However, how excitatory amino acids, released from photoreceptors, and GABA, released from horizontal cells, interact in possible asymmetric synaptic arrangements to produce directional selective properties, remains to be demonstrated. In the inner retina, studies have shown that glutamate and serotonin are likely transmitter candidates for bipolar cells and that GABA and

glycine are likely transmitter candidates for more than half the amacrine cells in the turtle retina. While only a subpopulation of bipolar cells has been shown to contain serotonin (Weiler and Schiitte, 1985a), virtually all turtle bipolar cells are strongly immunoreactive for glutamate (Ehinger et al., 1988). This is consistent with the evidence that both ON and OFF bipolar cells use an excitatory neurotransmitter in the vertebrate retina. In addition to bipolar cell terminals, significant glutamate-immunoreactivity in the inner synaptic layer has been associated with amacrine cell processes (Ehinger et al., 1988). These have been observed making contact with bipolar cell axon terminals, other amacrine cell neurites, and ganglion cell dendrites. Since reciprocal feedback onto bipolar cells is presumed to be inhibitory (probably GABAergic), the IPL pathways in which glutamate is involved are unknown. Like other vertebrate retinas, the turtle retina contains a population of GABAergic amacrine cells which make up nearly 30°7o of proximal inner nuclear layer somata. These cells are immunoreactive for GABA and possess a high-affinity uptake mechanism for [3H]-GABA. The cells are maximally labelled by autoradiography when exposed to [3H]-GABA or its analogue, [3Hl-muscimol, in the dark adapted retina, presumably when these cells are hyperpolarized. In thin sections, the density of [3H]-GABAlabelled processes revealed four substrata which co-stratify with the largest terminals of bipolar cells; B2 and B 6 - 9 in inner synaptic layer substrata $5, B3 in $4, B4 in $2/3, and B5 in SI (Kolb, 1982). As in other vertebrate retinas, the neurites of GABAergic amacrine cells make reciprocal synapses with bipolar ceils, and are probably responsible for the antagonistic centresurround receptive field properties of turtle ganglion cells (Ariel and Adolph, 1985). In the fish retina, it has been suggested that the surround component of double colour opponent ganglion cell receptive fields could be formed by input from a GABAergic amacrine cell subtype (Marc, 1989). In general, however, ganglion cells with simple concentric receptive fields are not affected by GABA or its agonists and antagonists as much as cells with more complex receptive fields (Daw

R. WEILERet al. et al., 1989). Presumably this is because the simple

cell receptive fields are mostly formed by bipolar cell input, while those of complex cells are formed by inner synaptic layer pathways involving one or more types of inhibitory GABAergic amacrine cell. The turtle retina contains a large number of directionally selective ganglion cells whose properties appear to be formed by a mechanism similar to the well-studied directionally-selective ganglion cell in the rabbit retina. The excitatory input to directionally-selective ganglion cells may be from cholinergic amacrine cells (Criswell and Brandon, 1987; Ariel and Adolph, 1985), while GABA is the transmitter mediating inhibition in the null direction. Both picrotoxin (a GABA antagonist) and physostigmine (an acetylcholine potentiator) reduce directional selectivity in these cells. In other retinas, GABA has been implicated as the transmitter responsible for forming transient responses in ganglion cells. GABA, acting at GABA-B receptors in the inner synaptic layer, may be responsible for mediating the transient responses in these cells (Maguire et al., 1989; Slaughter et al., 1989). Sustained cells, which carry primarily colour and spatial information, may become more phasic when the GABA-B agonist, baclofen, is applied. Activation of the GABA-B receptor may relegate sustained cells to code information related to orientation or direction selectivity. Although the turtle retina contains a large number of ganglion cells with these trigger features, evidence for such a model of selective attention (Slaughter et al., 1989) remains to be gathered in this retina. GABAergic amacrine cells may also provide inhibitory input to inner synaptic layer pathways not leading directly to the formation of ganglion cell receptive fields. Dopaminergic cells in the turtle retina consist of a single type of tristratified amacrine cell (Witkovsky et al., 1984). This cell, which has been suggested to exert a modulatory action in the turtle retina and regulate events related to adaptation, has been shown to be affected by GABA (Piccolino et al., 1987). GABA antagonists have a strong uncoupling effect on horizontal cells. GABA itself, however, has only a small coupling effect on horizontal cells, suggesting that GABA is tonically released to keep

the rate of dopamine release low (Piccolino et al., 1982). This is supported by the finding that bicuculline, a GABA antagonist, causes a large release of dopamine from the turtle retina, but that only high concentrations of GABA can inhibit its basal release (Kolbinger, 1990). There is evidence in the turtle retina for some GABAergic amacrine cells also containing the neuropeptide met-enkephalin. Doublelabelling immunocytochemistry has demonstrated that a small proportion (9.5°70) of met-enkephalinimmunoreactive amacrines in the turtle retina are also GAD-immunoreactive (Zucker and Adolph, 1988). In the chick retina, 81°70 of enkephalinimmunoreactive amacrines also accumulate either [3H]-glycine or -GABA, giving rise to the suggestion that enkephalin-containing amacrines may have an autoregulatory mechanism involving the release of these co-existing neurotransmitters (Watt and Su, 1988). However, evidence for a similar mechanism in the turtle retina is lacking (Weiler and Ball, 1989). A correlation between GABAergic amacrine cells and the cell types identified in Golgi preparations (Kolb et al., 1988) is lacking because of the large number of GABAergic cells localized using either uptake or immunocytochemistry. However, the met-enkephalin-immunoreactive amacrine cells in which GAD has been colocalized (Zucker and Adolph, 1988) are morphologically similar, but not identical (Weiler and Bali, 1990), to the dopaminergic amacrine cell which has been correlated to cell type A28 (Kolb et al., 1988). 1.3. Glycine

Glycinergic amacrine cells comprise a population of cells which are as numerous as the GABAergic amacrine cell population. Approximately 30070 of inner nuclear layer somata accumulate [3H]-glycine. Their somas are small and are located in the first and second tier of cells which are adjacent to the inner synaptic layer - inner nuclear layer border. These cells have also been localized using immunocytochemical techniques to label endogenous glycine (Eldred and Cheung, 1989). Although the neurites of these cells are located throughout the IPL, there is

NEUROTRANSMITTER SYSTEMS IN THE TURTLE RETINA

evidence of stratification in $2 and $3 above the visual streak. Like the GABAergic amacrine cell population, the population of glycinergic amacrine cells consists of at least three subtypes, each probably involved in a different inner synaptic layer pathway (Eldred and Cheung, 1989). One of the small cells among the amacrine cell somata may be an interplexiform cell or a centrifugal bipolar cell (Eldred and Cheung, 1989). Although there is good evidence for glycine acting as a neurotransmitter in the retina, the actual number of glycinergic neurons which have been localized immunocytochemically or autoradiographically may be artifactually high (Massey and Redburn, 1987). Since glycine is a ubiquitous amino acid, endogenous glycine may be localized in a large number of retinal cells using immunocytochemistry. In addition, glycine is such a small molecule that it could diffuse through gap junctions, electrotonicaUy coupling cells which use glycine as a neurotransmitter and cells which do not. Such diffusion could account for glycine being localized to cells, such as bipolars, which would be expected to use an excitatory neurotransmitter in some species. Such diffusion has been cited as a possible explanation for the labelling of turtle ganglion cells in uptake experiments (Marc, 1985) and by immunocytochemistry (Eldred and Cheung, 1989). One subtype of glycinergic amacrine cell has been co-localized with the neuropeptide neurotensin in one of the two subtypes of amacrine cells which are immunoreactive for neurotensin (Weiler and Ball, 1984). This cell is a small-field amacrine cell which is broadly stratified in sublamina b of the inner synaptic layer (turtle amacrine cell type Al0) (Kolb, 1982; Kolb et al., 1988). The A10 amacrine is identical to cells which have been intracellularly stained, and recorded from; they respond maximally to long wavelengths with sustained depolarizations, suggesting that it is invoived~in the red ON pathway. In the mudpuppy retina, where the effects of glycine and neurotensin on ganglion cell activity have been studied, glycine inhibits both ON and OFF ganglion cells (Miller et al., 1981) while neurotensin excites them (Dick and Miller, 1981). If these transmitters have the same effect on turtle ganglion cells, it suggests

5

that this cell is capable of inhibiting cells by releasing glycine under some illumination conditions, and exciting cells under others. Alternatively, the synaptic release sites for glycine and neurotensin may be segregated along the neurites of this cell, or if they are co-released, glycine may act as a neurotransmitter and neurotensin may act as a neuromodulator. Glycine does not appear to be a transmitter involved in regulating dopamine release in the turtle retina. Strychnine, an antagonist of glycine and taurine has no effect on the release of dopamine in the turtle retina (Kolbinger, 1990). Although glycine is involved in the pathways leading to directionally selective ganglion cells in the turtle retina, it is not likely that there is a direct input to these cells either. Glycinergic amacrine cells may be presynaptic to bipolar cells, or perhaps inhibit the cholinergic arnacrine cell which, in turn, provides excitatory input to the directionally selective ganglion cell (Ariel and Adolph, 1985). There are no studies to date implicating other amino acids such as cyesteine sulphinic acid, serine, or taurine are transmitters in the turtle retina.

2. BIOGENIC A M I N E S

Among the biogenic amines serotonin and dopamine have been localized in the turtle retina. Whereas physiological data about the functional role of serotonin are lacking, the functional role of dopamine has recently been highlighted.

2.1. Serotonin

Evidence has accumulated in recent years that the indoleamine 5-hydroxytryptamine (serotonin; 5HT) is a possible neurotransmitter in the turtle retina. The evidence, however, is based purely on anatomical and pharmacological experiments; physiological studies, exploring the functional role of this indoleamine, are unfortunately completely lacking. This is especially regrettable, since the anatomical data emphasize multiple roles for this compound, as will be seen.

R. WEILERet al. Already the first report describing the existence of indoleamine containing neurons in the turtle pointed to the fact that serotonin is present in two cell classes of the retina: amacrine cells and bipolar cells (Witkovsky et al., 1984). This was a very interesting finding since in other species serotonin was previously attributed mainly to amacrine cells. The finding found some support by reports which also localized serotonin in bipolar cells of the skate and carp retina (Marc, 1982; Bruun et al., 1984). The identification of serotoninergic elements in these first studies was achieved using an antibody directed against serotonin obtained according to a protocol of Steinbusch et al. (1978). This immunohistochemical approach was combined with an uptake study of 3H-5HT at doses (1 /aM) where an unspecific uptake by catecholaminergic neurons does not yet occur. Both procedures labelled somata within the inner nuclear layer; larger, round ones along the inner border of the inner nuclear layer and smaller or pear-shaped ones in the outer third of the inner nuclear layer. Label was also found in the inner plexiform layer mainly along the inner and outer border. The larger somata along the inner border of the inner nuclear layer obviously belonged to amacrine cells and the somata within the outer third most likely to bipolar cells. While the immunocytochemical procedure allowed a more detailed analysis of the morphology of the labelled amacrine cells, such an analysis was not possible for the bipolar cells. Intraocular injection of 10/ag serotonin improved the immunocytochemical labelling thus allowing single Landolt clubs, a feature typical of bipolar cells in the turtle retina, to be distinguished and therefore identified. Endogenous fluorescence, however, was never observed, not even after intraocular injection of serotonin. A likely explanation of this observation might be that serotonin is rapidly converted into a nonfluorescent moiety which, however, is still recognized by the antibody. These first studies of serotoninergic neurons in the turtle retina were followed by a series of papers focusing on the functional morphology of these neurons. Pargyline, which blocks the breakdown of serotonin by monoaminoxidase, drastically increased the intensity of the

immunocytochemical label, especially in bipolar cells. A detailed analysis of the ramification pattern of the labelled neurons within the inner plexiform layer led to the classification of two amacrine cell types and three bipolar cell types (Weiler and Schiitte, 1985a). The distinction of two types of amacrine cells was based on their soma size and the initial ramification of their primary dendrites. The final ramification of the primary dendrites within the inner plexiform layer did not differ significantly between the two types. Both spread their fine processes throughout the entire IPL in a rather diffuse manner. They both belong to the class of diffuse amacrine cells with narrow fields (Marchiafava and Weiler, 1982; Kolb, 1982). The existence of the two types was not region-specific; both were observed across the retina and they were often paired. From their ramification pattern one might expect that they would respond to light stimulation with a graded, sustained depolarization of their membrane potential (Weiler and Marchiafava, 1981; Marchiafava and Weiler, 1982). So far, no direct localization of serotonin-immunoreactivity in an intracellularly recorded and dye-injected amacrine cell has been described and the above assumption is based on anatomical correlations. A comparison with Golgi-impregnated amacrine cells is somewhat difficult, but most likely these cells belong to a class of cells described as A5 amacrines by Kolb (1982). The somata of both A5 and serotonin-reactive amacrine cells have also been observed in the ganglion cell layer. Bipolar cells were distinguished according to their stratification within the IPL. All three types had a ramification within the outer border of the IPL (S1). Two types additionally ramified at layers 4 and 5. All three types of bipolars had Landolt clubs. The horizontal extension of the axonal ramification at all levels of the IPL was rather small (20- 30/am) and did not vary much with respect to the retinal location of the cell. All three types were found across the retina with a slightly greater density of the monostratified cells in the retinal periphery. A correspondence with Golgi-impregnated bipolar cells could not be established. However, comparison with intracellularly recorded bipolar cells (Weiler, 1981) led to the conclusion that all three types of serotonin-

NEUROTRANSMITTER SYSTEMS IN THE TURTLE RETINA

reactive bipolars must belong to the functional class of centre-OFF bipolars which are characterized by having at least some ramification within the outermost level of the IPL. The identification of these immunocytochemically labelled neurons as serotoninergic was further established by the observation of their selective destruction by the serotoninergic neurotoxins 5,7dihydroxytryptamine and 5,6-dihydroxytryptamine (Weiler and Schtitte, 1985a; Witkovsky et al., 1987). The question remained, however, whether any of these cells use serotonin as a neurotransmitter or whether they just take it up, store it, or metabolize it to some other compound. It has been claimed that the serotonin-reactive bipolar cells most likely acquire serotonin through gap junctions with serotoninergic amacrine cells. In the carp retina, where bipolar cells take up 3H-5HT but show little or no labelling with 5HT-antibodies (Teranishi et aL, 1987; Marc, 1982), several mechanisms for possible interactions leading to 5HT-labelling of bipolars were proposed (Marc et al., 1988), and of all labelled neurons (bipolar and amacrine) only one type of amacrine cell was considered to be truly serotoninergic. The situation in the turtle retina is different. Here, endogenous serotonin has been immunocytochemically detected in bipolar cells without any preloading (Weiler, unpublished). In addition, it was demonstrated that both cell classes, amacrine and bipolar cells, release their endogenous serotonin upon depolarization in a calciumdependent manner (Weiler and Schiitte, 1985a,b). Since one would not expect such a release of a substance which has just arbitrarily leaked into a neuron through gap junctions, this finding thus supported the view that both amacrine and bipolar cell classes use serotonin as a neurotransmitter. Further evidence came from a study which analyzed the release of serotonin under different light conditions (Weiler and Schfitte, 1985b). Light and dark conditions were emulated by using agonists and antagonists of the cone transmitter, glutamate. The results clearly demonstrated that under conditions similar to darkness, the bipolar cells released their endogenous serotonin, while the amacrine cells did not. These results suggest that the serotoninergic bipolars are centre-OFF

-k

FIG. 1. During light (left) serotonin (5HT) is tonically released by amacrine cells and during darkness (right) by bipolar cells. (Modified from Weiler and Schiitte, 1985a.)

(hyperpolarizing) and that serotoninergic amactines are centre-ON (depolarizing) cells. These observations confirmed the functional classification suggested by the morphological analysis of the ramification pattern (Weiler and Schtitte, 1985a). The differential release behaviour also further emphasizes the existence of two different serotoninergic systems in the turtle retina. Both systems have their highest density of branching in layers 1, 4 and 5 and we can therefore assume that high serotoninergic activity occurs within these layers through bipolars during dark stimulation and through amacrines during light stimulation (Fig. 1). Since serotonin most likely acts as a slow neurotransmitter which probably activates second messenger systems in its target neurons, we might expect to find a more or less constant total serotoninergic activity in these layers, irrespective of the stimulus situation. While bipolar cells receive a direct input from the photoreceptors, amacrine cells do not and their excitation involves at least one additional neuron. This, of course, opens additional possibilities for modulatory interactions and it might be that the serotoninergic activity during light stimulation is more fine tuned. The recent finding that perhaps all of the bipolar cells in the turtle retina contain glutamate as a neurotransmitter (Ehinger et aL, 1988) suggests that glutamate and serotonin may coexist in some bipolar cells.

8

R. WEILERet al.

L

1 0 0 IJm

J

II FIG. 2. Drawings of serotoninergic bipolar cells which were drawn from frozen sections at the respective locations indicated by the arrows. NO, optic nerve; V, ventral; D, dorsal; VS, visual streek. (From Schiitte and Weiler, 1987.)

The immunocytochemical labelling of the serotoninergic bipolar cells allowed for the first time the labelling of an entire group of bipolar cells that were most likely functionally homogenous, and the subsequent morphometric analysis of this group. Such an analysis (Schiitte and Weiler, 1987) not only resulted in density profiles but also revealed that some of the morphological parameters of bipolar cells are strictly correlated with eccentricity, with respect to the visual streak, and others are not. Figure 2 is an overview of one section through the turtle retina perpendicular to the visual streak. The morphology of the serotoninergic bipolar cells at different locations within this section is depicted. Several characteristics show a clear dependence on the retinal locations, while others do not. Among these the constancy of the ramification pattern within the IPL and the constancy of its size are the most prominent. This is in sharp contrast to the behaviour of the ramifications within the outer plexiform layer. Their lateral spread increases

gradually from the visual streak towards the periphery. This increase is paralleled by an increase of the soma size and the size of the Landolt club, that part of the cell which protrudes into the photoreceptor layer. Another remarkable location-dependent feature is the course of the bipolar cell axon across the inner nuclear layer. While this course is perpendicular at the visual streak and at about ll0 ° eccentricity, it takes an oblique course at intermediate regions of the retina. The different behaviour of input and output ramifications leads to a distorted projection of the outer retina onto the inner. The surface of the visual streak is already drastically overrepresented in the inner retina. That means that the existence of a magnification factor is not limited to the central projection of the retina, but also exists within the retina itself. This distortion will affect visual acuity and perhaps motion detection. The occurrence of both, perpendicular and oblique courses of bipolar cell axons within the same population, and the dependence of the

NEUROTRANSMITTER SYSTEMS IN THE TURTLE RETINA

axon course on retinal location is not in favour of the explanation for motion detection suggested by Barlow and Levick 0965). For their model one would need at each location an input from a bipolar with a straight axon and one from a more distant bipolar through an oblique axon. As already mentioned, serotonin was also localized in displaced amacrine cells (Weiler and Schiitte, 1985a). But not all of the labelled somas within the ganglion cell layer were in fact displaced amacrine cells. Some of them, having a larger soma and a slightly different branching pattern within the IPL, turned out to be ganglion cells. In a study combining intracellular recording and dye-injection with serotonin immunocytochemistry, it was possible to demonstrate unequivocally the existence of ganglion cells with endogenous serotonin (Weiler and Ammermiiller, 1986). The serotoninergic ganglion cells ramify in layers 2 and 3 of the IPL. Their density was extremely low, about 5 cells per mm 2 retina and they had a mid-sized anatomical receptive field of about 800 ~m. Their physiology was quite unusual and interesting. Generally they were of the ON - OFF type with only a weak, if any, centre-surround organization of their receptive field. They were excited by blue light stimulation, whereas they were inhibited by green light and less by red light. The physiology of these ganglion cells does not fit into the schemes so far developed for turtle ganglion cells and they might in fact represent a separate physiological group. Evidence has emerged that serotonin not only exists in intrinsic and output retinal neurons but also in a retinopetal system (Schiitte and Weiler, 1988). This system consists of only one fibre per retina. The fibre originates in the contralateral caudal mesencephalon. In the retina this fibre, after entering through the optic disc, runs in a slight bow within the ganglion cell axon layer to the dorsal temporal third of the eye. Several collaterals leave the fibre and run a short way parallel with it before they bend and take a direction orthogonal to the main fibre. These collaterals are extremely delicate and protrude into the retina where they ramify extensively and form a very fine and delicate network which spans the IPL and sometimes enters the inner nuclear layer. This web of serotoninergic processes, which

originates from a single efferent fibre, covers a substantial part of the retina, but it is restricted to a dorsotemporal location. Mapping of the innervated area showed that it lies within those parts of the retina that are suitable for binocular vision. This opens the possibility that efferent control using serotonin might be involved in retinal processes underlying binocularity. 2.2. Dopamine

Dopamine was the first neuroactive substance which was localized in the vertebrate retina by making use of its ability to form fluorescent compounds. It is therefore rather surprising that dopaminergic neurons in the turtle retina have only recently been morphologically analyzed, especially in the light of the fact that physiological effects of dopamine had already been reported in this retina (Gerschenfeld et al., 1982). The first two reports on dopaminergic neurons in the turtle retina were mainly addressed to clarify whether dopamine is located in an interplexiform cell as in the fish and other retinas (Witkovsky et al., 1984; Nguyen-LeGros et al., 1985). The rationale behind this search was the fact that the reported physiological action of dopamine appeared to be similar in turtle and fish retinas (Negishi and Drujan, 1979; Gerschenfield et al., 1982) and directed onto horizontal cells. Although dopaminergic interplexiform cells synapse onto horizontal cells in teleost fish (Dowling and Ehinger, 1975), the existence and cellular origin of dopamine in the turtle retina was not known. No dopaminergic interplexiform cells were found in the turtle retina, but dopamine was localized in a set of amacrine cells. The identification of dopaminergic neurons was achieved using an antibody directed against tyrosine hydroxylase, the rate limiting enzyme of dopamine metabolism (Witkovsky et ai., 1984; Nguyen-LeGros et al., 1985) in combination with autoradiographical localization of 3H-dopamine uptake and fluorescence techniques after preloading with dopamine (Witkovsky et al., 1984). All three markers labelled an amacrine cell population which was subsequently referred to as dopaminergic. The techniques requiring uptake of dopamine sometimes also labelled bipolar-like cells.

10

R. WEILERet al.

Since the labelling clearly depended on the concentration of dopamine in the incubation medium and appeared only with concentrations above the apparent saturation concentration of the highaffinity uptake system for dopamine, these bipolar-like cells were not considered to be dopaminergic. The restriction of the localization of tyrosine hydroxylase to a population of amacrine cells was confirmed in subsequent studies (Kolb et al., 1987; Weiler eta/., 1988). An antibody directed against dopamine also labelled an amacrine cell population which corresponded in number of labelled somas and branching pattern within the IPL to the one labelled with tyrosine hydroxylase antibody (Weiler, unpublished). Furthermore, the injection of the dopamine neurotoxin 6-hydroxydopamine destroyed presumptive amacrine cells without any effect in the outer plexiform layer (Witkovsky eta/., 1987). Subsequent immunocytochemistry revealed that the neurotoxin had selectively destroyed tyrosine hydroxylase-positive amacrine cells (Weiler eta/., 1988). Therefore, it seems reasonable that the only dopaminergic neuronal element in the turtle is an amacrine cell. The somata of tyrosine hydroxylase-positive amacrine cells are located either within the innermost row of the inner nuclear layer or slightly deeper within the inner nuclear layer. In the former case two to five primary dendrites leave the soma, and in the latter case there is usually one primary dendrite which ramifies shortly after reaching the inner plexiform layer (Witkovsky e t a / . , 1984; Nguyen-LeGros et al., 1985; Kolb et al., 1987). Three major layers of dendritic branching exist within the IPL. The outermost band is within layer 1, the middle one along the border between layers 2 and 3, and the innermost along the border between layers 4 and 5. Dopaminergic amacrine cells therefore seem to ramify within these layers of the IPL where many amacrine cells containing neuromodulatory substances such as indoleamines and neuropeptides also ramify (see this chapter). This opens the possibility of direct synaptic interactions among such modulatory amacrine cells, an idea which is supported by morphological and physiological observations (Weiler and Ball, 1989).

Dopaminergic amacrine cells belong to medium field amacrines (Kolb, 1982). Their anatomical receptive field has a size of about 200-700/Jm depending on the retinal location. They most likely correspond to the A28-type (Kolb et al., 1988). Intracellular recordings from amacrine cells with a similar morphology have shown that these cells respond to light stimulation of the retina with a rather complex waveform. The major components of the response are transient hyperpolarizations at the onset and offset of the light stimulus. The amplitude and the time course of the two hyperpolarizing components seems to depend on the intensity of the stimulus light (Ammermiiller and Weiler, 1989). Isodensity maps showed that the density peaks within the visual streak with about 60 cells per mm 2 and falls towards the periphery to about 10 cells per mm 2. The cells are distributed in a nonrandom pattern (Kolb et al., 1987). Dopaminergic cells therefore belong to the group of amacrine cells with a low density, like other amacrine cell populations containing neuromodulatory substances. Despite their low density, dopaminergic amacrine cells seem to have a rather pronounced effect on neuronal information processing within the turtle retina. The discovery of the functional role of dopamine in the vertebrate retina is certainly one of the most exciting aspects of retinal research in recent years. There is, in the meantime, overwhelming evidence that dopamine modulates electrical transmission via gap junctions between horizontal cells in fish and turtle retina (Dowling, 1986; Piccolino and Demontis, 1988). Evidence for such an effect of dopamine in the turtle came from intracellular recordings from horizontal cells (Gerschenfeld et al., 1982; Piccolino et ai., 1984). In Ll-horizontal cells light stimuli evoke graded hyperpolarizations whose amplitude depends on the size of the stimulated retinal area. This dependence results from the strong electrical coupling of these cells (Lamb, 1976; Witkovsky e t a / . , 1983). Small spots, even though they may stimulate all presynaptic photoreceptors which are presynaptic to the recorded horizontal cell, induce only a small voltage deflection in this cell because most of the induced synaptic currents spread through the gap junctions into adjacent cells. This tight coupling, on the other hand, makes it

NEUROTRANSMITTER SYSTEMS IN THE TURTLE RETINA

possible to record a voltage drop in a horizontal cell as a result of an annular stimulation, which directly stimulates only distant horizontal cells. The addition of dopamine to the superfusion medium affected the response to a small spot of light and to an annulus, but in the opposite direction. The response to a small spot was increased and that to an annulus was decreased. A very likely explanation for this was that dopamine altered the coupling resistance between Ll-horizontal cells. This hypothesis was supported by mathematical analysis of current injection experiments (Piccolino et al., 1984). It was further supported by the observation that the transfer between neighbouring cells of the dye Lucifer Yellow, which can pass through gap junctions, can be prevented by dopamine. Similar results had been reported from the fish retina (Teranishi et al., 1983). The application of several antagonists and agonists of the dopamine receptors finally revealed that D I receptors were involved (Piccolino et al., 1984). Elsewhere in the brain this receptor is linked to adenylate cyclase and its interaction with dopamine leads to a stimulation of the formation of cAMP. In fact, the action of dopamine could be mimicked by agents which increase the intracellular amounts of cAMP. It was therefore concluded that dopamine, by increasing cAMP in horizontal cells, increases the coupling resistance of the gap junctions (Piccolino et al., 1984). This conclusion was supported by the localization of D1 receptors on horizontal cells in the fish retina and their linkage to adenylate cyclase (for a review, see Dowling, 1986). It was recently confirmed by experiments performed on isolated fish horizontal cells, where the effect of cAMP and its dependent protein kinases on the coupling resistance was directly measured (Lasater, 1987), and by experiments where the effect of dopamine on single connexon channel properties were analyzed (McMahon et al., 1989). But dopamine affects not only the properties of a single connexon but also the overall density of connexons within a given gap junction area. This was shown by comparison of freeze fracture replicas of retinas in which the dopaminergic amacrine cells had been destroyed by 6-hydroxydopamine with control retinas (Weiler et al., 1988). The connexon density increased by 45°70 in

11

the dopamine-depleted retinas. Dopamine depletion was verified by biochemical analysis of the total endogenous dopamine content and by tyrosine-hydroxylase immunoreactivity. It is not yet clear whether the observed increase of connexon density is due to a shift of existing connexons or whether new connexons are inserted into the membrane. It is also not clear to what extent the observed effect of dopamine depends on the activation of cAMP. While in the fish retina dopaminergic interplexiform cells synapse directly onto the horizontal cells and dopaminergic action onto these neurons is anatomically plausible, the anatomy in the turtle retina does not favour such an action. Attempts were therefore made to clarify whether an increase of endogenous dopamine release would also affect horizontal cell coupling. Several substances known to affect dopamine release without interfering with dopamine receptors were tested and they all affected horizontal cell coupling (Piccolino et al., 1987). They did not affect the coupling if dopaminergic neurons were destroyed prior to the experiments, indicating that endogenous dopaminergic activity can indeed modulate the coupling resistance. These data, together with the observation that dopamine could affect dye transfer between horizontal cells even when these neurons were synaptically isolated by cobalt ions, emphasized the existence of a diffusion dependent action of dopamine. Dopamine, released from the dopaminergic amacrine cells must diffuse over a distance of about 10-30/am to reach the horizontal cells, a distance which is within the limits of other known long-distance acting neuromodulatory compounds in the somatic and autonomic nervous system. On the other hand, this would require that the local uptake and/or binding of dopamine within the IPL is quickly saturated or has a low affinity. Only with a low efficacy of the uptake systems could a diffusion process from the IPL to the horizontal cells in the outer plexiform layer be warranted. Further biochemical analysis is needed to verify the diffusion hypothesis which, however, at the moment is the only reasonable one. Dopaminergic neurons appear to be under tonic inhibition by a GABAergic pathway. Blockade of

12

R. WEILER et al. TABLE 1. Effects of Different Neuroactive

Substances on the Basal Release of Endogenous Dopamine in the Turtle Retina Excitatory amino acids Glutamate Kainate NMDA Inhibitory amino acids GABA Bicuculline Bicuculline, Ca-free ringer Picrotoxin Baclofen Glycine Strychnine Strychnine (on high potassium induced release)

10 pM--

10 b/M-10 laM10 pM-10 ,uM-10 /AM-

Amines Serotonin Serotonin, Ca-free ringer Neuropeptides Met-Enkephalin Naloxon Glucagon

10 /AM 10 JIM 10 JIM

0 + 0

1 mM 10 ,aM 10 /.aM 50 hiM 100 I.,IM 1 mM 50 JAM 50 JAM

0 + 0 0 0 0 0

50 laM 50 /.aM

+

10 ,caM-- 100 /aM 10 /aM 10 /aM-- 100 hIM

0

0 0

+

Data are taken from Kolbinger (1990).

this tonic inhibition by the GABA antagonist bicuculline leads to an increased dopamine release, which then modulates horizontal cell coupling (Piccolino et al., 1982). In a recent study it was possible to directly measure the release of endogenous dopamine from the turtle retina (Kolbinger, 1990). Basal release of dopamine was increased about three times when bicuculline was added to the superfusion medium. Several other neuroactive substances of known activity in the turtle retina were investigated in this study. Table 1 gives a summary of the observed effects. The threefold increase of endogenous dopamine release caused by naloxone confirmed the results of a recent study where the interactions between enkephalin and dopamine were analyzed (Weiler and Ball, 1989). In this study the potassium induced release of 3H-dopamine from the turtle retina was monitored. This release was decreased by a third when the neuropeptide Met-enkephalin was added to the superfusion medium. Naloxone blocked the inhibitory action of Met-enkephalin and doubled the baseline release of 3H-dopamine. These results demonstrate a tonic inhibition of the

dopaminergic amacrine cells through an opiate system of about the same magnitude as the tonic inhibition by the GABAergic system. Since Met-enkephalin does not affect the release of 3H-GABA (Weiler and Ball, 1989), the opiate system does not act via the GABAergic system. The opiate system consists of two neuronal populations in the turtle retina (Weiler, 1985a,b; see also this chapter). One population is formed by amacrine cells which stratify within the same layers of the IPL as do the dopaminergic amacrine cells. Co-localization of dopamine and Metenkephalin, however, was not found, but the dopaminergic and met-enkephalinergic amacrine cells make very close contacts within the IPL, suggesting direct synaptic interactions (Weiler and Ball, 1989). The other part of the opiate system is formed by efferent fibres originating in the caudal mesencephalon. Efferent fibres project onto amacrine cells (Marchiafava, 1976; Weiler, 1985b), some of which may be dopaminergic amacrine cells. The opiate efferent fibres ramify predominantly in layers 3 and 4 of the IPL, neither of which contain massive ramifications of the dopaminergic amacrine cells. The existing overlap, however, might be sufficient for direct synaptic interactions. This opens the possibility that the release of endogenous dopamine is under direct central control. Since dopamine regulates the gap junctions between the horizontal cells and consequently the spatial contrast sensitivity, this important aspect of retinal processing might be modulated within the retina by central influence. It is quite interesting to see that efferent systems in the turtle retina contain neuroactive substances with known modulatory capabilities like Metenkephalin, serotonin and glucagon (see this chapter) and the possibility that these neuromodulators may act by controlling the release of other neuromodulators within the retina.

3. NEUROPEPTIDES Numerous peptides which appear to be neurotransmitter candidates, have been detected in a variety of vertebrate retinas (Stell et aL, 1980;

NEUROTRANSMITTER SYSTEMS IN THE TURTLE RETINA

Brecha, 1983; Brecha and Karten, 1985; Karten and Brecha, 1983; Marc, 1986; Massey and Redburn, 1987). Many of these have also been localized by histochemical and biochemical techniques in the turtle retina. Up to now, these include: corticotropin-releasing factor (CRF), methionine enkephalin (M-ENK), glucagon (GLU), neuropeptide Y (NPY), neurotensin (NT), a NT-related hexapeptide, LANT-6, which resembles the biologically active portion of NT, substance P (SP), and somatostatin (SRIF).

3.1. Corticotropin-Releasing Factor (CRF) CRF is composed of 41 amino acids and was first identified from ovine hypothalamus. It regulates the release of adrenocorticotropic hormone and beta-endorphins frotn the anterior pituitary (Vale et al., 1981). In the turtle retina there is only one immunocytochemical study of the distribution of CRF-like immunoreactivity, using antisera against bovine CRF (Williamson and Eldred, 1989). The authors counted an average of approximately 2700 labelled amacrine cells per retina. However, the density of labelled amacrine cells was much higher in the visual streak region (145 mm 2) than more peripherally (57 per mm 2) and - - more importantly -- anatomically different amacrine cells were restricted to confined regions of the retina; there was no labelling dorsal to the visual streak. Two anatomically distinct amacrine cell types were labelled for CRF. Type A, whose cell body diameter varied from 8 - 12 t~m, was described as mainly bistratified, arborizing in sublayer 1 and at the border between strata 2/3. Occasionally, processes extended to stratum 5 (see Fig. 3, which is a summary of the immunocytochemical findings in turtle retina described in this chapter). The field covered by the processes was extremely elongated, with the long axis running parallel to the visual streak. Amacrine cells of this type were found exclusively in the visual streak region. The type B amacrine cells with CRF-like immunoreactivity, on the other hand, were only found in the ventral part of the retina. They were separated from the type A cells by a region with little or no CRF-like immunoreactivity. Type B

13

cells arborized in a wide band in the IPL including sublayers 4 and 5. Sparse arborizations in sublayer 1 were described (Fig. 3). The processes in stratum 5 seemed to surround cell bodies in the ganglion cell layer. In tangential view, processes extended more or less radially in all directions. In addition to these two types of amacrine cells with CRF-like immunoreactivity, Williamson and Eldred (1989) also found labelled cells in the ganglion cell layer (GCL). Those which were located in the visual streak region were believed to be displaced amacrine cells of type A, because they ramified in the same sublayers and were labelled equally well. At least some of the other labelled cells in the ganglion cell layer, which were more lightly labelled and which were distributed throughout the retina, were shown to be ganglion cells. This was proven by a combination of immunochemistry with retrograde labelling of many ganglion cells from the optic tectum with rhodamine. In several cases the markers co-localized. It is difficult to correlate the amacrine cells showing CRF-like immunoreactivity with the present amacrine cell classification of Kolb (1982) and Kolb et al. (1988). Neglecting the occasional processes in sublayer 5 and assuming that cells in Golgi stained material often lack fine processes, then the type A cell could correspond to the amacrine cell termed A 14 by Kolb (1982). This amacrine type is also oriented parallel to and along the border of the visual streak, has similar dimensions of cell body and processes, and arborizes in the sublayers 1 and 3. Ammermtiller and Weiler (1988), Kolb et al. (1988) and Guiloff et al. (1988) suggested that the type A amacrine cell with CRF-like immunoreactivity could correspond to the 'giant' OFF-centre amacrine cell (A 27) which is also oriented parallel to the visual streak. In view of the illustrations in the study of Williamson and Eldred (1989), this correlation seems now very unlikely. However, the ramification levels of the type A cell suggest indeed, that this cell type shows OFFcentre light responses, because the functional subdivision into distal OFF sublayers and proximal ON sublayers has been shown to be valid in turtle retina too (Weiler, 1981; Marchiafava and Weiler, 1980, 1982; Ammermtiller and Wefler,

14

R. WEILERet al. INL

IPL

GCL

INL

GCL

IPL

type A, vis. streak aRE

NPY

type B, only ventral displ, type A? ganglion cell type A

~ ~ ~

--

__

type B

LANT-6

_ ? ~ ~-~

m, j w

~

~

type C, peripheral type A, OFF~ A 12 GLU

~

type B, (?) displaced type A ? 0-4 efferent fibers

~:,

20

NT

SRIF

SP _ _

celffypes 10

~

v

~.

~

4JJJP

T f

%

g a r ~ i o n cell F G-prolein

rI --J

type B, sustained ON interstitial type B ?

displaced type B ?

, ~ ~.,.~ ~ ~

~

like NT-type B displaced gc ? ganglion cell A ganglion cell B gon~on cellC ganglion cell D

~.~ ~

numbero(

type B, non-vis,slreak 3-6 efferent fibers type A. 0N-OFF

~

~

~

J

~>~.~~ 'r

v->~

lypeA, vis.slreak ENK

like NT-lype A ~

sublayer 1

2

3

4

5

GCL

only visual streak gang~ncell ?

type A, nol vis.slreak type B g ( r ~ o n cels

FIG. 3. Schematic representation of the laminar distribution pattern in the turtle IPL of cell types containing neuropeptidelike immunoreactivity. See text for a more exact description o f the different cell types. A short classification is given on the right side. Dotted areas indicate levels of ramification inside IPL. Heavily dotted areas indicate main levels of arborizations, light dotted areas strata where only sparse arborizations occurred. INL is to the left, GCL to the right. Filled circles indicate localizations of cell bodies. The last distribution pattern shows the occurrence o f G-protein immunoreactivity in the 1PL. Contrary to the above representations, this row does not represent a cell type (see Terashima et al., 1987). The histogram at the bottom of this figure represents the total number of cell types with neuropeptide-like immunoreactivity, which ramify in the different sublayers. Each sublayer was divided into a distal and a proximal half.

1988, 1989). Williamson and Eldred (1989) suggested that type A cells may influence the OFF responses of ganglion cells in the visual streak. This hypothesis could be tested physiologically. The type B cells, on the other hand, may be amacrine cells that influence ON responses at the ganglion cell level in the ventral half of the retina. A correlation of the type B cell to the Golgi stained material of Kolb (1982) is again very difficult. From cell body size, ramification level, field size of arborizations and beaded appearance of secondary processes, Kolb's (1982) amacrine cell A l0 could resemble the type B cell with CRF-

like immunoreactivity. However, A 10 has been correlated with a sustained amacrine cell type, showing co-localization of neurotensin-like immunoreactivity and 3H-glycine uptake system (Kolb et al., 1988; Weiler and Ball, 1984). Doublelabelling experiments may help determine whether amacrine cells with CRF-like immunoreactivity are the same type as those with NT-like immunoreactivity and glycine uptake. The function of CRF in the turtle retina is completely unknown. Williamson and Eldred (1989) suggested a role in mediating CRF-induced hyperglycaemia by modulating glucagon levels in

NEUROTRANSMITTER SYSTEMS IN THE TURTLE RETINA

the retina. Amacrine cells with GLU-like immunoreactivity have been found in the turtle retina (Eldred and Karten, 1983; Weiler et al., 1989; see below).

3.2. Neuropeptide Y (NPY)

Neuropeptide Y is a 36 amino-acid peptide and is found in high concentrations in brain tissue as well as in the peripheral nervous system and other organs. It regulates hormone release and shows vascular action (O'Donohue et al., 1985). As in other vertebrate retinas, only amacrine cells were labelled when immunocytochemistry with an antiserum against NPY was performed in turtle retina (Adolph and Bruun, 1987; Zucker and Adolph, 1987; Isayama and Eldred, 1988; Isayama et al., 1988). Three types of amacrine cells with NPY-like immunoreactivity could be discerned (Isayama and Eldred, 1988). Type A was numerous and had large somata with a diameter of approximately 11/am and well stained processes ramifying in sublayers 1, 3, and at the 4/5 border (Fig. 3). The appearance and number of boutons were different in the sublayers 1, 3, and 4/5. This type of amacrine cells gave rise to processes which entered the ganglion cell layer (Zucker and Adolph, 1987; Isayama and Eldred, 1988). The size of these cells varied with retinal eccentricity. Smaller cells were located nearer to the visual streak region. The somata of the less numerous type B amacrine cells with NPY-like immunoreactivity were slightly smaller (9/am) and the processes were described to ramify primarily in sublayers 2 and 4 (Fig. 3). These cells appeared more lightly labelled than type A cells. Isayama and Eldred (1988) suggested that many of the processes of the type A cells may contact processes of type B cells, because both types were often found in close proximity. Support for this suggestion was obtained in an ultrastructural study (Isayama et al., 1988; see below). Some examples of putative displaced amacrine cells of type B, labelled in the GCL, were also reported by Isayama and Eldred (1988). The type C cells were restricted to the periphery of the retina (Isayama and Eldred, 1988). These were large, well-labelled cells with large somata

15

(12/am) and elongated, straight processes running away from the soma for several millimeters without branching. Arborizations were reported to ramify in strata 1 and 4 or 5 (Fig. 3). A total of approximately 6300 cells with NPYlike immunoreactivity yielded a mean overall density of approximately 31 cells per mm 2. Isayama and Eldred (1988) found significant differences of cell densities in different regions within the visual streak. The temporal portion had higher densities than the central or nasal portions. Additionally, there were significant differences between several other combinations of retinal areas. This unusual distribution of densities, which differs from the overall density of ganglion cells and cones (Peterson and Ulinski, 1982; Kolb and Jones, 1982), and of other neuropeptides (Eldred and Karten, 1983), may indicate an unusual physiological role of NPY in the turtle retina. Isayama and Eldred (1988) suggested a functional role in the rod-driven pathways, because rods are found predominantly outside the visual streak, where NPY immunoreactivity was quite high. It was not possible to correlate any of the three cell types with NPY-like immunoreactivity with previously described morphological cell types. Only A 12, A 5 , and perhaps A28 of the classification of Kolb et al. (1988) ramify at approximately the same levels as the type A in the study of Isayama and Eldred (1988). However, A 28 as well as A 12 and A 5 differ in their tangential appearance from the putative NPYcontaining A type cell. The diameter of A 5 is much smaller than that of type A NPY cells. Differences in location and size of synaptic boutons on the arborizations of A 12 and A 28 compared to the type A amacrine cells also speak against such a correlation. Therefore, it is likely that the A, B, and C type amacrine cells with NPY-like immunoreactivity represent three morphological types in the turtle retina which have not been described in Golgi stained material. Again, nothing is known about the physiological role of NPY in the turtle retina. Isayama and Eldred (1988) suggested a possible interaction with catecholaminergic amacrine cells, because arborization levels are within the same strata and NPY was found to possess modulatory effects on

16

R. WEILER el al.

catecholaminergic neurons (Witkovksy et al., 1984; Nguyen-LeGros et al., 1985; Kolb et al., 1987; Wahlestedt et al., 1986), This possible functional role was supported by an ultrastructural study where NPY-like immunoreactivity was found within large vesicles of NPYpositive amacrine cells -- probably of the A type (Isayama et al., 1988). No reaction product was found by the authors in small synaptic vesicles inside the same amacrine cells. This suggests that at least one type of amacrine cells with NPY-like immunoreactivity contains another neurotransmitter. Quantification of synaptic contacts yielded a predominant amacrine cell input (87°70) compared to bipolar cell input (13070). Synaptic output occurred mainly onto unknown, mostly vesiclecontaining profiles (67%), onto other, unlabelled amacrine cells (25°7o) and to a lesser extent onto bipolar cells (7o7o) (Isayama et al., 1988). Although input from amacrine cells and bipolar cells occurred in all three strata of the IPL, output onto bipolar cells occurred only in the region between strata 4/5. Output onto amacrine cells and unknown profiles was again in all three strata. A small percentage of output in layer 4/5 was found to occur onto other labelled processes, perhaps of type B amacrine cells with NPY-like immunoreactivity.

3.3. Glucagon (GLU)

Glucagon is a 29 amino-acid pancreatic hormonal peptide, occurring mainly in the gut and central nervous system. Its hormonal action is mainly the release of glucose from hepatic cells and interactions with the insulin system (Saskai et al., 1985). The concentration of GLU in the turtle retina was measured by Weiler et al. (1989) using a radioimmunoassay and ranged from 50-280 pg per mg of protein. The concentration seemed to depend on the age of the animals. Antisera against GLU labelled approximately 2500 amacrine cells in one turtle retina (Eldred and Karten, 1983). Density of labelled neurons was higher in the visual streak region and decreased from approximately 100 cells per mm 2

to less than 50 cells per mm 2 towards the periphery. The amacrine cells with GLU-like immunoreactivity were tristratified in sublayers 1, the 2/3 border and the 4/5 border (Eldred and Karten, 1985; Weiler et al., 1989). Additionally, putative displaced amacrine cells which ramified in the same strata of the IPL, but had their somata in the GCL were described. Weiler et al. (1989) suggested that amacrine cells with GLU-like immunoreactivity form two subpopulations, defined on the basis of their initial ramification patterns. Type A has one principal process leaving the soma, which splits off into secondary and tertiary processes covering an asymmetric field. This type seemed to be larger than the type B, which had a more circular field covered by the processes. Several principal branches were seen leaving the soma of this type. It seems to be difficult to distinguish both subpopulations exactly, however, Eldred and Karten (1983) also suggested that two subpopulations might exist. The amacrine cells with GLUlike immunoreactivity are similar to A 12 of Kolb's (1982) classification. Kolb et al. (1988) also correlated the GLUergic amacrine cells with A 12, based on their own, unpublished results. At the periphery of the retina another subpopulation of amacrine cells with GLU-like immunoreactivity could exist, because Eldred and Karten (1983) described a bundle of immunoreactive fibres in the ora serrata. At least some of these fibres were originating from nearby amacrine cells with GLUlike immunoreactivity. In addition to amacrine cells, Weiler et al. (1989) described a few ( 1 - 4 ) efferent, beaded fibres with GLU-like immunoreactivity running within the axon bundles of the ganglion cell axon layer. These fibres entered the retina via the optic nerve, ramified in the GCL, and terminated with swellings near unlabelled somata in the GCL, probably ganglion cells. Amacrine cells with GLU-like immunoreactivity are up to now the only example, where a physiological and morphological characterization of a neuropeptide-containing retinal neuron has been successful (Weiler et al., 1989). An amacrine cell with sustained, hyperpolarizing light responses during light-ON (OFF-centre) was stained with Lucifer yellow after physiological

NEUROTRANSMITTER SYSTEMS IN THE TURTLE RETINA

characterization and the Lucifer yellow fluorescence in the soma co-localized with the GLUlike immunoreaction endproduct in the same cell. The responses to light stimulation indicated a relatively small receptive field centre of approximately 200/am diameter. The centre-surround organization was not very prominent, but some anatagonistic surround was suggested, because the responses became smaller with increasing spot size. No depolarization was observed during annular stimulation. The responses to stimuli of different wavelengths yielded a pure luminositytype amacrine cell. The morphological appearance of the Lucifer yellow-stained cell resembled A 12 of Kolb's (1982) classification. The field covered by the processes was very asymmetrical and branches occurred at three levels of the IPL; at approximately 15% (sublayer 1), 50°70 (sublayer 3) and 80070 (4/5 border). This unusual light response for a tristratified amacrine cell was confirmed in several other recorded and Lucifer yellow-labelled, putative A 12 amacrine cells (Ammermiiller and Weiler, 1989). The polarity and sustained character of the photoresponses suggest that synaptic input occurs mainly in sublayer 1, where OFF-cells predominantly ramify (Weiler, 1981; Ammermtiller and Weiler, 1989). The branches in the other strata might be mainly responsible for synaptic output. It would be interesting to test this hypothesis at the ultrastructural level. If this were true, then this amacrine cell would be an important link between the OFF- and ON-pathways in the turtle IPL. The physiological light response indicates that amacrine cells with GLU-like immunoreactivity are indeed involved in retinal signal processing. The polarity of the light response indicates that GLU is probably released during darkness and that release is inhibited by light. This suggestion was supported by release experiments (Weiler et al., 1989). The authors found, that GLU release was below detection limit under constant light conditions and that enhanced potassium concentration led to a release of approximately 10 - 20% of the total GLU content. Additionally, Weiler et al. (1989) showed that cAMP formation in turtle retina could be stimulated by GLU in a dose-dependent manner, similar to turtle pancreas

17

extract. These results support the idea that GLUlike immunoreactivity in turtle retina indeed represents GLU content in amacrine cells, and that GLU acts at least partially on other neurons via a second messenger system. GLU was about 100 times more potent than dopamine, which also stimulated cAMP formation in the turtle retina (Weiler et al., 1989).

3.4. Methionine Enkephalin (M-ENK)

Methionine enkephalin is a five amino-acid opiate neuropeptide, first isolated from brain. Its role as a brain neurotransmitter is now well established (Snyder, 1980). In a single turtle retina, antisera against M-ENK labelled approximately 7 3 0 0 amacrine cells (Eldred and Karten, 1983). The density of labelled cells decreased from the visual streak region (300 cells per mm z) towards the periphery (100 cells per ram2). Eldred and Karten (1983) described two morphological types of amacrine cells with M-ENK-Iike immunoreactivity. Type A was found only in a narrow band within the visual streak region. These cells had an elongated and narrow field of arborizations, whose long axis, which measured about 300 ~m compared to about 50 ~m of the short axis, extended parallel to the visual streak. The levels of ramification within the IPL were strata 1 and a wide band in sublayers 4 and 5. The type B amacrine cells with M-ENK-like immunoreactivity arborized at the same levels of IPL as the type A cells. However, the field covered by their processes, as seen in tangential view, was not oriented. Type B cells occurred in all parts of the retina outside the visual streak region (Eldred and Karten, 1983, 1985; Weiler, 1985a). The type B amacrine with M-ENK-like immunoreactivity seemed to be quite similar to the Golgi-stained amacrine cell A 11 (Kolb, 1982; Kolb et al., 1988). In addition to the amacrine cells, Weiler (1985a,b) found 3 - 6 efferent fibres per retina with M-ENK-Iike immunoreactivity, originating in the mesencephalon. These beaded, unmyelinated fibres entered the retina through the optic disk extended near to the visual streak region, where they ramified and entered the IPL. In the

18

R. WEILER et al.

IPL the fibres terminated in strata 3 and 4. The origin of the fibres was confirmed by retrograde Nuclear yellow tracing from the eyes and doublelabelling experiments with antisera against M-ENK. About 25 - 50°7o of retrogradely labelled cells in the mesencephalon showed also M-ENKlike immunoreactivity. The absolute number of double-labelled cell somata was in good agreement with the number of fibres found in the optic nerve (Weiler, 1985b). Zucker and Adolph (1988) reported that about 10°70 of amacrine cells with M-ENK-Iike immunoreactivity contain co-localized GABA, and that M-ENK immunoreactive material containing processes in the IPL synapse onto GABAergic profiles, which again synapse onto ganglion cells. These results were based on light microscopic GAD immunocytochemistry and on GABA immunocytochemistry at the ultrastructural level. The finding that M-ENK might be colocalized with a second neurotransmitter, which might in some cases be GABA (Zucker and Adolph, 1988), was supported by an ultrastrucrural study of the type B amacrine cells with M-ENK-Iike immunoreactivity (Eldred and Karten, 1985). There, numerous small, unlabelled vesicles had been found inside type B cells in addition to labelled, dense-core vesicles containing M-ENK-Iike material. This suggested that these cells might contain a second transmitter. The unlabelled small synaptic vesicles were concentrated at synaptic release sites, whereas labelled dense-core vesicles were not associated with anatomically defined synaptic release sites. The labelled amacrine cells received conventional synaptic contact from unlabelled amacrine cells and ribbon synaptic contact from bipolar cells in both the distal and proximal sublayers. They made conventional synaptic contact onto bipolars in the distal IPL and onto ganglion cells in the proximal IPL. Some indication for reciprocal synaptic connections between these amacrines and bipolars was also obtained by Eldred and Karten (1985). Nothing is known about the light responses of the putative M-ENKergic amacrine cells. However, Ariel and Adolph (1985) reported some effects of M-ENK or related agonists, and of the broad spectrum opiate antagonist naloxone, on directionally selective ganglion cells. They showed

a small increase in light responsiveness of some directionally selective ganglion cells during superfusion of the retina with micromolar concentrations of methionine enkephalin and (D-AIa 2) methionine enkephalinamide (DALA). Naloxone decreased light responsiveness and had no effects onto directional or velocity tuning of these cells. Adolph (1989) also investigated the action of DALA onto spatial response characteristics of ganglion cells by focal application of DALA within the receptive fields of recorded ganglion cells, and showed an increase in light-evoked and spontaneous activity. Additionally, Adolph (1989) found a DALA-induced change from transient OFF-response pattern to sustained ON- and OFFresponses. Under the influence of DALA, responses to stimuli with high spatial frequencies were enhanced compared to untreated conditions. Naloxone had the opposite effect. These changes were hypothetically explained by changes of the receptive field organization; DALA was supposed to decrease the receptive field centre diameter. The modulatory actions of DALA were enhanced with concurrent application of the GABA antagonist picrotoxin. Adolph (1989) suggested that part of the opiate action was mediated by GABA effects -especially the effects onto spatial transfer functions. The increased spontaneous and lightevoked ganglion cell activity was supposed to result from direct DALA action. For an explanation of these effects, efferent input also has to be considered, although nothing is known at the moment about the transmitter(s) causing the slow depolarizations which could be induced by optic nerve stimulation in slow transient amacrine cells (Marchiafava, 1976; Weiler, 1985a). M-ENKergic efferent fibres could play some role in such a system (see above; Weiler, 1985b).

3.5.

Neurotensin (NT)

Neurotensin is a tridecapeptide first discovered in extracts of bovine hypothalamus; it occurs mainly in the central nervous system and the small intestine. NT produces many cardiovascular effects including hypotension, hyperglycaemia, and vasodilatation (Emson et al., 1985).

NEUROTRANSMITTER SYSTEMS IN THE TURTLE RETINA

Antisera against NT labelled approximately 12,800 amacrine cells in a single turtle retina. Density of labelled cells decreased from over 350 cells per mm 2 in the visual streak region to less than 150 cells per mm z in the periphery (Eldred and Karten, 1983). These authors described two types of amacrine cells with NT-like immunoreactivity. Type A had a large, vertically elongated cell body (I0 × 14/am) with a single stout primary process which gave rise to 3 - 4 stout secondary processes. These processes divided again to produce 12- 15 tertiary processes, which gradually decreased in diameter. These secondary and tertiary branches stratified in sublayer 3 of the IPL. There they extended radially, covering an area in excess of 500/am in diameter. No apparent synaptic swellings were observed on these processes (Eldred and Carraway, 1987). Type B amacrine cells with NT-like immunoreactivity were more heavily labelled than type A cells. The type B cells had smaller somata (8/am) located more distal in the INL than type A cell bodies. The somata gave rise to several delicate, tortuous and relatively short processes with apparent synaptic swellings. Levels of ramification of type B cells were mainly strata 3 and 4 of the IPL. Occasional processes in sublayer 5 were also observed (Eldred and Karten, 1983). The diameter of the field covered by the processes was estimated to range around 250/am (Eldred and Carraway, 1987). The same two types of amacrine cells with NTlike immunoreactivity were also observed in a study of Weiler and Ball (1984). Additionally, these authors described two further types which occurred less frequently. Type C was an interstitial amacrine cell with its large soma located within the IPL. Type D was a displaced amacrine cell with its soma located in the GCL. The stratification levels of both additional types, C and D, seemed to be similar to the ones of type B cells. In a study of Weiler and Ball (1984) co-localization of NT-like immunoreactivity with 3H-glycine uptake system had been shown. About 7% of 3H-glycine accumulating amacrine cells were also NT immunoreactive. All of these cells belonged to the type B amacrine cells with NT-like immunoreactivity. This finding was supported by a recent study investigating the localization of glycine in

19

the turtle retina with immunocytochemical methods (Eldred and Cheung, 1989). The authors confirmed the statement of Weiler and Ball (1984) that type B NT-containing amacrine cells comprise a small percentage of glycinergic amacrine cells in turtle retina. Eldred and Carraway (1987) tried a morphological correlation of the amacrine cells with NT-like immunoreactivity with the Golgi stained cells of Kolb's (1982) classification. They suggested that type B cells correlate with the A 10 Golgi stained type, and that type A cells correlate with the A 22 Golgi stained type. However, intracellular stainings and recordings from A 22 type amacrine cells make this correlation unlikely (unpublished results). A 22 cells seem to stratify slightly more distal, in sublayer 2, than type A amacrine cells with NT-like immunoreactivity. Additionally soma shapes seem to differ, and the physiological responses of A 22 cells were purely OFF, which would be very surprising for a cell ramifying monostratified in the middle of IPL as the type A cells do (unpublished results; Kolb, personal communication). Therefore, the correlation of type A cells with A 22 has to be judged very cautiously at the moment. Another possible candidate for type A cells with NT-like immunoreactivity would be A 24 of Kolb's (1982) Golgi classification. Weiler and Ball (1984) and Weiler (1985a) tried to correlate type A and B amacrine cells with physiological recordings from intracellularly stained amacrine cells which appeared morphologically similar to type A and B cells. They suggested that type A correlates to a fast, transient ON - OFF type with similar tangential appearance and a narrow level of stratification in sublayer 3. Type B cell was correlated with a similar, ONsustained amacrine cell ramifying predominantly in sublayers 3 - 5. These neurochemical - electrophysiological correlations are very likely in view of the distributions of the branches within IPL. Ammerm~iller and Weiler (1989) showed that monostratified amacrine cells branching narrowly in the middle of the IPL are ON - OFF type cells, and that cells which exclusively ramify in the inner half of the IPL are ON type cells. The O N - O F F amacrine cell which had been correlated with the NT type A cell responded with

20

R. WEILER el al.

fast transient depolarizations after light-ON and after light-OFF. Increasing the area of the stimulating light spot reduced response amplitudes and slowed their time course. No clear antagonistic centre-surround organization of the receptive field was detected (Weiler, 1985a). The ON amacrine cell type which correlated with the NT type B cell had a sustained ON response characteristic up to a stimulus diameter of 500/am. Increasing spot size led to a reduced and more transient ON depolarization, which indicated some inhibitory surround. The colour dependence was univariant with maximal sensitivity around 625 nm (Weiler and Ball, 1984). In an ultrastructural immunocytochemical study Eldred and Carraway (1987) found numerous small (62 nm diameter) and some large, dense-core vesicles in type A cells, which were unlabelled. Additionally, other large, dense-core vesicles (119 nm diameter) were clearly labelled. The cytoplasm of type A cell processes contained little diffuse reaction product, contrary to type B cells, where the cytoplasm was filled with electrondense reaction product. Additionally, in type B cells reaction product densely coated the outside of small synaptic vesicles which were devoid of immunoreactivity in the inside. Some large, labelled vesicles were also found, together with other, unlabelled large vesicles. Type A cells received approximately equal input from amacrine and bipolar cells. Overall, they had much more synaptic input than output. Conventional synaptic contacts onto bipolar, amacrine and ganglion cells were found. Eldred and Carraway (1987) also described recurrent input from type A cells back onto their bipolar input, which is in agreement with physiological findings, suggesting that transient amacrine cell responses might result from recurrent amacrine cell inhibition of excitatory bipolar input (Dowling, 1979). Specialized junctions between Type A cells with NT-like immunoreactivity supported physiological results from Jensen and DeVoe (1982) who found that fast, transient O N - O F F amacrine cells similar to type A cells were dye coupled to neighbouring amacrine cells of the same morphological type. Both findings provide some evidence for electrical coupling between type A amacrine cells with NT-

like immunoreactivity. Type B cells received primarily amacrine cell input and, to a lesser extent, bipolar cell input. Synaptic input and output contacts were approximately equal in number, and output was made conventionally onto amacrine cells, bipolar cells, and ganglion cells. Some of the contacts were made onto labelled type A amacrine cells (Eldred and Carraway, 1987). Levels of NT-like immunoreactivity in the turtle retina were measured using region-specific antisera (Eldred and Carraway, 1987). It was found that turtle NT seems to be similar to mammalian NT at the C-terminal ending, whereas the N-terminal ending seemed to differ. Both amacrine cell types, type A and type B, probably contained the same peptide, turtle neurotensin, indistinguishable from that in turtle brain. Retinal concentrations of NT lay in the order of 10 pmole per g retinal tissue (Eldred and Carraway, 1987; Eldred et al., 1987). Using HPLC, Eldred and Carraway (1987) found another peak in retinal extracts, which was supposed to represent the turtle counterpart to LANT-6 (see below). The actions of NT in the turtle retina have yet to be elucidated. Adolph (1986, 1989) found a slowonset, potent, sustained excitation of ganglion cell spontaneous and light-evoked activity. He supposed this action to be bimodally mediated via direct and indirect pathways.

3.6. L A N T - 6 LANT-6 is a hexapeptide isolated from chicken small intestine which resembles the COOHterminal of NT (Carraway and Ferris, 1983). It was shown to possess biological activity and has been found in a vesicle-like fraction of chicken brain (Kitabgi et al., 1984; Carraway et al., 1983). The content of LANT-6-1ike immunoreactivity in turtle retina seems to be about 10 times higher than the content of NT-like immunoreactivity (Eldred et al., 1987). The biochemical results of Eldred et al. (1988) indicated that extracts of turtle retina contained mainly a large molecular form of LANT-6. Immunocytochemistry performed in turtle retina with an antibody against synthetic LANT-6

NEUROTRANSMITTER SYSTEMS IN THE TURTLE RETINA

labelled cells with somata in the INL and GCL (Eldred et al., 1987, 1988). Two of the cell types with somata in the INL were most probably amacrine cells and were very similar to the cell types A and B with NT-like immunoreactivity, respectively. Eldred et al. (1987, 1988) suggested that NT and LANT-6 could co-localize in the same type A and B amacrine cells, because the LANT-6 antisera had less than 0.01 o70 cross-reactivity with NT, and they could not be blocked with bovine, chick or turtle NT in RIA studies. Many of the other somata in the INL with LANT-6 like immunoreactivity were supposed to be somata from displaced ganglion cells. Additionally, somata in the GCL were labelled with antisera against LANT-6. Among these, six morphological types of ganglion cells could be discerned (Eldred et al., 1988). Identity as ganglion cells was confirmed using doublelabelling techniques with retrogradely stained ganglion cells. Within the GCL, 1 0 - 15070 of the somata appeared to be labelled with LANT-6 antisera. Diameters of somata ranged from 6 - 2 2 ~m, with a maximum around 11 ~m. The processes of the six ganglion cell types could be followed inside IPL. Four types seemed to be monostratified - - two in sublayer 1, one in sublayer 5, and one in the middle of IPL. One type was bistratified around the 2/3 and 3/4 borders and another type seemed to be diffuse. One of the large, monostratified ganglion cells with processes in sublayer 1 was correlated with type B of Reiner (1981) and type I, which Ramon y Cajal (1933) observed in lizard retina, and also with a sustained OFF-centre ganglion cell recorded and stained by Weiler and Marchiafava (1981) (Eldred et al., 1988).

3.7. Somatostatin (SRIF)

Somatostatin is a g u t - brain neuropeptide first isolated from mammalian hypothalamus on the basis of its potent inhibitory action on growthhormone release. Mammalian hypothalamic SRIF is a tetradecapeptide. Several molecular forms of SRIF have been isolated from different species (Reichlin, 1983). Nothing is known about the

21

number of amino acids and the amino acid composition of turtle SRIF. Short reports of Zucker and Adolph (1987) and Adolph and Zucker (1988) described an exclusive localization of SRIF-like immunoreactive amacrine cells within the visual streak of the turtle retina. This region contained 4 5 0 - 5 0 0 cells per retina which ramified at the border between sublayers 2/3, the border between sublayers 4/5, and in the proximal part of sublayer 5 (Adolph and Zucker, 1988). The authors did not mention whether these plexuses were formed by a single, tristratified amacrine cell population or by two or more populations. Although somata were confined to the visual streak region, processes in the IPL covered the entire surface of the retina out to the far periphery. Zucker and Adolph (1987) described additionally labelled somata in the GCL. Application of SRIF-14 excited extracellularly recorded ganglion cells outside the visual streak region (Adolph, 1987; Adolph and Zucker, 1988). The action was dose-dependent and SRIF - - as M-ENK - - also converted transient light responses to sustained ones as the dose was increased (Adolph, 1988).

3.8. Substance P (SP)

Substance P is one of a group of related peptides called tachykinins that share common carboxy terminal sequences, which have been isolated from various vertebrate tissues. SP itself is composed of eleven amino acids. It has been detected in a variety of neurons throughout the central nervous system, the peripheral nervous system, and the gastrointestinal tract. There is evidence that SP may function as a neurotransmitter, neuromodulator and endocrine agent (Pernow, 1983; Otsuka and Konishi, 1983). The occurrence of SP in the turtle retina has been mentioned in passing in several studies (Reiner et al., 1984; Isayama and Eldred, 1988; Kolb et al., 1988). All these studies agreed that a monostratified amacrine cell ramifying in sublayer 3 was labelled with antisera against SP. Kolb et al. (1988) correlated this SP-containing amacrine cell with the A 20 type of her Golgi classification (Kolb, 1982). Recently, however, a short

22

R. WEILER et al.

communication described two different types of amacrine cells and a ganglion cell with SP-like immunoreactivity in turtle retina (Cuenca et al., 1989). Type A was described as a tristratified, wide-field cell ramifying in sublayers 1 and 3, with additional small, varicose terminals at the 4/5 border. This type was absent in the visual streak region. Type B was described as a small to medium field amacrine cell type with a single or two processes passing directly to sublayer 5, where an intimate network of processes was formed. The ganglion cells with SP-like immunoreactivity had large cell bodies. Their dendritic morphology was not described. Axons were labelled in the axon layer (Cuenca et al., 1989). At the moment it remains unclear whether there exists a monostratified amacrine cell ramifying in sublayer 3 or not. Further studies need to be carried out to answer this question. Reiner et al. (1984) obtained an SP content of approximately 12 pg per mg tissue using radioimmunoassay. This value was the lowest compared to other parts of the turtle nervous system in this study. Nothing is known about the function of SP in the turtle retina at the moment. 3.9. Summary

Figure 3 summarizes the laminar stratification pattern of the putative peptidergic neurons ramifying in the turtle IPL, which have been described in this chapter. The overall scheme is quite similar to neuropeptide distributions in the IPL of other vertebrate retinas, which have already been reviewed in the past (e.g. Karten and Brecha, 1983; Marc, 1986). However, several minor differences exist, which might represent group-specific adaptations. Although there may be some variation in the exact measurement of the strata among different authors, just counting the number of strata containing processes of peptidergic cells leads to an interesting result. This becomes even more evident, when the sublayers are divided into distal and proximal halves. The result is shown in the bottom part of Fig. 3. It seems to be quite evident that three peaks of neuropeptide distribution occur at sublayer 1, the distal half of sublayer 3, and the border between sublayers 4 and 5. Although this summing up of labelled strata tells

nothing about absolute numbers of cells or cell density or absolute neuropeptide concentration, it is a further indication for functional lamination in the IPL. Interestingly, the distribution of neuropeptide-like immunoreactivity is in close accordance with the distribution of a G-protein (Go), which is supposed to be related to calciumdependent intracellular processes via the membrane signal-transducing pathway (Terashima et al., 1987; Worley et al., 1986). The distal half of sublayer 4 and nearly the whole of sublayer 2 seem to be rather devoid of neuropeptide containing cell branches. Especially the distal half of sublayer 2 contains only processes of two types of cells, one of which is a ganglion cell. In the avian retina, these two sublayers are mostly occupied by acetylcholinecontaining processes (Marc, 1986). Guiloff et al. (1988) found that strata 2 and 4 contain the highest density of synaptic interactions in the turtle retina, both in the periphery and visual streak regions. This fact is an indication that these two strata are probably responsible for " f a s t " synaptic interactions with the classical " f a s t " neurotransmitters. Additionally, Guiloff et al. (1988) found the highest density of processes containing large, dense-cored vesicles in sublayers 1, 5, and to a lesser extent in sublayer 3. This is also in close accordance with the distribution pattern shown in Fig. 3. These findings further suggest that many of the neuropeptides may be released in a nonsynaptic manner. Although little is known about the function of neuropeptides in the turtle retina, it seems to be clear that they participate in the OFF-pathway (especially in sublayer 1), the ON-pathway (especially in sublayer 5), and also in O N - O F F interactions, which occur at the border between sublayers 2 and 3 (Ammermiiller and Weiler, 1989). This obvious and extensive participation of neuropeptide systems, which most probably modulate many -- if not all -- visual functions in the IPL, clearly needs still more attention. Nearly nothing is known about the physiological light responses of the involved neurons. The unusual distribution patterns of several cell types in tangential view and the strict localization of other types to the visual streak region or to other, distinct regions of the retina, are an

NEUROTRANSMITTER SYSTEMS IN THE TURTLE RETINA

important indication for a functional specialization of different parts of the retina. Regional specializations of the retina seem to exist in other species too (e.g. Tohyama et al., 1987). The function of this specialization remains obscure. Additionally, little or nothing is known about the functional significance of co-localizations of neurotransmitters -- some of which already have been shown, and others which are strongly indicated by the ultrastructural studies presented above. Even the classical morphological correlation is still unsatisfactory in many cases, because of the difficulties in amacrine cell classification. Therefore, studies combining several methodological approaches are more important than ever before. REFERENCES ADOLPH, A . R . (1986) Functional pharmacology of turtle retinal ganglion cells; Serotonin (5HT) and neurotensin (NT). Soc. Neurosci. Abstr. 12: 200. ADOLPH, A . R . (1988) Center-surround, orientation, and directional properties of turtle retinal horizontal cells. Biol. Cybern. 58: 373- 385. ADOLPH, A. R. (1989) Peptide and indoleamine modulation of spatial response properties in turtle retinal ganglion cells. In: Neurobiology o f the Inner Retina (R. Weiler and N. N. Osborne, eds) pp. 455-460. NATO ASI Series. Series H: Cell Biology, Vol. 31. Springer-Verlag, Berlin. ADOLPH, A . R . and BRUUN, A. (1987) Indoleamine and neuropeptide pharmacology of turtle retinal ganglion cells. Neurosci. 22: $412. ADOLPH, A . R . and ZUCKER, C . L . (1988) Exclusive localization of somatostatin-containing amacrine cells within the turtle visual streak. Invest. Ophthalmol. Vis. Sci. 29: 196. AMMERMULLER, J. and WEILER, R. (1988) Physiological and morphological characterization of OFF-center amacrine cells in the turtle retina. J. comp. Neurol. 273: 137- 148. AMMERMOLLER,J. and WEILER, R. (1989) Correlation between electrophysiological responses and morphological classes of turtle retinal amacrine cells. In: Neurobiology o f the Inner Retina (R. Weiler and N. N. Osborne, eds) pp. 117-132. NATO ASI Series. Series H: Cell Biology, Vol. 31. Springer-Verlag, Berlin. ARIEL, M. and ADOLPH, A. R. (1985) Neurotransmitter inputs to directionally sensitive turtle retinal ganglion cells. J. Neurophysiol. 54:1123 - 1143. BALL, A . K . , WEILER, R. and AMMERMOLLER, J. (1988) GABAergic amacrine cells in goldfish and turtle retinas. Proc. int. Soc. Eye Res. V: 19. BARLOW, H. B. and LEV~CK,W. R. (1965) The mechanism of directionally selective units in rabbit's retina. J. Physiol. 178: 477- 504. BRECHA, N. (1983) Retinal neurotransmitters: histochemical and biochemical studies. In: Chemical Neuroanatomy

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(P.C. Emson, ed.) pp. 85-129. Raven Press, New York. BRECHA, N. C. and KARIEN, H. J. (1985) Localization of biologically active peptides in retina. In: Retinal Transmitters and Modulators: Models for the Brain (W. H. Morgan, ed.) pp. 93-118, Vol. 1. CRC Press, Boca Raton, FL. BRUUN, A., EHINGER, B. and SYSTMA, V. (1984) Neurotransmitter localization in the skate retina. Brain Res. 295: 233- 248. CARRAWAY, R . E . and FERRIS, C . F . (1983) Isolation, biological and chemical characterization and synthesis of a neurotensin-related hexapeptide from chicken intestine. J. biol. Chem. 248: 2475- 2479. CARRAWAY,R. E., RUANE, S. E. and RITSEMA, R. S. (1983) Radioimmunoassay for Lys 8-Asn 9-neurotensin 8 - 13: tissue and subcellular distribution of immunoreactivity in chickens. Peptides 4:111 - 116. CR1SWELL,M. H. and DEVOE, R. D. (1986) Fourier analysis of directionally selective bipolar cells in the turtle retina. Invest. Ophthalmol. Vis. Sci. 27: 129. CRISWELL, M . H . and BRANDON, C . J . (1987) Immunocytochemical evidence that turtle cones are cholinergic. Invest. Ophthalmol. Vis. Sci. 28: 278. CUENCA, N., CLINE, C. and KOLB, H. (1989) Morphology and distribution of neurons immunoreactive to substance-P in the turtle retina. Invest. Ophthalmol. Vis. Sci. 30: 122. DAW, N. W., BRUNKEN,W. J. and PARKINSON, D. (1989) The function of synaptic transmitters in the retina. A. Rev. Neurosci. 12:205 - 226. DICK, E. and MILLER, R. F. (1981) Peptides influence retinal ganglion cells. Neurosci. Left. 26: 131- 135. DOWLING, J . E . (1979) Information processing by local circuits: The vertebrate retina as a model system. In: The Neurosciences (F. O. Schmidtt and P. G. Worden, eds) pp. 163 - 181. MIT Press, Cambridge, MA. DOWLING, J. E. (1986) Dopamine: a retinal neurotransmitter? TINS 5:236 - 240. DOWLING, J. E. and EHINGER, B. (1975) Synaptic organization of the amine-containing interplexiform cells of the goldfish and cebus monkey retina. Science 188: 270- 273. EH1NGER, B., OTTERSEN, O . P . , STORM-MATHISEN, J. and DOWLING, J. E. (1988) Bipolar cells in the turtle retina are strongly immunoreactive for glutamate. Proc. natl. Acad. Sci. U.S.A. 85:8321-8325. ELDRED, W. D. and KARTEN, H. J. (1983) Characterization and quantification of peptidergic amacrine cells in the turtle retina: enkephalin, neurotensin, and glucagon. J. comp. Neurol. 221: 371- 381. ELDRED, W. D. and KARTEN, H. J. (1985) Ultrastructure and synaptic contacts of enkephalinergic amacrine cells in the retina of turtle (Pseudemys scripta). J. comp. Neurol. 232: 36-42. ELDRED, W. D. and CARRAWAV,R. E. (1987) Neurocircuitry of two types of Neurotensin-containing amacrine cells in the turtle retina. Neurosci. 21:603-618. ELDRED, W. D. and WILLIAMSON, D. (1987) Corticotropic releasing factor-containing amacrine cells in turtle retina. Invest. Ophthalmol. Vis. Sci. 28: 351. ELDRED, W. D. and CHEUNG, K. (1989) Immunocytochemical localization of glycine in the retina of the turtle (Pseudemys scripta). Vis. Neurosci. 2: 331- 338.

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