Chapter 8 Enkephalins in the vertebrate retina

CHAPTER 8

Enkephalins in the Vertebrate Retina CARL B. W A T T , Y I N G - Y E T THOMAS SU a n d DOMINIC M A N - K I T LAM Cullen Eye Institute, Baylor College o f Medicine, Houston, Texas 77030, U.S.A.

CONTENTS 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

221

2. The 2.1. 2.2. 2.3.

Production and Partial Characterization of Monoclonal Antibodies Against Enkephalins . 222 Production of Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 Immunochemical Characterization of Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . 223 Immunocytochemical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

3. The 3.1. 3.2. 3.3.

Content, Biosynthesis and Release of MetS-Enkephalin in the Chicken Retina . . . . . . . . . . . Enkephalin Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enkephalin Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enkephalin Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

226 226 227 228

4. Synaptic Organization of Enkephalin-Immunoreactive Amacrine Cells in the Chicken R e t i n a . . 235 4.1. Light Microscopic Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 4.2. Electron Microscopic Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 5. Interactions Between Enkephalin and GABA in the Chicken Retina . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Inhibition Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Double-Label Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

237 237 238

6. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

239

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

239

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

240

I. INTRODUCTION

agents in the processing of neural information (Burnstock, 1976; Iverson et al., 1978; Burnstock et al., 1979; Hokfelt et al., 1980; Iverson, 1983). Utilizing the avian retina as a model system we have initiated studies to examine the functional roles played by a class of opioid peptides, Leu ~and MetS-enkephalin, in the processing of visual information. We chose the enkephalins for these studies because these important neuropeptides are not only well characterized both biochemically and pharmacologically (Loh and Ross, 1979), but have been localized to and shown to be physiologically active in the vertebrate retina. Recent studies utilizing immunochemical as well as immunocytochemical techniques provide evidence for the presence of opiate receptors (Medzhiradsky, 1976; Howells et al., 1980;

One of the most exciting developments in neuroscience in recent years has been the realization that in addition to classical transmitters such as acetylcholine, dopamine, glycine, gammaaminobutyric acid (GABA) and the catecholamines, a number of peptide-immunoreactive substances have been localized to and found to be physiologically active in many regions of the central nervous system (Hokfelt et al., 1980) including the retina (Stell et al., 1980; Ehinger, 1982; Brecha, 1983). The mechanisms by which the neuroactive peptides operate at the cellular and molecular level are to a large extent unknown, although there is increasing evidence that they serve a role as transmitter and/or modulatory 221

222

C. B. WATTet al.

Osborne et al., 1981; S u e t al., 1984b), assayable enkephalin immunoreactivity (Humbert et al., 1979; Eiden et al., 1980, Jackson et al., 1980; Su and Lam, 1982; Suet al., 1984a) and enkephalinlike immunoreactive neurons (Brecha et al., 1979; Stell el al., 1980, 1981; Tornqvist el al., 1981; Fukuda et al., 1982; Altschuler et al., 1982; Ishimoto et al., 1983; Brecha, 1983; Watt et al., 1983, 1984a, b; Eldred and Karten, 1984) in the retinas of some teleost, amphibian, reptilian, avian and/or mammalian species. A comparative analysis of previous immunohistochemical studies reveals that enkephalin-immunoreactivity, when present, is localized to one or more subpopulations of amacrine cells. Moreover, enkephalin-like immunoreactive amacrine cells of different species appear to have different patterns of ramifications of their processes in the inner plexiform layer. Only limited reports have appeared which address the electrophysiological properties of enkephalin in the vertebrate retina. Dick et al. (1980) noted in the mudpuppy retina that exogenously applied opiates generally inhibited both light- and aspartate-induced ganglion cell responses. In contrast to their actions in the amphibian retina exogenously applied opioid peptides and their analogues were shown to have quite different effects on certain ganglion cells of the teleost retina (Djamgoz el al., 1981). The application of exogenous morphine or D-Ala 2Met~-enkephalinamide in the goldfish retina enhanced both light responses and spontaneous activity of on-center ganglion cells but inhibited both light responses and spontaneous activity of off-center ganglion cells. In fact, the latter were sometimes converted to on-center. Furthermore, in the goldfish retina it was proposed that the opiate system effects the visual responses of ganglion cells through direct/indirect interactions which involve enkephalinergic and GABAergic amacrine cells. As a logical extension of previous studies on retinal enkephalinergic systems, we have adopted a multidisciplinary approach to examine the functional and morphological properties of the population of enkephalin immunoreactive amacrine cells in the chicken retina. The purpose of this paper is to review the current status of these studies and to elucidate, when possible, on how

this information adds to our understanding of the pre- and post-synaptic functions of enkephalins in the transmission and processing of visual information in the vertebrate retina.

2. THE PRODUCTION AND PARTIAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST ENKEPHALINS (WATT ETAL., 1983; TAVELLA ETAL., 1984) In our initial studies which examined the organization of the enkephalin system in the chicken retina, we used commonly available polyclonal antisera against enkephalins for immunochemical and immunohistochemical studies. During such studies it became apparent to us that these antibodies also reacted with a class of recently discovered opioid peptides, dynorphins 1 - 13 and 1 - 17 (Miller et al., 1978; Sar et al., 1978; Larson and Strengaard-Pederson, 1982; van Leeuwen et al., 1983; McGinty et al., 1983). Since dynorphins and enkephalins have been shown to be coded by different genes and localized in different cells (Noda et al., 1982; Gubler et al., 1982; Werber et al., 1983; McGinty et al., 1983), it is therefore especially crucial to distinguish between enkephalinergic and dynorphinergic neurons in any immunocytochemical study of opioid pathways. Furthermore, since the five amino acid sequence of LeuS-enkephalin is found in the various dynorphin molecules, it is unlikely that polyclonal antisera can be generated which distinguish between these peptides. A direct way of achieving this specificity is to use the hybridoma technology pioneered by Kohler and Milstein (1975). With these ideas in mind, the objectives of our study were three-fold. First we applied the hybridoma technology to produce monoclonal antibodies that were both well characterized and highly specific for the enkephalins. Second, we utilized these monoclonal antibodies to localize histochemically the enkephalin-immunoreactive neurons in the chicken retina. Finally, we compared the specificity and immunocytochemical staining characteristics of our monoclonal antibodies with a polyclonal antiserum against enkephalin (Batch A-206, K.-J. Chang) which has been used extensively in many

ENKEPHALINSIN THE VERTEBRATERETINA

laboratories including our own for immunochemical and immunohistochemical studies.

2.1. Production of Monoclonal Antibodies

Immunization in vivo and the procedures described by Kohler and Milstein (1975) were utilized to produce monoclonal antibodies against LeuS-enkephalin. Because small peptides such as the enkephalins are not by themselves immunogenic (Lerner, 1981; Sutcliffe, 1983), they were first conjugated to ovalbumin (Miller et al., 1978) before being used as the immunogen. The conjugate, LeuS-enkephalin - ovalbumin, that we prepared, had a molar ratio of enkephalin (2) to ovalbumin (1). Following the immunization protocol, the presence of anti-enkephalin antibodies in the sera of treated mice was determined by Ouchterlony double immunodiffusion tests. Spleen cells from the animal whose serum exhibited the strongest enkephalin immunoreactivity were fused with P3 × 63 Ag8 6.5.3. myeloma cells (Galfre et al., 1977). Seventy-seven percent of the hybridoma cultures, each containing one million spleen cells from the immunized mouse, produced viable hybridomas. The amount of anti-enkephalin antibodies from the cultures of viable hybridomas was subsequently quantitated. Six highly positive supernatants were identified by ELISA and the presence of the anti-enkephalin antibodies was confirmed by direct immunoassay. Two hybridomas, AD4 and DB4, secreted immunoglobulins highly specific for enkephalins. They also survived rigorous cloning and were grown in vivo as ascites tumors for all subsequent studies.

2.2. lmmunochemical Characterization of Monoclonal Antibodies

Monoclonal antibodies secreted by hybridomas AD4 and DB4 belong to the IgG type 1 class as

determined by Ouchterlony double immunodiffusion tests. Concentrated culture media from hybridomas AD4 and DB4 were further analyzed to determine their tendencies to cross-react with the carrier proteins BSA and Ova. These tests

223

showed that the monoclonal antibodies AD4 and DB4 recognize the Leu s- and MetS-enkephalin determinants in the conjugates but did not recognize the determinants of the carrier proteins. Furthermore, immunoglobulins from normal sera of mouse, rabbit and goat did not bind the enkephalins. Affinity determinations and crossreactivity studies were performed with monoclonal antibodies from hybridomas AD4 and DB4 which were purified by affinity chromatography. Binding studies were performed by RIA in the presence of 3H-LeuS-enkephalin concentrations ranging between 1 and 60 nM. Regression analyses of the resulting Scatchard plots (Fig. 1) showed that the dissociation constants of the monoclonal antibodies, AD4 and DB4, were 48.4 × 10-9M and 41.1 × 10-gM, respectively. Competitive binding studies performed with increasing concentrations of unlabeled enkephalin and 2.16 nM 3H-LeuSenkephalin, revealed the ICso (the 50o/o inhibiting concentration) of each monoclonal antibody to be approximately two orders of magnitude higher than that of a polyclonal antiserum (A206) which is routinely used in our laboratory (Fig. 2). Furthermore, the monoclonal antibody AD4 exhibited almost equal reactivity with either Leu sand MetS-enkephalin (Fig. 3, upper trace), while monoclonal antibody DB, exhibited only 20% crossreactivity with MetS-enkephalin (Fig. 3, lower trace). More interestingly, our monoclonal antibodies against enkephalin exhibited specific sequence requirements for binding enkephalin-related peptides. The amino-acid sequence Gly-Gly-Phe-Leu or Gly-Gly-Phe-Met was essential for enkephalin recognition by both AD4 and DB4 immunoglobulins, as demonstrated by the high reactivity of the monoclonal antibodies for these tetrapeptides (Fig. 3 and Table 1). On the other hand, Tyr-Gly-Gly-Phe, which lack Leu or Met in the fifth position, did not react with either monoclonal antibody (Table 1). It is also apparent that size was a critical factor for the immunoreactivity of the monoclonal antibodies, since both AD4 and DB4 failed to bind significantly to enkephalin-related peptides which contained the enkephalin sequence at the amino terminus and which contained six or more amino

224

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acids (Table 1). Clearly, our monoclonal antibodies did not react significantly with Tyr-Gly-Gly-Phe-Met-Arg, Tyr-Gly-Gly-Phe-MetArg-Phe, Tyr-Gly-Gly-Met-Arg-Gly-Leu. It is of particular interest to note that AD4 and DB, are the first antibodies against enkephalin that do not cross-react with the heptapeptide, Tyr-Gly-GlyPhe-Met-Arg-Phe, which is present at the Cterminus of the enkephalin-containing polypeptide (E.C.P.; 27.3 K daltons), now called proenkephalin A, (Gubler et al., 1982) and which has been identified as a naturally occurring opioid peptide by a number of investigators (Kojima et -~....~---x.,

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FIC. 2. Leu~-enkephalin competitive inhibition curves. These curves were obtained by using unlabeled Leuenkephalin in competition with 3H-Leu-enkephalin for the monoclonal antibodies, AD, (x..... x) and DB, (C)..... O), and polyclonal antiserum A206 (A ..... A). (From Tavella et al., 1984, with permission.)

FIG. 3. Competitive inhibition curves obtained by using enkephalin-related peptides in competition with 3H-Leu-

enkephalin for the monoclonal anti-enkephalin antibodies, AD4 (upper panel) and DB4 (lower panel). Leu-enkephalin (x..... x), met-enkephalin (D ..... [-]), gly-gly-phe-leu (A..... A) and gly-gly-phe-met(© ..... O). (From Tavella et al., 1984, with permission.) al., 1982; Girand et al., 1983; Costa et al., 1983). Furthermore, our monoctonal antibodies do not cross-react with the other enkephalin-related peptide, Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu, which is also part of E.C.P. and which has also been identified as a naturally occurring peptide by Kilpatrick et al. (1982). It was also apparent that the monoclonal antibodies do not react with dynorphin 1 - 6, dynorphin 1 - 13 or fl-endorphin (Table 1). However, it was not determined whether or not our monoclonal antibodies react with dynorphin 1 - 24 or other large enkephalinrelated peptides in which an enkephalin sequence is located at the carboxy terminus. Moreover, as shown in Table 1, under identical conditions the polyclonal antiserum A206 reacted with all the enkephalin-related peptides tested, with the exception of /3-endorphin. In particular, A-206 reacted significantly with the physiologically active and chemically distinct dynorphins 1 - 6 and 1 - 13 (Table 1). These results suggest that our monoclonal antibodies may be used to distinguish between the dynorphins tested and enkephalins in certain immunochemical and immunocytochemical studies. Accordingly, as shown in the forthcoming section we have used AD4 for the

225

ENKEPHALINS IN THE VERTEBRATE RETINA TABLE 1.

The Crossreactivities of Enkephalin-Related Peptides Against the Monoclonal Antibodies AD4 and DB, and the Polyclonal Antiserum A206 Radioactivity Bound (% of Control)

Unlabeled Peptide Added

Concentration (Molar)

AD4

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A206

100

100

100

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10-5 10-" 10-3

100 86 77

98 99 93

N.D. 80 50

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10-s

53

62

45

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10-5

52

59

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10-~

35

36

67

Tyr-Gly-Gly-Phe-Leu-Arg (Dynorphin 1 - 6)

10-5

91 87 83

49

10"4

92 92 92

10 -6 10 -3

100 100

10-4 10-3

88 35

97 94 87 33

79 36 N.D. N.D.

10"~

100 100 100 100

100 100 100 100

N.D. N.D. N.D.

100 100 100

100 100 100

60 N.D.

97 91 94

91 94 91

75 61 N.D.

100

98 98 99

94 88 N.D.

None

Tyr-Gly-Gly-Phe-Met-Arg

Tyr-Gly-Gly-Phe-Met-Arg-Phe

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10 -5 10 -s

10" Dynorphin ( 1 - 13)

10 -5 10 -5

10-4 /3-Endorphin

10 -5 10 -5

10-'

91 91

10

1

16

N.D.

N.D. - - not determined.

immunohistochemical localization of enkephalinergic neurons in the chicken retina.

2.3. lmmunocytochemical Studies

Enkephalin-immunoreactive neurons have been localized in the chicken retina using the polyclonal antiserum A206 (Brecha et al., 1979; Watt et al., 1983). Since this polyclonal antiserum, which has been used extensively in our laboratory, has been shown to cross-react with dynorphins and certain other enkephalin-related peptides (McGinty et ai., 1983; van Leeuwen et al., 1983), we used the monoclonal antibody AD4 to determine immunocytochemically whether this antibody, which is specifically directed against the enkephalins, stained a similar population of enkephalinergic amacrine cells in the chicken retina as the

polyclonal antiserum A206. As shown in Plate 1, AD4 labeled a subpopulation of pear-shaped cells which were observed exclusively in the inner nuclear layer in the second or third tier of cells from the border of the inner plexiform layer (Plate 1, small open block arrows). Processes of enkephalin-stained cells (Plate 1F, large open block arrow) entered the inner plexiform layer and were seen as punctate deposits in sublamina 1 (Plate 1, small block arrows) as well as throughout sublaminas 3 through 5 (Plate 1, asterisks and arrowheads). For comparison, the enkephalinimmunoreactive cells stained by the polyclonal antiserum A206 are shown in Plate 2. It was evident that the density of the labeled somas as well as the overall distribution of the enkephalinstained processes were quite comparable in retinas stained with either the monoclonal or the polyclonal antibodies. The results therefore indicate

226

C. B. WATT et al.

that the immunoreactive products visualized in Plates 4 and 5 as well as in forthcoming studies to be reviewed are not likely to be dynorphins (Table 1) but most probably belong to the enkephalins or peptides very similar to the enkephalins.

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3. THE CONTENT, BIOSYNTHESIS AND RELEASE OF METS-ENKEPHALIN IN THE CHICKEN RETINA (SU ET AL., 1983, 1984A) As stated earlier, immunochemical and immunocytochemical analyses utilizing both polyclonal and monoclonal antibodies against enkephalins point to the existence of enkephalins or closely related enkephalin-like peptides in a small subpopulation of amacrine cells. Moreover, other studies have alluded to possible functional roles of the enkephalins as neuromodulators or neurotransmitters. Two of the most important criteria in the establishment of any substance as a neurotransmitter or neuromodulator involve the demonstrations that the substance is present in an identified neuron at a significant concentration and is released from the cell upon its depolarization (Werman, 1966; Hebb, 1970). In our attempt to establish that the enkephalins act as neurotransmitter or neuromodulatory agents in the retina, we have used combined immunochemistry and high performance liquid chromatography (HPLC) to demonstrate (1) that authentic MetS-enkephalin, or a peptide very similar to enkephalin, is synthesized by the chicken retina and (2) that this peptide can be released from the retina in response to its depolarization in a calcium-dependent manner.

3.1. Enkephalin Content

The concentration of enkephalin-immunoreactive substances in the chicken retina was determined by the enzyme linked immunosorbent assay (ELISA) test. As a confirmation of the ELISA test, we compared the concentrations of enkephalin-immunoreactive substances in retinas and in rat brain as measured by ELISA and the more commonly employed technique of radioimmunoassay (Miller et al., 1978) and found

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FIG. 4. A standard curve for ELISA test. Each point represents at least two measurements that are assayed in triplicate. The enkephalin content in the chick retina was estimated. Points A, B and C represent different volumes of tissue extract with their protein concentrations corresponding to 0.1, 0.2 and 0.4 mg. The enkephalin concentrations for these points are 13, 25 and 47.5 pg, respectively. The average concentration of enkephalin in the retina is 126 pg/mg protein or approximately 2.5 × 10-SM. (From Suet al., 1984, with permission.)

that these two methods yield similar results. Additionally, the concentration of enkephalinimmunoreactivity in the chicken retina was in the same range as those reported for the rat cortex and whole brain using radioimmunoassays (about 5 riM; Miller et al., 1978). As shown in Fig. 4, each of the positions a, b and c corresponds to the ratio of optical density (B/Bo) in the presence of a different protein content (0.1, 0.2 and 0.4 mg, respectively) from a retinal homogenate. From the curve, the average concentration of enkephalinimmunoreactive substances in the chicken retina was estimated to be 126 pg/ml protein or 0.25 p mol/mg protein. Assuming that the protein content in the retina is 10% of its wet weight, the concentration of the enkephalin-immunoreactive substances is then in the order of 25mM. The content and biosynthesis of enkephalins have been demonstrated in a variety of neural tissues (Yang et al., 1978; Wilson et al., 1980; Lindberg et al., 1982; Su, 1982; S u e t al., 1982, 1983). Using the ELISA test, we have compared the concentrations of enkephalin-immunoreactivity in the avian retina with those in teleost

227

ENKEPHALINSIN THE VERTEBRATERETINA and mammalian retinas. Our results show that, among these retinas, the enkephalin concentration in the chicken retina (about 25 nM) was almost an order of magnitude higher than those for rabbit (about 4 nM) and goldfish (about 2.5 riM) retinas (Su and Lam, unpublished data). These concentration values may explain in part the difficulty encountered by our laboratory as well as others in visualizing enkephalin-immunoreactive neurons in the goldfish and rabbit retinas. It must, of course, be emphasized that in ELISA as in radioimmunoassays, the values determined represent not only the content of enkephalins alone but also of all the substances in the extract that are immunoreactive against the particular antibody used. Thus, the values that we obtained using these techniques should only be taken as an upper limit of the true enkephalin concentration. Finally, although an enkephalin concentration of 25 nM in the chicken retina may appear to be very low, this perhaps reflects the small number of enkephalin-containing retinal neurons rather than the low enkephalin content in these cells. Indeed, if one estimates from our immunocytochemical study (Watt et al., 1984a) that fewer than 1 070 of the retinal cells contain enkephalins, then the actual concentration of these peptides in enkephalinergic neurons of the chicken retina may be in the order of 2.5/aM, a value that is comparable to that found in adrenomedullary chromaffin cells which are known to contain enkephalins (about 5/aM, Wilson et al., 1980).

3.2. Enkephalin Biosynthesis Uptake a n d / o r biosynthesis are two common mechanisms by which a neuron accumulates a neurotransmitter or peptide. In an earlier study, we found by autoradiography that the chicken retina does not possess a mechanism for taking up exogenous enkephalins (Lam, unpublished data). In this study we used H P L C and immunoassays to examine the biosynthesis of enkephalin, by first incubating isolated chicken retinas in 0.2 ml of oxygenated (95070 02 and 5070 CO2) Ringer's solution containing 40/aCi of 3H-Met (sp. act. 50 Ci/mmole; from New England Nuclear Corp.) and the peptidase inhibitor, trasylol (250 KIU/ml)

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FIG. 5. Elusion profile of the tissue extract on a Bondapak C18 column. The column was eluted with a convexgradient of 12.5 mM phosphate, pH 6.4 (primary solvent) and acetonitrile (secondary solvent), with acetonitrile from 12 to 40% in 50 rain. The flow rate was set at 1 ml/min and fractions with 1 ml were collected. Radioactivity of each fraction was measured in a liquid scintillationcounter. The retention time corresponding to MetS-cnkephalin was indicated. Immunoassays of the fractions demonstrated that two peaks showed enkephalin-like immunoreactivity. (From Suet al., 1984, with permission.) for 30 rain. The tissue was washed three times in large volumes of Ringer's solution containing trasylol and then incubated in the same Ringer's solution containing 100/~g/ml of methionine for another hour (chase incubation). Following the incubation in the unlabeled medium for one hr, three major peaks of immunoreactivity were seen on H P L C . A typical elation profile is shown in Fig. 5. The peak that eluted at 28070 acetonitrile, with a retention time of 22 min (fraction no. 22), comigrated with authentic MetS-enkephalin. The peak (fraction no. 17) which eluted imme-

C. B. WATTet al.

228

diately before MetS-enkephalin comigrated with the synthetic peptide Gly-Gly-Phe-Met and may correspond to a degradative product of Met senkephalin. The identities of the products in the peak with the highest amount of radioactivity (fraction no. 8, eluted at 8 min) are not known. Under our experimental conditions, no other 3HMet-labeled enkephalin-related peptides were identified. Immunoassays of the fractions from HPLC showed that the peak that comigrated with Met senkephalin (fraction no. 22) cross-reacted with the polyclonal antibody against enkephalins (A206). Additionally, the peak at fraction no. 17 also possessed enkephalin-immunoreactivity. In fact, over 95°70 of the radioactivity present in the sample solutions from these two peaks was precipitated by the anti-enkephalin serum. In contrast, the peak with the highest radioactivity (fraction no. 8) did not cross-react with the antibody A206. These results indicate that Met senkephalin, or a peptide very similar to MetL enkephalin, is synthesized and accumulated by the

chicken retina. Moreover, studies are currently underway to determine the eDNA and amino acid sequence of this substance. The percentages of 3H-Met that were incorporated into Met-enkephalin after a 30 rain incubation in a medium containing 3H-Met and followed by different times of post-incubation (chase) in an unlabeled medium are shown on Fig. 6. Without any chase period (time 0), very little 3H-MetLenkephalin was detected in the retina. The amount of 3H-MetS-enkephalin in the retina increased linearly during the first hr of chase-incubation before gradually approaching the maximal level. This data suggests that, similar to reports of enkephalin biosynthesis in other tissues (Yang et al., 1978; Wilson et al., 1980; Lindberg et al., 1982), MetLenkephalin in the chicken retina is probably also synthesized as part of a larger precursor. Once again, the identity of this precursor and the mechanism of enkephalin biosynthesis in the retina must await further biochemical and molecular genetic studies.

3.3 Enkephalin Release

A piece of isolated retina was incubated in ringer's solution containing 3H-Methionine, using a procedure identical to that utilized for biosynthesis experiments. It was apparent that

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FIG. 6, The effect of chase incubation period on the 3HMet-enkephalin synthesized in the retinal tissue. The isolated chicken retina was cut radially into four pieces. The tissues were labeled with 40 pCi of 3H-met for 30 min (from - 30 to 0 min). After rinsing three t;mes in large volume of ringer's solution (containing the peptidase inhibitor), one piece of retina was removed for extraction (time 0). The other three pieces were incubated in the same ringer's solution containing 100 ~g/ml unlabeled Met for an additional 30, 60 and 120 min, respectively. The m o u n t of 3H-MetLenkephalin synthesized and accumulated in the retina at each time point was measured as described in Methods. The ratio of 3H-Met-enkephalin/total radioactivity in each extract was calculated and plotted against time of incubation. (From S u e t al., 1984, with permission.)

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Number

FIc. 7. The release of 3H-MetLenkephalin from the chicken retina. Each fraction represents a retina incubated for 3 rain with 1.5 ml of either normal or K+-rich ringer's solution. The eluates were individually collected, lyophilized, desalted and assayed for MetLenkephalin by HPLC. This figure shows that 3H-MetLenkephalin is released in response to high extracellular K÷ concentration, and that this release is greatly reduced by the presence of 5 mM Co 2. in the medium. (From Su et al., 1984, with permission.)

ENKEPHALINS IN THE VERTEBRATE RETINA

229

PLATE 1. Montage of enkephalin immunoreactive amacrine cells in the chicken retina as revealed with monoclonal antienkephalin antibody AD4. Small, open block arrows denote typical immunoreactive somas. Small block arrows point to punctate staining within sublamina 1. Large, open block arrow in F indicatesimmunostained process descending:through the inner plexiform layer. Asterisks denote dense immunoreactive bands within sublamina' 5. Arrowheads point to less intensely stained bands within sublaminas 3 and 4. (IN, inner nuclear layer; IP, inner plexi~orm layer; GC; ganglion cell layer). (A, B, C, D, E, F × 512; scale bars = 10 microns). (From Tavella et al., 1984, with permission.)

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C . B . WAIT e t a / .

nki

PLATE 2. Montage of enkephalin-immunoreactive amacrine cells in the chicken retina as revealed by polyclonal antiserum A206. Large, open block arrows point to typical immunoreactive cell bodies. Small block arrows denote punctate staining within sublamina 1. Asterisks indicate a broad band of immunoreactivity throughout sublaminas 3, 4 and 5. (IN, inner nuclear layer; IP, inner plexiform layer; GC, ganglion cell layer). (A, B, C × 512; scale bars = 10 microns). (From Tavella et al., 1984, with permission.)

ENKEPHALINS IN THE VERTEBRATE RETINA

231

PLATE 3. Montage of several enkephalinqike immunoreactive amacrine cells in the chicken retina (A, B and C). Arrowheads denote staining within sublamina 5 of the inner plexiform layer near the ganglion cell layer. D is a control section, incubated in the preimmune serum. (IN, inner nuclear layer; IP, inner plexiform layer; GC, ganglion cell layer). (A, B, C, D x 678). (From Watt et al., 1984a, with permission.)

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C . B . WATJ" et al.

PLATE 4. Electron micrograph demonstrating immunoreactive soma (open block arrow). The asterisk within the unstained soma denotes the position of stained cell in the third tier of cells from border of the inner plexiform layer (IP).( x 4000).

PLATE 5. Montage demonstrating synaptic relationships of enkephalin-amacrine cells in the chicken retina. A demonstrates amacrine to amacrine cell synapse (white open block arrow) in the chicken retina. The postsynaptic element in this example is enk-stalned (large open block arrow). Asterisk denotes the prcsynaptic element. B shows a bipolar to amacrine cell synapse in the chicken retina: in this example one dyadic member (open block arrow) in enk-positive. The white asterisk denotes the second element of the dyad which receives input from the bipolar cell terminal. (large asterisk). C to E demonstrate amacrine to amacrine cell synapses (small block arrows) in the chicken retina in which the presynaptic element in these examples is enk-labeled (large open block arrows). In C the postsynaptic element receives a dyad synapse from a bipolar cell terminal (asterisk). In D note the similarity in appearance between the postsynaptic element and the adjacent amacrine cell process (asterisk). In E the postsynaptic element contains a dense-core vesicle (open block arrow) and synapses onto a neighboring process (asterisk). In F an enk-positive varicosity (open block arrow) is prcsynaptic to a process (black asterisk) which lacks synaptic vesicles. So denotes soma in adjacent ganglion cell layer. Small block arrow points to the site of synaptic contact. (A, B, C, D, E × 34,000; F x 21,000)

ENKEPHALINS IN THE VERTEBRATE RETINA

Plate 5. See P. 232 for legend to this plate

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PLATE 6. Simultaneous visualization of enkephalins by enk-immunocytochemistry and GABA by 3H-GABA uptake autoradiography in a 2/~m thick plastic embedded transverse section of the chicken retina. Double-labeled cells in the inner nuclear layer are denoted by solid block arrows, while the solid white arrow and the hollow white arrow point to cells labeled only for GABA or enkephalin, respectively. Hollow black arrow denotes 3H-GABA uptake by horizontal cells in the outer region of the inner nuclear layer.

ENKEPHALINS IN THE VERTEBRATE RETINA

elevated K÷ in the medium depolarized the retina, and induced the release of the newly synthesized 3H-MetS-enkephalin Fig. 7. The identities of the radioactive substances in each eluate were determined by HPLC, and were shown to consist of approximately 95°7o MetS-enkephalin. Additionally, the K*-induced release of 3H-MetS-enkephalin was greatly reduced by the presence of 5 mM Co 2÷ in the medium, suggesting that, as at other conventional synapses, the release mechanism is dependent on the presence of extracellular Ca ÷2.

4. SYNAPTIC ORGANIZATION OF ENKEPHALIN-IMMUNOREACTIVE AMACRINE CELLS IN THE CHICKEN RETINA (WATT ET AL., 1983, 1984) As part of our overall study of functional role of enkephalinergic amacrine cells in the chicken retina, we proposed to examine the interrelationships between this population of retinal neurons and other transmitter-specific cell populations (see Section 5). A basic understanding of the distribution and synaptic relationships of processes of enkephalinergic amacrine cells is a prerequisite for studying their interactions with other neuronal populations. To this end, immunohistochemical studies were performed to investigate the morphology, distribution and synaptic organization of enkephalin (enk)-amacrine cells in the chicken retina.

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was observed occasionally in sublamina 5 near the ganglion cell layer (Plate 3, arrowheads). The existence of enkephalin-like immunoreactive amacrine cells in the chicken retina have recently been reported in several studies (Brecha et al., 1979; Tornqvist et ai., 1981; Fukuda, 1982; Watt et al., 1983; Ishimoto et al., 1983). Our light microscopic studies are generally consistent with these reports. For instance, all studies agree on the location and density of the somas of enkephalinamacrine cells and that processes of these cells project radially into the inner plexiform layer where they form a fine plexus of varicose fibers in sublamina 1. However, the pattern of immunoreactivity present in the proximal half (sublaminas 3, 4 and 5) of the inner plexiform layer varies somewhat between the various reports. In each of the previous investigations, enkephalin-immunoreactive fibers were observed in sublaminas 3 and 4 and thus, their distribution compares favorably with our studies. However, in two of these studies (Tornqvist et al., 1981; Ishimoto et al., 1983) enkephalin-stained fibers were not found in sublamina 5 of the inner plexiform layer. This observation is in contrast to our studies (Tavella et al., 1984; Watt et al., 1984a) in which both monoclonal and polyclonal antibodies revealed enkephalin immunoreactivity in sublamina 5. It is likely that these variations in the distribution of enk-immunoreactive processes arise in part from factors such as (1) differences in the antisera used; (2) differences in experimental protocols employed and/or (3) differences in personal interpretations of data (Childs, 1983; Petrusz, 1983).

4.1. Light Microscopic Observations An examination of transverse vibratome sections (20- 30/am) stained with FITC in the presence of Triton X- 100 revealed that enkephalinimmunoreactive, pear-shaped cell bodies were situated in the vitreal half of the inner nuclear layer in the second or third tier of cells from the border of the inner plexiform layer (Plate 3). These immunoreactive cells sent processes into the inner plexiform layer, where they formed a fine plexus of varicose fibers in sublamina 1 and a much broader band of labeling throughout sublaminas 3 and 4. In addition, immunoreactivity PRR4-N

4.2. Electron Microscopic Observations Ultrastructural features of enk-immunoreactive perikarya were studied in transverse retinal sections which were subjected to somewhat longer incubation times. Increasing the incubation times facilitated the penetration of the antibodies deeper into the tissue to a level at which adequately preserved and stained cell bodies could be detected. An enkephalin-positive soma exhibited a rather dense peroxidase reaction product distributed throughout its cytoplasm with the possible

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C.B. WA-rr

exception of the mitochondria and tubules of endoplasmic reticulum (Plate 4). On the other hand, the nucleus of an immunoreactive cell was less intensely stained, although the density of nuclear staining was considerably greater than that observed in surrounding unstained perikarya. Furthermore, the nucleus of an enk-amacrine cell was characterized by a round unindented nuclear membrane. At the EM level, the peroxidase reaction product was distributed throughout the matrix of stained processes with somewhat more dense accumulations associated with small clear vesicles and occasionally with large dense-core vesicles. In fact, large-enk positive varicosities encountered were often characterized by the presence of large dense-core vesicles in addition to typical small agranular synaptic vesicles. Other morphological features of enkephalin-labeled profiles were not readily apparent due to the disruptive effect of dense accumulations of reaction product within the immunoreactive profiles. An examination o f experimental tissue revealed that processes of enk-amacrine cells participate in several types of synaptic relationships in the chicken retina. When serving as the postsynaptic element, an enk-amacrine cell received synaptic input from either other unlabeled amacrine cells (Plate 5A) or less frequently from bipolar cells (Plate 5B). In the latter synaptic arrangement, one member of the dyad was immunoreactive, while the second postsynaptic element was not and usually contained synaptic vesicles. Furthermore, each of these synaptic arrangements was observed in sublamina 1 as well as throughout sublaminas 3 through 5. Enkephalin-containing amacrine cells participated in several synaptic arrangements in which they served as the presynaptic element. In most cases, the identity of the postsynaptic element was not entirely clear. First and most often, an immunoreactive varicosity was observed to form a single, conventional synaptic contact onto another unlabeled vesicle-filled profile tentatively suggested to originate from amacrine cells (Dowling and Cowan, 1966; Dubin, 1970; Rodieck, 1973) (Plate 5). Such postsynaptic targets were not observed to contain a synaptic ribbon although occasionally, they did receive input from a bipolar cell (Plate

el al.

Bb

Bo

INL

, AC

V

IPL

!"

i

2

,

V

J,

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i

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FIG 8. Summary diagram illustrating the synaptic connections of enk-amacrine cells (ENK) in the chicken retina. In sublaminas 1, 3, 4 and 5 enk-amacrine cells are pre and postsynaptic to non-enkephalinergic amacrine cells (AC). Enk-amacrine cells also receive input from bipolar cells (Ba and Bb) in sublaminas 1, 3, 4 and 5. In sublamina 5 enk-amacrine cells contact large basal processes of displaced amacrine cells (DAC) and/or ganglion cells (GC). Not illustrated are enk-amacrine cell inputs to non-vesiclecontaining processes of ganglion cells and/or amacrine cells which are observed throughout the innermost half of the inner plexiform layer (IPL). (Ba, off-center bipolar cell; Bb, on-center bipolar cell; INL, inner nuclear layer; GCL, ganglion cell layer; Numerals 1 through 5 denote sublaminas of the IPL). (From Watt et al., 1984a, with permission.) 5C). On occasions, a labeled varicosity which synapses onto a vesicle-filled profile served as the intermediate synaptic element in a serial synapse (Plate 5D). Furthermore, in some instances, vesicle-filled profiles post-synaptic to enkamacrine cells contained small dense-core vesicles in addition to typical agranular synaptic vesicles. In one example (Plate 5E), the dense-core vesicle containing profile formed a conventional synapse onto another unidentified process. Lastly, enkamacrine cells were observed to contact vesiclefilled profiles in sublamina 1 as well as throughout sublaminas 3 through 5. Enk-amacrine cells were also observed to contact unlabeled processes which did not contain synaptic vesicles and whose origin remains uncertain. Within sublamina 5 of the inner plexiform layer, a labeled varicosity was occasionally found to form a conventional synapse onto a large proximal process which most likely emanated from a cell in the nearby ganglion cell layer (Plate 5F). In addition to ganglion cells, displaced

ENKEPHALINS IN THE VERTEBRATE RETINA

amacrine cells have been described in the ganglion cell layer of the avian retina (Cajal, in Rodieck, 1973), and it would appear that their numbers are quite substantial (Binggeli and Paule, 1969). Therefore, in sublamina 5 the processes of enkamacrine cells may interact with the basal processes of ganglion cells and/or displaced amacrine cells. Furthermore, throughout the proximal half of the inner plexiform layer, enk-amacrine cells were occasionally observed to contact small profiles which lacked synaptic vesicles. Such profiles were usually void of identifiable organelles although in some instances they were characterized by the presence of vesiculated endoplasmic reticulum. Unfortunately, in the avian retina as well as in most other species, it is difficult to differentiate at the electron microscopic level small ganglion cell dendrites from small processes of amacrine cells which lack synaptic vesicles (Dubin, 1970). Therefore, it is possible that ganglion and/or amacrine cells give rise to such small distal processes which lack synaptic vesicles and are postsynaptic to enkamacrine cells. Some of the major synaptic relationships between enk-amacrine cells and other neurons in the chicken retina are summarized as a schematic diagram in Fig. 8. First, enk-amacrine cells are pre- and post-synaptic to other non-enk-amacrine cells in sublaminas I, 3, 4 and 5. Studies are presently underway to determine the identities of cells with which enk-amacrine cells interact in the chicken retina (see Section 5 for details). Our preliminary physiological studies indicate that enkephalin inhibits the K+-stimulated release of GABA and dopamine in the chicken retina (Suet al., 1983, 1984). Whether or not this inhibition is the result of direct interactions with enk-amacrine cells must await further studies in which dual histochemical and autoradiographic localizations are performed at the electron microscopic level in the same retina. Second, enk-amacrine cells also receive synaptic input from bipolar cells in sublaminas 1, 3, 4 and 5. In this regard, it is of interest that in most vertebrate retinas examined, the proximal region of the inner plexiform layer has been found to be associated mainly with the processing of on-center information, while the distal region concentrates predominantly on off-

237

center information (Famiglietti et al., 1977; Stell et al., 1977; Nelson et al., 1978). If this aspect of retinal organization is conserved in the chicken retina, then the enk-amacrine cells probably receive input from both on-center (Bb) and offcenter (Ba) bipolar cells (Fig. 8). Third, within sublamina 5, enk-amacrine cells also contact ganglion and/or displaced amacrine cells (Fig. 8). Direct anatomical proof of enk-amacrine cell input directly onto ganglion cells may be obtained in future studies by combining enk-immunocytochemistry with horseradish peroxidasebackfilling of ganglion cells in the same retina.

5. INTERACTIONS BETWEEN ENKEPHALIN AND GABA IN THE CHICKEN RETINA (SU ET AL., 1983; WATT ET AL., 1984d; LAM ET AL., 1984)

As detailed in the introduction, previous studies demonstrated that exogenous opioids affect both the GABA release and the firing patterns of ganglion cells in the goldfish retina. We began a systematic characterization of the opioid pathways in the chicken retina because, among vertebrate retinas, the avian retina has the highest concentration of enkephalins (Su and Lam, unpublished data). On the basis of our earlier discovery of an enkephalin-induced inhibition of GABA release in the goldfish retina, we proceeded to examine whether a similar interaction occurs in the chicken retina, and if it does, whether enkephalins and GABA might coexist the same amacrine cells. The results indicate that indeed exogenous enkephalin also inhibit GABA release in the chicken retina. Moreover, we found that although some amacrine cells contain either enkephalin or GABA, there are also amacrine cells in which both enkephalin and GABA are co-contained.

5.1. Inhibition Studies

The effects of opiates on the K+-stimulated release of preloaded GABA from GABAergic amacrine cells of the chicken retina was examined using methods reported earlier (Djamgoz et al.,

238

C.

B.

1981; Chin and Lam, 1980; Ayoub and Lam, 1984). Since exogenous G A B A is taken up primarily by both horizontal cells and amacrine cells in the chicken retina (Marshall and Voaden, 1974), the amount of 3H-GABA accumulated into GABAergic horizontal cells was minimized by injecting 3H-GABA directly into the vitreous of the eye and thus reducing the diffusion of the isotope to the outer regions of the retina. After one hour, over 90°70 of the accumulated 3HG A B A was localized in GABAergic amacrine cells (Watt, Su and Lam, unpublished result; Ayoub and Lam, 1984). The release of 3H-GABA from these cells by elevated K ÷ (50mM) in the absence or presence of opioid analogues could then be measured in isolated retina. As shown in Fig. 9, MetS-enkephalin inhibited the K+-stimulated release of 3H-GABA in a dose-responsive manner. This inhibition was mimicked by the enkephalin analogue D-Ala2-D-LeuS-enkephalin (DADL) and the opioid agonist morphine but was reversed by the opioid antagonist naloxone. The maximal inhibition of 3H-GABA release by opiates we tested was about 40070.

5.2. Double-Label Studies

A direct approach to visualize in the same retinal section putative enkephalinergic and GABAergic neurons was to process the retina sequentially for both G A B A - u p t a k e autoradiography in vitro (Lam et al., 1979; Brandon et al., 1980) and enk-immunocytochemistry (Watt et al., 1984a). If enkephalin and G A B A were colocalized in some amacrine cells, this would result in the accumulation of silver grains over immunoperoxidase-stained cell bodies. An examination of retinal sections processed sequentially for autoradiography and immunocytochemistry (Watt et al., 1984d) revealed that although some amacrine cells contain only G A B A (Plate 6, solid white arrows) or enkephalin (Plate 6, hollow white arrows), other amacrine cells were observed which indeed labeled for both enkephalin and G A B A (Plate 6, solid black arrows). The observation that some GABA-amacrine cells were not stained for enkephalin was not surprising, since m a n y of the GABA-amacrine cells are situated at levels of the

WATT

et al. JH-GABA Relea~;u (percent)

o

2 r

3

4

5

....

" ..........

control

2x 1O'SM m e t - E n k

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2 x 1 0 6 M met-Enk

16 x 10 ~ M DADL

2 x l o 7 M morDhine

2 x l O e M rnet-Enk 2 x 1 0 6 M nal(~xone

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FIG. 9. The effects of opioid agonists and antagonists on K%stimulated 3H-GABA release from GABAergic amacrine cells of the chicken retina. One hour after an intravitreal injection in vivo with 50/al ringer's solution containing 40/aCi 3H-GABA(sp. act. 82.6 Ci/mmole), the injected eye was removed and the posterior eye-cup bisected. The two pieces of retinae were washed with excess oxygenated ringer's solution for 30 minutes and incubated for 3 minutes with 1.5 ml of normal K+-rich (50 mM) ringer's solution in the absence or presence of an opioid analogue, using a method described in detail earlier. The percentages of 3H-GABA released (radioactivity released over radioactivity present in the retina) under different conditions were compared. Each histogram represents the mean and standard error from at least three independent experiments of duplicate samples. (From Watt et al., 1984d, with permission.) inner nuclear layer where enkephalin-amacrine cells are not found. If indeed all enkephalin-cells contained GABA, the inability to double-label these ceils might result from the deficiency of these techniques to label all enkephalinergic or GABAergic cells in any particular experiment. In any extent more comprehensive studies are presently underway to determine if there are regional differences within the chicken retina with respect to the relative number and distribution of cells which label for either enkephalin, G A B A or both putative transmitters.

ENKEPHALINS IN THE VERTEBRATE RETINA

The colocalization of GABA and enkephalin in the same amacrine cell is consistent with recent reports that certain peptides and transmitters coexist in some neurons in the peripheral and central nervous system (Hokfelt et al., 1980; Konishi et ai., 1981; Lundberg and Hokfelt, 1983; Louis et ai., 1983; Kawantani et al., 1983) and in the retina (Weiler et al., 1983). In particular enkephalins have been found in putative dopaminergic, serotoninergic and cholinergic neurons (Lundberg and Hokfelt, 1983). Recently, enkephalin-immunoreactive substances have also been detected in putative GABAergic neurons from embryonic chick cerebral hemisphere (Louis et al., 1983). The functional significance for peptidetransmitter coexistence is unknown, although numerous possibilities have been raised (Hokfelt et al., 1980; Lundberg and Hokfelt, 1983; Louis et al., 1983; Kawantani et al., 1983; Jan and Jan, 1983). For instance, in the parasympathetic system innervating the urinary bladder, it has been suggested that enkephalin, which is colocalized with acetylcholine (Kawantani et al., 1983), may function as an inhibitory transmitter by blocking in a presynaptic manner the release of acetylcholine from terminals in which enkephalin and acetylcholine coexist (Kawantani et al., 1983; Konishi et al., 1981). Our finding that enkephalin inhibits GABA release from GABAergic amacrine cells is consistent with this hypothesis. Our study is, however, complicated by the discovery that in addition to the presence of GABA and enkephalin in the same neuron, there are also GABAergic amacrine cells that do not contain enkephalin and vice versa. Consequently, it is possible that in addition to the type of presynaptic inhibition described above, enkephalin may also inhibit presynaptically GABA release from GABAergic amacrine cells that do not contain enkephalin. In this regard, double-label studies are presently underway at the ultrastructural level to examine the synaptic relationship between those amacrine cell populations which contain either enkephalin, GABA or both enkephalin and GABA.

239

6. CONCLUDING REMARKS Although there are as yet no published reports on the effects of enkephalins and other opiates in the processing of visual information in the avian retina, in the amphibian and teleost retinas, exogenously applied enkephalins have been shown to influence the responses of certain ganglion cells. Our results presented here and elsewhere demonstrate that in the chicken retina: (1) a substance immunologically and biochemically identical or very similar to enkephalin is present and synthesized; (2) this substance is released upon stimulation of the retina in a Ca+2-dependent manner; (3) enkephalin-immunoreactive amacrine cells are localized to and participate in several synaptic relationships; (4) exogenously applied enkephalins and opiates inhibit the release of GABA; (5) enkephalin and GABA are colocalized in some amacrine cells and (6) receptors for enkephalins have been identified biochemically ( S u e t al., 1984b). Recently, we have initiated studies which examine the organization of the enkephalinergic system in the larval tiger salamander retina. Similar to the chicken retina, enkephalin is localized to amacrine cells which provide input to both inner and outer regions of the inner plexiform layer (Watt et al., 1984b, c). Furthermore, intracellular recordings indicate that enkephalins and morphine affect the on-center pathways of ganglion cells in this retina (Watt et al., 1984c). Taken together, these findings points strongly to enkephalins or substances very similar to enkephalins as putative neurotransmitters or modulators in the vertebrate retina. Moreover, the retina may prove to be an excellent model of the central nervous system for investigating, at the cellular and molecular levels, the functional roles of these important neuroactive peptides. Acknowledgements - -

We wish to thank Valita Gaskie, Pat Glazebrook and Sarah Handlin for excellent technical assistance, Pat Cloud for typing the manuscript and Dr. K.-J. Chang for the antiserum (batch A206) against enkephaiins. This work was supported by grants from the U.S. National Institutes of Health (EY02423 to DMKL, EY03701 to YYTS and EY05690 to CBW), Research to Prevent Blindness, Inc. and the Retina Research Foundation.

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