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Peptide histidine-isoleucine (PHI)-immunoreactive amacrine cells in the retina of the rat
Neuroscience Letters, 79 (1987) 281-285 Elsevier ScientificPublishers Ireland Ltd.
281
NSL 04776
Peptide histidine-isoleucine (PHI)-immunoreactive amacrine cells in the retina of the rat J.D. M i k k e l s e n l, J . N . B . L a r s e n l, J. F a h r e n k r u g 2 a n d M. M o l l e r 1 Ilnstitute of Medical Anatomy, Department B, University of Copenhagen, Copenhagen (Denmark) and Department of Clinical Chemistry, BispebjergHospital, Copenhagen (Denmark) (Received 30 December 1986;Revisedversion received and accepted 29 April 1987) Key words: Peptidehistidine-isoleucine; Amacrinecell; Retina; Rat; Immunohistochemistry Peptide histidine-isoleucine (PHI) immunoreactivitywas located in amacrine-likecells in adult rat retina by use of immunohistochemical techniques. Immunoreactivesomata were found in the proximal part of the inner nuclear layer. From these somata, processes could be followed into the inner plexiform layer. The terminals of these processes were mainly found in the sublayers, 1, 2, and 3, but a few terminals were also present in the other inner plexiformsublaminae. The distribution of PHI-immunoreactivesomata and processes corresponds with the cellular distribution of vasoactive intestinal peptide (VIP) and indicates a possible co-localizationwith this peptide.
During recent years an increasing number of neuropeptide-immunoreactive substances have been shown in the retina of several species [2]. These neuropeptides are considered to act as neurotransmitters or neuromodulators in the retina itself. A m o n g the peptides of the glucagon-secretin family, glucagon [3] and vasoactive intestinal peptide (VIP) [5, 14, 15] have up to now been demonstrated in the rat retina by the use of immunohistochemistry and the presence of these peptides has further been substantiated by gelchromatography [4]. A m o n g several neuropeptides investigated, only glucagon, VIP and secretin, are able to stimulate the formation of cyclic A M P in the retina [9]. These investigations indicate that both glucagon and VIP are peptidergic neuromodulators in the retina. Recently a peptide, peptide histidine-isoleucine (PHI), has been isolated from porcine intestine [12] and later also from porcine brain [13]. This peptide has sequence homologies with members of the glucagon-secretin family and has thus been included in this family of peptides. The distribution of PHI-immunoreactivity in peripheral tissues paralleled that o f VIP and immunoreactivity for the two peptides was often seen in the same type of neural elements [1, 10, 19].
Correspondence: J.D. Mikkelsen, Institute of Medical Anatomy, Department B, The Panum Institute, 3, Blegdamsvej, DK-2200Copenhagen N, Denmark. 0304-3940/87/$ 03.50 © 1987 Elsevier ScientificPublishers Ireland Ltd.
282 The purpose of this study was by immunohistochemistry to determine whether PHI is present in the rat retina in addition to VIP, and if its localization gives evidence for co-existence with VIP. Eight male Wistar rats weighing 250-300 g were anaesthesized with nembutal and perfused intracardially with 200 ml 0.9 % saline to which heparine (15.000) IE/ml) was added, followed immediately by perfusion with 600 ml 4.0% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature. The eyes were then removed and postfixed in the above-mentioned fixative for 4 h at 4°C, followed by infiltration with a 30% sucrose solution in phosphate-buffered saline (PBS) at 4°C for a minimum of 3 days. Twenty-/tm thick sections were cut and placed on gelatinized glass slides. The sections were rinsed and reacted either with an antibody raised in rabbits against porcine PHI, lot. 3668-5 [6], diluted 1:800 or with an antibody against VIP diluted 1:600 (Cambridge Research Biochemicals, England) for 48 h at 4°C. Afterwards the sections were incubated for 60 min first in swine anti-rabbit IgG (Dakopatts, Copenhagen, Z 196), diluted 1:20, and then in peroxidase-conjugated rabbitanti-swine IgG (Dakopatts, Copenhagen Z 113), diluted 1:80. In between all of the steps, the sections were rinsed 3 times for 10 min in PBS (pH 7.4) with 1% Triton X-100 added. Finally they were incubated in 0.025% diamino-benzidine and 0.003% H202 in 0.05 M Tris-HCl buffer (pH 7.6) for 20 min. In control experiments the primary antisera were absorbed with 40 pg VIP or PHI per 1 ml antibody diluted 1:800 (obtained from Peninsula Laboratories, U.S.A.). The retinae from rats incubated with PHI-antiserum showed immunoreactivity within a subpopulation of ovoid or triangular amacrine-like cells located in the proximal part of the inner nuclear layer (INL) on the border to the inner plexiform layer (IPL) (Figs. 1, 2, 4 and 5). The PHI-immunoreactive cells were distributed in both central and peripheral retinal regions. No immunoreactive perikarya were found in other retinal layers, neither were PHl-immunoreactive fibres observed in the optic nerve. Some of the PHI-immunoreactive amacrine cells gave rise to a single process which extended into the IPL, where it dichotomized in sublamina 1 (Figs. 1 and 5). Another type of PHI-immunoreactive cell exhibited a Golgi-cell-like appearance with a short process which divided into 3 or 4 secondary processes located in sublamina 1, 2, and 3 (Fig. 2). Although the highest density of PHI-immunoreactive fibers was found in sublamina 1 to 3 (Figs. 4 and 5), fibres in sublamina 4 and 5 were occasionally observed (Fig. 3). No staining was seen after incubation with a preimmune serum or with antibodies to which natural PHI was added. Contrarily, no decrease in the PHI-like immunoreactivity could be observed if the antibody was absorbed with natural VIP. In this study, PHI-like immunoreactive cells were demonstrated in the retina of the rat. Based on morphological features, the PHI-immunoreactive cells were probably amacrine cells, because these cells were located in the proximal part of the INL with processes emerging into the IPL, in which layer the processes ramified primarily in the sublayers 1 to 3. This location of PHI-immunoreactive perikarya, and espe-
283
Fig. 1. PHI-immunoreactive cell located in the inner nuclear layer (INL) giving rise to a process which dichotomizes within sublamina 1 of the inner plexiform layer (IPL). ONL, outer plexiform layer, x 240. Fig. 2. PHI-immunoreactive cell located in the INL on the border of the IPL. The processes of the neuron arborize within sublamina 1, 2 and 3 of the IPL. x 220. Fig. 3. VIP-immunoreactive neuron located in the inner part of the INL with a long process dichotomizing in sublamina 1 and 3 of the IPL. x 364. Fig. 4. Many PHI-immunoreactive beaded nerve fibres within sublamina 1, 2 and 3 of the IPL. x 240. cially t h e i r p r o c e s s e s in t h e I P L , c o r r e s p o n d s w i t h i m m u n o s t a i n i n g f o r V I P - i m m u n o r e a c t i v e cells p e r f o r m e d in this s t u d y (Fig. 3) as well as t h e d i s t r i b u t i o n r e p o r t e d by o t h e r s [5, 14], s u g g e s t i n g a c o - l o c a l i z a t i o n o f t h e t w o p e p t i d e s as s h o w n in o t h e r n e u r o n a l s y s t e m s [1, 10, 19].
284
Fig. 5. PHl-immunoreactive cells located in the INL and processes which ramify primarily within sublamina 1, 2 and 3 of the IPL. Few PHI-positive processes are observed within sublamina 4 and 5. x 240.
Amacrine cells in the rat have been shown to contain different neurotransmitters and neuropeptides such as substance P [7], V I P [5, 14, 15], somatostatin [1 1], glucagon [3] and a variety o f classic transmitters [2]. A co-localization in the retina o f some o f these transmitters and neuropeptides has been demonstrated in the chicken [17] and in the turtle [18]. However, the striking similarity in m o r p h o l o g y between the VIP- and P H I - i m m u n o r e a c t i v e cells in the rat retina and the evidence o f a co-localization o f these two peptides is highly supported by the finding that V I P and P H I are part o f the same propeptide [8]. The function o f P H I in the retina o f m a m m a l s is at present u n k n o w n . In the carp, P H I has been shown to stimulate the production o f cyclic A M P , but the response was only 40% o f the response obtained after stimulation with V I P [16]. Further studies are needed in order to clarify the function o f P H I in the m a m m a l i a n retina and a possible co-localization with V I P or classical neurotransmitters. The authors would like to express their thanks to Mrs. G. H a h n for her excellent photographic assistance. This study was supported by the Carlsberg F o u n d a t i o n , the L u n d b e c k F o u n d a t i o n , the National Society for the Fight against Eye Diseases and Blindness (Denmark) and the Danish Medical Research Council (12-6429). 1 Bishop, A.E., Polak, J.M., Yiangou, Y., Christofides, N.D. and Bloom, S.R., The distribution of PHI and VIP in porcine gut and their co-localization to a proportion of intrinsic ganglion cells, Peptides, 5 (1984) 255-259. 2 Brecha, N., Retinal neurotransmitters: histochemical and biochemical studies. In P.C. Emson (Eds.), Chemical neuroanatomy, Raven, New York, 1983, pp. 85-129. 3 Das, A., Pansky, B. and Budd, G.C., Glucagon-like immunoreactivity in mouse and rat retina, Neurosci, lett., 60 (1985) 215 218.
285 4 Ekman, R. and Tornqvist, K., Glucagon and VIP in the retina, Invest. Ophthalmol. Vis. Sci., 26 (1986) 1405-1409. 5 Eriksen, E.F. and Larsson, L.I., Neuropeptides in the retina: evidence for different topographical localization, Peptides, 2 (1981) 153-157. 6 Fahrenkrug, J. and Pedersen, J.H., Development and validation of a specific radioimmunoasay for PHI in plasma, Clin. Chem. Acta, 143 (1984) 183-192. 7 Fukuda, M., Kuwayama, Y., Shiosaka, S., Ishimoto, I., Shimizu, Y., Takagi, H., Inagaki, S., Sakanaka, M., Semba, E., Takatsuhi, K. and Fokyama, M., Demonstration of a substance P-like immunoreactivity in retinal cells of the rat, Neurosci. Lett., 23 (1981) 239-242. 8 Itoh, N., Obata, K.-I., Yanaihara, N. and Okamato, H., Human preprovasoactive intestinal polypeptide contains a novel PHI-27-1ike peptide, PHM-27, Nature (London), 304 (1983) 547-549. 9 Longshore, M.A. and Makman, M.H., Stimulation of retinal adenylate cyclase by vasoactive intestinal peptide (VIP), Eur. J. Pharmacol., 70 (1981) 237-240. 10 Lundberg, J.M., Fahrenkrug, J., Hrkfelt, T., Martling, C.-R., Larsson, O., Tatemoto, K. and ,~ngg~rd, A., Co-existence of peptide HI (PHI) and VIP in nerves regulating blood flow and bronchial smooth muscle tone in various mammals including man, Peptides, 5 (1984) 593-606. 11 Sagar, S.M., Marshall, P.E. and Landis, D.M.D., Immunoreactive somatostatin in the rat retina: light immunocytochemistry and chromatographic characterization, Brain Res., 336 (1985) 235-242. 12 Tatemoto, K. and Mutt, V., Isolation and characterization of the intestinal peptide porcine PHI (PHI27), a new member of the glucagon-secretin family, Proc. Natl. Acad. Sci. USA, 78 (1981) 6603-6607. 13 Tatemoto, K., Carlquist, M., McDonald, T.J. and Mutt, V., Isolation of a brain peptide identical to the intestinal PHI (peptide HI), FEBS lett., 153 (1983) 248-252. 14 Terubayashi, H., Tsuto, T., Fukui, K., Obata, H.L., Okamura, H., Fujisawa, H., Itoi, M., Yanaihara, C., Yanaihara, N. and Ibata, Y., VIP (vasoactive intestinal polypeptide)-like immunoreactive amacrine cells in the retina of the rat, Exp. Eye Res., 36 (1983) 743-749. 15 Tornqvist, K. Uddman, R., Sundler, F. and Ehinger, B., Somatostatin and VIP neurons in the retina of different species, Histochemistry, 76 (1982) 137 152. 16 Watling, K.J. and Dowling, J.E., Effects of vasoactive intestinal peptide and other peptides on cyclic AMP accumulation in intact pieces and isolated horizontal cells of the teleost retina, J. Neurochem., 41 (1983) 1205-1213. 17 Watt, C.B., Su, Y.-y.T. and Lam, D.M.-K., Interactions between enkephalin and GABA in avian retina, Nature (London), 311 (1984) 761-763. 18 Weiler, R. and Ball, A.K., Co-localization of neurotensin-like immunoreactivity and 3H-glycine uptake system in sustained amacrine cells of turtle retina, Nature (London), 311 (1984) 759-761. 19 Yanaihara, N., Nokihara, K., Yanaihara, C., Iwanaga, T. and Fujita, T., Immunocytochemical demonstration of PHI and its co-existence with VIP in intestinal nerves of the rat and pig, Arch. Histol. Jpn., 46 (1983) 575-581.
281
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Peptide histidine-isoleucine (PHI)-immunoreactive amacrine cells in the retina of the rat J.D. M i k k e l s e n l, J . N . B . L a r s e n l, J. F a h r e n k r u g 2 a n d M. M o l l e r 1 Ilnstitute of Medical Anatomy, Department B, University of Copenhagen, Copenhagen (Denmark) and Department of Clinical Chemistry, BispebjergHospital, Copenhagen (Denmark) (Received 30 December 1986;Revisedversion received and accepted 29 April 1987) Key words: Peptidehistidine-isoleucine; Amacrinecell; Retina; Rat; Immunohistochemistry Peptide histidine-isoleucine (PHI) immunoreactivitywas located in amacrine-likecells in adult rat retina by use of immunohistochemical techniques. Immunoreactivesomata were found in the proximal part of the inner nuclear layer. From these somata, processes could be followed into the inner plexiform layer. The terminals of these processes were mainly found in the sublayers, 1, 2, and 3, but a few terminals were also present in the other inner plexiformsublaminae. The distribution of PHI-immunoreactivesomata and processes corresponds with the cellular distribution of vasoactive intestinal peptide (VIP) and indicates a possible co-localizationwith this peptide.
During recent years an increasing number of neuropeptide-immunoreactive substances have been shown in the retina of several species [2]. These neuropeptides are considered to act as neurotransmitters or neuromodulators in the retina itself. A m o n g the peptides of the glucagon-secretin family, glucagon [3] and vasoactive intestinal peptide (VIP) [5, 14, 15] have up to now been demonstrated in the rat retina by the use of immunohistochemistry and the presence of these peptides has further been substantiated by gelchromatography [4]. A m o n g several neuropeptides investigated, only glucagon, VIP and secretin, are able to stimulate the formation of cyclic A M P in the retina [9]. These investigations indicate that both glucagon and VIP are peptidergic neuromodulators in the retina. Recently a peptide, peptide histidine-isoleucine (PHI), has been isolated from porcine intestine [12] and later also from porcine brain [13]. This peptide has sequence homologies with members of the glucagon-secretin family and has thus been included in this family of peptides. The distribution of PHI-immunoreactivity in peripheral tissues paralleled that o f VIP and immunoreactivity for the two peptides was often seen in the same type of neural elements [1, 10, 19].
Correspondence: J.D. Mikkelsen, Institute of Medical Anatomy, Department B, The Panum Institute, 3, Blegdamsvej, DK-2200Copenhagen N, Denmark. 0304-3940/87/$ 03.50 © 1987 Elsevier ScientificPublishers Ireland Ltd.
282 The purpose of this study was by immunohistochemistry to determine whether PHI is present in the rat retina in addition to VIP, and if its localization gives evidence for co-existence with VIP. Eight male Wistar rats weighing 250-300 g were anaesthesized with nembutal and perfused intracardially with 200 ml 0.9 % saline to which heparine (15.000) IE/ml) was added, followed immediately by perfusion with 600 ml 4.0% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature. The eyes were then removed and postfixed in the above-mentioned fixative for 4 h at 4°C, followed by infiltration with a 30% sucrose solution in phosphate-buffered saline (PBS) at 4°C for a minimum of 3 days. Twenty-/tm thick sections were cut and placed on gelatinized glass slides. The sections were rinsed and reacted either with an antibody raised in rabbits against porcine PHI, lot. 3668-5 [6], diluted 1:800 or with an antibody against VIP diluted 1:600 (Cambridge Research Biochemicals, England) for 48 h at 4°C. Afterwards the sections were incubated for 60 min first in swine anti-rabbit IgG (Dakopatts, Copenhagen, Z 196), diluted 1:20, and then in peroxidase-conjugated rabbitanti-swine IgG (Dakopatts, Copenhagen Z 113), diluted 1:80. In between all of the steps, the sections were rinsed 3 times for 10 min in PBS (pH 7.4) with 1% Triton X-100 added. Finally they were incubated in 0.025% diamino-benzidine and 0.003% H202 in 0.05 M Tris-HCl buffer (pH 7.6) for 20 min. In control experiments the primary antisera were absorbed with 40 pg VIP or PHI per 1 ml antibody diluted 1:800 (obtained from Peninsula Laboratories, U.S.A.). The retinae from rats incubated with PHI-antiserum showed immunoreactivity within a subpopulation of ovoid or triangular amacrine-like cells located in the proximal part of the inner nuclear layer (INL) on the border to the inner plexiform layer (IPL) (Figs. 1, 2, 4 and 5). The PHI-immunoreactive cells were distributed in both central and peripheral retinal regions. No immunoreactive perikarya were found in other retinal layers, neither were PHl-immunoreactive fibres observed in the optic nerve. Some of the PHI-immunoreactive amacrine cells gave rise to a single process which extended into the IPL, where it dichotomized in sublamina 1 (Figs. 1 and 5). Another type of PHI-immunoreactive cell exhibited a Golgi-cell-like appearance with a short process which divided into 3 or 4 secondary processes located in sublamina 1, 2, and 3 (Fig. 2). Although the highest density of PHI-immunoreactive fibers was found in sublamina 1 to 3 (Figs. 4 and 5), fibres in sublamina 4 and 5 were occasionally observed (Fig. 3). No staining was seen after incubation with a preimmune serum or with antibodies to which natural PHI was added. Contrarily, no decrease in the PHI-like immunoreactivity could be observed if the antibody was absorbed with natural VIP. In this study, PHI-like immunoreactive cells were demonstrated in the retina of the rat. Based on morphological features, the PHI-immunoreactive cells were probably amacrine cells, because these cells were located in the proximal part of the INL with processes emerging into the IPL, in which layer the processes ramified primarily in the sublayers 1 to 3. This location of PHI-immunoreactive perikarya, and espe-
283
Fig. 1. PHI-immunoreactive cell located in the inner nuclear layer (INL) giving rise to a process which dichotomizes within sublamina 1 of the inner plexiform layer (IPL). ONL, outer plexiform layer, x 240. Fig. 2. PHI-immunoreactive cell located in the INL on the border of the IPL. The processes of the neuron arborize within sublamina 1, 2 and 3 of the IPL. x 220. Fig. 3. VIP-immunoreactive neuron located in the inner part of the INL with a long process dichotomizing in sublamina 1 and 3 of the IPL. x 364. Fig. 4. Many PHI-immunoreactive beaded nerve fibres within sublamina 1, 2 and 3 of the IPL. x 240. cially t h e i r p r o c e s s e s in t h e I P L , c o r r e s p o n d s w i t h i m m u n o s t a i n i n g f o r V I P - i m m u n o r e a c t i v e cells p e r f o r m e d in this s t u d y (Fig. 3) as well as t h e d i s t r i b u t i o n r e p o r t e d by o t h e r s [5, 14], s u g g e s t i n g a c o - l o c a l i z a t i o n o f t h e t w o p e p t i d e s as s h o w n in o t h e r n e u r o n a l s y s t e m s [1, 10, 19].
284
Fig. 5. PHl-immunoreactive cells located in the INL and processes which ramify primarily within sublamina 1, 2 and 3 of the IPL. Few PHI-positive processes are observed within sublamina 4 and 5. x 240.
Amacrine cells in the rat have been shown to contain different neurotransmitters and neuropeptides such as substance P [7], V I P [5, 14, 15], somatostatin [1 1], glucagon [3] and a variety o f classic transmitters [2]. A co-localization in the retina o f some o f these transmitters and neuropeptides has been demonstrated in the chicken [17] and in the turtle [18]. However, the striking similarity in m o r p h o l o g y between the VIP- and P H I - i m m u n o r e a c t i v e cells in the rat retina and the evidence o f a co-localization o f these two peptides is highly supported by the finding that V I P and P H I are part o f the same propeptide [8]. The function o f P H I in the retina o f m a m m a l s is at present u n k n o w n . In the carp, P H I has been shown to stimulate the production o f cyclic A M P , but the response was only 40% o f the response obtained after stimulation with V I P [16]. Further studies are needed in order to clarify the function o f P H I in the m a m m a l i a n retina and a possible co-localization with V I P or classical neurotransmitters. The authors would like to express their thanks to Mrs. G. H a h n for her excellent photographic assistance. This study was supported by the Carlsberg F o u n d a t i o n , the L u n d b e c k F o u n d a t i o n , the National Society for the Fight against Eye Diseases and Blindness (Denmark) and the Danish Medical Research Council (12-6429). 1 Bishop, A.E., Polak, J.M., Yiangou, Y., Christofides, N.D. and Bloom, S.R., The distribution of PHI and VIP in porcine gut and their co-localization to a proportion of intrinsic ganglion cells, Peptides, 5 (1984) 255-259. 2 Brecha, N., Retinal neurotransmitters: histochemical and biochemical studies. In P.C. Emson (Eds.), Chemical neuroanatomy, Raven, New York, 1983, pp. 85-129. 3 Das, A., Pansky, B. and Budd, G.C., Glucagon-like immunoreactivity in mouse and rat retina, Neurosci, lett., 60 (1985) 215 218.
285 4 Ekman, R. and Tornqvist, K., Glucagon and VIP in the retina, Invest. Ophthalmol. Vis. Sci., 26 (1986) 1405-1409. 5 Eriksen, E.F. and Larsson, L.I., Neuropeptides in the retina: evidence for different topographical localization, Peptides, 2 (1981) 153-157. 6 Fahrenkrug, J. and Pedersen, J.H., Development and validation of a specific radioimmunoasay for PHI in plasma, Clin. Chem. Acta, 143 (1984) 183-192. 7 Fukuda, M., Kuwayama, Y., Shiosaka, S., Ishimoto, I., Shimizu, Y., Takagi, H., Inagaki, S., Sakanaka, M., Semba, E., Takatsuhi, K. and Fokyama, M., Demonstration of a substance P-like immunoreactivity in retinal cells of the rat, Neurosci. Lett., 23 (1981) 239-242. 8 Itoh, N., Obata, K.-I., Yanaihara, N. and Okamato, H., Human preprovasoactive intestinal polypeptide contains a novel PHI-27-1ike peptide, PHM-27, Nature (London), 304 (1983) 547-549. 9 Longshore, M.A. and Makman, M.H., Stimulation of retinal adenylate cyclase by vasoactive intestinal peptide (VIP), Eur. J. Pharmacol., 70 (1981) 237-240. 10 Lundberg, J.M., Fahrenkrug, J., Hrkfelt, T., Martling, C.-R., Larsson, O., Tatemoto, K. and ,~ngg~rd, A., Co-existence of peptide HI (PHI) and VIP in nerves regulating blood flow and bronchial smooth muscle tone in various mammals including man, Peptides, 5 (1984) 593-606. 11 Sagar, S.M., Marshall, P.E. and Landis, D.M.D., Immunoreactive somatostatin in the rat retina: light immunocytochemistry and chromatographic characterization, Brain Res., 336 (1985) 235-242. 12 Tatemoto, K. and Mutt, V., Isolation and characterization of the intestinal peptide porcine PHI (PHI27), a new member of the glucagon-secretin family, Proc. Natl. Acad. Sci. USA, 78 (1981) 6603-6607. 13 Tatemoto, K., Carlquist, M., McDonald, T.J. and Mutt, V., Isolation of a brain peptide identical to the intestinal PHI (peptide HI), FEBS lett., 153 (1983) 248-252. 14 Terubayashi, H., Tsuto, T., Fukui, K., Obata, H.L., Okamura, H., Fujisawa, H., Itoi, M., Yanaihara, C., Yanaihara, N. and Ibata, Y., VIP (vasoactive intestinal polypeptide)-like immunoreactive amacrine cells in the retina of the rat, Exp. Eye Res., 36 (1983) 743-749. 15 Tornqvist, K. Uddman, R., Sundler, F. and Ehinger, B., Somatostatin and VIP neurons in the retina of different species, Histochemistry, 76 (1982) 137 152. 16 Watling, K.J. and Dowling, J.E., Effects of vasoactive intestinal peptide and other peptides on cyclic AMP accumulation in intact pieces and isolated horizontal cells of the teleost retina, J. Neurochem., 41 (1983) 1205-1213. 17 Watt, C.B., Su, Y.-y.T. and Lam, D.M.-K., Interactions between enkephalin and GABA in avian retina, Nature (London), 311 (1984) 761-763. 18 Weiler, R. and Ball, A.K., Co-localization of neurotensin-like immunoreactivity and 3H-glycine uptake system in sustained amacrine cells of turtle retina, Nature (London), 311 (1984) 759-761. 19 Yanaihara, N., Nokihara, K., Yanaihara, C., Iwanaga, T. and Fujita, T., Immunocytochemical demonstration of PHI and its co-existence with VIP in intestinal nerves of the rat and pig, Arch. Histol. Jpn., 46 (1983) 575-581.