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The development of amacrine cells containing somatostatin-like immunoreactivity in chicken retina
Developmental Brain Research, 8 (1983) 71- 76
71
Elsevier Biomedical Press
The Development of Amacrine Cells Containing Somatostatin-like Immunoreactivity in Chicken Retina I. G. MORGAN, J. OLIVER and I. W. CHUBB
Department of Behavioral Biology, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra City, 2601, and Centrefor Neuroscience, (J. 0.) Departments of Medicine and (!. W.C. Human Physiology, Flinders University, Bedford Park, S.A. 5032,A ustralia (Accepted October 18th, 1982)
Key words retina - somatostatin - amacrine cells
Mature chicken retinas contain significant amounts of somatostatin-like immunoreactivity, which appear to be located in a prominent population of amacrine cells. By radio immunoassay, somatostatin-like immunoreactivity can first be detected around day 7 in ovo, and the first cells detectable by immunohistochemistry on around day 11 in ovo. The cells appear to mature in 3 phases, with a small but rapid increase in levels of somatostatin-like immunoreactivity from day 7 to day 11 in ovo, followed by a period of more gradual increase up to day 17 in ovo. The final phase consists of a rapid pre-hatch increase in levels ofsomatostatin-like immunoreactivity to adult levels by hatching. Immunoreactive cells are detectable from day I 1 in ovo, but immunoreactive processes in the inner plexiform layer are not visible until day 19 in ovo. The rapid increase in levels ofsomatostatin-like immunoreactivity, and the appearance of immunoreactive processes in the inner plexiform layer coincide temporally with the onset of light-driven activities in the retina. INTRODUCTION
The morphological heterogeneity of the amacrine and ganglion cell classes of the avian retina has been known for almost 100 years 22. Recent work has demonstrated that there is a corresponding heterogeneity of amacrine cell putative transmitters, among which are a number of neuropeptides (for reviews see refs. 3,15,25). In the mature retina there are specific sub-classes of amacrine cells, characterized by specific morphologies and putative transmitters, with specific patterns of connection with sub-classes of bipolar cells, ganglion cells and amacrine cells, organized in laminar arrays through the thickness of the retinatL This precise organization must be produced by subtle inter-cellular interactions during retinal histogenesis, as the pluripotent germinal cells of the invaginated neuroepithelium give rise to the 6 major neuronal cell classes and their many sub-classes by a sequential process of withdrawal from cell division, cell migration, and morphological and biochemical cell differentiation. 0165-3806/83/0000 0000/$03.00 "~'1983 Elsevier Science Publishers
While precise timing of these processes is complicated due to the central-peripheral gradient of retinal maturation, some of the basic features of the histogenesis of the chicken retina have been described. The majority of ganglion cells and amacrine cells are formed on days 3-5 in ovo 12.~3.Ganglion cell axons appear from days 2 to 3 in ovo onwards 9,19,23, and their dendrites begin to appear around days 7-8 in ovo, and become progressively more elaborate up to hatching j6. Around 40% of the ganglion cells die between days 13 and 15 in ovo 2°. One sub-class of amacrine cells, the displaced amacrine cells8, is formed in parallel with ganglion cell differentiation, but little is known about the differentiation of most amacrine cell morphological sub-classes. Bipolar cells are formed a little later, and apparently over a much longer period up to around day 13 in ovo iT. Their axonal processes mature roughly in parallel with those of ganglion cells ~7.24,and thus the inner plexiform layer appears first at around day 8 in ovo in the central retina, and reaches its full thickness by around day 16 in ovo 6. Conventional synapses
72 are first detectable in the inner plexiform layer on day 13 in ovo and bipolar synapses are first detectable on day 14 in ovo ~. Synapse formation then proceeds rapidly in the inner plexiform layer from day 14 to day 20 in ovo 7. Biochemical studies concern almost exclusively the cholinergic amacrine cell subclass. The development of acetylcholine k4. choline acetyltransferaseH~.-'~-'L acetylcholinesteraso-~j -'4-'~. high-affinity choline uptake ~and nicotinit~'7 "'~ and muscariniC ~'.-'9 acetylcholine receptors has been followed, and proceeds throughout the period of establishment of the inner plexiform layer. The development of retinal acetylcholinesterase has been followed histochemicallf 4. The adult pattern of distribution of enzyme activity is established by day 16 in ovo. In this study, we report a parallel biochemical and immunohistochemical study of the development of the most numerous neuropeptide-containing amacrine cells so far studied, those which contain somatostatin-like immunoreactivit~ .5. MATERIALS AND METHODS
Fertilized eggs of a White Leghorn/Black Australorp cross were obtained from Research Poultry Farm. Victoria, Australia and incubated at 38 "C. Embryos were removed from day 7 in ovo onwards, and staged according to Hamilton ~°, to verify that development was normal. For radioimmunoassay, the eyeballs were removed. The anterior half of the eye was removed, and the neural retina plus the pigment epithelium was carefully dissected. The retinas were homogenized in 2 M acetic acid. The homogenates were transferred to a plastic tube and boiled for 15 min in a boiling salt-water bath. After standing overnight at 4 °C, the tubes were centrifuged and the supernatants frozen until assayed. An aliquot (10/d) of the supernatant was added to an assay tube which, after freezedrying, was made up with 0.2 ml of assay buffer (0.05 M sodium phosphate, pH 7.4 containing 0.2 g% bovine serum albumin, 10 mM EDTA and 500 kallikrein inactivator units trasylol/ml). Radio-labeled somatostatin and antiserum (fi-
nal dilution 1/10,000) in assay buffer were added to the tube so as to bring the final incubation volume to 0.40 ml. A standard curve was constructed using synthetic somatostatin. The contents of the tubes were incubated for 16 h at 4 °C, after which time the hormone bound to antibody was separated from free hormone by precipitation with polyethylene glycol. The radioactivity of the bound fraction was determined by counting in a Nuclear Enterprises NENI600 gamma-counter. The sensitivity of the assay under these conditions was 3 pg ofsomatostatin. For somatostatin immunohistochemistry, the eyeballs were removed, hemisected, and the posterior portions fixed by immersion in 4% formaldehyde in 0.1 M phosphae buffer (pH 7.2) for 9 12 h at 4°C. The retinas were then removed, washed in 8% sucrose, 0.1 M phosphate buffer (pH 7.2) for at least 6 h, mounted in TissueTek II OCT compound (Miles Labs., Australia), and frozen in isopentane cooled with liquid nitrogen. Sections of 10/~m were cut from the central retina on a cryostat at - 1 5 °C. The frozen sections were mounted on chrom-alum-treated slides, dried in air for 15 min and washed in 10 mM PBS for 10 min. The sections were incubated overnight in anti-somatostatin antiserum diluted (1:75) with sheep serum:PBS (1:5) in a humid chamber at room temperature. After a brief wash in PBS, the sections were incubated for 60 min at room temperature in fluoroscein isothicyanate-sheep anti-rabbit immunoglobulin (Wellcome) diluted with sheep serum: PBS (1: 5). Following a final rinse in PBS the sections were mounted in glycerol: 0.5 M bicarbonate buffer (1: 1) (pH 8.6). Sections were examined under ultraviolet light using a Leitz Orthoplan microscope with a BG 12 excitation filter. Antiserum to somatostatin was produced in the rabbit by repeated subcutaneous, multiple site injections of synthetic somatostatin (Ayerst Laboratories, Montreal, Canada), conjugated to bovine thyroglobulin. The antiserum (2L-13), when tested against other peptides and hormones, demonstrated the following cross-reactivities: gastrin 1.5%; C-peptide 0.007%: and less than 0.001% for human/3-endorphin, angiotensins I and II, rat and human growth hormones,
73
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HATCHING AGE OF EMBRYO (DAYS) Fig. I. Development of wet weight, protein and levels ofsomatostatin-like immunoreactivity in chicken retina. Results are given as total activity per retina. E 7 - 21 refers to the number of days of incubation, H I - 7 to the number ofdays after hatching. A: mg protein/retina. B: mg wet weight/retina. C: ng somatostatin-like immunoreactivity/retina.
glucagon, thyrotropin-releasing factor, substance P, arginine- and lysine-vasopressin, eledoisin, bradykinin, adrenocorticotropin and insulin. RESULTS
As shown in Fig. 1, somatostatin-like immunoreactivity was first detectable by radioimmunoassay in the chicken retina by day 7 in ova, and increased significantly in concentration between days 9 and 11 in ova. The rate of increase then diminished from day 11 in ova, until days 17-18 in ova. Around days 18-19 in ova there was a sudden increase in the rate of accumulation of somatostatin-like immunoreactivity and adult levels were attained around hatching. By contrast, retinal protein content and wet weight
increased rapidly from day 7 until day 13 in ova, then the rate of increase diminished until hatching. Thus the specific activity of somatostatinlike immunoreactivity decreased up to day 13, then increased rapidly prior to hatching. Immunohistochemically (Fig. 2), somatostatin-positive cell bodies were first detectable around day 11 in ova, and were already displaced from the border between the nascent inner nuclear and inner plexiform layers, in a position which roughly corresponds to that of the somatostatin-positive amacrine cells of the adult retina. By day 13 in ova, prominent descending dendrites could also be seen, but no clear dendritic arborizations could be detected in the inner plexiform layer. This picture remained essentially the same for days 15 and 17 in ova, al-
74
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Fig. 2. Development of somatostatin-like immunoreactivity in chick retina as revealed by immunohistochemistry, a: day I I in ovo. b: day 13 in ovo. c: day 15 in ovo. d" day 17 in ovo. c: day 18 in ovo. f: one day after hatching.
75 though the fluorescence of the somatostatinpositive cell bodies increased relative to the background. On day 17 in ovo the first signs of punctate fluorescence were seen in the inner plexiform layer, and these had increased markedly by day 19 in ovo. By one day after hatching, the characteristic laminar pattern of the dendritic arborizations had become clear, and the cell bodies had become so fluorescent that the perinuclear staining visible at earlier times was obscured. DISCUSSION
These results indicate that the levels of somatostatin-like immunoreactivity, and the cells which contain it, do not develop simply in parallel with the gross development of the neural retina. Rather, there appear to be 3 phases of development of the somatostatin-positive system. From days 7 to 9 in ovo, there is a small, but rapid, increase in levels of somatostatin-like immunoreactivity, at around the time that inner plexiform layer begins to appear in the central retina. By day 11 in ovo, immunoreactive cell bodies are visible in the inner nuclear layer, and there is a gradual increase in levels of somatostatin-like immunoreactivity over the period during which ganglion cells, displaced amacrine cells, bipolar cells, and presumably amacrine cells elaborate their dendrites. This period is then followed from days 18 to 19 in ovo by a rapid increase in the levels of somatostatin-like immunoreactivity which reach adult levels by hatching. This rapid increase in the levels ofsomatostatin-like immunoreactivity roughly coincides with the final development of the photoreceptor outer segments, and the beginnings of light-driven electrical activity in the retina s . Thus the pattern of development of somatostatin-like immunoreactivity and the cells which contain it is consistent with the localization of the somatostatin-like immunoreactivity in amacrine cells. The appearance of somatostatinlike immunoreactivity parallels the way in which choline acetyltransferase activity ~.29develops, in that there is an accumulation of activity around from day 7 to day 13 in ovo, followed by a lag
period, then a rapid pre-hatch increase in activity. Other activities characteristic of the cholinergic system, namely high-affinity choline uptake I, nicotinic and muscarinic acetylcholine receptors, and acetylcholinesterase develop with different patterns 29. Thus, although there is a parallel between the development of some amacrine cell presynaptic components, such as somatostatin-like immunoreactivity and choline acetyltransferase K,29and in less detailed studies, acetylcholine14, other presynaptic components such as high-affinity choline uptake ~and GABA (unpublished results) have distinct developmental profiles, as does glycine (unpublished resuits), which is likely to be located in part in amacrine cell synapses. Little somatostatin-like immunoreactivity is detected in the inner plexiform layer until just prior to hatching. This contrasts with the early appearance of two bands of acetylcholinesterase activity in the inner plexiform layer from day 8 in ovo, with the adult 4 bands being distinctly visible by day 16 in ovo24. The lack of immunoreactivity in the inner plexiform layer is unlikely to be due to late morphological maturation of the dendritic arborizations of that amacrine cell sub-class. It is more likely to be related to low rates of synthesis of whatever compounds are responsible for the somatostatin-like immunoreactivity detected by radioimmunoassay and immunohistochemistry. It is not clear whether the rapid pre-hatch increase in levels of somatostatin-like immunoreactivity is triggered by the functional maturation of the retina 2, or whether it is simply a contemporaneous event. The former possibiity is supported by the observation that there are marked diurnal variations in levels of somatostatin-like immunoreactivity in the retinas of chickens held on 12:12 light-dark cycles, with maximum values attained at the end of the light phase twice those at the end of the dark phase (unpublished results). In fact, it is possible that the rapid pre-hatch increase is just the first occurrence of a light-entrained accumulation of somatostatin-like immunoreactivity, which then occurs every day under normal circumstances.
76 REFERENCES 1 Bader, C. R., Baughman, R. W. and Moore, J. L., Different time-course of development for high-affinity choline uptake and choline acetyltransferase in the chick retina. Proc. nat. Acad Sci. U.S.A., 75 (1978) 2525 2529. 2 Blozovski, D. and Blozovski, M., Developpement compare de l'electroretinogrammc et des potenticls evoques visuels du toit optique, du cervelet et du telencephale chez le Poussin, J. Phvsiol. (Paris), 60 (1968) 33 50. 3 Bonting, S. L. (Ed.) Transmitters in the Visual Process. Pergamon Press, Oxford, 1976. 4 Brecha. N., Karten. H. J. and Schenker, C. S.. Neurotensin-like and somatostatin-like immunoreactivity within amacrine cells of the retina, Neuroscieme. 6 ( 19811 1329 1340. 5 Buckerfield, M., Oliver, J., Chubb, I. W. and Morgan, I. G., Somatostatin-like immunoreactivity in amacrine cells of the chicken retina, Neuroscience. 6 (1981) 685 693. 6 Coulombre, A. J., Correlations of structural and biochemical changes in the developing retina of the chick. Amer. J. Anat., 96 (1955) 153- 189. 7 Fisher, L. J., Synaptic development in chicken retina, Invest. Ophthalmol. vis. Sci., Suppl. 20(1981) 203. 8 Galvez, J. E. G., Puelles, L. and Prada, C., Inverted (displaced) retinal amacrine cells and their embryonic development in the chick, Exp. Neurol., 56 (1977) 151 157. 9 Goldberg, S. and Coulombre, A. J., Topographical development of the ganglion cell layer in the chick retina. A whole mount study. J. comp. Neurol.. 146 (1972) 507 518. 10 Hamilton, H. L., l,illie~ Development of the Chick. An Introduction to Embr~'ologv, Holt, Rinehart and Winston, New York, 1952. I 1 Hughes, W. F. and LaVelle, A., On the synaptogenic sequence in the chick retina, Anat. Rec., 179 (1974) 297-302. 12 Kahn, A. J., Ganglion cell formation in the chick neural retina, Brain Res., 63 (1973) 285 290. 13 Kahn, A. J., An autoradiographic analysis of the time of appearance of neurons in the developing chick neural retina, Develop. BioL, 38 (1974) 30 40. 14 l,indeman, V. F., The cholinesterase and acetylcholine content of the chick retina, with special relevance to functional activity as indicated by the pupillary constrictor reflex. Amer. J. Physiol., 148 (1947) 40 44. 13 Morgan, 1. G., The organization of amacrine cell types which use different transmitters in chicken retina. In J. P.
Changeux, J. Glowinski, M. Imberhard, F. E. Bloom (Eds.], Molecular and Cellular Interactions Underlying Higher Brain Functions, Progress in Brain Research. Vol. 58, Elsevier, Amsterdam, 1983, in press. 16 Nishimura, Y., lnoue, Y. and Shimai, K., Morphological development of retinal ganglion cells in the chick embryo, Exp. Neurol., 64(1979)44 60. 17 Nishimura, Y. and Shimai, K., Determination of thc development of bipolar cells in embryonic chick retina, Neurosci. l,ett.. Suppl. 6 ( 1981 ) S 109. 18 Puro, D. G., Demello, F. G. and Nirenberg, M., Synapse turnover: the formation and termination of transient synapses, Proc. nat. Acad, Sci. U.S.A., 79 (1979) 4977 4981. 19 Rager. (.;., Morphogenesis and physiogenesis of the retino-tectal conection in the chicken. I. The rctinal ganglion cells and their axons, Proc. roy. Sot'. B, 192 (1976) 331 352. 20 Rager. G. and Rager. ()., Systems-matching by degeneration I. A quantitative electron microscopic study of the generation and degeneration of retinal ganglion cells in the chicken. Exp. Brain Res., 33 (1978) 65 78. 21 Ramirez, G., Cholinergic development in chick optic tectum and retina reaggregated cell cultures, Neurochem. Res., 2 (1977) 427-438. 22 Ramon y Cajal, S., La retine des vertebres, La Cellule, 9 (1893) 17- 27. 23 Rogers. K. T., Early development of the optic nerve in the chick, Anato Rec., 127 (1957) 97 107. 24 Shen, S.-C..Greenfield, P. and Boell, E. J., Localization of acetylcholinesterase in chick retina during histogenesis. J. comp. Neurol., 106 (1956) 433 46 I. 25 Stell, W., Marshak, D., Yamada, T., Brecha, N. and Karten, H., Peptides are in the eye of the beholder. Trends Neurosci., 3 (1980) 292 295. 26 Sugiyama, H., Daniels, M. P. and Nirenberg, M., Muscarinic acetylcholine receptors of the developing retina, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 5524-5528. 27 Vogel, Z. and Nirenberg, M., Localization ofacetylcholine receptors during synaptogenesis in retina. Proc. nat. Acad Sci. U.S.A.,73(1976) 1806 1810. 28 Wang, G. K. and Schmidt, J., Receptors for a-bungarotoxin in the developing visual system of the chick, Brain Res.. 114 (1976) 524-529. 29 Woolston, M. E., Hambley, J. W., Rose, S.P. R, and Morgan. I. G., Development of cholinergic transmitter systems in chicken retina, optic lobe and forebrain, Develop. Neurosci.. in preparation.
71
Elsevier Biomedical Press
The Development of Amacrine Cells Containing Somatostatin-like Immunoreactivity in Chicken Retina I. G. MORGAN, J. OLIVER and I. W. CHUBB
Department of Behavioral Biology, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra City, 2601, and Centrefor Neuroscience, (J. 0.) Departments of Medicine and (!. W.C. Human Physiology, Flinders University, Bedford Park, S.A. 5032,A ustralia (Accepted October 18th, 1982)
Key words retina - somatostatin - amacrine cells
Mature chicken retinas contain significant amounts of somatostatin-like immunoreactivity, which appear to be located in a prominent population of amacrine cells. By radio immunoassay, somatostatin-like immunoreactivity can first be detected around day 7 in ovo, and the first cells detectable by immunohistochemistry on around day 11 in ovo. The cells appear to mature in 3 phases, with a small but rapid increase in levels of somatostatin-like immunoreactivity from day 7 to day 11 in ovo, followed by a period of more gradual increase up to day 17 in ovo. The final phase consists of a rapid pre-hatch increase in levels ofsomatostatin-like immunoreactivity to adult levels by hatching. Immunoreactive cells are detectable from day I 1 in ovo, but immunoreactive processes in the inner plexiform layer are not visible until day 19 in ovo. The rapid increase in levels ofsomatostatin-like immunoreactivity, and the appearance of immunoreactive processes in the inner plexiform layer coincide temporally with the onset of light-driven activities in the retina. INTRODUCTION
The morphological heterogeneity of the amacrine and ganglion cell classes of the avian retina has been known for almost 100 years 22. Recent work has demonstrated that there is a corresponding heterogeneity of amacrine cell putative transmitters, among which are a number of neuropeptides (for reviews see refs. 3,15,25). In the mature retina there are specific sub-classes of amacrine cells, characterized by specific morphologies and putative transmitters, with specific patterns of connection with sub-classes of bipolar cells, ganglion cells and amacrine cells, organized in laminar arrays through the thickness of the retinatL This precise organization must be produced by subtle inter-cellular interactions during retinal histogenesis, as the pluripotent germinal cells of the invaginated neuroepithelium give rise to the 6 major neuronal cell classes and their many sub-classes by a sequential process of withdrawal from cell division, cell migration, and morphological and biochemical cell differentiation. 0165-3806/83/0000 0000/$03.00 "~'1983 Elsevier Science Publishers
While precise timing of these processes is complicated due to the central-peripheral gradient of retinal maturation, some of the basic features of the histogenesis of the chicken retina have been described. The majority of ganglion cells and amacrine cells are formed on days 3-5 in ovo 12.~3.Ganglion cell axons appear from days 2 to 3 in ovo onwards 9,19,23, and their dendrites begin to appear around days 7-8 in ovo, and become progressively more elaborate up to hatching j6. Around 40% of the ganglion cells die between days 13 and 15 in ovo 2°. One sub-class of amacrine cells, the displaced amacrine cells8, is formed in parallel with ganglion cell differentiation, but little is known about the differentiation of most amacrine cell morphological sub-classes. Bipolar cells are formed a little later, and apparently over a much longer period up to around day 13 in ovo iT. Their axonal processes mature roughly in parallel with those of ganglion cells ~7.24,and thus the inner plexiform layer appears first at around day 8 in ovo in the central retina, and reaches its full thickness by around day 16 in ovo 6. Conventional synapses
72 are first detectable in the inner plexiform layer on day 13 in ovo and bipolar synapses are first detectable on day 14 in ovo ~. Synapse formation then proceeds rapidly in the inner plexiform layer from day 14 to day 20 in ovo 7. Biochemical studies concern almost exclusively the cholinergic amacrine cell subclass. The development of acetylcholine k4. choline acetyltransferaseH~.-'~-'L acetylcholinesteraso-~j -'4-'~. high-affinity choline uptake ~and nicotinit~'7 "'~ and muscariniC ~'.-'9 acetylcholine receptors has been followed, and proceeds throughout the period of establishment of the inner plexiform layer. The development of retinal acetylcholinesterase has been followed histochemicallf 4. The adult pattern of distribution of enzyme activity is established by day 16 in ovo. In this study, we report a parallel biochemical and immunohistochemical study of the development of the most numerous neuropeptide-containing amacrine cells so far studied, those which contain somatostatin-like immunoreactivit~ .5. MATERIALS AND METHODS
Fertilized eggs of a White Leghorn/Black Australorp cross were obtained from Research Poultry Farm. Victoria, Australia and incubated at 38 "C. Embryos were removed from day 7 in ovo onwards, and staged according to Hamilton ~°, to verify that development was normal. For radioimmunoassay, the eyeballs were removed. The anterior half of the eye was removed, and the neural retina plus the pigment epithelium was carefully dissected. The retinas were homogenized in 2 M acetic acid. The homogenates were transferred to a plastic tube and boiled for 15 min in a boiling salt-water bath. After standing overnight at 4 °C, the tubes were centrifuged and the supernatants frozen until assayed. An aliquot (10/d) of the supernatant was added to an assay tube which, after freezedrying, was made up with 0.2 ml of assay buffer (0.05 M sodium phosphate, pH 7.4 containing 0.2 g% bovine serum albumin, 10 mM EDTA and 500 kallikrein inactivator units trasylol/ml). Radio-labeled somatostatin and antiserum (fi-
nal dilution 1/10,000) in assay buffer were added to the tube so as to bring the final incubation volume to 0.40 ml. A standard curve was constructed using synthetic somatostatin. The contents of the tubes were incubated for 16 h at 4 °C, after which time the hormone bound to antibody was separated from free hormone by precipitation with polyethylene glycol. The radioactivity of the bound fraction was determined by counting in a Nuclear Enterprises NENI600 gamma-counter. The sensitivity of the assay under these conditions was 3 pg ofsomatostatin. For somatostatin immunohistochemistry, the eyeballs were removed, hemisected, and the posterior portions fixed by immersion in 4% formaldehyde in 0.1 M phosphae buffer (pH 7.2) for 9 12 h at 4°C. The retinas were then removed, washed in 8% sucrose, 0.1 M phosphate buffer (pH 7.2) for at least 6 h, mounted in TissueTek II OCT compound (Miles Labs., Australia), and frozen in isopentane cooled with liquid nitrogen. Sections of 10/~m were cut from the central retina on a cryostat at - 1 5 °C. The frozen sections were mounted on chrom-alum-treated slides, dried in air for 15 min and washed in 10 mM PBS for 10 min. The sections were incubated overnight in anti-somatostatin antiserum diluted (1:75) with sheep serum:PBS (1:5) in a humid chamber at room temperature. After a brief wash in PBS, the sections were incubated for 60 min at room temperature in fluoroscein isothicyanate-sheep anti-rabbit immunoglobulin (Wellcome) diluted with sheep serum: PBS (1: 5). Following a final rinse in PBS the sections were mounted in glycerol: 0.5 M bicarbonate buffer (1: 1) (pH 8.6). Sections were examined under ultraviolet light using a Leitz Orthoplan microscope with a BG 12 excitation filter. Antiserum to somatostatin was produced in the rabbit by repeated subcutaneous, multiple site injections of synthetic somatostatin (Ayerst Laboratories, Montreal, Canada), conjugated to bovine thyroglobulin. The antiserum (2L-13), when tested against other peptides and hormones, demonstrated the following cross-reactivities: gastrin 1.5%; C-peptide 0.007%: and less than 0.001% for human/3-endorphin, angiotensins I and II, rat and human growth hormones,
73
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HATCHING AGE OF EMBRYO (DAYS) Fig. I. Development of wet weight, protein and levels ofsomatostatin-like immunoreactivity in chicken retina. Results are given as total activity per retina. E 7 - 21 refers to the number of days of incubation, H I - 7 to the number ofdays after hatching. A: mg protein/retina. B: mg wet weight/retina. C: ng somatostatin-like immunoreactivity/retina.
glucagon, thyrotropin-releasing factor, substance P, arginine- and lysine-vasopressin, eledoisin, bradykinin, adrenocorticotropin and insulin. RESULTS
As shown in Fig. 1, somatostatin-like immunoreactivity was first detectable by radioimmunoassay in the chicken retina by day 7 in ova, and increased significantly in concentration between days 9 and 11 in ova. The rate of increase then diminished from day 11 in ova, until days 17-18 in ova. Around days 18-19 in ova there was a sudden increase in the rate of accumulation of somatostatin-like immunoreactivity and adult levels were attained around hatching. By contrast, retinal protein content and wet weight
increased rapidly from day 7 until day 13 in ova, then the rate of increase diminished until hatching. Thus the specific activity of somatostatinlike immunoreactivity decreased up to day 13, then increased rapidly prior to hatching. Immunohistochemically (Fig. 2), somatostatin-positive cell bodies were first detectable around day 11 in ova, and were already displaced from the border between the nascent inner nuclear and inner plexiform layers, in a position which roughly corresponds to that of the somatostatin-positive amacrine cells of the adult retina. By day 13 in ova, prominent descending dendrites could also be seen, but no clear dendritic arborizations could be detected in the inner plexiform layer. This picture remained essentially the same for days 15 and 17 in ova, al-
74
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IF
ii
Fig. 2. Development of somatostatin-like immunoreactivity in chick retina as revealed by immunohistochemistry, a: day I I in ovo. b: day 13 in ovo. c: day 15 in ovo. d" day 17 in ovo. c: day 18 in ovo. f: one day after hatching.
75 though the fluorescence of the somatostatinpositive cell bodies increased relative to the background. On day 17 in ovo the first signs of punctate fluorescence were seen in the inner plexiform layer, and these had increased markedly by day 19 in ovo. By one day after hatching, the characteristic laminar pattern of the dendritic arborizations had become clear, and the cell bodies had become so fluorescent that the perinuclear staining visible at earlier times was obscured. DISCUSSION
These results indicate that the levels of somatostatin-like immunoreactivity, and the cells which contain it, do not develop simply in parallel with the gross development of the neural retina. Rather, there appear to be 3 phases of development of the somatostatin-positive system. From days 7 to 9 in ovo, there is a small, but rapid, increase in levels of somatostatin-like immunoreactivity, at around the time that inner plexiform layer begins to appear in the central retina. By day 11 in ovo, immunoreactive cell bodies are visible in the inner nuclear layer, and there is a gradual increase in levels of somatostatin-like immunoreactivity over the period during which ganglion cells, displaced amacrine cells, bipolar cells, and presumably amacrine cells elaborate their dendrites. This period is then followed from days 18 to 19 in ovo by a rapid increase in the levels of somatostatin-like immunoreactivity which reach adult levels by hatching. This rapid increase in the levels ofsomatostatin-like immunoreactivity roughly coincides with the final development of the photoreceptor outer segments, and the beginnings of light-driven electrical activity in the retina s . Thus the pattern of development of somatostatin-like immunoreactivity and the cells which contain it is consistent with the localization of the somatostatin-like immunoreactivity in amacrine cells. The appearance of somatostatinlike immunoreactivity parallels the way in which choline acetyltransferase activity ~.29develops, in that there is an accumulation of activity around from day 7 to day 13 in ovo, followed by a lag
period, then a rapid pre-hatch increase in activity. Other activities characteristic of the cholinergic system, namely high-affinity choline uptake I, nicotinic and muscarinic acetylcholine receptors, and acetylcholinesterase develop with different patterns 29. Thus, although there is a parallel between the development of some amacrine cell presynaptic components, such as somatostatin-like immunoreactivity and choline acetyltransferase K,29and in less detailed studies, acetylcholine14, other presynaptic components such as high-affinity choline uptake ~and GABA (unpublished results) have distinct developmental profiles, as does glycine (unpublished resuits), which is likely to be located in part in amacrine cell synapses. Little somatostatin-like immunoreactivity is detected in the inner plexiform layer until just prior to hatching. This contrasts with the early appearance of two bands of acetylcholinesterase activity in the inner plexiform layer from day 8 in ovo, with the adult 4 bands being distinctly visible by day 16 in ovo24. The lack of immunoreactivity in the inner plexiform layer is unlikely to be due to late morphological maturation of the dendritic arborizations of that amacrine cell sub-class. It is more likely to be related to low rates of synthesis of whatever compounds are responsible for the somatostatin-like immunoreactivity detected by radioimmunoassay and immunohistochemistry. It is not clear whether the rapid pre-hatch increase in levels of somatostatin-like immunoreactivity is triggered by the functional maturation of the retina 2, or whether it is simply a contemporaneous event. The former possibiity is supported by the observation that there are marked diurnal variations in levels of somatostatin-like immunoreactivity in the retinas of chickens held on 12:12 light-dark cycles, with maximum values attained at the end of the light phase twice those at the end of the dark phase (unpublished results). In fact, it is possible that the rapid pre-hatch increase is just the first occurrence of a light-entrained accumulation of somatostatin-like immunoreactivity, which then occurs every day under normal circumstances.
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