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The release of Leu5-enkephalin-like immunoreactivity from chicken retina is reduced by light in vitro
Brain Research, 488 (1989) 43-48 Elsevier
43
BRE 14498
The release of LeuS-enkephalin-like immunoreactivity from chicken retina is reduced by light in vitro Meeuwis K. Boelen, Mark Dowton and Ian W. Chubb The Neuroscience Unit, Department of Biology, Universityof Wollongong, Wollongong, N.S. W. (Australia) (Accepted 1 November 1988)
Key words: Enkephalin; Release; Retina; Chick; Amacrine cell
A superfusion system was established to examine the efflux of endogenous LeuS-enkephalin-like immunoreactivity (LE-LI) from isolated chicken retinas. Superfusion with buffer containing high concentrations of K ÷ (60 mM KCI) increased the efflux of LE-LI by 96%. This effect was not observed when Co2÷ (4 mM CoCi2) was present. Exposing the retinas to light decreased the efflux of LE-LI by 59% compared to that observed during superfusion in the dark. No effect of ambient light could be detected in the presence of Co2÷. Upon reverse-phase high-performance liquid chromatography the material released by the retina comigrated with synthetic LeuS-enkephalin. These results demonstrate that the release of LE-LI from retinal neurons is increased during the dark, and it is concluded that the lighting conditions exert their effects by modifying the state of polarization of the LE-LI amacrine cells and hence the release of LE-LI from these neurons. INTRODUCTION There is strong evidence that an opiate-mediated neurotransmitter system is present in vertebrate retina (for reviews see refs. 3 and 29). This has been examined extensively in avian retina, which is rich in assayable enkephalin-like immunoreactivity 29. Immunohistochemical studies have revealed that the enkephalin-like immunoreactivity is located in amacrine cells 4. Biochemical studies have demonstrated the synthesis of (radiolabelled) MetS-enkephalin in the retina of goldfish and chicken, and that this newly synthesized enkephalin can be released by a high-K + stimulus in vitro 25'27. In addition, opiate receptors 2°'24 have been demonstrated in the retina of several species. However, little is known about the participation of such an enkephalinergic transmitter system in visual processing. Enkephalin has been shown to decrease the high-K + evoked release of [3H]7-aminobutyric acid ( G A B A ) 28 and [3H]dopamine26 in avian retina and to decrease the electrically induced release of [3H]dopamine in rabbit retina 7, and opiate-affected ganglion cell
responses have been demonstrated in goldfish 6 and amphibia 5. In chicken, the retinal levels of L E - L I vary during the light/dark cycle, and it has been suggested that the decrease of L E - L I during the dark may indicate an increase in the release and subsequent breakdown of this neurotransmitter candidate during the dark 17. To determine whether ambient light modulates the release of L E - L I we have established a technique for superfusing the chicken retina in vitro and for measuring its release of L E - L I by radioimmunoassay (RIA). Here we present direct evidence that the efflux of L E - L I from superfused chicken retina is inhibited by exposure to light. Parts of these results have been published in preliminary form 1'2. MATERIALS AND METHODS
Materials The antiserum to LeuS-enkephalin was obtained from Dr. Oliver, Flinders University, Australia. The cross-reactivity of the antibody with MetS-enkeph alin was less than 1%, and less than 0.1% with
Correspondence: M.K. Boelen, The Neuroscience Unit, Department of Biology, Bendigo C.A.E., P.O. Box 199, Bendigo, Vic. 3550, Australia. 0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
44 LeuS-enkephalin-Arg 6 and LeuS-enkephalin-Lys 6. It did not cross-react with [des-Leu]-LeuS-enkephalin. Bacitracin and peptides were from Sigma.
Tissue preparation Two-day-old chickens (White Leghorn x Black Australorp, Gallus Domesticus) were obtained from a local hatchery and kept on a 12-h light/dark cycle with ample access to food and water. Chickens at least 6 weeks old were used for all experiments. All retinas were obtained from chickens previously exposed to 12 h light, as at this time LE-LI levels are highest 17. Chickens were killed with ether, the eyes removed and the retinas (with minimal amounts of pigment epithelium and without choroid) isolated and immersed in physiological buffer containing 118.1 mM NaC1, 4.7 mM KCI, 1.2 mM MgCI2, 24.9 mM NaHCO3, 10 mM CaCI2, 10 mM glucose and 100/~g/liter bacitracin. The buffer was bubbled with carbogen (95% Oa and 5% CO2) for at least 30 min before use and throughout the experiment. The pH of the buffer was adjusted to 7.3 with 1 M HCI.
Superfusion The retinas were layered onto polysuifone filters (Gellman Sciences, GA6-S) and transferred to superfusion chambers (Whatman Millex filter holders). The total volume of a superfusion chamber including tubing was 3.0 ml. Within 30 min of killing, retinas were superfused with physiological buffer at a flow rate of 0.60 ml/min and 3.0-ml fractions collected. The physiological buffer was modified in some instances by increasing the concentration of KCI to 60 mM (replacing an equimolar amount of NaCI) or by adding 4.0 mM CoCI 2. For dark/light modified release, retinas were isolated under red dim illumination (Phillips, PF712B; less than 0.5 lux) and the superfusion chambers wrapped tightly in aluminium foil. To expose the retinas to the light, the chambers were unwrapped and illuminated by diffuse light (about 5500 lux) from an overhead projector. To minimize heat from the light source, light was passed through 4 cm of water at ambient temperature. Superfusate fractions were collected into 500/~1 of 14 M acetic acid in ice-cold polypropylene tubes. At the end of the experiment the fractions were boiled for 10 min. The superfusate fractions were stored at
-80 °C before measurement of LE-L1 by RIA. The amount of released LE-LI was normalized by expressing the amount in each fraction as a percentage of the total amount of LE-LI recovered from the first 10 superfusate fractions. The R I A was carried out as described elsewhere 17. For standards, synthetic LeuS-enkephalin was added to 3 ml of physiological buffer, mixed with 500 #1 of 14 M acetic acid and boiled for 10 min.
Identification of released LeuS-enkephalin-like immunoreactivity by high-performance liquid chromatography LE-LI was extracted from 10 pooled superfusate fractions with acidified acetone 27 or, in some cases, loaded onto Sep-Pak C18-cartridges (Waters). Extracts and material eluted from the cartridges with 80% acetonitrile in 0.1% triftuoroacetic acid, were analyzed by reverse-phase (aquapore RP300 column) HPLC using 0.1% trifluoroacetic acid (solvent A) and 60% acetonitrile in 0.1% trifluoroacetic acid (solvent B). The material was eluted at a flow rate of 1 ml/min (40 °C) with a gradient of 0-22% solvent B over 18 min and then isocratically at 22% solvent B for up to 30 rain. One- to 4-ml fractions were collected, rotary evaporated until dry (Savant SpeedVac Concentrator), and LE-LI measured by R I A as described previously w. RESULTS
High-K+-evoked release of LeuS-enkephalin-like immunoreactivity A small basal release of LE-LI (corresponding to 0.12% of tissue content per min) from the retina was detected during the initial 15 min of superfusion (Fig. 1). Upon depolarization by 60 mM K ÷, the effiux of LE-LI from the retinas approximately doubled. On return to normal levels of K + (4.7 mM KC1), the effiux decreased to basal levels. A second depolarization with 60 mM KCI elicited a smaller increase in the effiux of LE-LI. In the presence of 4 mM COC12, the basal effiux of LE-LI decreased to below the detection limit (<2 pg LE-LI per min), and the addition of high-K + did not increase the effiux of LE-LI (Fig. 1). After the Co 2+ had been removed, superfusion with buffer containing 60 mM K + elicited an increase in the effiux of LE-LI from
45 60
all retinas, indicating that all were able to respond to depolarizing conditions. W h e n the peptidase inhibitor bacitracin was omitted from the superfusing m e d i u m , there was n o
30
detectable L E - L I in the superfusates. E v e n in the
er
presence of bacitracin, the recovery of L E - L I in the superfusates accounted for only 3 6 - 7 2 % of the loss of LE-LI in the tissue during the experiment. It has 0
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Time (rain) Fig. 1. High-K + evoked release of LeuS-enkephalin-like immunoreactivity from chicken retina. The retinas were superfused with either normal (4.7 mM KCI) or K+-rich (60 mM KCI) buffer in vitro, 3.0-ml fractions collected and assayed for LE-LI by RIA. In some cases the released immunoreactivity was analyzed by reverse-phase HPLC and comigrated with synthetic LeuLenkephalin (see text). The amount of released LE-LI was normalized by expressing the amount in each fraction as a percentage of the total amount of LE-LI recovered from the first 10 superfusate fractions; one unit corresponds to one pg LE-LI. II, Retinas superfused in the absence of Co2+ (n = 12). I-q, Retinas superfused in the presence of 4 mM CoC12 during the first 30 min (n = 11). Vertical bars indicate S.E.M.s. LE-LI was released in response to high extracellular K÷ concentration and this release was blocked by the presence of Co2+ in the medium.
its intracellular storage sites, iting degradation after the leased ~3. W h e n exogenous superfused over the retina,
but is crucial in inhibpeptide has b e e n reLeuS-enkephalin was the recovery was less
than 50%. Although bacitracin inhibited this degradation completely (results not shown), it is possible that bacitracin does not completely protect enkephalin at the site of release 22. Therefore, the actual amounts of released L E - L I could be considerably higher than those recovered in the superfusates 16.
Light modulated release of LeuS-enkephalin-like immunoreactivity The effect of changes in a m b i e n t lighting on the spontaneous efflux of L E - L I from the isolated
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Fig. 2. Light modulated release of LeuLenkephalin-like immunoreactivity from chicken retina. A: retinas were superfused in darkness for 25 min by keeping the superfusion chambers wrapped in aluminium foil. Then, the superfusion chambers were unwrapped and exposed to diffuse light from an overhead projector for 25 min (n = 11), and, in some cases (n = 5) wrapped in foil again and superfused for another 25 min. At the end of the experiment all retinas were superfused with buffer containing 60 mM KCI. Three ml fractions were collected and assayed for LE-LI by RIA. One unit corresponds to one pg LE-LI. S.E.M.s are indicated by vertical bars. B: as for A, except that the retinas were superfused in darkness during the entire experiment (n = 5). C: as for A, except that 4 mM CoCI2 was present in the superfusing medium during the first 50 rain of superfusion (n = 4). A relatively high basal release of LE-LI during darkness was observed, which was depressed by light. This effect was greatly reduced by the presence of Co2+ in the medium.
46 retinas was measured. The basal effiux of LE-L1 decreased only gradually during superfusion in the dark (Fig. 2B). The total effiux of LE-LI during 25 min in the light was reduced by 59% when compared to the efflux during the previous 25 min in the dark (Fig. 2A). On returning to the dark, the efflux increased to initial rates. Light had no detectable effect on the spontaneous release when 4 mM CoC12 was present (Fig. 2C). However, as the baseline effiux during exposure to Co 2÷ was beyond the detection limit of the RIA, small effects cannot be excluded. All retinas responded to depolarizing concentrations of K ÷ after removal of the CoCI 2.
Identification of released immunoreactivity The nature of the immunoreactivity present in the superfusate fractions was examined after extraction with acidified acetone followed by reversed-phase HPLC (results not shown). Two peaks of immunoreactivity were detected. The first peak had the same retention time as synthetic LeuS-enkephalin and was increased in fractions obtained by superfusing retinas with high-K +. The second peak of immunoreactivity was present in acetone-extracts of fresh superfusion buffer and therefore not of retinal origin. Only one peak of immunoreactivity was detected in HPLC-profiles of material retained on Sep-Pak Cls-cartridges. This peak had the same retention time as synthetic LeuS-enkephalin; the increase in immunoassayable material in fractions from retinas which had been superfused in the dark or with buffer containing high-K + was reflected by an increase in the immunoreactive material eluting from the column with this retention time. It is concluded that the increase in LE-LI in the superfusates is due to increased amounts of LeuS-enkeph alin. DISCUSSION The results reported here show that recoverable LE-LI in the superfusate of the retina was elevated in the dark and reduced in the light. The effiux of LE-LI, whether spontaneous or induced either by light/dark or by elevated potassium, was blocked if Co 2÷ was present in the superfusing medium. Since in avian retina, LE-LI has only been observed in amacrine cells (including their processes
in the inner plexiform layer) 4 (for reviews see refs. 3 and 29), it is reasonable to conclude that the immunoreactive material released from the retina in the present study originates from these cells or their processes. As the LE-LI had the same retention time as synthetic LeuS-enkephalin upon reverse-phase HPLC, this demonstrates that the retina releases true LeuS-enkephalin. In addition, as Co 2+ blocked release, it is also likely that the mechanism for release of LeuS-enkephalin is C a 2+ dependent and may thus involve release from intracellular organelles. It has been demonstrated in goldfish and chicken retina that newly synthesized radiolabelled Met 5enkephalin can be released in vitro by exposure to depolarizing concentrations of K + (refs. 25, 27). Apart from the demonstration of high-K* evoked release of endogenous LeuS-enkephalin-like material from the retina, the results presented here clearly demonstrate that this release can be modulated by a more physiological stimulus. Based on extracellular recordings of ganglion cell activity in goldfish retina, it was noted that tight-off rather than light-on may trigger the release of endogenous opiates 6. Our data is consistent with this notion, but suggests that there is a sustained shift in input balance during the dark towards net excitation of the LE-LI amacrine cell. The results of the present study provide a plausible explanation for the observed depletion of retinal levels of LE-LI during the dark 17. Histological studies in the chicken retina revealed arborizations of LE-LI processes in both distal (sublamina 1) and proximal (sublaminas 3 and 5) regions of the inner plexiform layer 4,9A1A7,3°. Although still controversial in published literature 11,30, latest observations seem to agree that the LE-LI amacrine cells receive direct input from bipolar cells (Millar, personal communications). Since the distal and proximal regions of vertebrate retina are generally associated with off-center and on-center information respectively 19, the LE-LI amacrine cells could receive both off- and on-center input. Our data suggests that there is a sustained shift in input balance towards net inhibition of the LE-LI amacrine cell during the light. Preliminary findings indicate an involvement of glycinergic, but not GABAergic transmission in this light-modulated
47 shift in input TM. R e t i n a l levels of various o t h e r biologically active c o m p o u n d s have been shown to be affected by light 1°A2,zl"z3, and the effects of light on the release of [3H]dopamine 14, [3H]glycine8 and [3H]acetylcholine 15 have been studied. In these studies, m a x i m u m release was o b t a i n e d by stimulation with flickering light. L E - L I is the only neuroactive comp o u n d which has b e e n shown to exhibit a sustained release r e l a t e d to a m b i e n t lighting. In conclusion, it was shown that the chicken retina releases m o r e L E - L I during the d a r k than when the r e t i n a is e x p o s e d to light. This further supports the hypothesis that the L E - L I amacrine cells are active during the dark. Retinal L E - L I was shown to be
r e l e a s e d u p o n d e p o l a r i z a t i o n b y e x p o s u r e to high concentrations of potassium. Thus, the variations in the l i g h t - d a r k conditions m a y m o d i f y the state of depolarization of the L E - L I a m a c r i n e cells and hence affect the release of L E - L I . ACKNOWLEDGEMENTS We thank Dr. John Oliver for the supply of antibodies to LeuS-enkephalin, R i c h a r d Smith, Irene B a c k e n and M a r d i Silburn for technical assistance, and Dr. Ian M o r g a n for valuable discussions. The work was s u p p o r t e d by a grant from the National H e a l t h and Medical R e s e a r c h F o u n d a t i o n of Australia.
REFERENCES 1 Boelen, M.K., Dowton, M., Smith, R. and Chubb, I., Comparison of the effects of light on the release of leu-enkephalin-like immunoreactivity and 3H-dopamine from chicken retina in vitro, Neurosci. Lett., Suppl. 30 (1988) $49. 2 Boelen, M.K., Smith, R., Dowton, M. and Chubb, I., Increased release of leu-enkephalin-like immunoreactivity during the dark from chicken retinas in vitro, IUPHAR Satellite Symposium -- Tools for Tachykinin and Neuropeptide Research, Salamander Bay, August 29-30, 1987, (Proceedings). 3 Brecha, N.C., Eldred, W., Kuljis, R.O. and Karten, H.J., Identification and localization of biologically active peptides in the vertebrate retina. In N.N. Osborne and G.J. Chader (Eds.), Progress in Retinal Research, Vol. 3, Pergamon, New York, 1984, pp. 185-226. 4 Brecha, N., Karten, H.J. and Laverack, C., Enkephalincontaining amacrine cells in the avian retina: immunohistochemical localization, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 3010-3014. 5 Dick, E. and Miller, R.E, Peptides influence retinal ganglion cells, Neurosci. Lett., 26 (1981) 131-135. 6 Djamgoz, M.B.A., SteU, W.K., Chin, C.-A. and Lam, D.M.K., An opiate system in the goldfish retina, Nature (Lond.), 292 (1981) 620-623. 7 Dubocovich, M.L. and Weiner, N., Enkephalins modulate [3H]dopamine release from rabbit retina in vitro, J. Pharmacol. Exp. Ther., 224 (1983) 634-639. 8 Ehinger, B. and Lindberg-Bauer, B., Light-evoked release of glycine from cat and rabbit retina, Brain Research, 113 (1976) 535-549. 9 Fukuda, M., Localization of neuropeptides in the avian retina: an immunohistochemical analysis, Cell. Mol. Biol., 28 (1982) 275-283. 10 Ishimoto, I., Millar, T., Chubb, I.W. and Morgan, I.G., Somatostatin-immunoreactive amacrine cells of chicken retina: retinal mosaic, ultrastructural features, and lightdriven variations in peptide metabolism, Neuroscience, 17 (1986) 1217-1233. 11 Ishimoto, I., Shiosaka, S., Shimizu, Y., Kuwayama, Y.,
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13 14
15
16 17
18
19
20 21 22
Fukuda, M., Inagaki, S., Takagi, H., Sakanaka, M., Sasaoka, A., Senba, E., Sakiyama, T. and Tohyama, M., Leucine-enkephalin-like immunoreactivity in the chicken retina with a special reference to its fine structures, Invest. Ophthalmol. Vis. Sci., 24 (1983) 879-885. Iuvone, P.M., Galli, C.L., Garrison-Gund, C.K. and Neff, N.H., Light stimulates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine neurons, Science, 202 (1978) 901-902. Iversen, L.L., Iversen, S.D., Bloom, EE., Vargo, T. and Guillemin, R., Release of enkephalin from rat globus pallidus in vitro, Nature (Lond.), 271 (1978) 679-681. Kramer, S.G., Dopamine: a retinal neurotransmitter. I. Retinal uptake, storage, and light-stimulated release of 3H-dopamine in vivo, Invest. Ophthalmol., 10 (1971) 438-452. Masland, R.H. and Livingstone, C.J., Effect of stimulation with light on synthesis and release of acetylcholine by an isolated mammalian retina, J. NeurophysioL, 39 (1976) 1210-1219. Meyer, D.K. and Feuerstein, T., The problem of recoveries in peptide release experiments, Trends Pharmacol. Sci., 5 (1985) 220. Millar, T.J., Salipan, N., Oliver, J.O., Morgan, I.G. and Chubb, I.W., The concentration of enkephalin-like material in the chick retina is light dependent, Neuroscience, 13 (1984) 221-226. Morgan, I.G., Millar, T.J., Ishimoto, I., Boelen, M.K., Dowton, M. and Chubb, I.W., Peptides in the retina. In R. Weiler and N. Osborne (Eds.), The Neurobiology of the Inner Retina, in press. Nelson, R., Famiglietti, J.R. and Kolb, H., Intracellular staining reveals different levels of stratification for on- and off-center ganglion cells in cat retina, J. Neurophysiol., 41 (1978) 472-483. Osborne, H. and Herz, A., Opioid binding properties in bovine retina, Neurochem. Int., 3 (1981) 143-148. Parkinson, D. and Rando, R.R., Effects of light on dopamine metabolism in the chick retina, J. Neurochem., 40 (1983) 39-46. Patey, G., de la Baume, S. and Schwartz, J.-C., Selective protection of methionine enkephalin released from brain
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24 25 26
slices by enkephalinase inhibition, Science, 212 (1981) 1153-1155. Schaeffer, J.H., Brownstein, M.J. and Axelrod, J., Thyrotropin-releasing hormone-like material in the rat retina: changes during environmental lighting, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 3579-3581. Slaughter, M.M., Mattler, J.A. and Gottlieb, D.I., Opiate binding sites in the chick, rabbit and goldfish retina, Brain Research, 339 (1985) 39-47. Su, Y.-Y.T., Fry, K.R., Lam, D.M.-K. and Watt, C.B., Enkephalin in the goldfish retina, Cell. Mol. Neurobiol., 6 (1986) 331-347. Su, Y.-Y.T. and Watt, C.B., Interaction between enkephalin and dopamine in the avian retina, Brain Research, 423 (1987) 63-70.
27 Su, Y.-Y.T., Watt, C.B. and Lam, D.M.-K.. Opioid pathways in an avian retina. I. The content biosynthesis, and release of MetS-enkephalin, J. Neurosei., 5 (1985) 851-856. 28 Watt, C.B., Su, Y.-Y.T. and Lain, D.M.-K., Interactions between enkephalin and GABA in avian retina, Nature (Lond.), 311 (1984) 761-763. 29 Watt, C.B., Su, Y.-Y.T. and Lam, D.M.-K., Enkephalins in the vertebrate retina. In N.N. Osborne and GA. Chader (Eds.), Progress in Retinal Research, Vol. 4, Pergamon, New York, 1985, pp. 221-246. 30 Watt, C.B., Su, Y.-Y.T. and Lam, D.M.-K., Opioid pathways in an avian retina. II. Synaptic organization of enkephalin-immunoreactive amacrine cells, J. Neurosci., 5 (1985) 857-865.
43
BRE 14498
The release of LeuS-enkephalin-like immunoreactivity from chicken retina is reduced by light in vitro Meeuwis K. Boelen, Mark Dowton and Ian W. Chubb The Neuroscience Unit, Department of Biology, Universityof Wollongong, Wollongong, N.S. W. (Australia) (Accepted 1 November 1988)
Key words: Enkephalin; Release; Retina; Chick; Amacrine cell
A superfusion system was established to examine the efflux of endogenous LeuS-enkephalin-like immunoreactivity (LE-LI) from isolated chicken retinas. Superfusion with buffer containing high concentrations of K ÷ (60 mM KCI) increased the efflux of LE-LI by 96%. This effect was not observed when Co2÷ (4 mM CoCi2) was present. Exposing the retinas to light decreased the efflux of LE-LI by 59% compared to that observed during superfusion in the dark. No effect of ambient light could be detected in the presence of Co2÷. Upon reverse-phase high-performance liquid chromatography the material released by the retina comigrated with synthetic LeuS-enkephalin. These results demonstrate that the release of LE-LI from retinal neurons is increased during the dark, and it is concluded that the lighting conditions exert their effects by modifying the state of polarization of the LE-LI amacrine cells and hence the release of LE-LI from these neurons. INTRODUCTION There is strong evidence that an opiate-mediated neurotransmitter system is present in vertebrate retina (for reviews see refs. 3 and 29). This has been examined extensively in avian retina, which is rich in assayable enkephalin-like immunoreactivity 29. Immunohistochemical studies have revealed that the enkephalin-like immunoreactivity is located in amacrine cells 4. Biochemical studies have demonstrated the synthesis of (radiolabelled) MetS-enkephalin in the retina of goldfish and chicken, and that this newly synthesized enkephalin can be released by a high-K + stimulus in vitro 25'27. In addition, opiate receptors 2°'24 have been demonstrated in the retina of several species. However, little is known about the participation of such an enkephalinergic transmitter system in visual processing. Enkephalin has been shown to decrease the high-K + evoked release of [3H]7-aminobutyric acid ( G A B A ) 28 and [3H]dopamine26 in avian retina and to decrease the electrically induced release of [3H]dopamine in rabbit retina 7, and opiate-affected ganglion cell
responses have been demonstrated in goldfish 6 and amphibia 5. In chicken, the retinal levels of L E - L I vary during the light/dark cycle, and it has been suggested that the decrease of L E - L I during the dark may indicate an increase in the release and subsequent breakdown of this neurotransmitter candidate during the dark 17. To determine whether ambient light modulates the release of L E - L I we have established a technique for superfusing the chicken retina in vitro and for measuring its release of L E - L I by radioimmunoassay (RIA). Here we present direct evidence that the efflux of L E - L I from superfused chicken retina is inhibited by exposure to light. Parts of these results have been published in preliminary form 1'2. MATERIALS AND METHODS
Materials The antiserum to LeuS-enkephalin was obtained from Dr. Oliver, Flinders University, Australia. The cross-reactivity of the antibody with MetS-enkeph alin was less than 1%, and less than 0.1% with
Correspondence: M.K. Boelen, The Neuroscience Unit, Department of Biology, Bendigo C.A.E., P.O. Box 199, Bendigo, Vic. 3550, Australia. 0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
44 LeuS-enkephalin-Arg 6 and LeuS-enkephalin-Lys 6. It did not cross-react with [des-Leu]-LeuS-enkephalin. Bacitracin and peptides were from Sigma.
Tissue preparation Two-day-old chickens (White Leghorn x Black Australorp, Gallus Domesticus) were obtained from a local hatchery and kept on a 12-h light/dark cycle with ample access to food and water. Chickens at least 6 weeks old were used for all experiments. All retinas were obtained from chickens previously exposed to 12 h light, as at this time LE-LI levels are highest 17. Chickens were killed with ether, the eyes removed and the retinas (with minimal amounts of pigment epithelium and without choroid) isolated and immersed in physiological buffer containing 118.1 mM NaC1, 4.7 mM KCI, 1.2 mM MgCI2, 24.9 mM NaHCO3, 10 mM CaCI2, 10 mM glucose and 100/~g/liter bacitracin. The buffer was bubbled with carbogen (95% Oa and 5% CO2) for at least 30 min before use and throughout the experiment. The pH of the buffer was adjusted to 7.3 with 1 M HCI.
Superfusion The retinas were layered onto polysuifone filters (Gellman Sciences, GA6-S) and transferred to superfusion chambers (Whatman Millex filter holders). The total volume of a superfusion chamber including tubing was 3.0 ml. Within 30 min of killing, retinas were superfused with physiological buffer at a flow rate of 0.60 ml/min and 3.0-ml fractions collected. The physiological buffer was modified in some instances by increasing the concentration of KCI to 60 mM (replacing an equimolar amount of NaCI) or by adding 4.0 mM CoCI 2. For dark/light modified release, retinas were isolated under red dim illumination (Phillips, PF712B; less than 0.5 lux) and the superfusion chambers wrapped tightly in aluminium foil. To expose the retinas to the light, the chambers were unwrapped and illuminated by diffuse light (about 5500 lux) from an overhead projector. To minimize heat from the light source, light was passed through 4 cm of water at ambient temperature. Superfusate fractions were collected into 500/~1 of 14 M acetic acid in ice-cold polypropylene tubes. At the end of the experiment the fractions were boiled for 10 min. The superfusate fractions were stored at
-80 °C before measurement of LE-L1 by RIA. The amount of released LE-LI was normalized by expressing the amount in each fraction as a percentage of the total amount of LE-LI recovered from the first 10 superfusate fractions. The R I A was carried out as described elsewhere 17. For standards, synthetic LeuS-enkephalin was added to 3 ml of physiological buffer, mixed with 500 #1 of 14 M acetic acid and boiled for 10 min.
Identification of released LeuS-enkephalin-like immunoreactivity by high-performance liquid chromatography LE-LI was extracted from 10 pooled superfusate fractions with acidified acetone 27 or, in some cases, loaded onto Sep-Pak C18-cartridges (Waters). Extracts and material eluted from the cartridges with 80% acetonitrile in 0.1% triftuoroacetic acid, were analyzed by reverse-phase (aquapore RP300 column) HPLC using 0.1% trifluoroacetic acid (solvent A) and 60% acetonitrile in 0.1% trifluoroacetic acid (solvent B). The material was eluted at a flow rate of 1 ml/min (40 °C) with a gradient of 0-22% solvent B over 18 min and then isocratically at 22% solvent B for up to 30 rain. One- to 4-ml fractions were collected, rotary evaporated until dry (Savant SpeedVac Concentrator), and LE-LI measured by R I A as described previously w. RESULTS
High-K+-evoked release of LeuS-enkephalin-like immunoreactivity A small basal release of LE-LI (corresponding to 0.12% of tissue content per min) from the retina was detected during the initial 15 min of superfusion (Fig. 1). Upon depolarization by 60 mM K ÷, the effiux of LE-LI from the retinas approximately doubled. On return to normal levels of K + (4.7 mM KC1), the effiux decreased to basal levels. A second depolarization with 60 mM KCI elicited a smaller increase in the effiux of LE-LI. In the presence of 4 mM COC12, the basal effiux of LE-LI decreased to below the detection limit (<2 pg LE-LI per min), and the addition of high-K + did not increase the effiux of LE-LI (Fig. 1). After the Co 2+ had been removed, superfusion with buffer containing 60 mM K + elicited an increase in the effiux of LE-LI from
45 60
all retinas, indicating that all were able to respond to depolarizing conditions. W h e n the peptidase inhibitor bacitracin was omitted from the superfusing m e d i u m , there was n o
30
detectable L E - L I in the superfusates. E v e n in the
er
presence of bacitracin, the recovery of L E - L I in the superfusates accounted for only 3 6 - 7 2 % of the loss of LE-LI in the tissue during the experiment. It has 0
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Time (rain) Fig. 1. High-K + evoked release of LeuS-enkephalin-like immunoreactivity from chicken retina. The retinas were superfused with either normal (4.7 mM KCI) or K+-rich (60 mM KCI) buffer in vitro, 3.0-ml fractions collected and assayed for LE-LI by RIA. In some cases the released immunoreactivity was analyzed by reverse-phase HPLC and comigrated with synthetic LeuLenkephalin (see text). The amount of released LE-LI was normalized by expressing the amount in each fraction as a percentage of the total amount of LE-LI recovered from the first 10 superfusate fractions; one unit corresponds to one pg LE-LI. II, Retinas superfused in the absence of Co2+ (n = 12). I-q, Retinas superfused in the presence of 4 mM CoC12 during the first 30 min (n = 11). Vertical bars indicate S.E.M.s. LE-LI was released in response to high extracellular K÷ concentration and this release was blocked by the presence of Co2+ in the medium.
its intracellular storage sites, iting degradation after the leased ~3. W h e n exogenous superfused over the retina,
but is crucial in inhibpeptide has b e e n reLeuS-enkephalin was the recovery was less
than 50%. Although bacitracin inhibited this degradation completely (results not shown), it is possible that bacitracin does not completely protect enkephalin at the site of release 22. Therefore, the actual amounts of released L E - L I could be considerably higher than those recovered in the superfusates 16.
Light modulated release of LeuS-enkephalin-like immunoreactivity The effect of changes in a m b i e n t lighting on the spontaneous efflux of L E - L I from the isolated
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B
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Fig. 2. Light modulated release of LeuLenkephalin-like immunoreactivity from chicken retina. A: retinas were superfused in darkness for 25 min by keeping the superfusion chambers wrapped in aluminium foil. Then, the superfusion chambers were unwrapped and exposed to diffuse light from an overhead projector for 25 min (n = 11), and, in some cases (n = 5) wrapped in foil again and superfused for another 25 min. At the end of the experiment all retinas were superfused with buffer containing 60 mM KCI. Three ml fractions were collected and assayed for LE-LI by RIA. One unit corresponds to one pg LE-LI. S.E.M.s are indicated by vertical bars. B: as for A, except that the retinas were superfused in darkness during the entire experiment (n = 5). C: as for A, except that 4 mM CoCI2 was present in the superfusing medium during the first 50 rain of superfusion (n = 4). A relatively high basal release of LE-LI during darkness was observed, which was depressed by light. This effect was greatly reduced by the presence of Co2+ in the medium.
46 retinas was measured. The basal effiux of LE-L1 decreased only gradually during superfusion in the dark (Fig. 2B). The total effiux of LE-LI during 25 min in the light was reduced by 59% when compared to the efflux during the previous 25 min in the dark (Fig. 2A). On returning to the dark, the efflux increased to initial rates. Light had no detectable effect on the spontaneous release when 4 mM CoC12 was present (Fig. 2C). However, as the baseline effiux during exposure to Co 2÷ was beyond the detection limit of the RIA, small effects cannot be excluded. All retinas responded to depolarizing concentrations of K ÷ after removal of the CoCI 2.
Identification of released immunoreactivity The nature of the immunoreactivity present in the superfusate fractions was examined after extraction with acidified acetone followed by reversed-phase HPLC (results not shown). Two peaks of immunoreactivity were detected. The first peak had the same retention time as synthetic LeuS-enkephalin and was increased in fractions obtained by superfusing retinas with high-K +. The second peak of immunoreactivity was present in acetone-extracts of fresh superfusion buffer and therefore not of retinal origin. Only one peak of immunoreactivity was detected in HPLC-profiles of material retained on Sep-Pak Cls-cartridges. This peak had the same retention time as synthetic LeuS-enkephalin; the increase in immunoassayable material in fractions from retinas which had been superfused in the dark or with buffer containing high-K + was reflected by an increase in the immunoreactive material eluting from the column with this retention time. It is concluded that the increase in LE-LI in the superfusates is due to increased amounts of LeuS-enkeph alin. DISCUSSION The results reported here show that recoverable LE-LI in the superfusate of the retina was elevated in the dark and reduced in the light. The effiux of LE-LI, whether spontaneous or induced either by light/dark or by elevated potassium, was blocked if Co 2÷ was present in the superfusing medium. Since in avian retina, LE-LI has only been observed in amacrine cells (including their processes
in the inner plexiform layer) 4 (for reviews see refs. 3 and 29), it is reasonable to conclude that the immunoreactive material released from the retina in the present study originates from these cells or their processes. As the LE-LI had the same retention time as synthetic LeuS-enkephalin upon reverse-phase HPLC, this demonstrates that the retina releases true LeuS-enkephalin. In addition, as Co 2+ blocked release, it is also likely that the mechanism for release of LeuS-enkephalin is C a 2+ dependent and may thus involve release from intracellular organelles. It has been demonstrated in goldfish and chicken retina that newly synthesized radiolabelled Met 5enkephalin can be released in vitro by exposure to depolarizing concentrations of K + (refs. 25, 27). Apart from the demonstration of high-K* evoked release of endogenous LeuS-enkephalin-like material from the retina, the results presented here clearly demonstrate that this release can be modulated by a more physiological stimulus. Based on extracellular recordings of ganglion cell activity in goldfish retina, it was noted that tight-off rather than light-on may trigger the release of endogenous opiates 6. Our data is consistent with this notion, but suggests that there is a sustained shift in input balance during the dark towards net excitation of the LE-LI amacrine cell. The results of the present study provide a plausible explanation for the observed depletion of retinal levels of LE-LI during the dark 17. Histological studies in the chicken retina revealed arborizations of LE-LI processes in both distal (sublamina 1) and proximal (sublaminas 3 and 5) regions of the inner plexiform layer 4,9A1A7,3°. Although still controversial in published literature 11,30, latest observations seem to agree that the LE-LI amacrine cells receive direct input from bipolar cells (Millar, personal communications). Since the distal and proximal regions of vertebrate retina are generally associated with off-center and on-center information respectively 19, the LE-LI amacrine cells could receive both off- and on-center input. Our data suggests that there is a sustained shift in input balance towards net inhibition of the LE-LI amacrine cell during the light. Preliminary findings indicate an involvement of glycinergic, but not GABAergic transmission in this light-modulated
47 shift in input TM. R e t i n a l levels of various o t h e r biologically active c o m p o u n d s have been shown to be affected by light 1°A2,zl"z3, and the effects of light on the release of [3H]dopamine 14, [3H]glycine8 and [3H]acetylcholine 15 have been studied. In these studies, m a x i m u m release was o b t a i n e d by stimulation with flickering light. L E - L I is the only neuroactive comp o u n d which has b e e n shown to exhibit a sustained release r e l a t e d to a m b i e n t lighting. In conclusion, it was shown that the chicken retina releases m o r e L E - L I during the d a r k than when the r e t i n a is e x p o s e d to light. This further supports the hypothesis that the L E - L I amacrine cells are active during the dark. Retinal L E - L I was shown to be
r e l e a s e d u p o n d e p o l a r i z a t i o n b y e x p o s u r e to high concentrations of potassium. Thus, the variations in the l i g h t - d a r k conditions m a y m o d i f y the state of depolarization of the L E - L I a m a c r i n e cells and hence affect the release of L E - L I . ACKNOWLEDGEMENTS We thank Dr. John Oliver for the supply of antibodies to LeuS-enkephalin, R i c h a r d Smith, Irene B a c k e n and M a r d i Silburn for technical assistance, and Dr. Ian M o r g a n for valuable discussions. The work was s u p p o r t e d by a grant from the National H e a l t h and Medical R e s e a r c h F o u n d a t i o n of Australia.
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27 Su, Y.-Y.T., Watt, C.B. and Lam, D.M.-K.. Opioid pathways in an avian retina. I. The content biosynthesis, and release of MetS-enkephalin, J. Neurosei., 5 (1985) 851-856. 28 Watt, C.B., Su, Y.-Y.T. and Lain, D.M.-K., Interactions between enkephalin and GABA in avian retina, Nature (Lond.), 311 (1984) 761-763. 29 Watt, C.B., Su, Y.-Y.T. and Lam, D.M.-K., Enkephalins in the vertebrate retina. In N.N. Osborne and GA. Chader (Eds.), Progress in Retinal Research, Vol. 4, Pergamon, New York, 1985, pp. 221-246. 30 Watt, C.B., Su, Y.-Y.T. and Lam, D.M.-K., Opioid pathways in an avian retina. II. Synaptic organization of enkephalin-immunoreactive amacrine cells, J. Neurosci., 5 (1985) 857-865.