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Enkephalin and zinc in the hippocampal mossy fiber system
230
Brain Research, 212 (1981) 230-233 © Elsevier/North-HollandBiomedical Press
Enkephalin and zinc in the bippocampal mossy fiber system
K. STENGAARD-PEDERSEN, K. FREDENS and L.-I. LARSSON Institute of Medical Biochemistry and (K.F.) Institute of Anatomy, University of Aarhus, Aarhus (Denmark)
(Accepted December 18th, 1980) Key words: enkephalin - zinc - opiate receptor - hippocampus - immunocytochemistry- Timm
staining
The hippocampus has been reported to contain opiate receptors 1 and endogenous opioid peptides (enkephalins) s. We report that the staining pattern for enkephalin (by immunocytochemistry) and for zinc (by the Timm method) is congruent and confined to the hippocampal mossy fiber system. In parallel neurochemical studies we have been able to show that zinc ions have profound effects on opiate receptor bindinglL Hence, these studies suggest a possible functional interaction between zinc ions and opioids in the hippocampal mossy fiber system. Both the immunocytochemical methods10,14 and the Timm staining procedure 5 have been detailed elsewhere. Briefly, cryostat sections from brains of formaldehyde perfused guinea pigs were treated with the primary antiserum KA-3 at its optimal dilution (1:500) for 24 h at 4 °C. This antiserum reacts with both Met- and Leu-enkephalin. The site of antigen-antibody reaction was revealed by the peroxidase-antiperoxidase (PAP) procedure of Sternberger 16. Sections were lightly counterstained with haematoxylin for identification of background details. Controls used included conventional staining controls (Sternberger, 1979) as well as specificity controls. The latter were performed by allowing the antisera to react (at 4 °C for 24 h prior to use in staining experiments) with 100/~g per ml diluted antiserum of the following peptides: (1) synthetic Met-enkephalin: (2) synthetic Leu-enkephalin; (3) synthetic a-endorphin; (4) highly purified polcine ACTH (1-39); (5) syothetic ACTH (1-24); (6) synthetic aMSH; (7) synthetic human gastrin 1; (8) synthetic caerulein (a CCK-8 analog); and (9) highly purified porcine CCK-33. Peptides 1 + 2 were obtained from Bioproducts Peptide Department, peptides 3 + 6 from Peninsula, peptide 4 was a generous gift from Dr. H. Vilhardt (Ferring AB, Malta6, Sweden), peptide 6 was Synacten from Ciba-Geigy, peptide 7 was generously donated by Dr. J. S. Morley (ICI, Alderley Park, Cheshire, U.K.), peptide 8 was a kind gift from Farmitalia and peptide 9 a kind gift from Professor V. Mutt (Gastrointestinal Hormone Lab., Karolinska Institutet, Stockholm, Sweden). Antiserum KA-3 reacted equally with both Met- and Leu-enkephalin, but was unaffected by absorptions against the remaining peptides. Briefly, the
231
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Figs. 1 and 2. The pattern of Timm staining (Fig. 1) and the enkephalin immunoreactivity (Fig. 2) show identical localization confined to the mossy fiber system in horizontal sections at approximately the same septo-temporal level from two individual guinea pigs. mr, mossy fibers; fd, fascia dentata. Magnification: ;."15.2 (Fig. 1) and × 20 (Fig. 2). localization of heavy metals involved sulphide precipitation of the metals by petfusion of the animals with a solution of 11.7 g NazS and 11.9 g Na2H2PO4.HzO in 10O0 ml distilled water. The brains were frozen in COs gas. Cryostat sections of 30 # m were dried, postfixed in ethanol and the argyrophilic sulphide was visualized by physical development 5. Enkephalin immunoreactivity was seen in a band parallel to the granule cells, but separated from these cells by the plexiform subzone of Cajal. A few fiber-like ramifications, reminiscent of supragranular mossy fibers were seen between the granule cells. The suprapyramidal band of enkephalin immunoreactivity was seen throughout region CA3 and ended with a typical end-bulb where CA3 borders on CA1. (Fig. 2). No enkephalin was detected in the remaining hippocampus. Thus, it is likely that all enkephalin in the hippocampal formation is localized in the mossy fiber system. The Timm staining pattern of the mossy fiber system was identical to the distribution of enkephalin immunoreactive nerve fibers and terminals (Fig. 1). Electron micrographs of Timm stained mossy fibers show a heavy staining of the Timm positive substance in the mossy fiber boutons v,9. By atomic absorption spectrophotometry it has been shown that the zinc content of the hippocampal formation far exceeds the average content of the whole brain 2,~ and zinc is thought to be responsible for the Timm staining in the mossy fiber system4, 5. The coexistence of zinc ions and enkephalin in the mossy fiber system is very likely to be of physiological importance. Although the hippocampus contains a low density of opiate receptors lz and a low total enkephalin content s, opioid peptides can
232 have p r o f o u n d influences on the h i p p o c a m p a l neurons. Opiates a n d o p i o i d p e p t i d e s have been shown to excite h i p p o c a m p a l p y r a m i d a l cells in culture. This effect was reversible and could be b l o c k e d by naloxone. F u r t h e r evidence p o i n t e d to a p r e s y n a p tic enkephalinergic action on synaptic transmission 17. M i c r o i o n t o p h o r e t i c studies have shown t h a t o p i o i d peptides can have a p r o f o u n d excitatory influence o n the electrical activity o f h i p p o c a m p a l p y r a m i d a l neurons in situ 11. The function o f zinc ions in the h i p p o c a m p u s has r e m a i n e d unclear. However, yon Euler 3 showed t h a t sulphide t r e a t m e n t o f the h i p p o c a m p u s a b o l i s h e d t r a n s m i s s i o n t h r o u g h these synapses within a few minutes, p r e s u m a b l y by b i n d i n g the zinc. Recently, we have been able to show that zinc ions can inhibit the stereospecific b i n d i n g o f [aH]enkephalinamide (2-D-Ala-5-L-methionine) to opiate receptor sites a n d this effect is m o s t likely due to a redox r e a c t i o n between opiate r e c e p t o r thiols a n d zinc ions 15. It can thus be c o n c l u d e d t h a t e n k e p h a l i n a n d zinc display similar t o p o g r a p h i c a l localization in the h i p p o c a m p a l m o s s y fiber layer o f the guinea pig. F u r t h e r , zinc ions can inhibit the binding o f e n k e p h a l i n to opiate receptors. A l t o g e t h e r this suggests that the zinc ion m a y be a physiologically i m p o r t a n t m o d u l a t o r o f opiate receptor function in the h i p p o c a m p a l mossy fiber system. G r a n t s u p p o r t was o b t a i n e d from the D a n i s h M e d i c a l Research Council a n d " F o n d e n til L~egevidenskabens F r e m m e " . Excellent technical assistance was delivered by L. Laursen, E. D o r g e a n d A. Meyer.
1 Atweh, S. F. and Kuhar, M., Autoradiographic localization of opiate receptors in rat brain. III. The telencephalon, Brain Research, 134 (1977) 393 405. 2 Danscher, G., Fjerdingstad, E. J., Fjerdingstad, E. and Fredens, K., Heavymetal content in subdivisions of the rat hippocampus (zinc, lead and copper), Brain Research, 112 (1976) 442M46. 3 Euler, C. von, On the significance of the high zinc content in the hippocampal formation. In P. Passouant (Ed.), Physiologic de l'Hippocampe, CNRS, Paris, 1962, pp. 135-145. 4 Fjerdingstad, E., Danscher, G. and Fjerdingstad, E. J., Zinc content in hippocampus and whole brain of normal rats, Brain Research, 79 (1974) 338-342. 5 Geneser-Jensen, F. A., Haug, F.-M. S. and Danscher, G., Distribution of heavy metals in the hippocampal region of the guinea pig, Z. Zellforsch., 147 (1974) 441-478. 6 Gfihwiler, B. H., Excitatory action of opioid peptides and opiates on cultured hippocampal pyramidal cells, Brain Research, 194 (1980) 193 203. 7 Haug, F. -M. S., Electron microscopical localization of the zinc in hippocampal mossy fiber synapses by a modified sulphide silver procedure, Histochemie 8 (1967) 355-368. 8 Hong, J. S., Yang, H. Y., Fratta, W. and Costa, E., Determination of methionine-enkephalin in discrete regions of rat brain, Brabz Research, 134 (1977) 383-386. 9 lbata, Y. and Otsuka, N., Electron microscopic demonstration of zinc in the hippocampal formation using Timm's sulphide silver technique, J. Histochem. Cytochem., 17 (1969) 171-175. 10 Larsson, L.-I., Childers, S. and Snyder, S. H . , Met- and Leu-enkephalin immunoreactivity in separate neurons, Nature (Lond.), 282 (1979) 407-410. 11 Nicoll, R. A., Siggins, G. R., Ling, N., Bloom, F. E. and Guillemin, R., Neuronal actions of endorphins and enkephalins among brain regions: a comparative microiontophoretic study, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 2584-2588. 12 Simon, E. J. and Hiller, J. M., The opiate receptors, Ann. Rev. Pharmacol. Toxicol., 18 (1978) 371-394. 13 Stengaard-Pedersen, K. and Larsson, L.-I., Interaction of putative opioid peptides with opiate receptors, Actapharmacol. (Kbh.), in press.
233 14 Stengaard-Pedersen, K. and Larsson, L.-I., Comparative immunocytochemical localization of putative opioid ligands in the central nervous system, Neuroscience, (1980) submitted. 15 Stengaard-Pedersen, K.,Zincasinhibitor of enkephalin binding to opiate receptors in the hippocampus, J. Neurochem., (1980) submitted. 16 Sternberger, L. A., Immunocytochemistry, Prentice Hall, Englewood Cliffs, N. J., 1974. 17 Zieglg~insberger,W., French, E. D., Siggins, G. R. and Bloom, F. E., Opioid peptides may excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory interneurons, Scietlce, 205 (1979) 414 417.
Brain Research, 212 (1981) 230-233 © Elsevier/North-HollandBiomedical Press
Enkephalin and zinc in the bippocampal mossy fiber system
K. STENGAARD-PEDERSEN, K. FREDENS and L.-I. LARSSON Institute of Medical Biochemistry and (K.F.) Institute of Anatomy, University of Aarhus, Aarhus (Denmark)
(Accepted December 18th, 1980) Key words: enkephalin - zinc - opiate receptor - hippocampus - immunocytochemistry- Timm
staining
The hippocampus has been reported to contain opiate receptors 1 and endogenous opioid peptides (enkephalins) s. We report that the staining pattern for enkephalin (by immunocytochemistry) and for zinc (by the Timm method) is congruent and confined to the hippocampal mossy fiber system. In parallel neurochemical studies we have been able to show that zinc ions have profound effects on opiate receptor bindinglL Hence, these studies suggest a possible functional interaction between zinc ions and opioids in the hippocampal mossy fiber system. Both the immunocytochemical methods10,14 and the Timm staining procedure 5 have been detailed elsewhere. Briefly, cryostat sections from brains of formaldehyde perfused guinea pigs were treated with the primary antiserum KA-3 at its optimal dilution (1:500) for 24 h at 4 °C. This antiserum reacts with both Met- and Leu-enkephalin. The site of antigen-antibody reaction was revealed by the peroxidase-antiperoxidase (PAP) procedure of Sternberger 16. Sections were lightly counterstained with haematoxylin for identification of background details. Controls used included conventional staining controls (Sternberger, 1979) as well as specificity controls. The latter were performed by allowing the antisera to react (at 4 °C for 24 h prior to use in staining experiments) with 100/~g per ml diluted antiserum of the following peptides: (1) synthetic Met-enkephalin: (2) synthetic Leu-enkephalin; (3) synthetic a-endorphin; (4) highly purified polcine ACTH (1-39); (5) syothetic ACTH (1-24); (6) synthetic aMSH; (7) synthetic human gastrin 1; (8) synthetic caerulein (a CCK-8 analog); and (9) highly purified porcine CCK-33. Peptides 1 + 2 were obtained from Bioproducts Peptide Department, peptides 3 + 6 from Peninsula, peptide 4 was a generous gift from Dr. H. Vilhardt (Ferring AB, Malta6, Sweden), peptide 6 was Synacten from Ciba-Geigy, peptide 7 was generously donated by Dr. J. S. Morley (ICI, Alderley Park, Cheshire, U.K.), peptide 8 was a kind gift from Farmitalia and peptide 9 a kind gift from Professor V. Mutt (Gastrointestinal Hormone Lab., Karolinska Institutet, Stockholm, Sweden). Antiserum KA-3 reacted equally with both Met- and Leu-enkephalin, but was unaffected by absorptions against the remaining peptides. Briefly, the
231
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.
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.... ,.~,~-
"
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"~'''':"'" ~ i)
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Figs. 1 and 2. The pattern of Timm staining (Fig. 1) and the enkephalin immunoreactivity (Fig. 2) show identical localization confined to the mossy fiber system in horizontal sections at approximately the same septo-temporal level from two individual guinea pigs. mr, mossy fibers; fd, fascia dentata. Magnification: ;."15.2 (Fig. 1) and × 20 (Fig. 2). localization of heavy metals involved sulphide precipitation of the metals by petfusion of the animals with a solution of 11.7 g NazS and 11.9 g Na2H2PO4.HzO in 10O0 ml distilled water. The brains were frozen in COs gas. Cryostat sections of 30 # m were dried, postfixed in ethanol and the argyrophilic sulphide was visualized by physical development 5. Enkephalin immunoreactivity was seen in a band parallel to the granule cells, but separated from these cells by the plexiform subzone of Cajal. A few fiber-like ramifications, reminiscent of supragranular mossy fibers were seen between the granule cells. The suprapyramidal band of enkephalin immunoreactivity was seen throughout region CA3 and ended with a typical end-bulb where CA3 borders on CA1. (Fig. 2). No enkephalin was detected in the remaining hippocampus. Thus, it is likely that all enkephalin in the hippocampal formation is localized in the mossy fiber system. The Timm staining pattern of the mossy fiber system was identical to the distribution of enkephalin immunoreactive nerve fibers and terminals (Fig. 1). Electron micrographs of Timm stained mossy fibers show a heavy staining of the Timm positive substance in the mossy fiber boutons v,9. By atomic absorption spectrophotometry it has been shown that the zinc content of the hippocampal formation far exceeds the average content of the whole brain 2,~ and zinc is thought to be responsible for the Timm staining in the mossy fiber system4, 5. The coexistence of zinc ions and enkephalin in the mossy fiber system is very likely to be of physiological importance. Although the hippocampus contains a low density of opiate receptors lz and a low total enkephalin content s, opioid peptides can
232 have p r o f o u n d influences on the h i p p o c a m p a l neurons. Opiates a n d o p i o i d p e p t i d e s have been shown to excite h i p p o c a m p a l p y r a m i d a l cells in culture. This effect was reversible and could be b l o c k e d by naloxone. F u r t h e r evidence p o i n t e d to a p r e s y n a p tic enkephalinergic action on synaptic transmission 17. M i c r o i o n t o p h o r e t i c studies have shown t h a t o p i o i d peptides can have a p r o f o u n d excitatory influence o n the electrical activity o f h i p p o c a m p a l p y r a m i d a l neurons in situ 11. The function o f zinc ions in the h i p p o c a m p u s has r e m a i n e d unclear. However, yon Euler 3 showed t h a t sulphide t r e a t m e n t o f the h i p p o c a m p u s a b o l i s h e d t r a n s m i s s i o n t h r o u g h these synapses within a few minutes, p r e s u m a b l y by b i n d i n g the zinc. Recently, we have been able to show that zinc ions can inhibit the stereospecific b i n d i n g o f [aH]enkephalinamide (2-D-Ala-5-L-methionine) to opiate receptor sites a n d this effect is m o s t likely due to a redox r e a c t i o n between opiate r e c e p t o r thiols a n d zinc ions 15. It can thus be c o n c l u d e d t h a t e n k e p h a l i n a n d zinc display similar t o p o g r a p h i c a l localization in the h i p p o c a m p a l m o s s y fiber layer o f the guinea pig. F u r t h e r , zinc ions can inhibit the binding o f e n k e p h a l i n to opiate receptors. A l t o g e t h e r this suggests that the zinc ion m a y be a physiologically i m p o r t a n t m o d u l a t o r o f opiate receptor function in the h i p p o c a m p a l mossy fiber system. G r a n t s u p p o r t was o b t a i n e d from the D a n i s h M e d i c a l Research Council a n d " F o n d e n til L~egevidenskabens F r e m m e " . Excellent technical assistance was delivered by L. Laursen, E. D o r g e a n d A. Meyer.
1 Atweh, S. F. and Kuhar, M., Autoradiographic localization of opiate receptors in rat brain. III. The telencephalon, Brain Research, 134 (1977) 393 405. 2 Danscher, G., Fjerdingstad, E. J., Fjerdingstad, E. and Fredens, K., Heavymetal content in subdivisions of the rat hippocampus (zinc, lead and copper), Brain Research, 112 (1976) 442M46. 3 Euler, C. von, On the significance of the high zinc content in the hippocampal formation. In P. Passouant (Ed.), Physiologic de l'Hippocampe, CNRS, Paris, 1962, pp. 135-145. 4 Fjerdingstad, E., Danscher, G. and Fjerdingstad, E. J., Zinc content in hippocampus and whole brain of normal rats, Brain Research, 79 (1974) 338-342. 5 Geneser-Jensen, F. A., Haug, F.-M. S. and Danscher, G., Distribution of heavy metals in the hippocampal region of the guinea pig, Z. Zellforsch., 147 (1974) 441-478. 6 Gfihwiler, B. H., Excitatory action of opioid peptides and opiates on cultured hippocampal pyramidal cells, Brain Research, 194 (1980) 193 203. 7 Haug, F. -M. S., Electron microscopical localization of the zinc in hippocampal mossy fiber synapses by a modified sulphide silver procedure, Histochemie 8 (1967) 355-368. 8 Hong, J. S., Yang, H. Y., Fratta, W. and Costa, E., Determination of methionine-enkephalin in discrete regions of rat brain, Brabz Research, 134 (1977) 383-386. 9 lbata, Y. and Otsuka, N., Electron microscopic demonstration of zinc in the hippocampal formation using Timm's sulphide silver technique, J. Histochem. Cytochem., 17 (1969) 171-175. 10 Larsson, L.-I., Childers, S. and Snyder, S. H . , Met- and Leu-enkephalin immunoreactivity in separate neurons, Nature (Lond.), 282 (1979) 407-410. 11 Nicoll, R. A., Siggins, G. R., Ling, N., Bloom, F. E. and Guillemin, R., Neuronal actions of endorphins and enkephalins among brain regions: a comparative microiontophoretic study, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 2584-2588. 12 Simon, E. J. and Hiller, J. M., The opiate receptors, Ann. Rev. Pharmacol. Toxicol., 18 (1978) 371-394. 13 Stengaard-Pedersen, K. and Larsson, L.-I., Interaction of putative opioid peptides with opiate receptors, Actapharmacol. (Kbh.), in press.
233 14 Stengaard-Pedersen, K. and Larsson, L.-I., Comparative immunocytochemical localization of putative opioid ligands in the central nervous system, Neuroscience, (1980) submitted. 15 Stengaard-Pedersen, K.,Zincasinhibitor of enkephalin binding to opiate receptors in the hippocampus, J. Neurochem., (1980) submitted. 16 Sternberger, L. A., Immunocytochemistry, Prentice Hall, Englewood Cliffs, N. J., 1974. 17 Zieglg~insberger,W., French, E. D., Siggins, G. R. and Bloom, F. E., Opioid peptides may excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory interneurons, Scietlce, 205 (1979) 414 417.