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Distribution of dynorphin B and methionine-enkephalin in the mouse hippocampus: Influence of genotype
Neuroscience Letters, 97 (1989) 241 244
24 I
Elsevier Scientific Publishers Ireland Ltd.
NSL 05896
Distribution of dynorphin B and methionine-enkephalin in the mouse hippocampus: influence of genotype J.H.H.M. van Daal, H.E.A. Zanderink, B.G. Jenks and J.H.F. van Abeelen Department ~[A nimal Physiology, University o['Nijmegen, Nijmegen ( The Netherlands) (Received 2 May 1988; Revised version received 10 October 1988; Accepted 22 October 1988)
Key wortZ~. Dynorphin B; Methionine-enkephalin; Hippocampus; Mossy fiber; Interneuron: Genotype; Mouse
Immunohistochemical techniques were used to localize dynorphin B and methionine-enkephalin in the mouse hippocampus. Methionine-enkephalin-like immunoreactivity was found within the somata of interneurons distributed mainly in and around the CA1 stratum pyramidale and stratum granulosum as well as in the mossy fibers. Dynorphin B appeared to be confined to the mossy fiber pathway. In addition, we observed a difference between the inbred mouse strains DBA/2 and C57BL/6 with regard to the areas of the dynorphinergic mossy fiber projections: the intra- and infrapyramidal terminal fields were larger in the latter group.
In the last few years there has been an increasing interest in hippocampal opioids. Studies of these peptides have been largely confined to rats and guinea pigs [1, 4, 6, 7, 9 11, 17]. The opioids act to disinhibit cholinergically innervated pyramidal cells by blocking inhibitory GABAergic interneurons [2, 9]. Our previous research was directed at the role of neuropeptides in the regulation of exploratory behavior in the house mouse. We found evidence that exposure of mice to environmental novelty induces the release of hippocampal dynorphin B and methionine-enkephalin in a genotype-dependent manner [15, 16]. Although these studies have shed light upon the function of the peptides, it is of importance to elucidate which hippocampal neuronal networks participate in the control of behavior by dynorphin B and methionineenkephalin. The location of hippocampal opioid peptides in rats and guinea pigs [4, 6, 7, 10, 11] and, very recently, in mice [5] has been described. We now extend this observation in mice by demonstrating that the distribution of hippocampal immunoreactive dynorphin B in this species depends on the genotype. Male C57BL/6JKunNmg and DBA/2JNmg mice [13] were bred in our laboratory Correspondence." J.H.H.M. van Daal, Department of Animal Physiology, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands. 0304-3940,,89, $ 03.50 ',~ 1989 Elsevier Scientific Publishers Ireland Ltd.
242
under standard conditions [14] and used when 3-4 months of age. Untreated and colchicine-treated animals were used to investigate the location of immunoreactive fibers and perikarya, respectively. The latter groups received 10/~g of colchicine dissolved in 2/A of saline, which was injected under ether anesthesia into the right lateral ventricle 48 h before the animals were sacrificed. Tissue preparation was carried out as described [12]. The immunohistochemistry was performed on 25/lm transverse cryostat brain sections mounted on gelatine-coated glass slides. The antisera used had been raised by us in rabbits against synthetic methionine-enkephalin and dynorphin B (Bachem, Bubendorf, Switzerland) which had been coupled to thyroglobulin with carbodiimide. The specificity of immunostaining obtained with these antisera was tested according to the paper strip method described [8] and by preadsorption with 100/tM of both the homotypic and heterotypic antigens. The anti-dynorphin B antiserum recognized neither methionine-enkephalin nor leucine-enkephalin and showed a low affinity to dynorphin A(1-7) and dynorphin A(1-13); the latter crossreactivity was less than 1%. The anti-methionine-enkephalin antiserum cross-reacted 2-4% with leucine-enkephalin but bound neither dynorphin B nor the dynorphin A fragments. For the immunohistochemistry, both antisera were used at a 1:5000 dilution in Tris-buffered saline containing 1% normal goat serum and 0.5% Triton X-100. Incubation was at 4°C for 48 h. Tissues, processed by the peroxidase-antiperoxidase (PAP) technique, were treated with goat anti-rabbit v-globulin (1:50; Nordic, Tilburg, The Netherlands) for 2 h and with PAP-complex (1:300; UCB-Bioproducts, Braine-L'Alleud, Belgium) for 2.5 h at room temperature. Diaminobenzidine (0,25 mg/ml, dissolved in 50 mM Tris buffer, pH 7.6, containing 0.01% H202 and 5 mg/ml ammonium nickel sulphate) served as an electron donor. Incubation was at room temperature for l0 rain. The mounted tissue sections were dehydrated and sealed in Entellan. The results showed that two neuronal populations were stained for methionineenkephalin: the mossy fibers and cell bodies. The latter were diffusely spread within the hippocampus but showed some concentration in and around the stratum pyramidale of the regio superior and the stratum granulosum (Fig. 1). Their overall morphology indicates that they are probably interneurons. Although such a hippocampal distribution has been seen in the mouse [5] and in the rat [6], we also observed an aggregation of large enkephalin-immunoreactive somata at the transition between the CA! and CA3 pyramidal cell layers (Fig. 2). Dynorphin B immunoreactivity was restricted to the mossy fibers (Figs. 3 and 4); moreover, in colchicine-treated animals it was limited to the perikarya of the granule cells of the dentate area. We conclude that in the mouse hippocampus the dynorphins are confined to this major intrinsic pathway, which confirms a recent finding in this species [5] and is in line with findings in rats [1, 4, 11]. As compared to methionineenkephalin-immunoreactive mossy fibers, the dynorphin-immunoreactive mossy fiber projection zone appeared to be much denser, suggesting that a smaller proportion of the granule cells produces the pentapeptide. A most interesting observation was the difference observed between strains C57BL/6 and DBA/2 with regard to the sizes of the dynorphin B-positive intra- and
243
L
,.
i
Figs. 1 and 2. Methionine-enkephalin-containingneurons, diffusely spread within the hippocampal CA I region (1) and clustered at the transition between the CAI and CA3 pyramidal cell layers (2), in mouse brain sections. Scale bar= 100 ,urn. Figs. 3 and 4. Dynorphinergic hippocampal mossy fibers in C57BL/6 mice (3) possessing intra- and infrapyramidal projection areas, as indicated by arrowheads, that were considerably larger than those in DBA' 2 animals (4). Representative brain sections. Scale bar ~ 500 l~m.
infrapyramidal mossy fiber terminal fields: they were larger in C57BL/6 (see Figs. 3 and 4). This agrees with differences between these mouse strains as found by others [3] using an entirely different staining method, namely Timm's silver sulphide technique (intra- and infrapyramidal mossy fibers containing methionine-enkephalin were rare). The above neuroanatomical between-strain variation strengthens the earlier conclusion [16] that the functioning of the dynorphinergic pathway in the regulation of mouse exploratory behavior depends on the genotype. We are grateful to Prof. Dr. R. Nieuwenhuys and Henk Joosten (Nijmegen) and Dr. H.W.M. Steinbusch and John Bol (Amsterdam) for advice and for allowing us to use their laboratory facilities. We thank Anton Buis and Tony Coenen for biotechnical assistance. Chavkin, C., Shoemaker, W.J., McGinty, J.F., Bayon, A. and Bloom, F.E., Characterization of the prodynorphin and proenkephalin neuropeptide systems in rat hippocampus, J. Neurosci., 5 (1985) 808 816. 2 Corrigall, W.A., Opiates and the hippocampus: A review of the functional and morphological evidence, Pharmacol. Biochem. Behav, 18 (1983) 255 262. 3 Crusio, W.E., Schwegler, H. and Lipp, H.-P., Radial-maze performance and structural variation of the hippocampus in mice: a correlation with mossy fibre distribution, Brain Res., 425 (1987) 182 185.
244 4 Fallon, J.H. and Leslie, F.M., Distribution of dynorphin and enkephalin peptides in rat brain, .t. Comp. Neurol., 249 (1986) 293 336. 5 Gall, C., Seizures induce dramatic and distinctly different changes in enkephalin, dynorphin, and CCK immunoreactivities in mouse hippocampal mossy fibers, J. Neurosci., 8 (1988) 1852 1862. 6 Gall, C., Brecha, N., Karten, H.J. and Chang, K.-J., Localization of enkephalin-like immunoreactivity to identified axonal and neuronal populations of the rat hippocampus, J. Comp. Neurol., 198 (1981j 335 350. 7 Hoffman, D.W., Altschuler, R.A. and Gutierrez, J., Multiple molecular forms of enkephalins in the guinea pig hippocampus, J. Neurochem., 41 (1983) 1641 1647. 8 Larsson, L.-I., A novel immunocytochemical model system for specificity and sensitivity screening of antisera against multiple antigens, J. Histochem. Cytochem., 29 (1981) 408410. 9 Masukawa, L.M. and Prince, D.A., Enkephalin inhibition of inhibitory input to CA1 and CA3 pyramidal neurons in the hippocampus, Brain Res., 249 (1982) 271-280. 10 Merchenthaler, I., Maderdrut, J.L., Altschuler, R.A. and Petrusz, P., Immunocytochemical localization of proenkephalin-derived peptides in the central nervous system of the rat, Neuroscience, 17 (1986) 325 348. 11 Morris, B.J., Haarmann, l., Kempter, B., H611t, V. and Herz, A., Localization of prodynorphin messenger RNA in rat brain by in situ hybridization using a synthetic oligonucleotide probe, Neurosci. Lett., 69 (1986) 104-108. 12 Somogyi, P. and Takagi, H., A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry, Neurosciencc. 7 (1982) 1779 1783. 13 Staats, J., Standardized nomenclature for inbred strains of mice: Eighth listing, Cancer Res., 45 (1985) 945 977. 14 Van Abeelen, J.H.F. and Boersma, H.J.L.M., A genetically controlled hippocampal transmitter system regulating exploratory behavior in mice, J. Neurogenet., 1 (1984) 153 -158. 15 Van Abeelen, J.H.F. and Gerads, H.J.M.I., Role of hippocampal Met-enkephalin in the genotype-dependent regulation of exploratory behavior in mice, J. Neurogenet., 3 (1986) 183-186. 16 Van Daai, J.H.H.M., De Kok, Y.J.M., Jenks, B.G., Wendelaar Bonga, S.E. and Van Abeelen, J.H.F.. A genotype-dependent hippocampal dynorphinergic mechanism controls mouse exploration, Pharmacol. Biochem. Behav,, 28 (1987) 465~,68. 17 White, J.D., Gall, C.M. and McKelvy, J.F,, Enkephalin biosynthesis and enkephalin gene expression are increased in hippocampal mossy fibers following a unilateral lesion of the hilus, J, Neurosci., 7 (1987) 753 759.
24 I
Elsevier Scientific Publishers Ireland Ltd.
NSL 05896
Distribution of dynorphin B and methionine-enkephalin in the mouse hippocampus: influence of genotype J.H.H.M. van Daal, H.E.A. Zanderink, B.G. Jenks and J.H.F. van Abeelen Department ~[A nimal Physiology, University o['Nijmegen, Nijmegen ( The Netherlands) (Received 2 May 1988; Revised version received 10 October 1988; Accepted 22 October 1988)
Key wortZ~. Dynorphin B; Methionine-enkephalin; Hippocampus; Mossy fiber; Interneuron: Genotype; Mouse
Immunohistochemical techniques were used to localize dynorphin B and methionine-enkephalin in the mouse hippocampus. Methionine-enkephalin-like immunoreactivity was found within the somata of interneurons distributed mainly in and around the CA1 stratum pyramidale and stratum granulosum as well as in the mossy fibers. Dynorphin B appeared to be confined to the mossy fiber pathway. In addition, we observed a difference between the inbred mouse strains DBA/2 and C57BL/6 with regard to the areas of the dynorphinergic mossy fiber projections: the intra- and infrapyramidal terminal fields were larger in the latter group.
In the last few years there has been an increasing interest in hippocampal opioids. Studies of these peptides have been largely confined to rats and guinea pigs [1, 4, 6, 7, 9 11, 17]. The opioids act to disinhibit cholinergically innervated pyramidal cells by blocking inhibitory GABAergic interneurons [2, 9]. Our previous research was directed at the role of neuropeptides in the regulation of exploratory behavior in the house mouse. We found evidence that exposure of mice to environmental novelty induces the release of hippocampal dynorphin B and methionine-enkephalin in a genotype-dependent manner [15, 16]. Although these studies have shed light upon the function of the peptides, it is of importance to elucidate which hippocampal neuronal networks participate in the control of behavior by dynorphin B and methionineenkephalin. The location of hippocampal opioid peptides in rats and guinea pigs [4, 6, 7, 10, 11] and, very recently, in mice [5] has been described. We now extend this observation in mice by demonstrating that the distribution of hippocampal immunoreactive dynorphin B in this species depends on the genotype. Male C57BL/6JKunNmg and DBA/2JNmg mice [13] were bred in our laboratory Correspondence." J.H.H.M. van Daal, Department of Animal Physiology, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands. 0304-3940,,89, $ 03.50 ',~ 1989 Elsevier Scientific Publishers Ireland Ltd.
242
under standard conditions [14] and used when 3-4 months of age. Untreated and colchicine-treated animals were used to investigate the location of immunoreactive fibers and perikarya, respectively. The latter groups received 10/~g of colchicine dissolved in 2/A of saline, which was injected under ether anesthesia into the right lateral ventricle 48 h before the animals were sacrificed. Tissue preparation was carried out as described [12]. The immunohistochemistry was performed on 25/lm transverse cryostat brain sections mounted on gelatine-coated glass slides. The antisera used had been raised by us in rabbits against synthetic methionine-enkephalin and dynorphin B (Bachem, Bubendorf, Switzerland) which had been coupled to thyroglobulin with carbodiimide. The specificity of immunostaining obtained with these antisera was tested according to the paper strip method described [8] and by preadsorption with 100/tM of both the homotypic and heterotypic antigens. The anti-dynorphin B antiserum recognized neither methionine-enkephalin nor leucine-enkephalin and showed a low affinity to dynorphin A(1-7) and dynorphin A(1-13); the latter crossreactivity was less than 1%. The anti-methionine-enkephalin antiserum cross-reacted 2-4% with leucine-enkephalin but bound neither dynorphin B nor the dynorphin A fragments. For the immunohistochemistry, both antisera were used at a 1:5000 dilution in Tris-buffered saline containing 1% normal goat serum and 0.5% Triton X-100. Incubation was at 4°C for 48 h. Tissues, processed by the peroxidase-antiperoxidase (PAP) technique, were treated with goat anti-rabbit v-globulin (1:50; Nordic, Tilburg, The Netherlands) for 2 h and with PAP-complex (1:300; UCB-Bioproducts, Braine-L'Alleud, Belgium) for 2.5 h at room temperature. Diaminobenzidine (0,25 mg/ml, dissolved in 50 mM Tris buffer, pH 7.6, containing 0.01% H202 and 5 mg/ml ammonium nickel sulphate) served as an electron donor. Incubation was at room temperature for l0 rain. The mounted tissue sections were dehydrated and sealed in Entellan. The results showed that two neuronal populations were stained for methionineenkephalin: the mossy fibers and cell bodies. The latter were diffusely spread within the hippocampus but showed some concentration in and around the stratum pyramidale of the regio superior and the stratum granulosum (Fig. 1). Their overall morphology indicates that they are probably interneurons. Although such a hippocampal distribution has been seen in the mouse [5] and in the rat [6], we also observed an aggregation of large enkephalin-immunoreactive somata at the transition between the CA! and CA3 pyramidal cell layers (Fig. 2). Dynorphin B immunoreactivity was restricted to the mossy fibers (Figs. 3 and 4); moreover, in colchicine-treated animals it was limited to the perikarya of the granule cells of the dentate area. We conclude that in the mouse hippocampus the dynorphins are confined to this major intrinsic pathway, which confirms a recent finding in this species [5] and is in line with findings in rats [1, 4, 11]. As compared to methionineenkephalin-immunoreactive mossy fibers, the dynorphin-immunoreactive mossy fiber projection zone appeared to be much denser, suggesting that a smaller proportion of the granule cells produces the pentapeptide. A most interesting observation was the difference observed between strains C57BL/6 and DBA/2 with regard to the sizes of the dynorphin B-positive intra- and
243
L
,.
i
Figs. 1 and 2. Methionine-enkephalin-containingneurons, diffusely spread within the hippocampal CA I region (1) and clustered at the transition between the CAI and CA3 pyramidal cell layers (2), in mouse brain sections. Scale bar= 100 ,urn. Figs. 3 and 4. Dynorphinergic hippocampal mossy fibers in C57BL/6 mice (3) possessing intra- and infrapyramidal projection areas, as indicated by arrowheads, that were considerably larger than those in DBA' 2 animals (4). Representative brain sections. Scale bar ~ 500 l~m.
infrapyramidal mossy fiber terminal fields: they were larger in C57BL/6 (see Figs. 3 and 4). This agrees with differences between these mouse strains as found by others [3] using an entirely different staining method, namely Timm's silver sulphide technique (intra- and infrapyramidal mossy fibers containing methionine-enkephalin were rare). The above neuroanatomical between-strain variation strengthens the earlier conclusion [16] that the functioning of the dynorphinergic pathway in the regulation of mouse exploratory behavior depends on the genotype. We are grateful to Prof. Dr. R. Nieuwenhuys and Henk Joosten (Nijmegen) and Dr. H.W.M. Steinbusch and John Bol (Amsterdam) for advice and for allowing us to use their laboratory facilities. We thank Anton Buis and Tony Coenen for biotechnical assistance. Chavkin, C., Shoemaker, W.J., McGinty, J.F., Bayon, A. and Bloom, F.E., Characterization of the prodynorphin and proenkephalin neuropeptide systems in rat hippocampus, J. Neurosci., 5 (1985) 808 816. 2 Corrigall, W.A., Opiates and the hippocampus: A review of the functional and morphological evidence, Pharmacol. Biochem. Behav, 18 (1983) 255 262. 3 Crusio, W.E., Schwegler, H. and Lipp, H.-P., Radial-maze performance and structural variation of the hippocampus in mice: a correlation with mossy fibre distribution, Brain Res., 425 (1987) 182 185.
244 4 Fallon, J.H. and Leslie, F.M., Distribution of dynorphin and enkephalin peptides in rat brain, .t. Comp. Neurol., 249 (1986) 293 336. 5 Gall, C., Seizures induce dramatic and distinctly different changes in enkephalin, dynorphin, and CCK immunoreactivities in mouse hippocampal mossy fibers, J. Neurosci., 8 (1988) 1852 1862. 6 Gall, C., Brecha, N., Karten, H.J. and Chang, K.-J., Localization of enkephalin-like immunoreactivity to identified axonal and neuronal populations of the rat hippocampus, J. Comp. Neurol., 198 (1981j 335 350. 7 Hoffman, D.W., Altschuler, R.A. and Gutierrez, J., Multiple molecular forms of enkephalins in the guinea pig hippocampus, J. Neurochem., 41 (1983) 1641 1647. 8 Larsson, L.-I., A novel immunocytochemical model system for specificity and sensitivity screening of antisera against multiple antigens, J. Histochem. Cytochem., 29 (1981) 408410. 9 Masukawa, L.M. and Prince, D.A., Enkephalin inhibition of inhibitory input to CA1 and CA3 pyramidal neurons in the hippocampus, Brain Res., 249 (1982) 271-280. 10 Merchenthaler, I., Maderdrut, J.L., Altschuler, R.A. and Petrusz, P., Immunocytochemical localization of proenkephalin-derived peptides in the central nervous system of the rat, Neuroscience, 17 (1986) 325 348. 11 Morris, B.J., Haarmann, l., Kempter, B., H611t, V. and Herz, A., Localization of prodynorphin messenger RNA in rat brain by in situ hybridization using a synthetic oligonucleotide probe, Neurosci. Lett., 69 (1986) 104-108. 12 Somogyi, P. and Takagi, H., A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry, Neurosciencc. 7 (1982) 1779 1783. 13 Staats, J., Standardized nomenclature for inbred strains of mice: Eighth listing, Cancer Res., 45 (1985) 945 977. 14 Van Abeelen, J.H.F. and Boersma, H.J.L.M., A genetically controlled hippocampal transmitter system regulating exploratory behavior in mice, J. Neurogenet., 1 (1984) 153 -158. 15 Van Abeelen, J.H.F. and Gerads, H.J.M.I., Role of hippocampal Met-enkephalin in the genotype-dependent regulation of exploratory behavior in mice, J. Neurogenet., 3 (1986) 183-186. 16 Van Daai, J.H.H.M., De Kok, Y.J.M., Jenks, B.G., Wendelaar Bonga, S.E. and Van Abeelen, J.H.F.. A genotype-dependent hippocampal dynorphinergic mechanism controls mouse exploration, Pharmacol. Biochem. Behav,, 28 (1987) 465~,68. 17 White, J.D., Gall, C.M. and McKelvy, J.F,, Enkephalin biosynthesis and enkephalin gene expression are increased in hippocampal mossy fibers following a unilateral lesion of the hilus, J, Neurosci., 7 (1987) 753 759.