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Neurokinin B and substance P genes are co-expressed in a subset of neurons in the rat habenula
0143-4179/89/0013-0165/%10.00
Neuropeptides (1989) 13,165169 0 Longman Group UK Ltd 1989
Neurokinin Expressed Habenula J-M. BURGUNDER
B and Substance P Genes are Coin a Subset of Neurons in the Rat
and W. S. YOUNG
Ill
Laboratory of Cell Biology, National institute of Mental Health, Building 36, Room 2010, Bethesda, MD 20892 (Reprint requests to J. M. B.).
Abstract-Colocalization of neurokinin B (NKB) and substance P (SP) mRNAs in neurons of the habenula wasxamirt$ on thin, adjacent sections in the rat. Extensive colocalization was found in the medial habenula. In its dorsolateral part, most of the neurons contained both transcripts, with high levels of SP, while in the dorsomedial part fewer instances of colocalization were found. The ventral half could be divided into two parts, a dorsal part with most of the neurons containing both messages, having low levels of SP mRNA, and a ventral part with most of the cells containing only NKB mRNA. Cells in the medial habenula had low levels of NKB mRNA. These results suggest a structural and functional heterogeneity of the medial habenula.
Introduction The tachykinins are a family of peptides present in several species, including mammals (1). In mammals, three kinds of tachykinins have so far been demonstrated: substance P (SP), neurokinin A (NKA) and neurokinin B (NKB). While a common gene encodes SP and NKA, from which several different mRNA species are formed by differential splicing (1, 2), NKB is encoded by a separate gene (3-4). These mammalian tachykinins share common antigenic markers, which have impaired the immunocytochemical localization of neurons containing them until recently
Date received 3 November 1988 Date accepted 8 November 1988
(1). In contrast, the specificities of probes generated against segments of the two mRNA species have allowed studies using in situ hybridization histochemistry to address the localization of neurons expressing SP and NKB in the rat brain (3, 4). A combined mapping study of cells containing SP or NKB mRNA (5) revealed several areas of the rat brain contained both messages, suggesting that they might be colocalized in individual neurons. Recent studies from our laboratory have demonstrated colocalization of SP and NKB mRNAs in the caudate-putamen (6). In contrast, no instance of colocalization was found in the magnocellular basal nucleus (7). In the present study, we demonstrate colocalization of NKB and SP mRNAs in cells of the rat habenula using adjacent thin sections. 165
NEUROPEPTIDES
Ffg 1 a: Section through the medial habenula studied with a probe recognizing NKB mRNA. b: adjacent section studied with a probe against SP mRNA. Double arrows: cells containing both peptides, single, filled arrows: cells containing NKB but not SP mRNA, single, empty arrows: cells containing SP but not NKB mRNA. Large arrows points dorsally, V3D: dorsal part of the third ventricle. Bar equals 30pm.
Methods Serial, 6 pm coronal sections were cut through the habenula of an adult male Sprague-Dawley rat. Alternate sections were hybridized with probes recognizing NKB and SP mRNA, respectively. Hybridization histochemistry was performed as described previously (6-8). After fixation with buffered formaldehyde solution, the sections were acetylated and delipidated. The sections were covered with 45 p,l of 4 x SSC (1 x SSC is 0.15M NaCl/O.OlSM sodium citrate, pH 7.2), 50% (v/v)
formamide, 10% (w/v) dextran sulfate, 5OO&ml yeast tRNA, 1 x Denhardt’s medium and 1OOmM dithiothreitol containing 1 x 106dpm of 35Slabeled oligodeoxyribonucleotide probe under a parafilm coverslip, and incubated at 37°C for 20 hours. The NKB ,probe was complementary to bases 361-408 and the SP probe to bases 124-171 of rat NKB (4) and SP (9) mRNAs, respectively. The specificity of these probes has been studied and described previously (4, 8) and studies of similar sections through the habenula with other probes (e.g. message sense vasopressin) resulted only in
CO-EXPRESSION OF NKB AND SP IN SUBSET OF NEURONS IN THE RAT HABENULA
167
Fig 1 b
background levels of grains. After four 15 min washes in 2 X SSC containing 50% formamide at 40°C followed by two 30 min washes in 1 x SSC at room temperature, the slides were dipped in nuclear emulsion (N7B3, 1:l in water, Kodak) and exposed for 8 weeks. After development, the sections were counterstained with 0.2% toluidine blue and analyzed by brightfield and darkfield microscopy. Cells present on 2 adjancent sections were examined for their content of &I3 mRNA and/or SP mRNA. Results
Low levels of NKB mRNA were found in many
B
cells distributed along the whole rostrocaudal and dorsoventral extent of the medial habenula (Fig la). SP mRNA was found in high levels in cells of the dorsal half of the medial habenula and in low levels in scattered cells of the ventral half of the medial habenula (Fig lb). No cell containing NKEI mRNA was observed in the lateral habenula, whereas scattered cells contained low levels of SP mRNA. Among neurons containing high level of SP mRNA, many also had NKB mRNA. These were more numerous in the dorsolateral than in the dorsomedial part of the medial habenula (Fig 1). Many neurons with.low levels of SP transcripts also contained NKB mRNA. These were located ven-
NEUROPEPTIDES
n
SP
o NKB
* SP hi h + NK 8
l
;pKtw+
Fig 2
Schematic drawing of the medial habenula with a proposed subdivision according to the relative densities of cells containing SP or NKB mRNA alone and cells containing high or low levels of SP mRNA colocalized with NKB mRNA, respectively. DG: dentate gyrus of the hippocampus, LH: lateral habenula, sm: stria medularis thalami, V3D: dorsal part of the third ventricle.
tral to the neurons containing high levels of SP mRNA. Most of the remaining medial habenula neurons in the ventral part of the nucleus had low levels of NKB mRNA and were devoid of SP mRNA (Fig 1). Discussion Histological examination of Nissl stained sections of the habenula reveals that its medial portion is quite homogenous and it is usually not further parcellated (10). Immunohistochemical studies, however, have demonstrated differences between a dorsal part, containing SP (11-13) and a ventral part, containing choline acetyltransferase (11,14). In addition, our results suggest a further division of the dorsal portion into medial and lateral parts and of the ventral portion into dorsal and ventral parts (Fig 2). Neurons in the medial habenula innervate the interpenduncular nucleus (15-16). Recent studies
have pointed to some particularities of this projection. The dorsal portion of the medial habenula projects ipsilaterally to the peripheral borders of the interpeduncular nucleus (17) where SP fibers are found (11, 18, 19). The ventral portion sends fibers to the central core of the same nucleus (17) where abundant cholinergic fibers are located (11, 14, 20). Our results demonstrate that NKB is colocalized with SP and most probably with choline acetyltransferase. They also suggest that, since NKB mRNA is contained in neurons of all subdivisions of the medial habenula, this nucleus provides an additional, diffuse NKB projection to the whole interpeduncular nucleus. Recently tachykinin binding sites in the interpeduncular nucleus have beens studied in detail (21). SP binding sites are situated primarily in the central core of the interpeduncular nucleus. Eledoisin binding sites, probably NKB receptors (22), in contrast, are much more widespread in the interpeduncular nucleus (21), and our proposed projection of NKB neurons to the interpeduncular nucleus coincides with this distribution. Since the physiological effects of NKB are poorly understood at present, it is not yet possible to understand why SP and NKB are present in the same neurons.
References I. Maggio, J. E. (1988). Tachykinins. Ann. Rev. Neurosci., 11: 13-28. 2. Nakanishi. S., (1987) Substance P precursor and kininogens: their structures, gene organizations, and regulation. Physiol. Rev., 67: 1117-1142. 3. Kotani. H., Hoshimary, M., Nawa. H. and Nakanishi, S. (1986). Structure and gene organization of bovine neuromedin K precursor. Proc. Natl. Acad. Sci. (USA), 83: 7074-7078. 4. Bonner, T. I., Affolter. H. U., Young, A. C. and Young, W. S. III (1987). A cDNA encoding the precursor of the rat neuropeptide neurokinin B. Mol. Brain Res., 2: 243-249. 5. Warden M. K. and Young, W. S. III (1988). Distribution of cells containing mRNAs encoding substance P and neurokinin B in the rat central nervous system. J. Comp. Neural.. 272: 90-113. 6. Burgunder J-M. and Young. W. S. III (1988). Neurons with neurokinin B mRNA in the rat magnocellular basal nucleus: distribution. projection and colocalization studies. submitted. 7. Burgunder J-M. and Young W. S. III. Distribution, projection and dopaminergic regulation of the neurokinin
CO-EXPRESSION
OF NKB AND SP IN SUBSET OF NEURONS IN THE RAT HABENULA
B mRNA-containing neurons of the rat caudate-putamen. submitted. 8. Young, W. S. III, Bonner, T. I. and Brann, M. R. (1986). Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proc. Natl. Acad. Sci. USA, 83: 9827-9831. 9. Krause, J. E.. Chirgwin, J. M.. Carter, M. S., Xu, 2. S. and Hershey, A. D. (1987). Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A. Proc. Nat]. Acad. Sci. USA, 84: 881-885. 10. Paxinos, G. and Watson, C. (1986). The rat brain in stereotaxic coordinates. Academic Press Orlando. 11. Constantible A., Villani, L., Fasolo, A., Franzoni, M. F.. Gribaudo, L.. Oktedalen, 0. and Fonnum. F. (1987). Topography of cholinergic and substance P pathways in the habenulo-interpeduncular system of the rat. An immunocytochemical and microchemical approach. Neuroscience, 21: 253-270. 12. Cuello, A. C. and Kanazawa, I. (1978). The distribution of substance P immunoreactive fibers in the rat central nervous system. J. Comp. Neurol., 178: 129-156. 13. Shinoda, K.. Inagaki. S.. Shiosaka. S.. Kohno, J. and Tohyama, M. (1984). Experimental immunohistochemical studies on the substance P neuron system in the lateral habenular nucleus of the rat: distribution and origins. J. Comp. Neurol.. 222: 578-588. 14. Houser, C. R., Crawford. G. D., Barber. R. P.. Salvaterra. P. M. and Vaughn, J. E. (1983). Organization and morphological characteristics of cholinergic neurons: an immunocytohistochemical study with a monoclonal antibody to choline acetyltransferase. Brain Res., 266: 97- 119.
169
15. Herkenham, M. and Nauta. W. J. H. (1979). Efferent connections of the habenular nuclei in the rat. J. Comp. Neurol.. 187: 19-48. 16. Marchand E. R., Riley, J. N. and Moore, R. Y. (1980). Interpeduncular nucleus afferent in the rat. Brain Res.. 1983: 339-352. 17. Kawaja M. D., Flumerfelt, B. A. and Hrycyshyn. A. W. (1988). Topographical and ultrastructural investigation of the habenulo-interpeduncular pathway in the rat: a wheat germ agglutinin-horseradish peroxidase anterograde study. J. Comp. Neurol., 275: 117-127. 18. Hamill, G. S.. Olschowka, J. A.. Lenn. N. J. and Jacobowitz, D. M. (1984). The subnuclear distribution of substance P. cholecystokinin. vasoactive intextinal peptide. somatostatin, leu-enkephalin. dopamine-betahydroxylase, and serotonin in the rat interpenduncular nucleus. J. Comp. Neurol., 226: 580-596. 19. Artymyshyn R. and Murray, M. (1985). Substance Pin the interpeduncular nucleus of the rat: normal distribution and the effects of deafferentation. J. Comp. Neural.. 231: 7X-90. 20. Eckenrode, T. C., Barr, G. A., Battisti, W. P., and Murray. M. (1987). Acetylchoiine in the interpenducular nucleus of the rat: normal distribution and effects of deafferentation. Brain Res.. 418: 273-286. 21. Murray. M., Saffroy, M. Torrens, Y., Beujouan. J. C.. and Glowinski. J. (1988). Tachykinin binding sites in the interpeduncular nucleus of the rat: normal distribution, postnatal; development and the effects of lesions. Brain Res., 459: 76-92. 22. Quirion, R. and Dam, T. V. ( 1988). Multiple neurokinin receptors: recent developments. Reg. Peptides. 22: 18-25.
Neuropeptides (1989) 13,165169 0 Longman Group UK Ltd 1989
Neurokinin Expressed Habenula J-M. BURGUNDER
B and Substance P Genes are Coin a Subset of Neurons in the Rat
and W. S. YOUNG
Ill
Laboratory of Cell Biology, National institute of Mental Health, Building 36, Room 2010, Bethesda, MD 20892 (Reprint requests to J. M. B.).
Abstract-Colocalization of neurokinin B (NKB) and substance P (SP) mRNAs in neurons of the habenula wasxamirt$ on thin, adjacent sections in the rat. Extensive colocalization was found in the medial habenula. In its dorsolateral part, most of the neurons contained both transcripts, with high levels of SP, while in the dorsomedial part fewer instances of colocalization were found. The ventral half could be divided into two parts, a dorsal part with most of the neurons containing both messages, having low levels of SP mRNA, and a ventral part with most of the cells containing only NKB mRNA. Cells in the medial habenula had low levels of NKB mRNA. These results suggest a structural and functional heterogeneity of the medial habenula.
Introduction The tachykinins are a family of peptides present in several species, including mammals (1). In mammals, three kinds of tachykinins have so far been demonstrated: substance P (SP), neurokinin A (NKA) and neurokinin B (NKB). While a common gene encodes SP and NKA, from which several different mRNA species are formed by differential splicing (1, 2), NKB is encoded by a separate gene (3-4). These mammalian tachykinins share common antigenic markers, which have impaired the immunocytochemical localization of neurons containing them until recently
Date received 3 November 1988 Date accepted 8 November 1988
(1). In contrast, the specificities of probes generated against segments of the two mRNA species have allowed studies using in situ hybridization histochemistry to address the localization of neurons expressing SP and NKB in the rat brain (3, 4). A combined mapping study of cells containing SP or NKB mRNA (5) revealed several areas of the rat brain contained both messages, suggesting that they might be colocalized in individual neurons. Recent studies from our laboratory have demonstrated colocalization of SP and NKB mRNAs in the caudate-putamen (6). In contrast, no instance of colocalization was found in the magnocellular basal nucleus (7). In the present study, we demonstrate colocalization of NKB and SP mRNAs in cells of the rat habenula using adjacent thin sections. 165
NEUROPEPTIDES
Ffg 1 a: Section through the medial habenula studied with a probe recognizing NKB mRNA. b: adjacent section studied with a probe against SP mRNA. Double arrows: cells containing both peptides, single, filled arrows: cells containing NKB but not SP mRNA, single, empty arrows: cells containing SP but not NKB mRNA. Large arrows points dorsally, V3D: dorsal part of the third ventricle. Bar equals 30pm.
Methods Serial, 6 pm coronal sections were cut through the habenula of an adult male Sprague-Dawley rat. Alternate sections were hybridized with probes recognizing NKB and SP mRNA, respectively. Hybridization histochemistry was performed as described previously (6-8). After fixation with buffered formaldehyde solution, the sections were acetylated and delipidated. The sections were covered with 45 p,l of 4 x SSC (1 x SSC is 0.15M NaCl/O.OlSM sodium citrate, pH 7.2), 50% (v/v)
formamide, 10% (w/v) dextran sulfate, 5OO&ml yeast tRNA, 1 x Denhardt’s medium and 1OOmM dithiothreitol containing 1 x 106dpm of 35Slabeled oligodeoxyribonucleotide probe under a parafilm coverslip, and incubated at 37°C for 20 hours. The NKB ,probe was complementary to bases 361-408 and the SP probe to bases 124-171 of rat NKB (4) and SP (9) mRNAs, respectively. The specificity of these probes has been studied and described previously (4, 8) and studies of similar sections through the habenula with other probes (e.g. message sense vasopressin) resulted only in
CO-EXPRESSION OF NKB AND SP IN SUBSET OF NEURONS IN THE RAT HABENULA
167
Fig 1 b
background levels of grains. After four 15 min washes in 2 X SSC containing 50% formamide at 40°C followed by two 30 min washes in 1 x SSC at room temperature, the slides were dipped in nuclear emulsion (N7B3, 1:l in water, Kodak) and exposed for 8 weeks. After development, the sections were counterstained with 0.2% toluidine blue and analyzed by brightfield and darkfield microscopy. Cells present on 2 adjancent sections were examined for their content of &I3 mRNA and/or SP mRNA. Results
Low levels of NKB mRNA were found in many
B
cells distributed along the whole rostrocaudal and dorsoventral extent of the medial habenula (Fig la). SP mRNA was found in high levels in cells of the dorsal half of the medial habenula and in low levels in scattered cells of the ventral half of the medial habenula (Fig lb). No cell containing NKEI mRNA was observed in the lateral habenula, whereas scattered cells contained low levels of SP mRNA. Among neurons containing high level of SP mRNA, many also had NKB mRNA. These were more numerous in the dorsolateral than in the dorsomedial part of the medial habenula (Fig 1). Many neurons with.low levels of SP transcripts also contained NKB mRNA. These were located ven-
NEUROPEPTIDES
n
SP
o NKB
* SP hi h + NK 8
l
;pKtw+
Fig 2
Schematic drawing of the medial habenula with a proposed subdivision according to the relative densities of cells containing SP or NKB mRNA alone and cells containing high or low levels of SP mRNA colocalized with NKB mRNA, respectively. DG: dentate gyrus of the hippocampus, LH: lateral habenula, sm: stria medularis thalami, V3D: dorsal part of the third ventricle.
tral to the neurons containing high levels of SP mRNA. Most of the remaining medial habenula neurons in the ventral part of the nucleus had low levels of NKB mRNA and were devoid of SP mRNA (Fig 1). Discussion Histological examination of Nissl stained sections of the habenula reveals that its medial portion is quite homogenous and it is usually not further parcellated (10). Immunohistochemical studies, however, have demonstrated differences between a dorsal part, containing SP (11-13) and a ventral part, containing choline acetyltransferase (11,14). In addition, our results suggest a further division of the dorsal portion into medial and lateral parts and of the ventral portion into dorsal and ventral parts (Fig 2). Neurons in the medial habenula innervate the interpenduncular nucleus (15-16). Recent studies
have pointed to some particularities of this projection. The dorsal portion of the medial habenula projects ipsilaterally to the peripheral borders of the interpeduncular nucleus (17) where SP fibers are found (11, 18, 19). The ventral portion sends fibers to the central core of the same nucleus (17) where abundant cholinergic fibers are located (11, 14, 20). Our results demonstrate that NKB is colocalized with SP and most probably with choline acetyltransferase. They also suggest that, since NKB mRNA is contained in neurons of all subdivisions of the medial habenula, this nucleus provides an additional, diffuse NKB projection to the whole interpeduncular nucleus. Recently tachykinin binding sites in the interpeduncular nucleus have beens studied in detail (21). SP binding sites are situated primarily in the central core of the interpeduncular nucleus. Eledoisin binding sites, probably NKB receptors (22), in contrast, are much more widespread in the interpeduncular nucleus (21), and our proposed projection of NKB neurons to the interpeduncular nucleus coincides with this distribution. Since the physiological effects of NKB are poorly understood at present, it is not yet possible to understand why SP and NKB are present in the same neurons.
References I. Maggio, J. E. (1988). Tachykinins. Ann. Rev. Neurosci., 11: 13-28. 2. Nakanishi. S., (1987) Substance P precursor and kininogens: their structures, gene organizations, and regulation. Physiol. Rev., 67: 1117-1142. 3. Kotani. H., Hoshimary, M., Nawa. H. and Nakanishi, S. (1986). Structure and gene organization of bovine neuromedin K precursor. Proc. Natl. Acad. Sci. (USA), 83: 7074-7078. 4. Bonner, T. I., Affolter. H. U., Young, A. C. and Young, W. S. III (1987). A cDNA encoding the precursor of the rat neuropeptide neurokinin B. Mol. Brain Res., 2: 243-249. 5. Warden M. K. and Young, W. S. III (1988). Distribution of cells containing mRNAs encoding substance P and neurokinin B in the rat central nervous system. J. Comp. Neural.. 272: 90-113. 6. Burgunder J-M. and Young. W. S. III (1988). Neurons with neurokinin B mRNA in the rat magnocellular basal nucleus: distribution. projection and colocalization studies. submitted. 7. Burgunder J-M. and Young W. S. III. Distribution, projection and dopaminergic regulation of the neurokinin
CO-EXPRESSION
OF NKB AND SP IN SUBSET OF NEURONS IN THE RAT HABENULA
B mRNA-containing neurons of the rat caudate-putamen. submitted. 8. Young, W. S. III, Bonner, T. I. and Brann, M. R. (1986). Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proc. Natl. Acad. Sci. USA, 83: 9827-9831. 9. Krause, J. E.. Chirgwin, J. M.. Carter, M. S., Xu, 2. S. and Hershey, A. D. (1987). Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A. Proc. Nat]. Acad. Sci. USA, 84: 881-885. 10. Paxinos, G. and Watson, C. (1986). The rat brain in stereotaxic coordinates. Academic Press Orlando. 11. Constantible A., Villani, L., Fasolo, A., Franzoni, M. F.. Gribaudo, L.. Oktedalen, 0. and Fonnum. F. (1987). Topography of cholinergic and substance P pathways in the habenulo-interpeduncular system of the rat. An immunocytochemical and microchemical approach. Neuroscience, 21: 253-270. 12. Cuello, A. C. and Kanazawa, I. (1978). The distribution of substance P immunoreactive fibers in the rat central nervous system. J. Comp. Neurol., 178: 129-156. 13. Shinoda, K.. Inagaki. S.. Shiosaka. S.. Kohno, J. and Tohyama, M. (1984). Experimental immunohistochemical studies on the substance P neuron system in the lateral habenular nucleus of the rat: distribution and origins. J. Comp. Neurol.. 222: 578-588. 14. Houser, C. R., Crawford. G. D., Barber. R. P.. Salvaterra. P. M. and Vaughn, J. E. (1983). Organization and morphological characteristics of cholinergic neurons: an immunocytohistochemical study with a monoclonal antibody to choline acetyltransferase. Brain Res., 266: 97- 119.
169
15. Herkenham, M. and Nauta. W. J. H. (1979). Efferent connections of the habenular nuclei in the rat. J. Comp. Neurol.. 187: 19-48. 16. Marchand E. R., Riley, J. N. and Moore, R. Y. (1980). Interpeduncular nucleus afferent in the rat. Brain Res.. 1983: 339-352. 17. Kawaja M. D., Flumerfelt, B. A. and Hrycyshyn. A. W. (1988). Topographical and ultrastructural investigation of the habenulo-interpeduncular pathway in the rat: a wheat germ agglutinin-horseradish peroxidase anterograde study. J. Comp. Neurol., 275: 117-127. 18. Hamill, G. S.. Olschowka, J. A.. Lenn. N. J. and Jacobowitz, D. M. (1984). The subnuclear distribution of substance P. cholecystokinin. vasoactive intextinal peptide. somatostatin, leu-enkephalin. dopamine-betahydroxylase, and serotonin in the rat interpenduncular nucleus. J. Comp. Neurol., 226: 580-596. 19. Artymyshyn R. and Murray, M. (1985). Substance Pin the interpeduncular nucleus of the rat: normal distribution and the effects of deafferentation. J. Comp. Neural.. 231: 7X-90. 20. Eckenrode, T. C., Barr, G. A., Battisti, W. P., and Murray. M. (1987). Acetylchoiine in the interpenducular nucleus of the rat: normal distribution and effects of deafferentation. Brain Res.. 418: 273-286. 21. Murray. M., Saffroy, M. Torrens, Y., Beujouan. J. C.. and Glowinski. J. (1988). Tachykinin binding sites in the interpeduncular nucleus of the rat: normal distribution, postnatal; development and the effects of lesions. Brain Res., 459: 76-92. 22. Quirion, R. and Dam, T. V. ( 1988). Multiple neurokinin receptors: recent developments. Reg. Peptides. 22: 18-25.