Brain Research. 558 ( 1991) 33t)-334 © 1991 Elsevier Sciencc Publishers B.V. All rights reserved. 1~X)6-8993/91/$03.51t .4 DONIS 000689939124831( ?
330
BRES 24831
Calcitonin gene-related peptide in monkey spinal cord and medulla oblongata Ulf Arvidsson a, Brun Ulfhake 1, Staffan Cullheim l, Lars Terenius 2 and Tomas H6kfelt
3
Departments of 1Anatomy, 2Experimental Drug Dependence Research and 3Histology and Neurobiology, Karolinska Institute. Stockholm (Sweden) (Accepted 4 June 1991)
Key words: Motoneuron; Raphe nucleus; Bulbospinal pathway; Peroxidase-antiperoxidase technique; Radioimmunoassay; Macaca fascicularis
The distribution of calcitonin gene-related peptide (CGRP)-immunoreactive (IR) fibers and cell bodies was studied in the spinal cord and the medulla oblongata of the grey monkey (Macaca fascicularis) using peroxidase-antiperoxidase (PAP) immunohistochemistry. At all levels of the spinal cord many CGRP-IR motoneurons and fibers were seen in the motor nuclei. In the medulla, CGRP-IR cell bodies were encountered in nucleus raphe obscurus, nucleus raphe pallidus and nucleus raphe magnus, nucleus reticularis lateralis as well as in the area dorsal to the inferior olive. Bulbar motoneurons were much more intensely stained than spinal cord motoneurons, indicating higher levels of CGRP-Iike immunoreactivity (LI) at the medullary level. The concentration of CGRP-L! measured by radioimmunoassay showed higher levels in the dorsal quadrants as compared to the ventral quadrants, but the dorsal/ventral ratio was lower than has previously been reported from the rat. The present results demonstrate that using the PAP technique CGRP-LI can be visualized in a larger number of spinal cord motoneurons of the monkey than earlier revealed by immunofluorescence. Moreover, the finding supports the view that the CGRP-IR nerve endings in the spinal motor nuclei originate from cell bodies in the medullary raphe nuclei. In an earlier paper 3 using the indirect immunofluorescence technique, we have described the presence of calcitonin gene-related peptide T M (CGRP)-Iike immunoreactivity (LI) in 5-hydroxytryptamine (5-HT)immunoreactive (IR) nerve endings in the ventral horn of the m o n k e y (Macaca fascicularis) spinal cord. Since expression of C G R P m R N A could be demonstrated in preprotachykinin m R N A positive, midline raphe neurons using in situ hybridization and since substance P and 5-HT coexist in this region, C G R P may be present in the 5-HT bulbospinal pathway in the monkey. In contrast to cat 2 and rat 9 only a very limited number of C G R P - I R motoneurons could be seen with immunofluorescence in the m o n k e y ventral horn, and C G R P m R N A levels were low. In motoneurons of man C G R P - L I is low but C G R P m R N A levels are high 7. Thus, the distribution of C G R P - L I in the ventral horn of the m o n k e y spinal cord differs in several ways from other species. In the present study we have therefore reexamined the localization of C G R P - L I in the monkey spinal cord ventral horn with a more sensitive technique than indirect immunofluorescence, namely the peroxidase-antiperoxidase (PAP) method originally described by Sternberger et al. TM, and measured the levels of C G R P - L I by radioimmunoassay. Attempts have also
been made to demonstrate C G R P - L I in the raphe nuclei and other medullary nuclei which are known to project to the spinal cord. Four grey monkeys (Macaca fascicularis) were transcardially perfused with Tyrode's solution followed by a fixative containing 4% paraformaldehyde, 0.2% picric acid and 0.5% glutaraldehyde 3. The spinal cord and medulla oblongata were after postfixation (90 min) and saturation in 10% sucrose buffer solution rinsed in Tris buffered saline (TBS), sectioned in a Vibratome (40-50/am) and thereafter treated with 1% sodium borohydride 3. The sections were then processed for the P A P method 14 As primary antibodies against C G R P rabbit polyclonal antiserum (Terenius, unpublished) or a mouse monoclonal antibody (Galfre, unpublished) were used, both at a dilution of 1:2000. G o a t anti-rabbit or goat anti-mouse bridge-antibody (Dakopatts, Copenhagen, Denmark) and rabbit or mouse PAP-antibody complex (Sternberger-Meyer Immunocytochemical Inc., Jarrettsville, MD, U . S . A . ) were used at a dilution of 1:60 and 1:600, respectively. For the localization of peroxidase activity, sections were incubated with diaminobenzidine (Sigma, St. Louis, MO, U . S . A . ) and 0.3% H 2 0 2 dissolved in TBS. The sections were mounted on gelatinized glass and air dried and finally mounted in Entellan (Merck,
Correspondence: U. Arvidsson, Department of Anatomy, Karolinska Institute, P.O. Box 60400, S-104 01 Stockholm, Sweden.
331
Fig. 1. Micrographs from the spinal cord (A,B) and the medulla oblongata ( C - E ) of the monkey after processing for CGRP PAP-immunohistochemistry. A,B: sections are from the lateral (LMN: A) and medial (MMN: B) motor nuclei of the spinal cord. Several motoneurons and varicose fibers containing CGRP-LI are seen in the lateral motor nucleus (A). Labeled cells and fibers are demonstrated in the MMN (B). C-E: sections are from the nucleus raphe magnus (NRM;C), nucleus raphe obscurus (NRO:D), nucleus raphe pallidus (NRP;D) and the lateral reticular nucleus (LRN) and adjacent areas (E) of the medulla oblongata. Intensely CGRP-IR cell bodies can be seen in the raphe nuclei, LRN and in the area dorsal to the inferior olive (IO). Note also the presence of CGRP-IR fibers in these areas. Directions given in E (vent, ventral; lat, lateral) are valid for all plates. Bars = 50/~m ( A = B ; C = D = E ) .
332
Fig. 2. Micrographs from the nucleus hypoglossus (XII;A), nucleus ambiguus (AMB;B) and nucleus abducens (VI;C,D) in the monkey medulla oblongata after processing for CGRP PAP immunohistochemistry. A: many labeled cell bodies can be seen in the bypoglossal nucleus. B: CGRP-IR cell bodies as well as fibers are encountered in the nucleus ambiguus. C,D: many CGRP-positive cell bodies of relatively small size can be demonstrated in nucleus abducens. D: high magnification of area indicated by rectangle in C. Bars - 100 um in D (A=B~D) and 250/xm in C.
Darmstadt, F . R . G . ) and coverslipped. Fresh spinal cord from 3 monkeys were taken for radioimmunoassay (RIA). Each spinal cord segment was divided into ventral and dorsal quadrants. A detailed description of the extraction and assay is found in ref. 12. The specificity of the C G R P antisera was tested by absorption with synthetic h u m a n a - C G R P (Peninsula, Belmont, C A , U . S . A . ) at a concentration of 1 # M for 24 h at 4 °C. No staining was seen after absorption. The staining patterns of C G R P - L I were identical with the two primary antibodies used. A relatively dense terminal network of C G R P - I R fibers was found in the neuropil and in close apposition to large cell bodies in the motor nucleus at all levels of the cord (Fig. 1A). Large cell bodies containing C G R P - L I with a punctate staining pattern were encountered in the lateral motor nucleus at all levels (Fig. 1A). The most intensely stained cell bod-
CGRP-LI in the monkey spinal cord pmol/g w w 100.
80
• VentrAl quad:ant I~1 Dorsal quada'ant
6o
4o 2O 0
i m
C
Th
L
Fig. 3. Histogram illustrating the mean --+ S.D. (n = 3) of the concentration of CGRP-LI (pmol/g w.w.) in the ventral and dorsal quadrants in monkey, at lower cervical (C), mid thoracic (Th) and lower lumbar (L) spinal levels.
333 ies of presumably motoneuron origin were located in the medial motor nucleus, however (Fig. 1B). As a rough estimate, 50% or more of the large cell bodies were CGRP-IR. At the medullary level cell bodies containing CGRP-LI were encountered in the nucleus raphe magnus (Fig. 1C), nucleus raphe obscurus (Fig. 1D) and nucleus raphe pallidus (Fig. 1D), with the highest frequency of positive cell bodies in the nucleus raphe pallidus and nucleus raphe obscurus. In these regions there was also a rather dense network of I R fibers. C G R P - I R cell bodies were also found in the nucleus reticularis lateralis (Fig. 1E) as well as in the area dorsal to the inferior olive (Fig. 1E). A very intense C G R P labeling was encountered in cell bodies of the nucleus hypoglossus (Fig. 2A), nucleus ambiguus (Fig. 2B) and nucleus abducens (Fig. 2C,D). In nucleus ambiguus, a lot of C G R P - I R fibers could also be demonstrated (Fig. 2B). The levels of CGRP-LI as determined with R I A were 3.7-5.9 pmol/g w.w. in the ventral quadrants and 35.986.6 pmol/g w.w. in the dorsal quadrants (Fig. 3). The highest concentrations or CGRP-LI, both in the ventral and dorsal quadrants, were found in the lumbar spinal cord (Fig. 3). The present results show that many motoneurons as well as varicose fibers surrounding the motoneurons in the monkey spinal cord contain CGRP-LI. The frequent occurrence of C G R P - I R motoneurons in this study is in contrast to what we found in an earlier paper using indirect immunofluorescence 3. Thus, with the PAP method motoneurons in the monkey spinal cord contain CGRP-LI in about the same number as has been found in cat and rat 2"9. This may indicate that the amount of CGRP-LI in individual motoneurons in monkey spinal cord is lower and thus that the more sensitive PAP technique is needed to demonstrate these cell bodies. Interestingly, bulbar motoneurons stain much more intensely than spinal cord motoneurons, again suggesting high levels of CGRP-LI in neurons of medullary motor nuclei. Also the cell bodies in the medullary raphe nuclei could be easily visualized, and they thus seem to contain higher levels of C G R P peptide than spinal motoneurons. Neuroanatomical tracing experiments have shown that cell bodies in the raphe nuclei project to the spinal cord in many species including monkey 4-6's'13. Thus, as al-
1 Amara, S.G., Jonas, V., Rosenfeld, M.G., Ong, E.S. and Evans, R.M., Alternative RNA processing in caleitonin gene expression generates mRNAs encoding different polypeptide products, Nature, 298 (1982) 240-244. 2 Arvidsson, U., Cullheim, S., Ulfhake, B., H6kfelt, T. and Terenius, L., Altered levels of calcitonin gene-related peptide (CGRP)-Iike immunoreactivity of cat lumbar motoneurons after chronic spinal cord transection, Brain Research, 489 (1989)
ready suggested 3 C G R P seems to be a peptide present in the descending bulbospinal serotoninergic pathway, in particular the part projecting to the ventral horn. The presence of CGRP-LI in nucleus raphe magnus cell bodies indicates that bulbospinal C G R P - I R fibers may project also to the dorsal horn 13. However, in a previous study no clear coexistence between 5-HT and C G R P could be demonstrated in the dorsal horn 3. This may suggest the existence of nucleus raphe magnus neurons projecting to the dorsal horn which contain CGRP-LI but lack 5-HT, alternatively that C G R P levels in the 5-HT fibers in the dorsal horn contain too low C G R P levels to be detected with the technique used previously 3. The quantitative analysis demonstrated that the concentration of radioimmunoassayable CGRP-LI in the dorsal quadrants is about 10 times higher than in the ventral quadrants. In the rat spinal cord, using the same RIA, the corresponding ratio was about 50 (see ref. 10). Thus, in relation to the dorsal quadrant the monkey ventral quadrant contains 5 times more CGRP-LI than the rat. This difference in all probability reflects the fact that the bulbospinal 5-HT neurons projecting to the ventral horns contain CGRP-LI in the monkey but not in the rat. It should be noticed, however, that the overall concentration of CGRP-LI in the monkey spinal cord, as judged by the present R I A is only about 10% as compared to the levels in rat 1°. Whether this difference is real or whether, for example, monkey C G R P is different from the rat peptide, remains to be analyzed. In summary, our results suggest that in the ventral horn of the monkey C G R P is present in motoneurons and varicose fibers. The fibers probably originate from cell bodies in the raphe nuclei, presumably 5-HT neurons, and the content of CGRP-LI in these fibers should account for a considerable part of all CGRP-LI detected in the ventral spinal cord quadrant.
This study was supported by grants from the Swedish MRC (04X-2887, 04X-3766 and 12X-6815), Marcus and Amalia Wallenbergs Minnesfond, Goljes Minnesfond, Anders Otto Sw~irds Minnesfond and The Swedish Society for Medical Research. We are grateful to Pharmacia AB (Dr. A. H~irfstrand and Ms. A.-S. Hoist) for supply of monkeys, and G. Galfre (Celltech Limited, Berkshire, U.K.) for generous supply of mouse monoclonal CGRP antibody.
387-391. 3 Arvidsson, U., Schalling, M., Cullheim, S,, Ulfhake, B., Terenius, L., Verhofstad, A. and H6kfelt, T., Evidence for coexistence between caleitonin gene-related peptide and serotonin in the bulbospinal pathway in the monkey, Brain Research, 532 (1990) 47-57. 4 Bowker, R.M., Westlund, K.N., Sullivan, M.C., Wilber, J.E and Coulter, J.D., Descending serotonergic, peptidergic and
334
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6
7
8
9
cholinergic pathways from the raphe nuclei: a multiple transmitter complex, Brain Research, 288 (1983) 33-48. Carlton, S.M., Chung, J.M., Leonard, R.B. and Willis, W.D., Funicular trajectories of brainstem neurons projecting to the lumbar spinal cord in the monkey (Macaca fascicularis): a retrograde labeling study, J. Comp. Neurol., 241 (1985) 382-404. Dahlstr~m, A. and Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system-lI. Experimentally induced changes in the intraneuronal amine levels of the bulbospinal neuron systems, Acta Physiol. Scand., 62 (1965) 5-36. Gibson, S.J., Polak, J.M., Giaid, A., Hamid, Q.A., Kar, S., Jones, P.M., Denny, P., Legon, S., Amara, S.G., Craig, R.K., Bloom, S.R., Penketh, R.J.A., Rodek, C., Ibrahim, N.B.N. and Dawson, A., Calcitonin gene-related peptide messenger RNA is expressed in sensory neurones of the dorsal root ganglia and also in spinal motoneurones in man and rat, Neurosci. Lett., 91 (1988) 283-288. Holstege, J.C. and Kuypers, H.G.J.M., Brainstem projections to spinal motoneurons: an update, Neuroscience, 23 (1987) 809821. Kruger, L., Mantyh, P.W., Sternini, C., Brecha, N.C. and Mantyh, C.R., Calcitonin gene-related peptide (CGRP) in the rat
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11
12
13
14
central nervous system: patterns of immunoreactivity and receptor binding sites, Brain Research, 463 (1988) 223-244. Piehl, F., Arvidsson, U., Johnson, H., Cullheim, S., Villar, M., Dagerlind, A., Terenius, L., HOkfelt, T. and Ulfhake, B., Calcitonin gene-related peptide (CGRP)-like immunoreactivity and CGRP mRNA in rat spinal cord motoneurons after different types of lesions, Eur. J. Neurosci., in press. Rosenfeld, M.G., Mermod, J.-J., Amara, S.G., Swanson, L.W., Sawchenko, P.E., Rivier, J., Vale, W.W. and Evans, R.M., Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing, Nature. 304 (1983) 129-135. Sakurada, T., Le Grev6s, P. and Terenius, L., Fragmentation of calcitonin gene-related peptide and substance P in the rat central nervous system, Neurochem. Int., in press, Skagerberg, G. and Bj6rklund, A., Topographic principles in the spinal projections of serotonergic and non-serotonergic brainstem neurons in the rat, Neuroscience, 15 (1985) 445-480. Sternberger, L.A., Hardy, P.H., Cueulis, J.J. and Meyer, H.G., The unlabelled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish-peroxidase-anti-horseradisb peroxidase) and its use in the identification of spirochetes, J. Histochem. Cytochem.. 18 (1970) 315-333.
330
BRES 24831
Calcitonin gene-related peptide in monkey spinal cord and medulla oblongata Ulf Arvidsson a, Brun Ulfhake 1, Staffan Cullheim l, Lars Terenius 2 and Tomas H6kfelt
3
Departments of 1Anatomy, 2Experimental Drug Dependence Research and 3Histology and Neurobiology, Karolinska Institute. Stockholm (Sweden) (Accepted 4 June 1991)
Key words: Motoneuron; Raphe nucleus; Bulbospinal pathway; Peroxidase-antiperoxidase technique; Radioimmunoassay; Macaca fascicularis
The distribution of calcitonin gene-related peptide (CGRP)-immunoreactive (IR) fibers and cell bodies was studied in the spinal cord and the medulla oblongata of the grey monkey (Macaca fascicularis) using peroxidase-antiperoxidase (PAP) immunohistochemistry. At all levels of the spinal cord many CGRP-IR motoneurons and fibers were seen in the motor nuclei. In the medulla, CGRP-IR cell bodies were encountered in nucleus raphe obscurus, nucleus raphe pallidus and nucleus raphe magnus, nucleus reticularis lateralis as well as in the area dorsal to the inferior olive. Bulbar motoneurons were much more intensely stained than spinal cord motoneurons, indicating higher levels of CGRP-Iike immunoreactivity (LI) at the medullary level. The concentration of CGRP-L! measured by radioimmunoassay showed higher levels in the dorsal quadrants as compared to the ventral quadrants, but the dorsal/ventral ratio was lower than has previously been reported from the rat. The present results demonstrate that using the PAP technique CGRP-LI can be visualized in a larger number of spinal cord motoneurons of the monkey than earlier revealed by immunofluorescence. Moreover, the finding supports the view that the CGRP-IR nerve endings in the spinal motor nuclei originate from cell bodies in the medullary raphe nuclei. In an earlier paper 3 using the indirect immunofluorescence technique, we have described the presence of calcitonin gene-related peptide T M (CGRP)-Iike immunoreactivity (LI) in 5-hydroxytryptamine (5-HT)immunoreactive (IR) nerve endings in the ventral horn of the m o n k e y (Macaca fascicularis) spinal cord. Since expression of C G R P m R N A could be demonstrated in preprotachykinin m R N A positive, midline raphe neurons using in situ hybridization and since substance P and 5-HT coexist in this region, C G R P may be present in the 5-HT bulbospinal pathway in the monkey. In contrast to cat 2 and rat 9 only a very limited number of C G R P - I R motoneurons could be seen with immunofluorescence in the m o n k e y ventral horn, and C G R P m R N A levels were low. In motoneurons of man C G R P - L I is low but C G R P m R N A levels are high 7. Thus, the distribution of C G R P - L I in the ventral horn of the m o n k e y spinal cord differs in several ways from other species. In the present study we have therefore reexamined the localization of C G R P - L I in the monkey spinal cord ventral horn with a more sensitive technique than indirect immunofluorescence, namely the peroxidase-antiperoxidase (PAP) method originally described by Sternberger et al. TM, and measured the levels of C G R P - L I by radioimmunoassay. Attempts have also
been made to demonstrate C G R P - L I in the raphe nuclei and other medullary nuclei which are known to project to the spinal cord. Four grey monkeys (Macaca fascicularis) were transcardially perfused with Tyrode's solution followed by a fixative containing 4% paraformaldehyde, 0.2% picric acid and 0.5% glutaraldehyde 3. The spinal cord and medulla oblongata were after postfixation (90 min) and saturation in 10% sucrose buffer solution rinsed in Tris buffered saline (TBS), sectioned in a Vibratome (40-50/am) and thereafter treated with 1% sodium borohydride 3. The sections were then processed for the P A P method 14 As primary antibodies against C G R P rabbit polyclonal antiserum (Terenius, unpublished) or a mouse monoclonal antibody (Galfre, unpublished) were used, both at a dilution of 1:2000. G o a t anti-rabbit or goat anti-mouse bridge-antibody (Dakopatts, Copenhagen, Denmark) and rabbit or mouse PAP-antibody complex (Sternberger-Meyer Immunocytochemical Inc., Jarrettsville, MD, U . S . A . ) were used at a dilution of 1:60 and 1:600, respectively. For the localization of peroxidase activity, sections were incubated with diaminobenzidine (Sigma, St. Louis, MO, U . S . A . ) and 0.3% H 2 0 2 dissolved in TBS. The sections were mounted on gelatinized glass and air dried and finally mounted in Entellan (Merck,
Correspondence: U. Arvidsson, Department of Anatomy, Karolinska Institute, P.O. Box 60400, S-104 01 Stockholm, Sweden.
331
Fig. 1. Micrographs from the spinal cord (A,B) and the medulla oblongata ( C - E ) of the monkey after processing for CGRP PAP-immunohistochemistry. A,B: sections are from the lateral (LMN: A) and medial (MMN: B) motor nuclei of the spinal cord. Several motoneurons and varicose fibers containing CGRP-LI are seen in the lateral motor nucleus (A). Labeled cells and fibers are demonstrated in the MMN (B). C-E: sections are from the nucleus raphe magnus (NRM;C), nucleus raphe obscurus (NRO:D), nucleus raphe pallidus (NRP;D) and the lateral reticular nucleus (LRN) and adjacent areas (E) of the medulla oblongata. Intensely CGRP-IR cell bodies can be seen in the raphe nuclei, LRN and in the area dorsal to the inferior olive (IO). Note also the presence of CGRP-IR fibers in these areas. Directions given in E (vent, ventral; lat, lateral) are valid for all plates. Bars = 50/~m ( A = B ; C = D = E ) .
332
Fig. 2. Micrographs from the nucleus hypoglossus (XII;A), nucleus ambiguus (AMB;B) and nucleus abducens (VI;C,D) in the monkey medulla oblongata after processing for CGRP PAP immunohistochemistry. A: many labeled cell bodies can be seen in the bypoglossal nucleus. B: CGRP-IR cell bodies as well as fibers are encountered in the nucleus ambiguus. C,D: many CGRP-positive cell bodies of relatively small size can be demonstrated in nucleus abducens. D: high magnification of area indicated by rectangle in C. Bars - 100 um in D (A=B~D) and 250/xm in C.
Darmstadt, F . R . G . ) and coverslipped. Fresh spinal cord from 3 monkeys were taken for radioimmunoassay (RIA). Each spinal cord segment was divided into ventral and dorsal quadrants. A detailed description of the extraction and assay is found in ref. 12. The specificity of the C G R P antisera was tested by absorption with synthetic h u m a n a - C G R P (Peninsula, Belmont, C A , U . S . A . ) at a concentration of 1 # M for 24 h at 4 °C. No staining was seen after absorption. The staining patterns of C G R P - L I were identical with the two primary antibodies used. A relatively dense terminal network of C G R P - I R fibers was found in the neuropil and in close apposition to large cell bodies in the motor nucleus at all levels of the cord (Fig. 1A). Large cell bodies containing C G R P - L I with a punctate staining pattern were encountered in the lateral motor nucleus at all levels (Fig. 1A). The most intensely stained cell bod-
CGRP-LI in the monkey spinal cord pmol/g w w 100.
80
• VentrAl quad:ant I~1 Dorsal quada'ant
6o
4o 2O 0
i m
C
Th
L
Fig. 3. Histogram illustrating the mean --+ S.D. (n = 3) of the concentration of CGRP-LI (pmol/g w.w.) in the ventral and dorsal quadrants in monkey, at lower cervical (C), mid thoracic (Th) and lower lumbar (L) spinal levels.
333 ies of presumably motoneuron origin were located in the medial motor nucleus, however (Fig. 1B). As a rough estimate, 50% or more of the large cell bodies were CGRP-IR. At the medullary level cell bodies containing CGRP-LI were encountered in the nucleus raphe magnus (Fig. 1C), nucleus raphe obscurus (Fig. 1D) and nucleus raphe pallidus (Fig. 1D), with the highest frequency of positive cell bodies in the nucleus raphe pallidus and nucleus raphe obscurus. In these regions there was also a rather dense network of I R fibers. C G R P - I R cell bodies were also found in the nucleus reticularis lateralis (Fig. 1E) as well as in the area dorsal to the inferior olive (Fig. 1E). A very intense C G R P labeling was encountered in cell bodies of the nucleus hypoglossus (Fig. 2A), nucleus ambiguus (Fig. 2B) and nucleus abducens (Fig. 2C,D). In nucleus ambiguus, a lot of C G R P - I R fibers could also be demonstrated (Fig. 2B). The levels of CGRP-LI as determined with R I A were 3.7-5.9 pmol/g w.w. in the ventral quadrants and 35.986.6 pmol/g w.w. in the dorsal quadrants (Fig. 3). The highest concentrations or CGRP-LI, both in the ventral and dorsal quadrants, were found in the lumbar spinal cord (Fig. 3). The present results show that many motoneurons as well as varicose fibers surrounding the motoneurons in the monkey spinal cord contain CGRP-LI. The frequent occurrence of C G R P - I R motoneurons in this study is in contrast to what we found in an earlier paper using indirect immunofluorescence 3. Thus, with the PAP method motoneurons in the monkey spinal cord contain CGRP-LI in about the same number as has been found in cat and rat 2"9. This may indicate that the amount of CGRP-LI in individual motoneurons in monkey spinal cord is lower and thus that the more sensitive PAP technique is needed to demonstrate these cell bodies. Interestingly, bulbar motoneurons stain much more intensely than spinal cord motoneurons, again suggesting high levels of CGRP-LI in neurons of medullary motor nuclei. Also the cell bodies in the medullary raphe nuclei could be easily visualized, and they thus seem to contain higher levels of C G R P peptide than spinal motoneurons. Neuroanatomical tracing experiments have shown that cell bodies in the raphe nuclei project to the spinal cord in many species including monkey 4-6's'13. Thus, as al-
1 Amara, S.G., Jonas, V., Rosenfeld, M.G., Ong, E.S. and Evans, R.M., Alternative RNA processing in caleitonin gene expression generates mRNAs encoding different polypeptide products, Nature, 298 (1982) 240-244. 2 Arvidsson, U., Cullheim, S., Ulfhake, B., H6kfelt, T. and Terenius, L., Altered levels of calcitonin gene-related peptide (CGRP)-Iike immunoreactivity of cat lumbar motoneurons after chronic spinal cord transection, Brain Research, 489 (1989)
ready suggested 3 C G R P seems to be a peptide present in the descending bulbospinal serotoninergic pathway, in particular the part projecting to the ventral horn. The presence of CGRP-LI in nucleus raphe magnus cell bodies indicates that bulbospinal C G R P - I R fibers may project also to the dorsal horn 13. However, in a previous study no clear coexistence between 5-HT and C G R P could be demonstrated in the dorsal horn 3. This may suggest the existence of nucleus raphe magnus neurons projecting to the dorsal horn which contain CGRP-LI but lack 5-HT, alternatively that C G R P levels in the 5-HT fibers in the dorsal horn contain too low C G R P levels to be detected with the technique used previously 3. The quantitative analysis demonstrated that the concentration of radioimmunoassayable CGRP-LI in the dorsal quadrants is about 10 times higher than in the ventral quadrants. In the rat spinal cord, using the same RIA, the corresponding ratio was about 50 (see ref. 10). Thus, in relation to the dorsal quadrant the monkey ventral quadrant contains 5 times more CGRP-LI than the rat. This difference in all probability reflects the fact that the bulbospinal 5-HT neurons projecting to the ventral horns contain CGRP-LI in the monkey but not in the rat. It should be noticed, however, that the overall concentration of CGRP-LI in the monkey spinal cord, as judged by the present R I A is only about 10% as compared to the levels in rat 1°. Whether this difference is real or whether, for example, monkey C G R P is different from the rat peptide, remains to be analyzed. In summary, our results suggest that in the ventral horn of the monkey C G R P is present in motoneurons and varicose fibers. The fibers probably originate from cell bodies in the raphe nuclei, presumably 5-HT neurons, and the content of CGRP-LI in these fibers should account for a considerable part of all CGRP-LI detected in the ventral spinal cord quadrant.
This study was supported by grants from the Swedish MRC (04X-2887, 04X-3766 and 12X-6815), Marcus and Amalia Wallenbergs Minnesfond, Goljes Minnesfond, Anders Otto Sw~irds Minnesfond and The Swedish Society for Medical Research. We are grateful to Pharmacia AB (Dr. A. H~irfstrand and Ms. A.-S. Hoist) for supply of monkeys, and G. Galfre (Celltech Limited, Berkshire, U.K.) for generous supply of mouse monoclonal CGRP antibody.
387-391. 3 Arvidsson, U., Schalling, M., Cullheim, S,, Ulfhake, B., Terenius, L., Verhofstad, A. and H6kfelt, T., Evidence for coexistence between caleitonin gene-related peptide and serotonin in the bulbospinal pathway in the monkey, Brain Research, 532 (1990) 47-57. 4 Bowker, R.M., Westlund, K.N., Sullivan, M.C., Wilber, J.E and Coulter, J.D., Descending serotonergic, peptidergic and
334
5
6
7
8
9
cholinergic pathways from the raphe nuclei: a multiple transmitter complex, Brain Research, 288 (1983) 33-48. Carlton, S.M., Chung, J.M., Leonard, R.B. and Willis, W.D., Funicular trajectories of brainstem neurons projecting to the lumbar spinal cord in the monkey (Macaca fascicularis): a retrograde labeling study, J. Comp. Neurol., 241 (1985) 382-404. Dahlstr~m, A. and Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system-lI. Experimentally induced changes in the intraneuronal amine levels of the bulbospinal neuron systems, Acta Physiol. Scand., 62 (1965) 5-36. Gibson, S.J., Polak, J.M., Giaid, A., Hamid, Q.A., Kar, S., Jones, P.M., Denny, P., Legon, S., Amara, S.G., Craig, R.K., Bloom, S.R., Penketh, R.J.A., Rodek, C., Ibrahim, N.B.N. and Dawson, A., Calcitonin gene-related peptide messenger RNA is expressed in sensory neurones of the dorsal root ganglia and also in spinal motoneurones in man and rat, Neurosci. Lett., 91 (1988) 283-288. Holstege, J.C. and Kuypers, H.G.J.M., Brainstem projections to spinal motoneurons: an update, Neuroscience, 23 (1987) 809821. Kruger, L., Mantyh, P.W., Sternini, C., Brecha, N.C. and Mantyh, C.R., Calcitonin gene-related peptide (CGRP) in the rat
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