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Does cholecystokinin-like immunoreactivity in rat primary sensory neurons represent calcitonin gene-related peptide?
Neuroscience Letters, 68 (1986) 305 310
305
Elsevier Scientific Publishers Ireland Ltd.
NSL 04072
DOES CHOLECYSTOKININ-LIKE IMMUNOREACTIVITY IN RAT PRIMARY SENSORY N E U R O N S REPRESENT CALCITONIN GENE-RELATED PEPTIDE?
G O N G JU L, T O M A S H O K F E L T l'*, J A N A. F I S C H E R 2, P E T E R F R E Y 3, JENS F. R E H F E L D 4 and G R A H A M J. D O C K R A Y 5
1Department qf Histology, Karolinska Institutet, P.O. Box 60400, S-104 O1 Stockholm (Sweden); :Research Laboratoo'Jbr Calcium Metabolism, Department of Orthopedic Surgery and Medicine, University o['Ziirich, Ziirieh and 3Wander Research Institute (a Sandoz Research Unit), Wander LTD, Bern (Switzerland); 4Department of Clinical Chemistry, Rigshospitalet, University of Copenhagen, Copenhagen (Denmark) and 5The Physiological Laboratory, University of Liverpool, Liverpool ( U.K. ) (Received April 14th, 1986: Revised version received and accepted May 5th, 1986)
Key words:
dorsal root ganglia - spinal cord - immunohistochemistry cross-reactivity- rat
Using immunohistochemistry, calcitonin gene-related peptide (CGRP)- and cholecystokinin (CCK)-like immunoreactivity (LI) were found in m a n y of the same spinal and trigeminal ganglion cells and motoneurons in the spinal cord and hypoglossal nucleus, as well as in fibers with an overlapping distribution in the spinal cord (dorsal horn, bundle ventral to the central canal) and in the spinal trigeminal nucleus. CCK-LI in all these structures disappeared after preadsorption of C C K antiseva with C G R P at l0 4 M and almost completely at 10 5 M. C C K peptide in concentrations up to 10 4 M, on the other hand, did not influence C G R P staining. The present findings raise the possibility that some CCK-LI in primary sensory neurons in rat may represent C G R P or a similar peptide.
Using immunohistochemicai techniques, cholecystokinin (CCK)-like immunoreactivity (LI) has been demonstrated in the superficial layers in the dorsal horn of the spinal cord and in spinal ganglion cells, suggesting a localization in primary sensory neurons [2, 4, 9-11, 13-15, 17, 18, 22-24, 27, 28], a view supported by the immunohistochemical demonstration that CCK-LI, to a large extent, disappears from the superficial layers of the dorsal horn after capsaicin treatment [2, ! l, 17, 22-24]. The latter findings are, however, challenged by biochemical analysis with radioimmunoassay (RIA) showing that capsaicin treatment [12] does not appear to change CCK levels [7a, 18, 25]. More recently a novel peptide, calcitonin gene-related peptide (CGRP) [1, 21], has been demonstrated in many primary sensory neurons and in a dense fiber network in the superficial layers of the dorsal horn [8, 21]. In the present paper we have analysed, with immunohistochemistry, the relation between CGRP- and CCK-LI in primary sensory neurons of rat. *Author for correspondence.
0304-3940/86;$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
306
Male Sprague-Dawley rats ( n = 4 ; body wt. 150-200 g; ALAB, Stockholm, Sweden) were used. Colchicine (100 lag in 10 lal) was injected into the subarachnoidal space of the lumbar spinal cord. Twenty-four to 48 h after injection, the rats were perfused with formalin-containing picric acid in phosphate buffer [29]. The medulla oblongata, the spinal cord and spinal ganglia were dissected out and immersed in the same fixative, rinsed and cut on a cryostat (section thickness 8-14 Jam) and processed for the indirect immunofluorescence technique [3]. Briefly, serial sections were incubated with, respectively, goat antiserum raised against rat C G R P (antiserum 216; 1:400) [26], rabbit antisera against the CCK-analogue, non-sulfated gastrin-17 (antiserum 4562; 1:400) [19], non-sulfated CCK-8 (CCK-8NS) (antiserum 18: 1:400) [7], or CCK-4 (antiserum L112; 1:400) [6]. All 3 CCK antisera were directed towards the last 4 amino acids of the C-terminal of CCK-33. The sections were then rinsed, incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Amersham, Amersham, England), rinsed, mounted, examined in a fluorescence microscope and photographed. Incubations were also carried out with the antisera preadsorbed with varying concentrations of peptides. Thus, the C G R P antiserum was preadsorbed with rat C G R P (10 - 7 t o 10 - 4 M ) and with CCK-8NS or sulfated CCK-8 (CCK-8S) (10 7 t o 10 - 4 M); and CCK antisera were preadsorbed with CCK-8NS or CCK-8S ( 1 0 7 t o l0 4 M) and with rat C G R P (10 - 7 t o l 0 - 4 M ) for 2 h at room temperature or at +4:'C overnight. All peptides were purchased from Peninsula (Belmont, CA, U.S.A.). After incubation of sections of spinal and trigeminal ganglia of colchicine-treated rats with C G R P antiserum, numerous cell bodies of varying size (15-60 lam in diameter) were seen to be immunoreactive (Fig. la, c). Incubation with antisera to CCK (Fig. lb) revealed fluorescent cell bodies in similar numbers and of similar size. In fact, CGRP- and CCK-LI were in many cases present in the same cell bodies, as seen in adjacent sections (cf. Fig. la, b). Since no double staining analysis or elution-restaining analysis was performed, it was not possible to establish whether or not all cell bodies contained both immunoreactivities. In the dorsal horn of the lumbar spinal cord and in the superficial layers of the spinal trigeminal nucleus dense networks of C G R P - and CCK-positive fibers were observed in the superficial layers with lower numbers of fibers also in deeper layers. In lamina X of the spinal cord, single CCK-positive cell bodies and a patchy fiber network were observed, as well as a distinct, transversely cut fiber bundle ventral to
Fig. h a-j: immunofluorescence micrographs o f L3 spinal ganglion (a-e), dorsal horn of the spinal cord (L4 level) (f and g), and lamina X of the lumbar spinal cord (h-j) after incubation with antiserum to CGRP (a and c), to CCK-8 (b, l a n d h), to C G R P preadsorbed with C G R P (d) or with CCK-8 (e) and to CCK-8 preadsorbed with C G R P (g and j) or with CCK-8 (i). a-e: CGRP- and CCK-8-immunoreactive cell bodies show a similar distribution and presence in the same cell bodies (arrows and arrowheads) (a and b). CGRP-LI disappears after preadsorption with CGRP (cf. c and d) but not after preadsorption with CCK8 (cf. c and e). Many cell bodies can be identified in both sections (arrowheads in c and e) (c~e). f and g: a dense network of CCK-8-positive fibers can be seen in the external layer (f), and they disappear after preadsorption with CGRP (g). h-j: CCK-8-immunoreactive fibers and some cell bodies can be seen in
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i l a m i n a X (h), and these fibers d i s a p p e a r after p r e a d s o r p t i o n with C C K - 8 (i) but not with ( ' G R P (j). H o w ever. the small, l r a n s v e r s a l l y cut fiber b u n d l e ( a r r o w h e a d ) ventral to the central canal (asterisks) c a n n o t be seen after p r e t r e a t m e n t with C G R P . Bars = 50 lam; a and b on the one h a n d and c j, on the other, have the same magnification.
308 the central canal (Fig. lh). With CGRP antiserum only the latter fiber bundle was seen in lamina X. Many motoneurons in the spinal cord and medulla oblongata (hypoglossal nucleus) were both CGRP- and CCK-immunoreactive. All three CCK antisera revealed identical staining patterns. The preadsorption experiments revealed that pretreatment of CGRP antiserum with CGRP peptide (down to 10 -7 M) completely abolished the immunostaining (cf. Fig. lc and ld), whereas sulfated CCK octapeptide (CCK-8S) in concentrations up t o 10 4 M did not influence CGRP staining (Fig. le). These results were obtained both in spinal and trigeminal ganglia, spinal cord, spinal trigeminal nucleus and hypoglossal nucleus. Incubation with CCK antisera preadsorbed with CCK-8S peptide abolished staining in ganglia, spinal cord, spinal trigeminal and hypoglossal nuclei in concentrations down to 10 - 7 M (Fig. li). Pretreatment of the same antiserum with CGRP at 10 - 4 M completely abolished CCK staining in these structures (cf. Fig. If, g), except in lamina X where only the fiber bundle ventral to the central canal disappeared, whereas the patchy fiber network and cell bodies remained (cf. Fig. lh, j). At a concentration of 10 --5 M, CGRP almost completely abolished staining in these structures. No effect of CGRP at 10 - 6 M could be observed. The present results confirm many early studies demonstrating that primary sensory neurons contain CCK- and CGRP-LI when analysed with immunohistochemistry [2, 4, 8-l 1, 13-15, 17, 18, 21-24, 27, 28] and show that the two immunoreactivities often are found in the same cell bodies. Moreover, the present result raises the possibility that CCK-LI, visualized with antisera directed towards the C-terminal portion of CCK-33, cross-react with the newly discovered peptide CGRP [1, 21], or perhaps a so far unknown, CGRP-like peptide. Therefore the CCK immunostaining in rat can be artefactual. It should, however, be remembered that the concentrations of CGRP needed to block CCK staining are high and that it is not known whether such high concentrations are present in these sensory neurons. On the other hand, peptides are probably stored in high concentrations in vesicles. In RIA experiments both humanCGRP-I and rat-CGRP cross-react with the antiserum raised against CCK-8NS but at concentrations approximately 100,000 times higher than for CCK-8S (Frey, unpublished results). The present results may explain some discrepancies in the earlier literature. Thus, whereas CCK-Li disappears after capsaicin treatment when monitored with immunohistochemistry, no such effects on CCK-LI could be observed by RIA [7a, ! 8, 23]. Capsaicin-induced disappearance of CCK-L1 from primary sensory neurons seen with immunohistochemistry [2, 11, 17, 23, 24] could in fact reflect depletion of CGRP, or another cross-reacting peptide. The present findings do not exclude the possibility that some of the immunoreactivity seen after incubation with CCK antiserum, in fact, represents a CCK-like peptide or that these neurons contain both CGRP- and CCK-LI. However, the levels of CCK-8 in spinal ganglia are very low [23] or below the limit of detection [18]. In contrast, concentrations of CGRP in rat dorsal root ganglia range between 130 and 260 pmol/g wet weight of tissue [8]. Sensory systems in other species, such as the vagus nerve in the cat, may, however, contain genuine CCK [5, 20].
309 C C K - 8 S a n d the carboxyl t e r m i n u s o f C G R P - 3 7 exhibit limited structural h o m o logy, with glycine residues in positions 4 a n d 33, respectively, a n d the t e r m i n a l phenylalanines are a m i d a t e d in b o t h cases. This very low degree of identity in crucial positions m a y be sufficient to cause cross-reactivity. It should be n o t e d that specific antisera can be o b t a i n e d to small molecules such as 5 - h y d r o x y t r y p t a m i n e [25]. Moreover, extensive cross-reactivities have earlier been e n c o u n t e r e d , for example, a m o n g the family of pancreatic polypeptides, where several m e m b e r s share the same two Cterminal a m i n o acids with the t e r m i n a l tyrosine a m i d a t e d [16]. The present results further underline the risk o f unspecific reactions when using i m m u n o h i s t o c h e m i c a l techniques a n d focus particularly o n a m i d a t e d C - t e r m i n a l a m i n o acids as antigenic sites with a high potential for cross-reactivity. The present study was s u p p o r t e d by the Swedish Medical Research Council (04X2887), M a g n u s Bergvalls Stiftelse, Alice och K n u t W a l l e n b e r g s Stiftelse a n d the Swiss N a t i o n a l Science F o u n d a t i o n ( G r a n t 3.957-0.84). G.J., on leave from the D e p a r t m e n t of N e u r o b i o l o g y , The F o u r t h Military Medical College, Xian, People's R e p u b lic of China, was s u p p o r t e d by LKB, S t o c k h o l m , Sweden. The skillful technical assistance of Ms. W. Hiort a n d the expert secretarial help of Ms. E. B j 6 r k l u n d are gratefully acknowledged. 1 Amara, S.G., Jonas, V., Rosenfeld, M.G., Ong, E.S. and Evans, R.M., Alternative RNA-processing in calcitonin gene expression generates mRNAs encoding different polypeptide products, Nature (London), 298 (1982) 240-244. 2 Conrath-Verrier, M., Dietl, M. and Trarnu, G., Cholecystokinin-like immunoreactivity in the dorsal horn of the spinal cord of the rat: a light and electron microscopic study, Neuroscience, 13 (1984) 871 885. 3 Coons, A.H., Fluorescent antibody methods. In J.F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, 1958, pp. 399-422. 4 Dalsgaard, C.-J., Vincent, S.R., H6kfelt, T., Lundberg, J.M., Dahlstr6m, A., Schultzberg, M., Dockray, G.J. and Cuello, A.C., Coexistence of cholecystokinin- and substance P-like peptides in neurons of the dorsal root ganglia of the rat, Neurosci. Lett., 33 (1982) 159 164. 5 Dockray, G.J., Gregory, R.A., Tracy, H.J. and Zhu, W.-Y., Transport of cholecystokinin-octapeptide-like immunoreactivity toward the gut in afferent vagal fibres in cat and dog, J. Physiol. (London), 314(1981)501 511. 6 Dockray, G.J., Williams, R.G. and Zhu, W.-Y., Development of region-specificantisera for the Cterminal tetrapeptide of gastrin/cholecystokinin and their use in studies of immunoreactive forms of cholecystokinin in rat brain, Neurochem. Int., 3 (1981) 281-288. 7 Frey,P., Cholecystokinin octapeptide (CCK 26-33), nonsulfated octapeptide and tetrapeptide (CCK 30 33) in rat brain: analysis by high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA), Neurochem. Int., 5 (1985) 811-815. 7a Gibson, S.J., McGregor, G., Bloom, S.R., Polak, J.M. and Wall, P.D., Local application of capsaicin to one sciatic nerve of the adult rat induces a marked depletion in the peptide content of the lumbar dorsal horn, Neuroscience, 7 (1982) 3153-3162. 8 Gibson, S.J., Polak, J.M., Bloom, S.R., Sabate, I.M., Mulderry, P.M., Ghatei, M.A., McGregor, G.P., Morrison, J.F.B., Kelly, J.S., Evans, R.M. and Rosenfeld, M.G., Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species, J. Neurosci., 4 (1984) 3101 3111. 9 Gibson, J.G., Polak, J.M., Bloom, S.R. and Wall, P.D., The distribution of nine peptides in rat spinal cord with special cmphasis on the substantia gelatinosa and on the area around the central canal (lamina X), J. Comp. Neurol., 201 (1981) 65 79.
3 II~
10 H6kfelt, T., Skirboll, L., Everitt, B.J., Meister, t3., Brownstein, M., Jacobs, T., Faden, A., Kuga, S.. Goldstein, M., Markstein, R., Dockray, G. and Rehfeld, J., Distribution of cholecystokinin-like immunoreactivity ira the nervous system with special reference to coexistence with classical ncurotransmitters and other neuropeptides, Ann. N.Y. Acad. Sci., 448 (1985) 255-274. 11 Jancsd, G., H6kfelt, T., Lundberg, J.M.. Kiraly, E., Halasz, N., Nilsson, G., Terenius. L., Rehfeld. J., Steinbusch, H., Verhofstad, A., Elde, R., Said, S. and Brown, M., Immunohistochemical studies on the effect of capsaicin on peptide and monoamine neurons using antisera to substance P, gastrin, CCK, somatostatin, VIP, enkephalin, neurotensin and 5-hydroxytryptamine, J. Neurocytol., t0 (1981) 963 980. 12 Jancsd, G. and Kiraly, E., Distribution of chemosensitive primary sensory afferents in the central nervous system of the rat, J. Comp. Neurol., 190 (1980) 781 792. 13 Larsson, L.-I. and Rehfeld, J.F., Localization and molecular heterogeneity of cholecystokinin in the central and peripheral nervous system, Brain Res., 165 (1979) 201 218. 14 Leah, J.D., Cameron, A.A., Kelly, W.L. and Snow, P.J., Coexistence of peptide immunoreactivity in sensory neurons of the cat, Neuroscience, 16 (1985) 683 690. 15 Lorbn, I., Alumets, J., H'~kanson, R.H. and Sundler, F., Distribution of gastrin and cholecystokininlike peptides in rat brain, Histochemistry, 59 (1979) 249-257. 16 Ltmdberg, J.M., Terenius, L., H6kfelt, T. and Tatemoto, K., Comparative immunohistoehemical and biochemical analysis of pancreatic polypeptide Y in central and peripheral neurons, J. Neurosci., 4 (1984) 237(~ 2386. 17 Maderdrut, J.L., Yaksh, T.L., Petrusz, P. and Go, V.L.W., Origin and distribution of cholecystokinin-containing ncrve terminals in the lumbar dorsal horn and nucleus caudalis of the cat, Brain Res., 243 (1982) 363 368. 18 Marley, P.D., Nagy, J.E., Emson, P.C. and Rehfeld, J.F., Cholecystokinin in the rat spinal cord: distribution and lack of effect of neonatal capsaicin treatment and rhizotomy, Brain Res., 238 (1982) 494-498. 19 Rchfeld, J.F., A unique high-titer antiserum to gastrin, Scand. J. Clin. Lab. Invest., 41 (1981) 723 727. 20 Rehfeld, J.F. and Lundberg, J.M., Cholecystokinin in feline vagal and sciatic nerves: concentration, molecular form and transport velocity, Brain Res., 275 (1983) 341-347. 21 Rosenfeld, M.G., Mermod, J.-J., Amara, S.G., Swanson, L.W., Sawchanko, P.E., Rivier, J., Vale. W.W. and Evans, R.M., Production of a novel neuropeptide encoded by the ealcitonin gene via tissuespecific RNA processing, Nature (London), 304 (1983) 129-135. 22 Schroder, H.D., Localization of cholecystokinin-like immunoreactivity in the rat spinal cord, with particular reference to the autonomic innervation of the pelvic organs, J. Comp. Neurol., 217 (1983) 176186. 23 Schultzberg, M., Dockray, G.J. and Williams, R.G., Capsaicin depletes CCK-like immunoreactivity detected by immunohistochemistry, but not that measured by radioimmunoassay in rat dorsal spinal cord, Brain Res., 235 (1982) 198-204. 24 Skofitsch, G., Zamir, N., Helke, C.J., Savitt, J.M. and Jacobowitz, D.M., Corticotropin releasing factor-like immunoreactivity in sensory ganglia and capsaicin sensitive neurons of the rat central nervous system: colocalization with other neuropeptides, Peptides (Fayetteville), 6 (1985) 307-318. 25 Steinbusch, H.W.M., Verhofstad, A.A.J. and Joosten, H.W.J., Localization of serotonin in the central nervous system by immunohistochemistry: description of a specific and sensitive technique and some applications, Neuroscience, 3 (1978) 811-819. 26 Tschopp, F.A., Henke, H., Petermann, J.B., Tobler, P.H., Janzer, R., H6kfelt, T., Lundberg, J.M., Cuello, C.A. and Fischer, J.A., Calcitonin gene-related peptide and its binding sites in the human central nervous system and pituitary, Proc. Natl. Acad. Sci. USA, 82 (1985) 248-252. 27 Tuchscherer, M.M. and Seybold, V.S., Immunohistochemical studies of substance P, cholecystokininoctapeptide and somatostatin in dorsal root ganglia of the rat, Neuroscience, 14 (1985) 593-605. 28 Vanderhaeghen, J.-J., Lotstra, F., De Mey, J. and Gilles, C., Immunohistochemical localization of cholecystokinin- and gastrin-like peptides in the brain and hypophysis of the rat, Proc. Natl. Acad. Sci. USA, 77 (1980) 119(~ 1194. 29 Zamboni, L. and DeMartino, C., Buffered picric acid-formaldehyde: a new rapid fixative for electron-microscopy, J. Cell. Biol., 35 (1967) 148A.
305
Elsevier Scientific Publishers Ireland Ltd.
NSL 04072
DOES CHOLECYSTOKININ-LIKE IMMUNOREACTIVITY IN RAT PRIMARY SENSORY N E U R O N S REPRESENT CALCITONIN GENE-RELATED PEPTIDE?
G O N G JU L, T O M A S H O K F E L T l'*, J A N A. F I S C H E R 2, P E T E R F R E Y 3, JENS F. R E H F E L D 4 and G R A H A M J. D O C K R A Y 5
1Department qf Histology, Karolinska Institutet, P.O. Box 60400, S-104 O1 Stockholm (Sweden); :Research Laboratoo'Jbr Calcium Metabolism, Department of Orthopedic Surgery and Medicine, University o['Ziirich, Ziirieh and 3Wander Research Institute (a Sandoz Research Unit), Wander LTD, Bern (Switzerland); 4Department of Clinical Chemistry, Rigshospitalet, University of Copenhagen, Copenhagen (Denmark) and 5The Physiological Laboratory, University of Liverpool, Liverpool ( U.K. ) (Received April 14th, 1986: Revised version received and accepted May 5th, 1986)
Key words:
dorsal root ganglia - spinal cord - immunohistochemistry cross-reactivity- rat
Using immunohistochemistry, calcitonin gene-related peptide (CGRP)- and cholecystokinin (CCK)-like immunoreactivity (LI) were found in m a n y of the same spinal and trigeminal ganglion cells and motoneurons in the spinal cord and hypoglossal nucleus, as well as in fibers with an overlapping distribution in the spinal cord (dorsal horn, bundle ventral to the central canal) and in the spinal trigeminal nucleus. CCK-LI in all these structures disappeared after preadsorption of C C K antiseva with C G R P at l0 4 M and almost completely at 10 5 M. C C K peptide in concentrations up to 10 4 M, on the other hand, did not influence C G R P staining. The present findings raise the possibility that some CCK-LI in primary sensory neurons in rat may represent C G R P or a similar peptide.
Using immunohistochemicai techniques, cholecystokinin (CCK)-like immunoreactivity (LI) has been demonstrated in the superficial layers in the dorsal horn of the spinal cord and in spinal ganglion cells, suggesting a localization in primary sensory neurons [2, 4, 9-11, 13-15, 17, 18, 22-24, 27, 28], a view supported by the immunohistochemical demonstration that CCK-LI, to a large extent, disappears from the superficial layers of the dorsal horn after capsaicin treatment [2, ! l, 17, 22-24]. The latter findings are, however, challenged by biochemical analysis with radioimmunoassay (RIA) showing that capsaicin treatment [12] does not appear to change CCK levels [7a, 18, 25]. More recently a novel peptide, calcitonin gene-related peptide (CGRP) [1, 21], has been demonstrated in many primary sensory neurons and in a dense fiber network in the superficial layers of the dorsal horn [8, 21]. In the present paper we have analysed, with immunohistochemistry, the relation between CGRP- and CCK-LI in primary sensory neurons of rat. *Author for correspondence.
0304-3940/86;$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
306
Male Sprague-Dawley rats ( n = 4 ; body wt. 150-200 g; ALAB, Stockholm, Sweden) were used. Colchicine (100 lag in 10 lal) was injected into the subarachnoidal space of the lumbar spinal cord. Twenty-four to 48 h after injection, the rats were perfused with formalin-containing picric acid in phosphate buffer [29]. The medulla oblongata, the spinal cord and spinal ganglia were dissected out and immersed in the same fixative, rinsed and cut on a cryostat (section thickness 8-14 Jam) and processed for the indirect immunofluorescence technique [3]. Briefly, serial sections were incubated with, respectively, goat antiserum raised against rat C G R P (antiserum 216; 1:400) [26], rabbit antisera against the CCK-analogue, non-sulfated gastrin-17 (antiserum 4562; 1:400) [19], non-sulfated CCK-8 (CCK-8NS) (antiserum 18: 1:400) [7], or CCK-4 (antiserum L112; 1:400) [6]. All 3 CCK antisera were directed towards the last 4 amino acids of the C-terminal of CCK-33. The sections were then rinsed, incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Amersham, Amersham, England), rinsed, mounted, examined in a fluorescence microscope and photographed. Incubations were also carried out with the antisera preadsorbed with varying concentrations of peptides. Thus, the C G R P antiserum was preadsorbed with rat C G R P (10 - 7 t o 10 - 4 M ) and with CCK-8NS or sulfated CCK-8 (CCK-8S) (10 7 t o 10 - 4 M); and CCK antisera were preadsorbed with CCK-8NS or CCK-8S ( 1 0 7 t o l0 4 M) and with rat C G R P (10 - 7 t o l 0 - 4 M ) for 2 h at room temperature or at +4:'C overnight. All peptides were purchased from Peninsula (Belmont, CA, U.S.A.). After incubation of sections of spinal and trigeminal ganglia of colchicine-treated rats with C G R P antiserum, numerous cell bodies of varying size (15-60 lam in diameter) were seen to be immunoreactive (Fig. la, c). Incubation with antisera to CCK (Fig. lb) revealed fluorescent cell bodies in similar numbers and of similar size. In fact, CGRP- and CCK-LI were in many cases present in the same cell bodies, as seen in adjacent sections (cf. Fig. la, b). Since no double staining analysis or elution-restaining analysis was performed, it was not possible to establish whether or not all cell bodies contained both immunoreactivities. In the dorsal horn of the lumbar spinal cord and in the superficial layers of the spinal trigeminal nucleus dense networks of C G R P - and CCK-positive fibers were observed in the superficial layers with lower numbers of fibers also in deeper layers. In lamina X of the spinal cord, single CCK-positive cell bodies and a patchy fiber network were observed, as well as a distinct, transversely cut fiber bundle ventral to
Fig. h a-j: immunofluorescence micrographs o f L3 spinal ganglion (a-e), dorsal horn of the spinal cord (L4 level) (f and g), and lamina X of the lumbar spinal cord (h-j) after incubation with antiserum to CGRP (a and c), to CCK-8 (b, l a n d h), to C G R P preadsorbed with C G R P (d) or with CCK-8 (e) and to CCK-8 preadsorbed with C G R P (g and j) or with CCK-8 (i). a-e: CGRP- and CCK-8-immunoreactive cell bodies show a similar distribution and presence in the same cell bodies (arrows and arrowheads) (a and b). CGRP-LI disappears after preadsorption with CGRP (cf. c and d) but not after preadsorption with CCK8 (cf. c and e). Many cell bodies can be identified in both sections (arrowheads in c and e) (c~e). f and g: a dense network of CCK-8-positive fibers can be seen in the external layer (f), and they disappear after preadsorption with CGRP (g). h-j: CCK-8-immunoreactive fibers and some cell bodies can be seen in
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g
i l a m i n a X (h), and these fibers d i s a p p e a r after p r e a d s o r p t i o n with C C K - 8 (i) but not with ( ' G R P (j). H o w ever. the small, l r a n s v e r s a l l y cut fiber b u n d l e ( a r r o w h e a d ) ventral to the central canal (asterisks) c a n n o t be seen after p r e t r e a t m e n t with C G R P . Bars = 50 lam; a and b on the one h a n d and c j, on the other, have the same magnification.
308 the central canal (Fig. lh). With CGRP antiserum only the latter fiber bundle was seen in lamina X. Many motoneurons in the spinal cord and medulla oblongata (hypoglossal nucleus) were both CGRP- and CCK-immunoreactive. All three CCK antisera revealed identical staining patterns. The preadsorption experiments revealed that pretreatment of CGRP antiserum with CGRP peptide (down to 10 -7 M) completely abolished the immunostaining (cf. Fig. lc and ld), whereas sulfated CCK octapeptide (CCK-8S) in concentrations up t o 10 4 M did not influence CGRP staining (Fig. le). These results were obtained both in spinal and trigeminal ganglia, spinal cord, spinal trigeminal nucleus and hypoglossal nucleus. Incubation with CCK antisera preadsorbed with CCK-8S peptide abolished staining in ganglia, spinal cord, spinal trigeminal and hypoglossal nuclei in concentrations down to 10 - 7 M (Fig. li). Pretreatment of the same antiserum with CGRP at 10 - 4 M completely abolished CCK staining in these structures (cf. Fig. If, g), except in lamina X where only the fiber bundle ventral to the central canal disappeared, whereas the patchy fiber network and cell bodies remained (cf. Fig. lh, j). At a concentration of 10 --5 M, CGRP almost completely abolished staining in these structures. No effect of CGRP at 10 - 6 M could be observed. The present results confirm many early studies demonstrating that primary sensory neurons contain CCK- and CGRP-LI when analysed with immunohistochemistry [2, 4, 8-l 1, 13-15, 17, 18, 21-24, 27, 28] and show that the two immunoreactivities often are found in the same cell bodies. Moreover, the present result raises the possibility that CCK-LI, visualized with antisera directed towards the C-terminal portion of CCK-33, cross-react with the newly discovered peptide CGRP [1, 21], or perhaps a so far unknown, CGRP-like peptide. Therefore the CCK immunostaining in rat can be artefactual. It should, however, be remembered that the concentrations of CGRP needed to block CCK staining are high and that it is not known whether such high concentrations are present in these sensory neurons. On the other hand, peptides are probably stored in high concentrations in vesicles. In RIA experiments both humanCGRP-I and rat-CGRP cross-react with the antiserum raised against CCK-8NS but at concentrations approximately 100,000 times higher than for CCK-8S (Frey, unpublished results). The present results may explain some discrepancies in the earlier literature. Thus, whereas CCK-Li disappears after capsaicin treatment when monitored with immunohistochemistry, no such effects on CCK-LI could be observed by RIA [7a, ! 8, 23]. Capsaicin-induced disappearance of CCK-L1 from primary sensory neurons seen with immunohistochemistry [2, 11, 17, 23, 24] could in fact reflect depletion of CGRP, or another cross-reacting peptide. The present findings do not exclude the possibility that some of the immunoreactivity seen after incubation with CCK antiserum, in fact, represents a CCK-like peptide or that these neurons contain both CGRP- and CCK-LI. However, the levels of CCK-8 in spinal ganglia are very low [23] or below the limit of detection [18]. In contrast, concentrations of CGRP in rat dorsal root ganglia range between 130 and 260 pmol/g wet weight of tissue [8]. Sensory systems in other species, such as the vagus nerve in the cat, may, however, contain genuine CCK [5, 20].
309 C C K - 8 S a n d the carboxyl t e r m i n u s o f C G R P - 3 7 exhibit limited structural h o m o logy, with glycine residues in positions 4 a n d 33, respectively, a n d the t e r m i n a l phenylalanines are a m i d a t e d in b o t h cases. This very low degree of identity in crucial positions m a y be sufficient to cause cross-reactivity. It should be n o t e d that specific antisera can be o b t a i n e d to small molecules such as 5 - h y d r o x y t r y p t a m i n e [25]. Moreover, extensive cross-reactivities have earlier been e n c o u n t e r e d , for example, a m o n g the family of pancreatic polypeptides, where several m e m b e r s share the same two Cterminal a m i n o acids with the t e r m i n a l tyrosine a m i d a t e d [16]. The present results further underline the risk o f unspecific reactions when using i m m u n o h i s t o c h e m i c a l techniques a n d focus particularly o n a m i d a t e d C - t e r m i n a l a m i n o acids as antigenic sites with a high potential for cross-reactivity. The present study was s u p p o r t e d by the Swedish Medical Research Council (04X2887), M a g n u s Bergvalls Stiftelse, Alice och K n u t W a l l e n b e r g s Stiftelse a n d the Swiss N a t i o n a l Science F o u n d a t i o n ( G r a n t 3.957-0.84). G.J., on leave from the D e p a r t m e n t of N e u r o b i o l o g y , The F o u r t h Military Medical College, Xian, People's R e p u b lic of China, was s u p p o r t e d by LKB, S t o c k h o l m , Sweden. The skillful technical assistance of Ms. W. Hiort a n d the expert secretarial help of Ms. E. B j 6 r k l u n d are gratefully acknowledged. 1 Amara, S.G., Jonas, V., Rosenfeld, M.G., Ong, E.S. and Evans, R.M., Alternative RNA-processing in calcitonin gene expression generates mRNAs encoding different polypeptide products, Nature (London), 298 (1982) 240-244. 2 Conrath-Verrier, M., Dietl, M. and Trarnu, G., Cholecystokinin-like immunoreactivity in the dorsal horn of the spinal cord of the rat: a light and electron microscopic study, Neuroscience, 13 (1984) 871 885. 3 Coons, A.H., Fluorescent antibody methods. In J.F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, 1958, pp. 399-422. 4 Dalsgaard, C.-J., Vincent, S.R., H6kfelt, T., Lundberg, J.M., Dahlstr6m, A., Schultzberg, M., Dockray, G.J. and Cuello, A.C., Coexistence of cholecystokinin- and substance P-like peptides in neurons of the dorsal root ganglia of the rat, Neurosci. Lett., 33 (1982) 159 164. 5 Dockray, G.J., Gregory, R.A., Tracy, H.J. and Zhu, W.-Y., Transport of cholecystokinin-octapeptide-like immunoreactivity toward the gut in afferent vagal fibres in cat and dog, J. Physiol. (London), 314(1981)501 511. 6 Dockray, G.J., Williams, R.G. and Zhu, W.-Y., Development of region-specificantisera for the Cterminal tetrapeptide of gastrin/cholecystokinin and their use in studies of immunoreactive forms of cholecystokinin in rat brain, Neurochem. Int., 3 (1981) 281-288. 7 Frey,P., Cholecystokinin octapeptide (CCK 26-33), nonsulfated octapeptide and tetrapeptide (CCK 30 33) in rat brain: analysis by high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA), Neurochem. Int., 5 (1985) 811-815. 7a Gibson, S.J., McGregor, G., Bloom, S.R., Polak, J.M. and Wall, P.D., Local application of capsaicin to one sciatic nerve of the adult rat induces a marked depletion in the peptide content of the lumbar dorsal horn, Neuroscience, 7 (1982) 3153-3162. 8 Gibson, S.J., Polak, J.M., Bloom, S.R., Sabate, I.M., Mulderry, P.M., Ghatei, M.A., McGregor, G.P., Morrison, J.F.B., Kelly, J.S., Evans, R.M. and Rosenfeld, M.G., Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species, J. Neurosci., 4 (1984) 3101 3111. 9 Gibson, J.G., Polak, J.M., Bloom, S.R. and Wall, P.D., The distribution of nine peptides in rat spinal cord with special cmphasis on the substantia gelatinosa and on the area around the central canal (lamina X), J. Comp. Neurol., 201 (1981) 65 79.
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