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Substance P-like immunoreactivity at the frog neuromuscular junction
0306-4522/90 53.00 + 0.00 Pergamon Press plc c 1990 IBRO
Nwrosc~irnceVol. 37. No. I. pp. 271-275. 1990 Printed in Gear Britain
SUBSTANCE P-LIKE IMMUNOREACTIVITY AT THE FROG NEUROMUSCULAR JUNCTION M.
MATTEOLI,*
C. HAIhuNNt
and P. DE CAMILLI$
*CNR Istituto Fisiologia Centri Nervosi, via Mariobianco 9, Milano, Italy tCNR Center of Cytopharmacology, Department of Medical Pharmacology and Center for the Study of Peripheral Neuropathies and Neuromuscular Diseases, Milano, Italy ZDepartment of Cell Biology, Yale University School of Medicine, New Haven, 333 Cedar Street, U.S.A. Abstract-It
has been recently shown that frog motoneurons and their nerve terminals contain calcitonin gene-related peptide-like immunoreactivity in large dense-core vesicles (Matteoli er al., Proc. natn. Acad. Sci. U.S.A. 85, 73667370, 1988). We report here by an immunofluorescence approach that the same neurons and nerve terminals also contain substance P-like immunoreactivity. The demonstration of substance P-like immunoreactivity in the frog motor nerve endings supports previous data suggesting a physiological role for this peptide in the modulation of cholinergic transmission.
Tissues preparation
Substance P (SP) is an undecapeptide widely distributed in the nervous system which acts as an exci-
Spinal cord. Frogs were transcardially perfused first with cold 0.1 sodium nhosohate buffer IDH 7.2) and then with 4% formaldehydk (frkshly prepa& from paraformaldehyde) in 0.1 sodium phosphate buffer. The cervical spinal cord was dissected out and immersed in the same fixative for an additional 3 h. After rinsing in phosphate-buffered saline the tissues were infiltrated with 18% sucrose and frozen in isopentane chilled with liquid nitrogen. Ten-micron-thick frozen sections were cut on a Reichert-Jung (Vienna, Austria) cryostat and collected on gelatine-coated glass slides. Neuromuscular junctions. Cutaneous pectoris nervemuscle preparations were dissected out and pinned on a Sylgarded Petri dish. The preparations were then fixed with 4% cold formaldehyde in 0.1 M sodium phosphate buffer. In some cases single fibers were teased apart under the dissecting microscope and rinsed in buffer prior to immunostaining.
tatory neurotransmitter. lo.23 In addition, SP has been found to inhibit acetylcholine (ACh)-induced responses in a variety of preparations including embryonic chicken sympathetic and ciliary ganglion neurons,26 PC12 cell~‘~ and isolated bovine chromaffin cells.4 A physiological role of SP on cholinergic transmission is supported by immunohistochemical studies which have revealed the presence of SP-like immunoreactivity in the cholinergic nerve terminals which innervate these cells in sira3.” At the amphibian neuromuscular junction, SP has been reported to decrease the sensitivity of nicotinic acetylcholine receptors (nAChRs)2 and to potentiate the quanta1 release of ACh.29 However, no evidence has been so far reported for the presence of SP in motor nerve terminals.
Immunosraining
EXPERIMENTAL PROCEDURES Animals
Frogs (Runa pipiens) of 5 cm body length (Connecticut Valley Biological Supply, Southampton, MA) were used. They were anesthetized with MS222 (Sandoz Pharmaceutical) prior to surgical manipulations. Antisera
Rabbit antisera directed against SP and calcitonin gene-related peptide (CGRP) (kind gift of Dr J. M. Polak, London) were characterized as previously described.25.‘3 They were used at a final dilution of I:600 (v/v). An antiserum directed against synapsin 1 was raised in rabbits as previously described.6 A mouse antiserum directed against CGRP was the kind gift of Dr S. Amara (Yale University). Rhodamine-conjugated goat anti-rabbit IgGs and fluoresceineconjugated goat anti-mouse IgGs were from Cappel (Malvern, PA). ACh, acetylcholine; CGRP, calcitonin generelated peptide; nAChR, nicotinic acetylcholine receptor; SP, substance P.
Abbreviations:
and photography
Immunostaining of tissue sections, of nerve-muscle preparations and of single muscle fibers was performed by indirect immunofluorescence on specimens permeabilized by Triton X-100 as previously described.6.” At the end of the immunostaining procedure, muscle fibers were briefly incubated with fluoresceine-conjugated a-bungarotoxin (5 pg/ml) (prepared as described in Ref. 7 and a kind gift of Dr M. Vitadello) to visualize the nAChR and, therefore, the arborization of the synapse. The specificity of the immunostain for SP was controlled by using sera directed against non-relevant antigens and by preadsorbing the serum directed against SP with syn
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Fig. 1. Immunocytochemical localization of SP-like immunoreactivity in a frozen section of the cervical frog spinal cord stained by immunorhodamine. (A) Network of immunoreactive fibers in the dorsal spinal cord. (B) SP-like immunoreactivity in the perikaryon of a moto neuron (arrow). Arrowheads point to SP-immunoreactive fibers in the ventral horn. (A: 274 x ; B: 800 x .)
in the dorsal spinal cord (Fig. 1A). In the ventral horns, only a few, thin, immunoreactive fibers were visible in proximity of the perikarya of motoneurons (Fig. IB, arrowheads). In addition, at high power observation, punctate SP-like immunoreactivity in the cell bodies of motoneurons could be seen (Fig. IB). Overall intensity of immunoreactivity was highly variable from one motoneuron to another. However, most motoneurons appeared positive for the peptide.
al.
Consistent with this finding, SP-like immunoreactivity could also be found at frog motor endplates of a skeletal (cutaneous pectoris) muscle. Figure 2 shows the end plate region of an isolated muscle fiber double-stained for the nAChR by fluoresceineconjugated a-bungarotoxin (Fig. 2B) and for SP by rabbit antibodies directed against SP followed by rhodamine-conjugated goat anti-rabbit IgGs (Fig. 2A). Since in the adult frog the distribution of nAChRs precisely reflects the morphology of the synapse, the close correspondence between Mbungarotoxin binding and SP immunostain suggests that SP is present throughout the nerve terminal arborization. However, at high magnification, SP-like immunoreactivity was found to have a finely punctate appearance (Fig. 2C), similar to its appearance in the perikarya of motoneurons, while stain for abungarotoxin had a characteristic striped pattern due to the localization of the receptor on the shoulders of the postsynaptic infoldings19 (Fig. 2D and Refs 17 and 30). SP immunofluorescence also differed from the segmented fluorescence produced by antibodies directed against synapsin IM (Fig. 2E) and synaptophysin” (not shown), i.e. two marker proteins for small synaptic vesicles. 21~‘2Fluorescent segments may correspond to portions of the nerve terminal where small synaptic vesicles are clustered.30 The punctate appearance of SP-like immunoreactivity is that expected for a localization of this peptide in the large dense-core vesicles which sparsely populate the motor nerve terminal. In the case of other neuropeptides it has been shown that each punctum may represent an individual large dense-core vesicle.‘,” Pre-incubation of the antiserum directed against SP with synthetic SP (0.1 nmol/ml of diluted antiserum) completely abolished the immunostain observed in both the spinal cord and the motor end plate (not shown). Recently, another neuroactive peptide, CGRP,’ has been detected at the frog neuromuscular junction, where it has been shown to be localized in large dense-core vesicles. ” To compare the distribution of SP- and of CGRP-like immunoreactivity at the same motor end plates, we double-labeled muscle fibers with a rabbit antiserum directed against directed against SP and a mouse antiserum CGRP. Figure 3 shows that CGRP (Fig. 3A) and SP immunofluorescence (Fig. 3B) have a very similar distribution. Since each punctum is likely to represent a single large dense-core vesicle,‘.” the results suggest that SP and CGRP may be contained within the same large dense-core vesicles. In addition, all motor end plates examined in both isolated muscle fibers and in nerve preparations were positive for both peptides. These results were not due to a cross-reactivity between the SP antiserum and CGRP because in whole mount preparations CGRP- but not SP-like immunoreactivity was detected in some nerve fibers running along the blood vessels (Fig. 3C and D).
Substance P-like immuno~acti~ty
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Fig. 2. Frog neuromuscular junctions visualized by immunofluorescence (A and B). Double-staining of the same synapse for SP-like immunoreactivity by immuno-rhodamine (A) and for the nAChR by fluoresceine-conjugated a-bungarotoxin (B). The close correspondence between the two fluorescent stains at this low level of rnagni~~tion indicates that SP-like immuno~activity is present along the entire nerve terminal arborization (A and B: 440 x ). (C, D and E) Gallery of micrographs showing details of frog motor nerve endings stained with antibodies to SP (C), with fluoresoeine-conjugated a-bungarotoxin (D) and with antibodies to synapsin I (E). Note the different patterns of fluorescence produced by the three stains (C: 1440x; D: 1200x; E: 1600x). DISCUSSION
In this paper we present evidence for the presence of SP-like immunoreactivity both in the perikarya and nerve terminals of frog motoneurons. These
findings provide support for the hypothesis, so far based only on pharmacological studies, that SP may have a modulatory function on amphibian neuromuscular transmission. Frog motor nerve terminals are densely populated by ACh-containing small synaptic vesicles. In addi-
tion, they also contain a minor complement of large dense-core vesicles, which represent about 1% of the total vesicle population. We have recently shown that at least one neuroactive peptide, CGRP, is stored in these large dense-core vesicles.” The pattern of immunofluorescence now observed for SP in motor nerve terminals is virtually identical to that produced by antibodies directed against CGRP. This suggests that SP is also localized in large dense-core vesicles and probably in the same large dense-core vesicles.
Fig. 3. Double immunofiuorescence of the same frog nerv~muscle preparations stained for CGRP- and SP-like immunoreactivities. (A and B) CGRP (A) and SP (B) immunofluorescence have a very similar distribution in the same motor nerve terminal. (C and D) Prnence of CGRP (C) but not of SP (D) in a nerve fiber closely adjacent to a blood vessel visualized by the presence of autofluorescent nucleated erithrocytes (A and B: 1010x; C and D: 512x).
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In other neurons as well. SP has been shown to be localized in large granular vesiclesZ4.“j and colocalization of SP and CGRP in the same large
dense-core vesicles has been demonstrated in sensory ganglion cells.‘4 SP has been shown to decrease the sensitivity of the muscle nAChR to ACh.’ It has been proposed that this effect is due to a direct binding of SP to nAChR.34 However, most actions of SP in the nervous system are thought to be mediated by specific receptors (see Ref. IO) which are thought to be coupled to polyphosphoinositides breakdown’6.3’ and, possibly, to protein kinase C activation.” A protein kinase C-mediated desensitization of nAChR has been recently reported.‘,” In addition, it has been shown that an increase in the state of phosphorylation of nAChR increase its rate of desensitization.18 An increase of the rate of desensitization of nAChR by SP has been observed at a variety of cholinergic synapses including embryonic chicken sympathetic and ciliary ganglion neurons.” PC12 cells2’ and isolated bovine chromaffin cells4 Thus SP may represent the first messenger responsible for this protein kinase C-mediated modulation. A similar action of SP at the motor end plates has not been identified so far. In view of our present results, it will be of interest to further investigate whether SP may have such an effect at the frog neuromuscular synapse. SP has also been shown to exert a facilitatory action on the quanta1 release of ACh from frog motor nerve terminals.‘” Agents which activate protein kinase C induce, in the same preparation. a parallel
increase in the quanta1 content of neuromuscular transmission with a comparable time course.15.Z7 Thus, the presence of autoreceptors for SP in motor nerve terminals involved in presynaptic facilitation cannot be excluded. Presence of presynaptic autoreceptors for SP has been postulated also in the case of calyciform preganglionic in the chick ciliary ganglion, which contains SP in dense-core vesicles.9 The presence in motor nerve terminals of neuroactive peptides, such as CGRP and SP, in addition to ACh, may have two important roles. On the one hand these peptides may produce modulatory-trophic changes in muscle cells, an effect already demonstrated for CGRP.‘3*B On the other hand, they may modulate cholinergic neurotransmission. We have recently shown that under extreme conditions of stimulation (exposure to alpha-latrotoxin) exocytosis from large dense-core vesicles and from small synaptic vesicles can be aImost completely dissociated, suggesting that release of ACh and neuroactive peptides are controlled by different regulatory mechanisms.” The stimulatory conditions that lead to release of peptides from motor nerve endings, as well as the precise mechanism of action of SP, remain to be elucidated. Acknowledgements-We are indebted to Dr J. M. Polak (Hammersmith Hospital, London) for the gift of the antisera directed against SP and to Dr S. Amara (Yale University School of Medicine) for the gift of the antiserum directed against CGRP. We thank Dr J. Meldolesi for discussion and Mrs L. Aquino and Mr L. Chiumiento for the technical assistance.
REFERENCES I. Amara S. G., Jonas V., Rosenfeld M. G., Ong E. S. and Evans R. M. (1982) Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature 298, 240-244. 2. Akasu 7.. Kojima M. and Koketsu K. (1983) Substance P modulates the sensitivity of the nicotinic receptor in amphibian cholinergic transmission. Br. J. Pharmac. 80, 123-131. 3. Bucsics A., Saria A. and Lembeck F. (1981) Substance P in the adrenal gland: origin and species distribution. Neuropeptides 4. 5.
I, 329-34
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Clapham D. E. and Neher E. (1984) Substance P reduces acetylcholine induced currents in isolated bovine chromaffin cells. J. Physioi. 347, 255-277. Coulter H. D. (1988) Vesicular localization of immunoreactive (metS) enkephalin in the globus pallidus. Proc. nafn.
Acad. Sri. U.S.A. 85, 1028-7032. 6. De Camilli P., Cameron R. and Greengard
P. (1983) Synapsin 1 (protein I), a nerve terminal-specific phosphoprotein. Its localization in synapses of the central and peripheral nervous system demonstrated by immunofluorescence in frozen and elastic sections. J. Cc/i Biol. %. 1337-1354. I. De Camilli P., Vitadello M.. Cane&i M. P., Zanoni R., Jahn R. and Gorio A. (1988) The synaptic vesicle proteins synapsin I and synaptophysin (protein ~38) are concentrated both in efferent and afferent nerve endings of the skeletal muscle. J. Neurosci. 8, 1625- 163I 8. Downing J. E. G. and Role L. W. (1987) Activators of protein kinase C enhance acetylcholine receptor desensitization in sympathetic ganglion neurons. Proc. natn. Acud. Sci. U.S.A. 84, 7739-7743. 9. Dryer S. E. and Chiappinelli V. A. (1985) Substance P depolarizes nerve terminals in an autonomic ganglion. Brain Res. 336, 190-194. Actions in rhe VerrebruIe Nervous Sysrem (eds Rogawsky M. A. IO. Dun J. N. (1985) Substance P. In Neurolrunsmiller and Barker J. L.), pp. 385-410. Plenum Press, New York. II. Erichsen J. T., Reiner A. and Karten H. J. (1982) Co-occurrence of substance P-like and Leu-enkephalin-like immunoreactivities in neurones and fibres of avian nervous system. Nurure 295, 407-410. 12. Eusebi F.. Molinaro M. and Zani B. M. (1985) Agents that activate protein kinase C reduce acetylcholine sensitivity in cultured myotubes. J. CeN Biol. 100, 1339-1342. 13. Fontaine B.. Klarsfeld A. and Chanpux J. P. (1987) CGRP and muscle activity regulate acetylcholine mRNA levels by distinct intracellular pathways. J. Cell Biol. 105, 1337-1342. 14. Gulbenkian S.. Merighi A.. Wharton J., Vamdell I. M. and Polak J. M. (1986) Ultrastructural evidence for the coexistence of calcitonin gene-related peptide (CGRP) and substance P (SP) in secretory vesicles of peripheral nerves in the guinea pig. J. NeurorjYol. 15, 535-542.
Substance P-like immunoreactivity
at the frog neuromuscular junction
27s
I 5. Haimann c.. Meidolesi J. and Ceccarelli B. (1987) The phorbol ester, ~2-O-tetradecanoyi-phorbol-13-acetate, enhances the evoked quanta] release of acetylcholine at the frog neuromuscular junction. fflzTgers Arch. ges. Physid 408, 27-3 I. 16. Mantyh P. W.. Pinnock R. D.. Downes C. P., Goedert M. and Hunt S. P. (1984) Correlation between inositol phospholjpids hydrolysis and substance P receptors in rat CNS. Narure 30% 795-797. 17. Matteoli M.. Haimann C., Torri Tarelli F.. Polak J. M.. Ceccarelii B. and De Camilli P. (1988) Differential effect of alpha-latrotoxin on exocytosis from small synaptic vesicles and from large dense core vesicles containing calcitonin gene-related peptide at the frog neuromuscular junction. Proc. naln. Acad. Sci. U.S.A. 85, 7366-7370. 18. Miles K. and Huganir R. (1988) Regulation of nicotinic ACh receptor by protein phospho~lation. h4olec. Neurobjo/. 2, 91-124.
Matthew-Bellinger .I. and Salpeter M. M. (1978) Distribution of acetylcholine receptors at frog neuromuscular junctions with a discussion of some physiological implications. J. Physiol., Lond. 297, 197-213. 20. Mulle C.. Benoit P., Pinset C., Roa M. and Changeux J. P. (t988) Calcitonin gene related peptide enhances the rate of desensitization of the nicotinic ACh receptor in cultured mouse muscle cells. Proc. nafn. Acud. Sci. U.S.A. 85, 19.
5728-5732. 21.
22. 23. 24. 25. 26. 27.
28. 29.
30. 3 I.
32. 33. 34.
Navone F., Greengard P. and De Camilii P. (1984) Synapsin I in nerve terminals: selective association with small synaptic vesicles. Science 226, 1209-121 I. Navone F.. Jahn R.. DiGioia G., Stukenbrok H.. Greengard P. and De Camilli P. (1986) Protein ~38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells. J. Cell Biol. 103, 251 l-2527. Nicoll R. A.. Schenker C. and Leeman S. E. (1980) Substance Pas a transmitter candidate. A. Rev. Neurosri. 3,227-268. Probert L., DeMey J. and Polak J. M. (1981) Distinct subpopulations of enteric p-type neurones contain substance P and vasoactive intestinal enteric polypeptide. Nature 294, 470-471. Rodrigo J.. Polak J. M., Terenghi G.. Cervantes C.. Ghatei M. A.. Mulderry P. K. and Bloom S. R. (1985) Calcitonin gene-related peptide (CGRP) immunoreactive sensory and motor nerves of the mammalian palate. ~~sroc/fernjs~r~ 82, 67-74. Role L. W. (I 984) Substance P modulation of acetylcholine induced currents in embryonic chicken sympathetic and ciliary ganglion neurons. Proc. ~wm. Acud. Sri. U.S.A. 81, 2824-2928. Shapiro R., Silberberg S. D., Ginsburg S. and Rahamimoff R. (1987) Activation of protein kinase C augments evoked transmitter release. Nurure 325, 58-60. Stallcup W. B. and Patrick J. (1980) Substance P enhances cholinergic receptor desensitization in a clonal nerve cell line. Proc. natn. Acad. Sri. U.S.A. 17, 634-638. Steinacker A. (1977) Calcium-de~ndent presynaptic action of substance P at the frog neuromu~uiar junction. Narure 267, 268-270. Valtorta F.. Villa A., Jahn R.. De Camilli P., Greengard P. and Ceccarelli B. (1988) Localization ofsynapsin I at the frog neuromuscular junction. Neuroscience 24, 593-603. Valtorta F.. Jahn R.. Fesce R.. Greengard P. and Ceccarelli B. (1988) Synaptophysin (~38) at the frog neuromu~ular junction: its incorporation into the axolemma and recycling after intense quanta1 secretion. J. Celi. Bin/. 107,2717-2727. Watson S. P. and Downes C. P. (1983) Substance P induced hydrolysis of inositol phospholipids in guinea-pig ileum and rat hypothalamus. Eur. J. Pharnzur. 93, 245-253. Wharton J.. Polak J. M.. McGregor G. P.. Bishop A. E. and Bloom S. R. (1981) The distribution of substance P like immunoreactive nerves in the guinea pig heart. Neuroscience 6, 2193-2204. Wieland G. A.. Durkin J. R., Henley J. M. and Simasko S. M. (1988) Effects of substance P on the binding of ligands to nicotinic acetylchoiine receptors. Moiec. Pharmac. 32, 625-638. (Acceppred
I7
December
1989)
Nwrosc~irnceVol. 37. No. I. pp. 271-275. 1990 Printed in Gear Britain
SUBSTANCE P-LIKE IMMUNOREACTIVITY AT THE FROG NEUROMUSCULAR JUNCTION M.
MATTEOLI,*
C. HAIhuNNt
and P. DE CAMILLI$
*CNR Istituto Fisiologia Centri Nervosi, via Mariobianco 9, Milano, Italy tCNR Center of Cytopharmacology, Department of Medical Pharmacology and Center for the Study of Peripheral Neuropathies and Neuromuscular Diseases, Milano, Italy ZDepartment of Cell Biology, Yale University School of Medicine, New Haven, 333 Cedar Street, U.S.A. Abstract-It
has been recently shown that frog motoneurons and their nerve terminals contain calcitonin gene-related peptide-like immunoreactivity in large dense-core vesicles (Matteoli er al., Proc. natn. Acad. Sci. U.S.A. 85, 73667370, 1988). We report here by an immunofluorescence approach that the same neurons and nerve terminals also contain substance P-like immunoreactivity. The demonstration of substance P-like immunoreactivity in the frog motor nerve endings supports previous data suggesting a physiological role for this peptide in the modulation of cholinergic transmission.
Tissues preparation
Substance P (SP) is an undecapeptide widely distributed in the nervous system which acts as an exci-
Spinal cord. Frogs were transcardially perfused first with cold 0.1 sodium nhosohate buffer IDH 7.2) and then with 4% formaldehydk (frkshly prepa& from paraformaldehyde) in 0.1 sodium phosphate buffer. The cervical spinal cord was dissected out and immersed in the same fixative for an additional 3 h. After rinsing in phosphate-buffered saline the tissues were infiltrated with 18% sucrose and frozen in isopentane chilled with liquid nitrogen. Ten-micron-thick frozen sections were cut on a Reichert-Jung (Vienna, Austria) cryostat and collected on gelatine-coated glass slides. Neuromuscular junctions. Cutaneous pectoris nervemuscle preparations were dissected out and pinned on a Sylgarded Petri dish. The preparations were then fixed with 4% cold formaldehyde in 0.1 M sodium phosphate buffer. In some cases single fibers were teased apart under the dissecting microscope and rinsed in buffer prior to immunostaining.
tatory neurotransmitter. lo.23 In addition, SP has been found to inhibit acetylcholine (ACh)-induced responses in a variety of preparations including embryonic chicken sympathetic and ciliary ganglion neurons,26 PC12 cell~‘~ and isolated bovine chromaffin cells.4 A physiological role of SP on cholinergic transmission is supported by immunohistochemical studies which have revealed the presence of SP-like immunoreactivity in the cholinergic nerve terminals which innervate these cells in sira3.” At the amphibian neuromuscular junction, SP has been reported to decrease the sensitivity of nicotinic acetylcholine receptors (nAChRs)2 and to potentiate the quanta1 release of ACh.29 However, no evidence has been so far reported for the presence of SP in motor nerve terminals.
Immunosraining
EXPERIMENTAL PROCEDURES Animals
Frogs (Runa pipiens) of 5 cm body length (Connecticut Valley Biological Supply, Southampton, MA) were used. They were anesthetized with MS222 (Sandoz Pharmaceutical) prior to surgical manipulations. Antisera
Rabbit antisera directed against SP and calcitonin gene-related peptide (CGRP) (kind gift of Dr J. M. Polak, London) were characterized as previously described.25.‘3 They were used at a final dilution of I:600 (v/v). An antiserum directed against synapsin 1 was raised in rabbits as previously described.6 A mouse antiserum directed against CGRP was the kind gift of Dr S. Amara (Yale University). Rhodamine-conjugated goat anti-rabbit IgGs and fluoresceineconjugated goat anti-mouse IgGs were from Cappel (Malvern, PA). ACh, acetylcholine; CGRP, calcitonin generelated peptide; nAChR, nicotinic acetylcholine receptor; SP, substance P.
Abbreviations:
and photography
Immunostaining of tissue sections, of nerve-muscle preparations and of single muscle fibers was performed by indirect immunofluorescence on specimens permeabilized by Triton X-100 as previously described.6.” At the end of the immunostaining procedure, muscle fibers were briefly incubated with fluoresceine-conjugated a-bungarotoxin (5 pg/ml) (prepared as described in Ref. 7 and a kind gift of Dr M. Vitadello) to visualize the nAChR and, therefore, the arborization of the synapse. The specificity of the immunostain for SP was controlled by using sera directed against non-relevant antigens and by preadsorbing the serum directed against SP with syn
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Fig. 1. Immunocytochemical localization of SP-like immunoreactivity in a frozen section of the cervical frog spinal cord stained by immunorhodamine. (A) Network of immunoreactive fibers in the dorsal spinal cord. (B) SP-like immunoreactivity in the perikaryon of a moto neuron (arrow). Arrowheads point to SP-immunoreactive fibers in the ventral horn. (A: 274 x ; B: 800 x .)
in the dorsal spinal cord (Fig. 1A). In the ventral horns, only a few, thin, immunoreactive fibers were visible in proximity of the perikarya of motoneurons (Fig. IB, arrowheads). In addition, at high power observation, punctate SP-like immunoreactivity in the cell bodies of motoneurons could be seen (Fig. IB). Overall intensity of immunoreactivity was highly variable from one motoneuron to another. However, most motoneurons appeared positive for the peptide.
al.
Consistent with this finding, SP-like immunoreactivity could also be found at frog motor endplates of a skeletal (cutaneous pectoris) muscle. Figure 2 shows the end plate region of an isolated muscle fiber double-stained for the nAChR by fluoresceineconjugated a-bungarotoxin (Fig. 2B) and for SP by rabbit antibodies directed against SP followed by rhodamine-conjugated goat anti-rabbit IgGs (Fig. 2A). Since in the adult frog the distribution of nAChRs precisely reflects the morphology of the synapse, the close correspondence between Mbungarotoxin binding and SP immunostain suggests that SP is present throughout the nerve terminal arborization. However, at high magnification, SP-like immunoreactivity was found to have a finely punctate appearance (Fig. 2C), similar to its appearance in the perikarya of motoneurons, while stain for abungarotoxin had a characteristic striped pattern due to the localization of the receptor on the shoulders of the postsynaptic infoldings19 (Fig. 2D and Refs 17 and 30). SP immunofluorescence also differed from the segmented fluorescence produced by antibodies directed against synapsin IM (Fig. 2E) and synaptophysin” (not shown), i.e. two marker proteins for small synaptic vesicles. 21~‘2Fluorescent segments may correspond to portions of the nerve terminal where small synaptic vesicles are clustered.30 The punctate appearance of SP-like immunoreactivity is that expected for a localization of this peptide in the large dense-core vesicles which sparsely populate the motor nerve terminal. In the case of other neuropeptides it has been shown that each punctum may represent an individual large dense-core vesicle.‘,” Pre-incubation of the antiserum directed against SP with synthetic SP (0.1 nmol/ml of diluted antiserum) completely abolished the immunostain observed in both the spinal cord and the motor end plate (not shown). Recently, another neuroactive peptide, CGRP,’ has been detected at the frog neuromuscular junction, where it has been shown to be localized in large dense-core vesicles. ” To compare the distribution of SP- and of CGRP-like immunoreactivity at the same motor end plates, we double-labeled muscle fibers with a rabbit antiserum directed against directed against SP and a mouse antiserum CGRP. Figure 3 shows that CGRP (Fig. 3A) and SP immunofluorescence (Fig. 3B) have a very similar distribution. Since each punctum is likely to represent a single large dense-core vesicle,‘.” the results suggest that SP and CGRP may be contained within the same large dense-core vesicles. In addition, all motor end plates examined in both isolated muscle fibers and in nerve preparations were positive for both peptides. These results were not due to a cross-reactivity between the SP antiserum and CGRP because in whole mount preparations CGRP- but not SP-like immunoreactivity was detected in some nerve fibers running along the blood vessels (Fig. 3C and D).
Substance P-like immuno~acti~ty
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Fig. 2. Frog neuromuscular junctions visualized by immunofluorescence (A and B). Double-staining of the same synapse for SP-like immunoreactivity by immuno-rhodamine (A) and for the nAChR by fluoresceine-conjugated a-bungarotoxin (B). The close correspondence between the two fluorescent stains at this low level of rnagni~~tion indicates that SP-like immuno~activity is present along the entire nerve terminal arborization (A and B: 440 x ). (C, D and E) Gallery of micrographs showing details of frog motor nerve endings stained with antibodies to SP (C), with fluoresoeine-conjugated a-bungarotoxin (D) and with antibodies to synapsin I (E). Note the different patterns of fluorescence produced by the three stains (C: 1440x; D: 1200x; E: 1600x). DISCUSSION
In this paper we present evidence for the presence of SP-like immunoreactivity both in the perikarya and nerve terminals of frog motoneurons. These
findings provide support for the hypothesis, so far based only on pharmacological studies, that SP may have a modulatory function on amphibian neuromuscular transmission. Frog motor nerve terminals are densely populated by ACh-containing small synaptic vesicles. In addi-
tion, they also contain a minor complement of large dense-core vesicles, which represent about 1% of the total vesicle population. We have recently shown that at least one neuroactive peptide, CGRP, is stored in these large dense-core vesicles.” The pattern of immunofluorescence now observed for SP in motor nerve terminals is virtually identical to that produced by antibodies directed against CGRP. This suggests that SP is also localized in large dense-core vesicles and probably in the same large dense-core vesicles.
Fig. 3. Double immunofiuorescence of the same frog nerv~muscle preparations stained for CGRP- and SP-like immunoreactivities. (A and B) CGRP (A) and SP (B) immunofluorescence have a very similar distribution in the same motor nerve terminal. (C and D) Prnence of CGRP (C) but not of SP (D) in a nerve fiber closely adjacent to a blood vessel visualized by the presence of autofluorescent nucleated erithrocytes (A and B: 1010x; C and D: 512x).
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In other neurons as well. SP has been shown to be localized in large granular vesiclesZ4.“j and colocalization of SP and CGRP in the same large
dense-core vesicles has been demonstrated in sensory ganglion cells.‘4 SP has been shown to decrease the sensitivity of the muscle nAChR to ACh.’ It has been proposed that this effect is due to a direct binding of SP to nAChR.34 However, most actions of SP in the nervous system are thought to be mediated by specific receptors (see Ref. IO) which are thought to be coupled to polyphosphoinositides breakdown’6.3’ and, possibly, to protein kinase C activation.” A protein kinase C-mediated desensitization of nAChR has been recently reported.‘,” In addition, it has been shown that an increase in the state of phosphorylation of nAChR increase its rate of desensitization.18 An increase of the rate of desensitization of nAChR by SP has been observed at a variety of cholinergic synapses including embryonic chicken sympathetic and ciliary ganglion neurons.” PC12 cells2’ and isolated bovine chromaffin cells4 Thus SP may represent the first messenger responsible for this protein kinase C-mediated modulation. A similar action of SP at the motor end plates has not been identified so far. In view of our present results, it will be of interest to further investigate whether SP may have such an effect at the frog neuromuscular synapse. SP has also been shown to exert a facilitatory action on the quanta1 release of ACh from frog motor nerve terminals.‘” Agents which activate protein kinase C induce, in the same preparation. a parallel
increase in the quanta1 content of neuromuscular transmission with a comparable time course.15.Z7 Thus, the presence of autoreceptors for SP in motor nerve terminals involved in presynaptic facilitation cannot be excluded. Presence of presynaptic autoreceptors for SP has been postulated also in the case of calyciform preganglionic in the chick ciliary ganglion, which contains SP in dense-core vesicles.9 The presence in motor nerve terminals of neuroactive peptides, such as CGRP and SP, in addition to ACh, may have two important roles. On the one hand these peptides may produce modulatory-trophic changes in muscle cells, an effect already demonstrated for CGRP.‘3*B On the other hand, they may modulate cholinergic neurotransmission. We have recently shown that under extreme conditions of stimulation (exposure to alpha-latrotoxin) exocytosis from large dense-core vesicles and from small synaptic vesicles can be aImost completely dissociated, suggesting that release of ACh and neuroactive peptides are controlled by different regulatory mechanisms.” The stimulatory conditions that lead to release of peptides from motor nerve endings, as well as the precise mechanism of action of SP, remain to be elucidated. Acknowledgements-We are indebted to Dr J. M. Polak (Hammersmith Hospital, London) for the gift of the antisera directed against SP and to Dr S. Amara (Yale University School of Medicine) for the gift of the antiserum directed against CGRP. We thank Dr J. Meldolesi for discussion and Mrs L. Aquino and Mr L. Chiumiento for the technical assistance.
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