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Differential innervation of the goldfish tonic red muscles and twitch white muscles by neuropeptide-immunoreactive motoneurons
Brain Research Bulletin, Vol. 52, No. 6, pp. 547–552, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/00/$–see front matter
PII S0361-9230(00)00295-1
Differential innervation of the goldfish tonic red muscles and twitch white muscles by neuropeptideimmunoreactive motoneurons Kengo Funakoshi,* Tetsuo Kadota, Yoshitoshi Atobe, Masato Nakano, Midori Tsukagoshi, Richard C. Goris and Reiji Kishida Department of Anatomy, Yokohama City University School of Medicine, Yokohama, Japan [Received 18 December 1999; Revised 26 April 2000; Accepted 28 April 2000] ABSTRACT: Neuropeptides in the motor nerves innervating the red and white muscles of the goldfish Carassius auratus were examined. In the tonic red muscles, varicose nerve endings immunoreactive for both calcitonin gene-related peptide and substance P were found spread over the surface of the muscle fibers, but in the twitch white muscles only scattered nerve endings immunoreactive for calcitonin gene-related peptide were found. At the electron microscopic observation, dense electron products immunoreactive for calcitonin gene-related peptide and for substance P (SP) were detected in the motor nerve endings making synapses on the muscle fibers of the red muscles. In the spinal cord, all of the motor neurons showed immunoreactivity to calcitonin gene-related peptide, but the motor neurons immunoreactive for substance P were restricted to the ventrolateral group that has been shown to project predominantly to the red muscles. These results suggest that the motor neurons innervating the red and white muscles of the goldfish are distinct in their neuropeptide content. The present study also raises the possibility that SP might be related to the unique physiological properties of the tonic type red muscles, probably by direct binding to the acetylcholine receptors. © 2000 Elsevier Science Inc.
slow tonic fibers are reduced in number with the exception of specific regions, such as the extraocular muscles [30]. In fish, which has the most primitive muscle fiber composition among vertebrates, the morphologically typical slow muscle fibers are lacking. However, in most fish species, muscle fibers in the red muscles are tonic and those in the white muscles are twitch, and both are innervated by multiple grape-type endings scattered over the surface of the muscle cells [5,30]. In the trunk region of teleosts, the red muscles are restricted to a small lateral area immediately beneath the lateral line, while the white muscles occupy a large deeper area of the musculature [10]. The red muscles are well-vascularized, and specialized for aerobic metabolism. They are active during sustained swimming (cruising). On the other hand, the white muscles are active mainly during vigorous bursts of swimming. The innervation is also quite different in the red and white muscles. In the goldfish Carassius auratus, and the zebrafish Brachydanio rerio, the red muscles in the trunk region are innervated by the spinal motor neurons situated in the ventrolateral column of the ventral horn, while the white muscles are innervated by the dorsomedial column [9,35]. In previous electrophysiological studies, only junction potentials, but not action potentials, were recorded intracellularly from the red muscle fibers after stimulation of single spinal nerve fibers in some species of teleosts [10,16 –18]. The junction potentials recorded after stimulation were classified into three types, i.e., excitatory potentials, inhibitory potentials, and diphasic potentials that are composed of an excitatory potential followed by an inhibitory potential. These responses may be mediated exclusively by nicotinic acetylcholine (Ach) receptors [16 –19]. Recently, nerve fibers immunoreactive for calcitonin generelated peptide (CGRP) and substance P (SP) have been described in the muscular tissue of the eel, raising the possibility that these neuropeptides may be present in the motor nerve endings to modulate neuromuscular transmission in teleosts [33]. On this basis, we examined in the present study the distribution of these neuropeptides in the spinal somatic motoneurons and the motor endings in the red and white muscles of the goldfish.
KEY WORDS: Spinal cord, Motor endplate, Neuropeptide, Immunohistochemistry, Electron microscopy, Teleosts.
INTRODUCTION The skeletal muscle fibers of vertebrates are classified into fast twitch fibers and slow tonic fibers [30]. Slow tonic fibers are morphologically characterized by zigzag-shaped Z-lines, poorly developed sarcoplasmic reticulum, and multiple innervations by grape-type motor endings. On the other hand, the fast twitch fibers have straight Z-lines, well-developed sarcoplasmic reticulum, and single innervation by plate-type or linear type motor endings [15,30]. This type of fibers is subdivided into three categories: white, red, and intermediate fibers, according to the relative amounts of mitochondria. The composition of these muscle fiber types varies among the classes of vertebrates [30]. In amphibians, reptiles, and birds, the fast twitch and the slow tonic muscle fibers are mixed in the trunk muscles. In mammals, on the other hand,
* Address for correspondence: Kengo Funakoshi, Department of Anatomy, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004 Japan. Fax: ⫹81-45-7827251; E-mail: [email protected]
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We used a total of three adult goldfish Carassius auratus, bought commercially and maintained in an aquarium. Light Microscopic Immunohistochemistry In the light microscopic study, two fish were anesthetized with 0.01% tricaine methanesulfonate in water, and perfused transcardially with heparinized saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The rostral part of the spinal cord and the musculature of the trunk were removed, postfixed in the same solution for 5– 6 h at 4°C, and infiltrated overnight with 30% sucrose in 0.1 M PB at 4°C for cryoprotection. The specimens were frozen and cut serially into 30-m cross-sections on a cryostat and thaw-mounted on glass slides. The sections were then incubated in the fixative for 10 min, and washed in 0.3% Triton X-100 in 0.1 M phosphate-buffered saline (PBST). The sections were first incubated with 10% normal goat serum (NGS), 1% bovine serum albumin (BSA), and 0.2% sodium azide in PBST for 1 h at room temperature. After rinsing in PBST, the sections were incubated for 48 h at 4°C with one of the following primary antibodies: rabbit polyclonal antibody against CGRP (1: 1500; Chemicon, Temecula, CA, USA), rat monoclonal antibody against SP (1:100; Harlan sera-lab, Loughborough, UK). The antibody was diluted with 0.1 M PBST containing 1% NGS, 0.2% BSA, and 0.2% sodium azide. The sections were then incubated for 2 h with goat anti-rabbit IgG (1:200; Cappel, Aurora, OH, USA) or goat anti-rat IgG (1:100; Cappel), diluted with 0.1 M PBST containing 1% NGS, 0.2% BSA, and 0.2% sodium azide, and incubated for 2 h with rabbit peroxidase-anti peroxidase (PAP) complex (1:200; Jackson, West Grove, PA, USA) or rat PAP complex (1:100; Jackson), diluted with 0.1 M PBS containing 1% NGS, 0.2% BSA, and 0.1% thimerosal at room temperature. The peroxidase reactivity was demonstrated with 3,3⬘-diaminobenzidine (DAB; Sigma, St. Louis, MO, USA). The specificity of the staining was tested by omission of the antibody. To examine the co-localization of CGRP and SP in the peripheral nerve terminals, the sections of the musculature were incubated for 48 h at 4°C with a mixture of rabbit anti-CGRP IgG (1:250; Chemicon) and rat anti-SP IgG (1:50; Harlan sera-lab), diluted with 0.1 M PBST containing 1% NGS, 0.2% BSA, and 0.2% sodium azide. The sections were then incubated for 3 h with a mixture of fluorescein isothiocyanate-conjugated goat anti-rat IgG (1:100; Cappel) and rhodamine-conjugated goat anti-rabbit IgG (1:100; Cappel), diluted with 0.1 M PBST containing 1% NGS, 0.2% BSA, and 0.2% sodium azide at room temperature. Observation was made with a fluorescence microscope (Leica DMR, Welzlar, Germany), equipped with a double band-pass filter set. Immunoelectron Microscopy The remaining fish was fixed with 0.1 M PB (pH 7.4) containing 4% paraformaldehyde, 0.1% glutaraldehyde, and 0.2% picric acid. The musculature was then dissected out, postfixed in the same solution for 5– 6 h, and infiltrated overnight with 30% sucrose in 0.1 M PB at 4°C. The specimens were freeze-thawed by liquid nitrogen, embedded in gelatin gel, and then cut serially into 30-m sections in a Vibratome. The sections were treated freefloating in 1% sodium borohydride in distilled water for 30 min at 4°C, incubated for 48 h at 4°C with rabbit anti-CGRP IgG or rat anti-SP IgG (described above), diluted with 0.1 M PBS containing 1% NGS, 0.2% BSA, and 0.2% sodium azide, and then stained with the PAP-DAB method. The sections were postfixed subsequently in 1% glutaraldehyde and 1% osmium tetroxide in 0.1 M
PB at 4°C for 30 min. After dehydration in a graded ethanol series and dipping in propylene oxide, the sections were embedded in a mixture of Epon and Araldite. Ultrathin sections were cut on an ultratome (LKB; Leica, Welzlar, Germany). After counterstaining with uranyl acetate, the sections were examined under an electron microscope (H-7500; Hitachi, Tokyo, Japan). Electronic Imaging Primary images of immunofluorescence reactivity in the muscles and of immunoperoxidase reactivity in the spinal cord were acquired on films, which were then scanned into a Macintosh computer, through a film scanner (Nikon, Tokyo, Japan). Contrast and brightness were adjusted with the Adobe Photoshop software. RESULTS In the fixed specimen, the red muscles could be clearly distinguished from the white muscles in the trunk region, on the basis of their color and location. The red muscles occupied superficial small area along the side of the body, immediately beneath the lateral line. The white muscles occupied the deeper large area of the musculature. They could be subdivided into epaxial and hypaxial muscles. Light Microscopic Study of Musculature In the red muscles, many CGRP-immunoreactive varicose nerve fibers were found on the surface of the muscle cells (Figs. 1A,B). The CGRP-immunoreactive nerve fibers were more abundant in the surface region than in the deep layers of the red muscles. No CGRP-positive nerve fibers were found to terminate around the capillaries of the musculature. The distribution of the SP-immunoreactive varicose nerve fibers was similar. They were found on the surface of the muscle cells (Fig. 1C). In the double-labeling study of CGRP and SP, all varicose nerve terminals appeared to be positive for both CGRP and SP (Fig. 2). Using the double band-pass filter, virtually all fluorescent nerve fibers in the red muscles were shown to be double immunopositive. In the white muscles, CGRP-immunoreactive nerve terminals were also found to be apposed on the surface of the muscle cells, but were much scarcer than in the red muscles (Fig. 1D). On the other hand, no fibers immunoreactive for SP were found in the white muscles. Electron Microscopic Study of Musculature In the red muscles, both CGRP- and SP-immunoreactive varicosities were found to form morphologically identifiable synapses on the striated muscle cells (Figs. 3A,B). The presynaptic membrane of the varicosities did not show extensive foldings but exhibited the smooth surfaces typical of grape-type motor endings. There was a synaptic cleft of 100-nm width between the surface of the muscle cells and the presynaptic membranes. A basement membrane was also found in the synaptic clefts. Electron-dense peroxidase reaction products filled the varicosities. Light Microscopic Study of Spinal Motor Neurons In the rostral part of the spinal cord, virtually all of the motor neuronal cell bodies were strongly CGRP-immunoreactive (Figs. 4A,B). The staining intensity of immunoreactive neurons was homogeneous. In contrast, weakly SP-immunoreactive neurons were predominant in the ventrolateral column (Fig. 4C).
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FIG. 1. Nerve endings in the musculature. (A) Calcitonin gene-related peptide (CGRP)-immunoreactive varicose nerve endings are abundant in the red muscles, but scarce in the white muscles. (B) CGRP-immunoreactive varicose nerve endings are in contact with the surface of the muscle cells of the red muscles. (C) Many substance P (SP)-immunoreactive varicose nerve endings (small arrows) are also seen in the red muscles, but not in the white muscles. (D) SP-immunoreactive varicose nerve endings (small arrows) are in contact with the surface of the muscle cells of the red muscles. Abbreviation: W, white muscles. Scale bars: 100 m (A,C), 50 m (B,D).
DISCUSSION To date, little is known about neuropeptides expressed by the somatic motor neurons in fish. The present study in the goldfish showed that the spinal motor neurons innervating the tonic red muscles were immunoreactive to both CGRP and SP, while those innervating the twitch white muscles were immunoreactive only to CGRP. Immunoreactivity to these peptides was also detected in the motor endings that make synapses with the muscle cells. A total co-localization of immunoreactivity to CGRP and SP was shown in the present study in the varicose nerve endings in the red muscles. Therefore, there is distinct grouping of target-specific, phenotypically distinct somatic motor neurons in the goldfish. Immunoreactivity to CGRP in the spinal motor neurons and the motor endings has previously been reported in mammals and amphibians [14,25,31]. Unlike mammals, in which the amount of CGRP in the motor neurons or the motor endplates differed according to the muscle type and the contractile activity of the muscles innervated [4,7,13,32], the strong immunoreactivity to CGRP was homogeneous in virtually all motor neurons in the
spinal cord of goldfish. There was no difference in staining among those projecting to the white or red muscles. This finding might suggest that CGRP is essential for the activity of motor neurons in fish. It has been shown in vitro that CGRP enhances the rate of desensitization of the muscle Ach receptor channels, probably by cyclic adenosine monophosphate-mediated phosphorylation [26, 28], and regulates the sensitivity of Ach receptors [8]. It remains to be elucidated whether these effects are physiological [6]. On the other hand, CGRP immunoreactivity is especially high in the developing stage of the motor neurons and the neuromuscular junctions in mammals [2,24]. CGRP has also been suggested to promote development and remodeling of neuromuscular junctions by synthesizing the ␣ subunit of the Ach receptors and potentiating postsynaptic Ach channel activity [11,12,20,21,29,34]. The present findings in the goldfish lead us to hypothesize that CGRP is essential for constant remodeling of the fish neuromuscular junction, which is morphologically primitive and much simpler than those in other vertebrates. We found immunoreactivity to SP in the motor neurons and the motor endings only in the red muscles. Immunoreactivity to SP has
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FIG. 2. Double-labeled immunofluorescent image of the nerve fibers in the red muscles. Fluorescein isothiocyanate-labeled substance P-immunoreactive varicose nerve endings (A,C) appear identical to rhodamine-labeled calcitonin gene-related peptideimmunoreactive varicose nerve endings (B,D). Scale bars: 50 m.
been found in the motor endings in the frog, but not in mammals [2,25]. In adult frogs, SP has been suggested to suppress the sensitivity of nicotinic Ach receptors by direct binding to the receptor molecules [1]. The direct binding of SP to the Ach receptors was demonstrated in vitro [3,22,27,36]. In the tonic red muscles of teleosts, inhibitory and diphasic potentials, both of
which have a hyperpolarizing phase, have sometimes been recorded after stimulation of single motor nerve fibers [16 –19]. These excitatory and inhibitory potentials were both shown to be mediated by the nicotinic Ach receptors [17–19]. Therefore, it is possible that SP released from the motor endings of teleosts may hyperpolarize the postsynaptic membrane via direct binding to the
FIG. 3. Electron micrographs of the calcitonin gene-related peptide-immunoreactive (A) and substance P-immunoreactive (B) varicose nerve endings that contact the striated muscle cells. A basement membrane is interposed between the presynaptic membrane of the nerve terminals (small arrows) and the postsynaptic membrane (arrowheads) of the muscle cells. Scale bars: 0.5 m (A,B).
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FIG. 4. Cross sections of the spinal cord. (A,B) Calcitonin gene-related peptide (CGRP)-immunoreactive motor neurons in the rostral spinal cord. Neurons in both the dorsomedial group (small arrows) and the ventrolateral group (large arrows) show strong CGRP immunoreactivity. (C) Substance P (SP)-immunoreactive motor neurons in the rostral spinal cord. Neurons in the ventrolateral group (large arrows), but not in the dorsolateral group (small arrows), show SP immunoreactivity. Abbreviations: M, Mauthner axon. Scale bars: 200 m (A), 100 m (B,C).
nicotinic Ach receptors, to cause the peculiar inhibitory electrophysiological reactions. Thus, the present study raises the possibility that neuropeptides such as CGRP and SP play important roles in the neuromuscular transmission in teleosts. In particular, in the red muscles SP might be related to the unique physiological properties of the tonic type muscles. ACKNOWLEDGEMENTS
We are grateful to M. Kobayashi, C. Usami, and T. Hisajima for their technical aids.
REFERENCES 1. Akasu, T.; Kojima, M.; Koketsu, K. Substance P modulates the sensitivity of the nicotinic receptor in amphibian cholinergic transmission. Br. J. Pharmacol. 80:123–131; 1983. 2. Andreose, J. S.; Sala, C.; Fumagalli, G. Immunolocalization of chomogranin B, secretogranin II, calcitonin gene-related peptide and substance P at developing and adult neuromuscular synapses. Neurosci. Lett. 174:177–180; 1994. 3. Arias, H. R. Noncompetitive inhibition of nicotinic acetylcholine receptors by endogenous molecules. J. Neurosci. Res. 52:369 –379; 1998. 4. Blanco, C.; Popper, P.; Micevych, P. ␣-CGRP mRNA levels in motoneurons innervating specific rat muscles. Mol. Brain Res. 44:253– 261; 1997. 5. Bone, Q.; Dana Ono, R. Systematic implications of innervation patterns in teleost myotomes. Breviora 470:1–23; 1982. 6. Caratsch, C. G.; Eusebi, F. Effect of calcitonin gene-related peptide on synaptic transmission at the neuromuscular junction of the frog. Neurosci. Lett. 111:344 –350; 1990. 7. Csillik, B.; Tajti, L.; Kova´cs, T.; Kukla, E.; Rakic, P.; Knyiha´r-Csillik, E. Distribution of calcitonin gene-related peptide in vertebrate neuromuscular junctions: Relationship to the acetylcholine receptor. J. Histochem. Cytochem. 41:1547–1555; 1993. 8. Eusebi, F.; Farini, D.; Grassi, F.; Monaco, L.; Ruzzier, F. Effects of calcitonin gene-related peptide on synaptic acetylcholine receptorchannels in rat muscle fibers. Proc. R. Soc. Lond. B 234:333–342; 1988.
9. Fetcho, J. R. The organization of the motoneurons innervating the axial musculature of vertebrates. I. Goldfish (Carassius auratus) and mudpuppies (Necturus maculosus). J. Comp. Neurol. 249:521–550; 1986. 10. Fetcho, J. R. A review of the organization and evolution of motoneurons innervating the axial musculature of vertebrates. Brain Res. Rev. 12:243–280; 1987. 11. Fontaine, B.; Klarsfeld, A.; Ho¨kfelt, T.; Changeux J.-P. Calcitonin gene-related peptide, a peptide present in spinal cord motoneurons, increases the number of acetylcholine receptors in primary cultures of chick embryo myotubes. Neurosci. Lett. 71:59 – 65; 1986. 12. Fontaine, B.; Klarsfeld, A.; Changeux J.-P. Calcitonin gene-related peptide and muscle activity regulate acetylcholine receptor ␣-subunit mRNA levels by distinct intracellular pathways. J. Cell. Biol. 105: 1337–1342; 1987. 13. Forsgren, S.; Bergh, A.; Carlsson, E.; Thornell, L. E. Calcitonin gene-related peptide expression at endplates of different fibre types in muscles in rat hind limbs. Cell Tissue Res. 274:439 – 446; 1993. 14. Gibons, S. J.; Polak, J. M.; Bloom, S. R.; Sabata, I. M.; Mulderry, P. M.; Ghatei, M. A.; Morrison, J. F. B.; Kelly, J. S.; Rosenfeld, M. G. Calcitonin gene-related peptide (CGRP)-immunoreactivity in the spinal cord of man and of eight species. J. Neurosci. 4:3101–3111; 1984. 15. Hess, A. Vertebrate slow muscle fibers. Physiol. Rev. 50:40 – 62; 1970. 16. Hidaka, T.; Miyahara, T. Excitatory and inhibitory neuromuscular transmission in fish red muscle. Zool. Sci. 4:819 – 823; 1987. 17. Hidaka, T.; Yukiyama, S. Excitatory and inhibitory junction potentials recorded from the red muscle of marine teleosts, puffer fish. Zool. Sci. 5:947–950; 1988. 18. Hidaka, T.; Igata, H.; Kita, T. Distribution of the excitatory and inhibitory Ach receptors in red muscle of the fishes, Cyprinus carpio and Carassius auratus. Zool. Sci. 8:485– 489; 1991. 19. Hidaka, T.; Igata, H. Augmentation and suppression of the excitatory and inhibitory neuromuscular transmission induced by the ionophores, X-537A and A23187, in the red muscle of carp, Cyprinus carpio. Jpn. J. Physiol. 45:509 –518; 1995. 20. Lu, B.; Fu, W. M.; Greengard, P.; Poo, M. M. Calcitonin gene-related peptide potentiates synaptic responses at developing neuromuscular junction. Nature 363:76 –79; 1993. 21. Lu, B.; Fu, W. M. Regulation of postsynaptic responses by calcitonin gene related peptide and ATP at developing neuromuscular junctions. Can. J. Physiol. Pharmacol. 73:1050 –1056; 1994.
552 22. Lukas, R. J.; Eisenhour, C. M. Interactions between tachynins and diverse, human nicotinic acetylcholine receptor subtypes. Neurochem. Res. 21:1245–1257; 1996. 23. Matteoli, M.; Haimann, C.; Torri-Tarelli, F.; Polak, J. M.; Ceccarelli, B.; De Camilli, P. Differential effect of ␣-latrotoxin on exocytosis from small synaptic vesicles and from large dense-core vesicles containing calcitonin gene-related peptide at the frog neuromuscular junction. Proc. Natl. Acad. Sci. USA 85:7366 –7370; 1988. 24. Matteoli, M.; Balbi, S.; Sala, C.; Chini, B.; Cimino, M.; Vitadello, M.; Fumagalli, G. Developmentally regulated expression of calcitonin gene-related peptide at mammalian neuromuscular junction. J. Mol. Neurosci. 2:175–184; 1990. 25. Matteoli, M.; Haimann, C.; De Camilli, P. Substance P-like immunoreactivity at the frog neuromuscular junction. Neuroscience 37:271– 275; 1990. 26. Miles, K.; Greengard, P.; Huganir, R. L. Calcitonin gene-related peptide regulates phosphorylation of the nicotinic acetylcholine receptor in rat myotubes. Neuron 2:1517–1524; 1989. 27. Min, C. K.; Weiland, G. A. Effects of substance P on the binding of agonists to the nicotinic acetylcholine receptor of Torpedo electroplague. J. Neurochem. 60:2238 –2246; 1993. 28. Mulle, C.; Benoit, P.; Pinset, C.; Roa, M.; Changeux, J. -P. Calcitonin gene-related peptide enhances the rate of desensitization of the nicotinic acetylcholine receptor in cultured mouse muscle cells. Proc. Natl. Acad. Sci. USA 85:5728 –5732; 1988. 29. New, H. V.; Mudge, A. W. Calcitonin gene-related peptide regulates
FUNAKOSHI ET AL. muscle acetylcholine receptor synthesis. Nature 323:809 – 811; 1986. 30. Ogata, T. Structure of motor endplates in the different fiber types of vertebrate skeletal muscles. Arch. Histol. Cytol. 51:385– 424; 1988. ´ . Distribution of calcitonin gene-related peptide 31. Petko´, M.; Sa´nta, A immunoreactivity in the central nervous system of the frog, Rana esculenta. Cell Tissue Res. 269:525–534; 1992. 32. Piehl, F.; Arvidsson, U.; Ho¨kfelt, T.; Cullheim, S. Calcitonin generelated peptide-like immunoreactivity in motoneuron pools innervating different hind limb muscles in the rat. Exp. Brain Res. 96:291–303; 1993. 33. Radaelli, G.; Domeneghini, C.; Arrighi, S.; Mascarello, F.; Veggetti, A. Different putative neuromodulators are present in the nerves which distribute to the teleost skeletal muscle. Histol. Histopathol. 13:939 – 947; 1998. 34. Tarabal, O.; Caldero, J.; Ribera, J.; Sorribas, A.; Lopez, R.; Molgo, J.; Esquerda, J. E. Regulation of motoneuronal calcitonin gene-related peptide (CGRP) during axonal growth and neuromuscular synaptic plasticity induced by botulinum toxin in rats. Eur. J. Neurosci. 8:829 – 836; 1996. 35. Westerfield, M.; McMurrary, J. V.; Eisen, J. S. Identified motoneurons and their innervation of axial muscles in the zebrafish. J. Neurosci. 6:2267–2277; 1986. 36. Weiland, G. A.; Durkin, J. R.; Henley, J. M.; Simasko, S. M. Effects of substance P on the binding of ligands to nicotinic acetylcholine receptors. Mol. Pharmacol. 32:625– 638; 1988.
PII S0361-9230(00)00295-1
Differential innervation of the goldfish tonic red muscles and twitch white muscles by neuropeptideimmunoreactive motoneurons Kengo Funakoshi,* Tetsuo Kadota, Yoshitoshi Atobe, Masato Nakano, Midori Tsukagoshi, Richard C. Goris and Reiji Kishida Department of Anatomy, Yokohama City University School of Medicine, Yokohama, Japan [Received 18 December 1999; Revised 26 April 2000; Accepted 28 April 2000] ABSTRACT: Neuropeptides in the motor nerves innervating the red and white muscles of the goldfish Carassius auratus were examined. In the tonic red muscles, varicose nerve endings immunoreactive for both calcitonin gene-related peptide and substance P were found spread over the surface of the muscle fibers, but in the twitch white muscles only scattered nerve endings immunoreactive for calcitonin gene-related peptide were found. At the electron microscopic observation, dense electron products immunoreactive for calcitonin gene-related peptide and for substance P (SP) were detected in the motor nerve endings making synapses on the muscle fibers of the red muscles. In the spinal cord, all of the motor neurons showed immunoreactivity to calcitonin gene-related peptide, but the motor neurons immunoreactive for substance P were restricted to the ventrolateral group that has been shown to project predominantly to the red muscles. These results suggest that the motor neurons innervating the red and white muscles of the goldfish are distinct in their neuropeptide content. The present study also raises the possibility that SP might be related to the unique physiological properties of the tonic type red muscles, probably by direct binding to the acetylcholine receptors. © 2000 Elsevier Science Inc.
slow tonic fibers are reduced in number with the exception of specific regions, such as the extraocular muscles [30]. In fish, which has the most primitive muscle fiber composition among vertebrates, the morphologically typical slow muscle fibers are lacking. However, in most fish species, muscle fibers in the red muscles are tonic and those in the white muscles are twitch, and both are innervated by multiple grape-type endings scattered over the surface of the muscle cells [5,30]. In the trunk region of teleosts, the red muscles are restricted to a small lateral area immediately beneath the lateral line, while the white muscles occupy a large deeper area of the musculature [10]. The red muscles are well-vascularized, and specialized for aerobic metabolism. They are active during sustained swimming (cruising). On the other hand, the white muscles are active mainly during vigorous bursts of swimming. The innervation is also quite different in the red and white muscles. In the goldfish Carassius auratus, and the zebrafish Brachydanio rerio, the red muscles in the trunk region are innervated by the spinal motor neurons situated in the ventrolateral column of the ventral horn, while the white muscles are innervated by the dorsomedial column [9,35]. In previous electrophysiological studies, only junction potentials, but not action potentials, were recorded intracellularly from the red muscle fibers after stimulation of single spinal nerve fibers in some species of teleosts [10,16 –18]. The junction potentials recorded after stimulation were classified into three types, i.e., excitatory potentials, inhibitory potentials, and diphasic potentials that are composed of an excitatory potential followed by an inhibitory potential. These responses may be mediated exclusively by nicotinic acetylcholine (Ach) receptors [16 –19]. Recently, nerve fibers immunoreactive for calcitonin generelated peptide (CGRP) and substance P (SP) have been described in the muscular tissue of the eel, raising the possibility that these neuropeptides may be present in the motor nerve endings to modulate neuromuscular transmission in teleosts [33]. On this basis, we examined in the present study the distribution of these neuropeptides in the spinal somatic motoneurons and the motor endings in the red and white muscles of the goldfish.
KEY WORDS: Spinal cord, Motor endplate, Neuropeptide, Immunohistochemistry, Electron microscopy, Teleosts.
INTRODUCTION The skeletal muscle fibers of vertebrates are classified into fast twitch fibers and slow tonic fibers [30]. Slow tonic fibers are morphologically characterized by zigzag-shaped Z-lines, poorly developed sarcoplasmic reticulum, and multiple innervations by grape-type motor endings. On the other hand, the fast twitch fibers have straight Z-lines, well-developed sarcoplasmic reticulum, and single innervation by plate-type or linear type motor endings [15,30]. This type of fibers is subdivided into three categories: white, red, and intermediate fibers, according to the relative amounts of mitochondria. The composition of these muscle fiber types varies among the classes of vertebrates [30]. In amphibians, reptiles, and birds, the fast twitch and the slow tonic muscle fibers are mixed in the trunk muscles. In mammals, on the other hand,
* Address for correspondence: Kengo Funakoshi, Department of Anatomy, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004 Japan. Fax: ⫹81-45-7827251; E-mail: [email protected]
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We used a total of three adult goldfish Carassius auratus, bought commercially and maintained in an aquarium. Light Microscopic Immunohistochemistry In the light microscopic study, two fish were anesthetized with 0.01% tricaine methanesulfonate in water, and perfused transcardially with heparinized saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The rostral part of the spinal cord and the musculature of the trunk were removed, postfixed in the same solution for 5– 6 h at 4°C, and infiltrated overnight with 30% sucrose in 0.1 M PB at 4°C for cryoprotection. The specimens were frozen and cut serially into 30-m cross-sections on a cryostat and thaw-mounted on glass slides. The sections were then incubated in the fixative for 10 min, and washed in 0.3% Triton X-100 in 0.1 M phosphate-buffered saline (PBST). The sections were first incubated with 10% normal goat serum (NGS), 1% bovine serum albumin (BSA), and 0.2% sodium azide in PBST for 1 h at room temperature. After rinsing in PBST, the sections were incubated for 48 h at 4°C with one of the following primary antibodies: rabbit polyclonal antibody against CGRP (1: 1500; Chemicon, Temecula, CA, USA), rat monoclonal antibody against SP (1:100; Harlan sera-lab, Loughborough, UK). The antibody was diluted with 0.1 M PBST containing 1% NGS, 0.2% BSA, and 0.2% sodium azide. The sections were then incubated for 2 h with goat anti-rabbit IgG (1:200; Cappel, Aurora, OH, USA) or goat anti-rat IgG (1:100; Cappel), diluted with 0.1 M PBST containing 1% NGS, 0.2% BSA, and 0.2% sodium azide, and incubated for 2 h with rabbit peroxidase-anti peroxidase (PAP) complex (1:200; Jackson, West Grove, PA, USA) or rat PAP complex (1:100; Jackson), diluted with 0.1 M PBS containing 1% NGS, 0.2% BSA, and 0.1% thimerosal at room temperature. The peroxidase reactivity was demonstrated with 3,3⬘-diaminobenzidine (DAB; Sigma, St. Louis, MO, USA). The specificity of the staining was tested by omission of the antibody. To examine the co-localization of CGRP and SP in the peripheral nerve terminals, the sections of the musculature were incubated for 48 h at 4°C with a mixture of rabbit anti-CGRP IgG (1:250; Chemicon) and rat anti-SP IgG (1:50; Harlan sera-lab), diluted with 0.1 M PBST containing 1% NGS, 0.2% BSA, and 0.2% sodium azide. The sections were then incubated for 3 h with a mixture of fluorescein isothiocyanate-conjugated goat anti-rat IgG (1:100; Cappel) and rhodamine-conjugated goat anti-rabbit IgG (1:100; Cappel), diluted with 0.1 M PBST containing 1% NGS, 0.2% BSA, and 0.2% sodium azide at room temperature. Observation was made with a fluorescence microscope (Leica DMR, Welzlar, Germany), equipped with a double band-pass filter set. Immunoelectron Microscopy The remaining fish was fixed with 0.1 M PB (pH 7.4) containing 4% paraformaldehyde, 0.1% glutaraldehyde, and 0.2% picric acid. The musculature was then dissected out, postfixed in the same solution for 5– 6 h, and infiltrated overnight with 30% sucrose in 0.1 M PB at 4°C. The specimens were freeze-thawed by liquid nitrogen, embedded in gelatin gel, and then cut serially into 30-m sections in a Vibratome. The sections were treated freefloating in 1% sodium borohydride in distilled water for 30 min at 4°C, incubated for 48 h at 4°C with rabbit anti-CGRP IgG or rat anti-SP IgG (described above), diluted with 0.1 M PBS containing 1% NGS, 0.2% BSA, and 0.2% sodium azide, and then stained with the PAP-DAB method. The sections were postfixed subsequently in 1% glutaraldehyde and 1% osmium tetroxide in 0.1 M
PB at 4°C for 30 min. After dehydration in a graded ethanol series and dipping in propylene oxide, the sections were embedded in a mixture of Epon and Araldite. Ultrathin sections were cut on an ultratome (LKB; Leica, Welzlar, Germany). After counterstaining with uranyl acetate, the sections were examined under an electron microscope (H-7500; Hitachi, Tokyo, Japan). Electronic Imaging Primary images of immunofluorescence reactivity in the muscles and of immunoperoxidase reactivity in the spinal cord were acquired on films, which were then scanned into a Macintosh computer, through a film scanner (Nikon, Tokyo, Japan). Contrast and brightness were adjusted with the Adobe Photoshop software. RESULTS In the fixed specimen, the red muscles could be clearly distinguished from the white muscles in the trunk region, on the basis of their color and location. The red muscles occupied superficial small area along the side of the body, immediately beneath the lateral line. The white muscles occupied the deeper large area of the musculature. They could be subdivided into epaxial and hypaxial muscles. Light Microscopic Study of Musculature In the red muscles, many CGRP-immunoreactive varicose nerve fibers were found on the surface of the muscle cells (Figs. 1A,B). The CGRP-immunoreactive nerve fibers were more abundant in the surface region than in the deep layers of the red muscles. No CGRP-positive nerve fibers were found to terminate around the capillaries of the musculature. The distribution of the SP-immunoreactive varicose nerve fibers was similar. They were found on the surface of the muscle cells (Fig. 1C). In the double-labeling study of CGRP and SP, all varicose nerve terminals appeared to be positive for both CGRP and SP (Fig. 2). Using the double band-pass filter, virtually all fluorescent nerve fibers in the red muscles were shown to be double immunopositive. In the white muscles, CGRP-immunoreactive nerve terminals were also found to be apposed on the surface of the muscle cells, but were much scarcer than in the red muscles (Fig. 1D). On the other hand, no fibers immunoreactive for SP were found in the white muscles. Electron Microscopic Study of Musculature In the red muscles, both CGRP- and SP-immunoreactive varicosities were found to form morphologically identifiable synapses on the striated muscle cells (Figs. 3A,B). The presynaptic membrane of the varicosities did not show extensive foldings but exhibited the smooth surfaces typical of grape-type motor endings. There was a synaptic cleft of 100-nm width between the surface of the muscle cells and the presynaptic membranes. A basement membrane was also found in the synaptic clefts. Electron-dense peroxidase reaction products filled the varicosities. Light Microscopic Study of Spinal Motor Neurons In the rostral part of the spinal cord, virtually all of the motor neuronal cell bodies were strongly CGRP-immunoreactive (Figs. 4A,B). The staining intensity of immunoreactive neurons was homogeneous. In contrast, weakly SP-immunoreactive neurons were predominant in the ventrolateral column (Fig. 4C).
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FIG. 1. Nerve endings in the musculature. (A) Calcitonin gene-related peptide (CGRP)-immunoreactive varicose nerve endings are abundant in the red muscles, but scarce in the white muscles. (B) CGRP-immunoreactive varicose nerve endings are in contact with the surface of the muscle cells of the red muscles. (C) Many substance P (SP)-immunoreactive varicose nerve endings (small arrows) are also seen in the red muscles, but not in the white muscles. (D) SP-immunoreactive varicose nerve endings (small arrows) are in contact with the surface of the muscle cells of the red muscles. Abbreviation: W, white muscles. Scale bars: 100 m (A,C), 50 m (B,D).
DISCUSSION To date, little is known about neuropeptides expressed by the somatic motor neurons in fish. The present study in the goldfish showed that the spinal motor neurons innervating the tonic red muscles were immunoreactive to both CGRP and SP, while those innervating the twitch white muscles were immunoreactive only to CGRP. Immunoreactivity to these peptides was also detected in the motor endings that make synapses with the muscle cells. A total co-localization of immunoreactivity to CGRP and SP was shown in the present study in the varicose nerve endings in the red muscles. Therefore, there is distinct grouping of target-specific, phenotypically distinct somatic motor neurons in the goldfish. Immunoreactivity to CGRP in the spinal motor neurons and the motor endings has previously been reported in mammals and amphibians [14,25,31]. Unlike mammals, in which the amount of CGRP in the motor neurons or the motor endplates differed according to the muscle type and the contractile activity of the muscles innervated [4,7,13,32], the strong immunoreactivity to CGRP was homogeneous in virtually all motor neurons in the
spinal cord of goldfish. There was no difference in staining among those projecting to the white or red muscles. This finding might suggest that CGRP is essential for the activity of motor neurons in fish. It has been shown in vitro that CGRP enhances the rate of desensitization of the muscle Ach receptor channels, probably by cyclic adenosine monophosphate-mediated phosphorylation [26, 28], and regulates the sensitivity of Ach receptors [8]. It remains to be elucidated whether these effects are physiological [6]. On the other hand, CGRP immunoreactivity is especially high in the developing stage of the motor neurons and the neuromuscular junctions in mammals [2,24]. CGRP has also been suggested to promote development and remodeling of neuromuscular junctions by synthesizing the ␣ subunit of the Ach receptors and potentiating postsynaptic Ach channel activity [11,12,20,21,29,34]. The present findings in the goldfish lead us to hypothesize that CGRP is essential for constant remodeling of the fish neuromuscular junction, which is morphologically primitive and much simpler than those in other vertebrates. We found immunoreactivity to SP in the motor neurons and the motor endings only in the red muscles. Immunoreactivity to SP has
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FIG. 2. Double-labeled immunofluorescent image of the nerve fibers in the red muscles. Fluorescein isothiocyanate-labeled substance P-immunoreactive varicose nerve endings (A,C) appear identical to rhodamine-labeled calcitonin gene-related peptideimmunoreactive varicose nerve endings (B,D). Scale bars: 50 m.
been found in the motor endings in the frog, but not in mammals [2,25]. In adult frogs, SP has been suggested to suppress the sensitivity of nicotinic Ach receptors by direct binding to the receptor molecules [1]. The direct binding of SP to the Ach receptors was demonstrated in vitro [3,22,27,36]. In the tonic red muscles of teleosts, inhibitory and diphasic potentials, both of
which have a hyperpolarizing phase, have sometimes been recorded after stimulation of single motor nerve fibers [16 –19]. These excitatory and inhibitory potentials were both shown to be mediated by the nicotinic Ach receptors [17–19]. Therefore, it is possible that SP released from the motor endings of teleosts may hyperpolarize the postsynaptic membrane via direct binding to the
FIG. 3. Electron micrographs of the calcitonin gene-related peptide-immunoreactive (A) and substance P-immunoreactive (B) varicose nerve endings that contact the striated muscle cells. A basement membrane is interposed between the presynaptic membrane of the nerve terminals (small arrows) and the postsynaptic membrane (arrowheads) of the muscle cells. Scale bars: 0.5 m (A,B).
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FIG. 4. Cross sections of the spinal cord. (A,B) Calcitonin gene-related peptide (CGRP)-immunoreactive motor neurons in the rostral spinal cord. Neurons in both the dorsomedial group (small arrows) and the ventrolateral group (large arrows) show strong CGRP immunoreactivity. (C) Substance P (SP)-immunoreactive motor neurons in the rostral spinal cord. Neurons in the ventrolateral group (large arrows), but not in the dorsolateral group (small arrows), show SP immunoreactivity. Abbreviations: M, Mauthner axon. Scale bars: 200 m (A), 100 m (B,C).
nicotinic Ach receptors, to cause the peculiar inhibitory electrophysiological reactions. Thus, the present study raises the possibility that neuropeptides such as CGRP and SP play important roles in the neuromuscular transmission in teleosts. In particular, in the red muscles SP might be related to the unique physiological properties of the tonic type muscles. ACKNOWLEDGEMENTS
We are grateful to M. Kobayashi, C. Usami, and T. Hisajima for their technical aids.
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