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A comparative survey of the type of sympathetic neuro-melanophore transmission in catfishes
Camp. Eiochem.
Physid.
Vol. 8%
0306~4492/86 $3.00+ 0.00 Pergamon Journals Ltd
No. 1, pp. 115-120, 1986
Printedin Great Britain
A COMPARATIVE SURVEY OF THE TYPE OF SYMPATHETIC NEURO-MELANOPHORE TRANSMISSION IN CATFISHES HIROAKI KASUKAWA,NORIKO OSHIMAand RYOZO FUJII* Department of Biology, Faculty of Science, Toho University;Miyama, Funabashi, Chiba 274, Japan. Telephone: (0474) 72- 114 I (Received 13 January 1986)
Abstract-l. The types of innervation to integumental melanophores were surveyed comparatively within the order Siluriformes. 2. In fish of many families, pigment aggregation within melanophores was found to be under the control of adrenergic sympathetic postganglionic fibers as in other fish. 3. In silurid catfish, on the other hand, the cells were found to be regulated cholinergically, though the fibers concerned were sympathetic postganglionic. Muscarinic cholinoceptors mediate the melaninaggregating response. 4. In some catfish belonging to Bagridae, Pimelodidae and Callichthyidae, the melanophores strangely possessed cholinoceptors, notwithstanding the fact that they were under adrenergic nervous control. 5. These results were discussed in conjunction with the phylogeny of Siluriformes.
hope of finding some key for elucidating the phylogeny of this group of fish.
INTRODUffION It is now established that in many fish the rapid pigment-aggregating responses of integumental chromatophores are primarily controlled by sympathetic postganglionic fibers (Iwata and Fukuda, 1973; Fujii and Oshima, 1986). Peripheral transmission to the effector cells has naturally been proved to be adrenergic (Fujii, 1961; Scheline, 1963; Healey and Ross, 1966), and the receptor involved has now been characterized as being of an alpha adrenergic nature (Grove, 1969; Reed and Finnin, 1972; Fernando and Grove, 1974; Fujii and Miyashita, 1975). Meanwhile, Fujii and Miyashita (1976) discovered that in a siluroid catfish (Purusilurus mom) the neurotransmission to the melanophores was peculiarly cholinergic, though the nerve fibers concerned were sympathetic postganglionic as usual. Further pharmacological analyses enabled them to characterize the postsynaptic cholinoceptor mediating the pigment aggregation as of a muscarinic nature. The same type of melanophore control has lately been found in the translucent glass catfish Kryptopterus bicirrhi, a popular aquarium fish, which also belongs to the family Siluridae (Fujii et al., 1982). Recently, we found that, although they were controlled by adrenergic fibers, the melanophores of a mailed catfish (Corydorus paleatus; Callichthyidae) possess muscarinic chlolinoceptors of unknown physiological significance (Kasukawa and Fujii, 1985). To date, such unusual cases have not been found in any teleostean orders other than Siluriformes. We therefore decided to make a comparative survey of the type of peripheral neural mechanisms controlling melanophores among fish within the order with the *Author to whom correspondence should be addressed.
MATERIALSAND
METHODS
A number of fish categorized as so-called “catfish” in a broad sense or into the order Siluriformes were used. They are listed in Table 1. In this study, we adopted the taxonomy of Chardon (1968), wherein the family nomenclature and classification as proposed by Greenwood er al. (1966) were incorporated. All the fish, with the exception of the marine catfish (Plotosus lineatus), were purchased from local dealers and were maintained in freshwater aquaria. Specimens of PIotosus lineatus were collected near the Kominato Marine Biological Laboratory, Faculty of Science, Ciba University, Awa, Chiba Prefecture, and were reared in a seawater tank. When we used isolated scales as the skin preparation, they were taken from the dorsal surface of the trunk with a pair of finely pointed forceps. Split fin pieces were prepared according to a previously described method (Fujii, 1959). In mailed catfish, melanophores on the inner surface of an isolated scale were observed (Kasukawa and Fujii, 1985). In some scaleless species, pieces of the dorsal trunk skin were used (Fujii and Miyashita, 1976). In all cases, the skin specimens were prepared and irrigated in a perfusion chamber with a physiological saline solution of the following composition (mM): NaCl 128, KC1 2.7, CaCI, 1.8, MgCI, 1.8, o-glucose 5.6, Tris-HCl buffer 10.0 (PH 7.2). Experimental procedures were fundamentally the same as those described earlier (Fujii and Miyashita, 1975). We adopted an improved method of microscopic photometry, however, for recording quantitatively the motile responses of melanophores (Oshima and Fujii, 1984). For some measurements, the pigment-aggregating response of the melanophore was elicited via nervous stimulation. For this purpose, a modified saline containing an increased K+ concentration (50 mM) was employed (Fujii, 1959). When this was done, the same amount of Na+ was compensatorily withdrawn in order to keep the tonicity of the fluid constant. 115
116
HIROAKI KASUKAWA et
al.
Table I. Types of pigment-aggregating innervation and neurotransmitter receptors of melanophoresin catfishes studiedto date Melanosolre-aggregating receptor
We Family
of
Species
Ictaluridae
Ictatwm
Bagridae
Alpha Muscarinic 'nnervat'on adrenergic cholinergic
nebuZosw
A
Mystus vittatus
A
M&us
A
sp.
+ + + + + + + ?
A*
ICtaluruSplmotatuw
Lsiocassissimarsis
A
Pseudobagms aumtiacus
A
P8eudobagmS fu'k&iRzCO
A
Liobagm
?
North America North America
+ +
Xryptopterusbioirrhi~
C
~ptopte~E
c
A glass
C
+
Schilbeidae
Eutropiellussp.
A
Pangasiidae
PangasiuspoZyumnodon
A
Heteropneustidae
Heteropmustes mCcrops
A
Chacidae
chaca c??aca
A
Mochokidae
symodo?ltis nigriumtiis
A
Doradidae
Tmchycorystes galeatus
A
Plotosidae
Plotosus Zineatus
A
Neosilums gleocoensis
A
PimelodeZlapi&us
A
PimelodeZtasp.
A
Smebodus sp.
A
Corydmas pateatus3
A
corydorastriZZineatus
A
CoqjdorasjulZi
A
Corydorassp.
A
Dianema tongibarbis
A
Pamque sp.
A
QtoocincZus affSni8
A
catfish (unidentified)
Pimelodidae
Callichthyidae
Loricarfidae
+ f + + + + + + + + + + + + + + + +
Asia
Japan
c**
sp.
India Japan
Parasilurusa60tus1
Siluridae
India Southeast
? + + +
mini
Habitat
Japan
India
Japan India India
Africa Southeast Asia Southeast Asia India Africa South America Asia Australia
+ + + + + + +
South America South America South America South America South America South America South America South America South America South America
‘Adrenergic. l*ChoEnergic. Among the above species, three have already been described by us elsewhere: 1, Fujii and Miyashita (1976); 2, Fujii et al. (1982); 3, Kasukawa and Fujii (1985).
The drugs employed were norepinephrine hydrochloride (Sankyo, Tokyo), phentolamine mesylate (Ciba-Geigy, Basel). acetylcholine chloride (Daiichi Seiyaku, Tokyo), atropine sulfate (Tanabe Seiyaku, Tqkyo), scopolamine hydrobromide (Yamanouchi Pharmaceutical, Tokyo) and
melatonin (Nakarai Chemical, Kyoto). Experiments were performed at room (20-26°C).
temperature
RESULTS
The results obtained are summarized in Table 1. In the families Ictaluridae, Schilbeidae, Pangasiidae, Heteropneustidae, Chacidae, Mochokidae, Doradidae, Plotosidae and Loricariidae, all the fishes investigated were found to have melanophores quite orthodoxly regulated by adrenergic postganglionic fibers, although not many species were examined in each family. Typical examples of the responses of the melanophores of this type can be seen in Figs 1A and 2A,
which illustrate the recordings obtained on the marine catfish, Plotosus lineatus, belonging to Plotosidae. These pharmacological results clearly indicated the adrenergic nature of peripheral transmission to the effector cells. Of the four species of silurid catfish listed in Table 1, two, i.e. the Japanese common catfish Parasilurus usatus (Fujii and Miyashita, 1976), and the translucent catfish Kryptopterus bicirrhi (Fujii et nl., 1982), had already been examined for the nature of innervation to the melanophores. Figures 1B and 2B show a typical series of the responses of the melanophores of the Japanese catfish Parusilurus asotus, recorded again for this study. Confirming surely earlier observations, the present results indicate even more clearly that the melanophores are regulated through the mediation of cholinoceptors of a muscarinic nature. In addition, we employed another species of translucent glass catfish, which apparently belongs to the
Neuro-melanophore transmission in catfishes
117
Fig. 1. Photoelectric recordings showing the responses of melanophores of three catfish species: A, the marine catfish Plotosus lineatus; B, the Japanese common catfish Parasilurus asotus; and C, the peppered catfish Corydoras paleatus. Each fish is representative of those in which melanophores were found to be under one of three types of regulatory system, i.e. orthodox adrenergic (A), muscarinic cholinergic (B) and adrenergic, but in which the effector cells possess extra cholinoceptors (C). The pigment-aggregating effect of acetylcholine (ACh) or norepinephrine (NE) was first examined. If the effect was detected, its influence on an alpha adrenolytic agent, phentolamine (PA), or a muscarinic cholinolytic agent, atropine (AP), was examined. Melatonin (MT, 1O-5M) was finally employed to induce the full level of melanosome aggregation.
same genus Kryptopterus, and another larger glass catfish. Neither the genus nor species of the latter could be determined. Judging from its external form and gross morphology, however, it undoubtedly belongs to this family. The melanophores in these fish as well were proved pharmacologically to receive cholinergic innervation in the same manner as that of the above-mentioned identified species of silurids.
Fig. 2. Photoelectric recordings of the responses of melanophores of the marine catfish (A), the Japanese common catfish (B) and the peppered catfish (C). Melanosomeaggregating stimulation by K+ (50mM) was applied in order to examine whether phentolamine (PA), an alpha adrenolytic agent, or scopolamine @A), a muscarinic cholinolytic, might antagonize the neurally evoked response. Melatonin (MT, 10m5M) was used to induce the full level of pigment aggregation.
We have already shown that in the peppered catfish paleatus the melanophores possess muscarinic cholinoceptors of unknown significance, although they are under the control of adrenergic postganglionics as usual (Kasukawa and Fujii, 1985). In this study, therefore, we tried to broadly survey similar systems among varieties of catfish. As expected, we found that three Corydoras spp. in addition to C. paleatus possessed the same system. As typical examples of melanophores regulated in this manner, the responses to testing of pharmacological agents in Corydoras paleatus were examined again, and sections of the recordings are shown in Figs 1C and 2C. Another catfish of the genus Dianema (species unidentified) was then examined. Although this fish is included in the order Callichthyidae, to which Corydoras catfish also belong, its regulatory system for melanophores was found to be of an orthodox adrenergic type without extra cholinoceptors. We also found that all three South American pimelodid catfish tested had systems of the Corydorus type in which melanophores are adrenergically controlled but have extra muscarinic cholinoceptors. Furthermore, two Mystus spp. among the six species examined in this survey and which belong to four genera from the family Bagridae were also found to have the same regulatory mechanism for melanophore motility. Corydoras
DISCUSSION
In examining the present results, it can first be said that, even in the order Siluriformes, melanophores are usually under the regulation of the adrenergic postganglionic fibers of the sympathetic system. That is, the melanophores are quite orthodoxly regulated in the same way as they are in many of the tele-
118
H~ROAKI KASUKAWA et af.
osts examined and described to date (~5 Fujii and Oshima, 1986). As we have already shown, the melanophores of two silurids, i.e. the Japanese common catfish, Parasilurus astutus (Fujii and Miyashita, 1976) and the translucent glass catfish, Kryptopterus bicirrhi (Fujii et al., 1982), are strangely controlled by cholinergic fibers. In the present study, a Kryptopterus glass catfish (species unidentified), and another glass catfish, which, although even the genus has not been identified, apparently belongs to the same family, were added to this group of fish. To our knowledge, no further species have been studied within this family, but other silurids may possibly have an identical system of nervous control for melanophores. It should be emphasized here, however, that in families other than Siluridae no species have been found to possess such a mode of neurotransmission to the pigment cells, although there still remains the possibility that the melanophores of Amblycipitidae might also be regulated by cholinergic fibers. Unfortunately, to date, we have not been able to obtain materials for these species. Furthermore, we have as yet no info~ation regarding the presence of cholinergic nervous control of chromatophores in animals of other classes. Silurid catfish may thus be considered a very unique group among the vertebrate classes. We have also learned that the melanophores of the peppered catfish, Corydoras paleatus, possess cholin~eptors of ~usca~nic nature in addition to the common adrenergic nervous supply (Kasukawa and Fujii, 1985). In the present survey, a quite similar system was found in other mailed catfish (Corydoras spp.), and in other families rather sporadically, i.e. in
AFRICA
SOUTH
EURASIA I
7 Schifbeidae
I I
I
NORTH AMERICA
Doradidae
I ! Heteropneustidae
i
AMEZRICA
BAGROIDEI 1
I
Mochokidae
two bagrid catfish belonging to the genus Mystus and in all three pimelodid species studied so far. As near as we can guess, there are no catfish in which melanophores do not receive pigmentaggregating innervation, or in which the cells are controlled by a different kind of chemical transmitter. In Siluriformes species, therefore, there exist three types of peripheral neuronal systems for controlling melanophore movement: an orthodox adrenergic one, a cholinergic one known only in Siluridae, and a third type in which adrenergically controlled melanophores possess extra cholinoceptors. A number of opinions have been put forward to date regarding the taxonomy and phylogeny of teleost fishes. If we restrict ourselves to those concerning catfish, however, only a few proposals have been presented. Among these, Chardon’s concept (1968) seems to be currently the most popular, although it still remains rather controversial. Figure 3 illustrates an abstraction from his phylogenetical tree of Siluroformes with special attention to the families actually dealt with in this work. Siluriformes has generally been classified into three suborders. Chardon (1968) himself defined and designated these suborders as Siluroidei, Bagroidei and Loricarioidei, all of them being derived from the same prototype (Stock II in Fig. 3). Glancing over the tree, we find the family Siluridae, in which the unique cholinergic nervous system operates, situated close to the stock, or rather directly originating from the prototype of Siluriformes. Bagridae, Pimelodidae and ~alIichthyidae, in which at least in some forms muscarinic cholinoceptors of unknown functional significance coexist with the functioning alpha adrenoceptors, are also found not far from the root. The tree also indicates that
i,
Pangasiidae
I
i
Chacidae Plotosidae
1
/Ictaluridae
I 1 Pimelodidae**
i
kStock I -(Diplomystidae~
Fig. 3. A genealogical tree based essentially on the concepts of Chardon (1968) regarding the evolution of catfish within the order Siluriformes. The names of families with which the present work was concerned were selectively postered in this figure. In the families, whose names are without asterisks, the melanophores are adrenergically controlled as in most teleosts. *Melanophores are cholinergically controlled. **Melanophores are adrenergically controlled but have extra muscarinic cholinoceptors.
Nemo-melanophore transmission in catfishes Ictaluridae, Schilbeidae, Pangasiidae, Heteropneustidae, Chacidae, Mochokidae, Doradidae and Plotosidae, in which melanophores are controlled in the same way as in fish of other orders, have been derived from Pimelodidae or Bagridae. Based on the results from a few species of catfish, we have already speculated that the cholinergic controlling mechanism for melanophore motility may be of a more evolved type than the orthodox one, and that those cells having both adrenoceptors and cholinoceptors may be transitional forms (Fujii et al., 1982; Kasukawa and Fujii, 1985). After conducting the present wide-ranging survey of Siluriformes, we have found no reason to change our previous opinion. Actually, examples of “cholinalso exist in ergic” sympathetic postganglionics higher animals, i.e. the sympathetic innervation to sweat glands (Dale and Feldberg, 1934) or to blood vessels in some muscular tissues in mammals (Uvnas, 1954). Such fibers may safely be assumed to have derived from conventional adrenergic ones. In addition, Le Douarin et al. (1975) showed that intrinsically adrenergic avian sympathetic ganglion cells can be artificially differentiated into either adrenergic or cholinergic cells. Working on mammalian sympathetic ganglion cells in culture, Reichardt and Patterson (1977) have also recently demonstrated that adrenergic-to-cholinergic conversion of the neuronal output can take place rather easily by selecting a proper culture medium. The reverse conversion, however, has not been shown to be an easy process. Therefore, it seems to be quite reasonable to assume that cholinergic control of the pigment cells has been derived from adrenergic control, and that cholinergic control is a more evolved type of regulation. The first of two speculations which we wish to present in this article therefore is quite inconsistent with the conjecture presented by Chardon (1968), who put Siluridae, Pimelodidae and Bagridae near the stock of his genealogical tree. As has been indicated earlier, the transition of adrenergic fibers to chohnergic ones might not have been a very difficult task for fish during their long period of evolutionary history (Fujii et al., 1982). We may thus surmise that conversion may have occurred in an ancient silurid from which all recent ones derived, or, later, frequently and independently among many of the silurids already radiated adaptively. In any case, the induction or expression of muscarinic cholinoceptors on the postsynaptic membrane must have been an easier process for fish, since vertebrate cells are undoubtedly loaded with the gene of the receptor protein. Thus, the sporadic occurrence of cholinoceptors, being independent of phylogeny, may be rather easily explained. In this connection, we can point out here the finding that even outside the fish classes, cholinoceptors are present on the melanophore membrane. The described species was an anuran Rana #ens, although not all the specimens examined possessed the receptor (MBller and Lerner, 1966; Goldman and Hadley, 1968). All these observations seem to favor the view that the simpler cholinergic system may have been derived from the regular adrenergic one during the evolution of chromatophore controlling mechanisms. The second hypothesis on the phylogeny of Siluri-
119
formes which we would like to put forward here is in good conformity with that of Chardon (1968), or, in a manner of speaking, is essentially based on it. As pointed out above, Pimelodidae, Bagridae, Callichthyidae and Siluridae can be found near the stock of the genealogical tree shown in Fig. 3. Chardon (1968) further regarded the former two families to be ancestral groups within the suborder Bagroidei (Chardon, 1968). In his tree, therefore, the families in which all the species studied in this survey were shown to possess an orthodox adrenergic melanosome-aggregating nervous system, are located more peripherally, being derived from ancient forms of the abovementioned two families. In conclusion, it may be put forward that, on the adrenergically innervated melanophores of the common ancestral catfish (Stock II in Fig. 3), muscarinic cholinoceptors may have come into existence by chance or by mistake. Thereafter, the adrenergic-tochohnergic conversion may have taken place in the melanophore system of primitive silurids. Radiating adaptively, their descendants may have formed the several recent silurid species found in Eurasia. In a few recently discovered bagrids and pimelodids, on the other hand, the original regulatory system for the pigment cell movements may have survived until today. When coexisting with alpha adrenoceptors, such cholinoceptors can naturally be thought to be practically useless, as has been suggested earlier (Fujii et al., 1982; Kasukawa and Fujii, 1985). As a matter of course, they may have disappeared from the melanophore membrane in many families assumed by Chardon (1968) to have been derived from the abovementioned two families. In Loricarioidei, melanophores of the genus Corydoras (family: Callichthyidae) were found to possess muscarinic cholinoceptors, while those of Dianema (family: Callichthyidae) did not. Since only two families have been examined within this order, it may ‘not be appropriate at this point to discuss the phyletic status further. In order to strengthen the hypothesis of this line, we should explain why and how such an unusual system, in which both alpha adrenoceptors and muscarinic cholinoceptors coexist, appeared in Stock I or II. Although the above-mentioned observations of the sporadic presence of cholinoceptors in melanophores of a Rana frog may be of a help, available information is still very meager. Further comparative surveys are therefore required. In any case, we hope that our results on the types of innervation to effector cells of one sort, the chromatophores, will provide some clue to a better understanding of the phylogeny of not only catfish but also of teleostean orders, which may be closely related to the order dealt with in this article. Acknowledgements-We wish to express our gratitude to Dr R. Arai, Department of Zoology, National Science Museum, Tokyo, for classifying some materials employed in this study. Our thanks are also due to Dr Y. Miyashita for interest and providing us some information relevant to our present study, to Dr S. Kikuchi and the staff of the Kominato Marine Biological Laboratory, Faculty of Science, Chiba University, for the supply of marine catfish, and to .I. Igarashi for technical assistance. This work was
120
H~R~AKI
supported in part by grants from the Ministry Science and Culture of Japan.
KAS~JKAWA
ofEducation,
REFERElVCES
Chardon M. (1968) Anatomie comparee de l’appreil de Weber et des structures connexes chez les Siluriformes. Ann. Mus. roy. Afr. centr. Ser. 8” 169, l-281. Dale II. H. and Feldberg W. (1934) The chemical transmission of secretory impulses to the sweat glands of the cat. J. Physiol. 82, 121-128. Fernando M. M. and Grove D 3. (1974) ~elanophor~ aggregation in the plaice {Pleuronecres xlralefsaL.)-II. in vitro effects of adrenergic drugs. Camp. Biochem. Physiol. 48A, 123-732. Fujii R. (1959) Mechanism of ionic action in the melanophore system of fish--I. Melanophore-concentrating action of potassium and some other ions. Annomes zooE. jap. 32,47-59. Fujii R. (1961) D~o~stmtion of the adrenergic naiure of transmission at the junction between meianophorecancentrating nerve and melanophore in bony fish. J. Fat. Sci. Univ. Tokyo Sect. IV 9, 111-196.
Fujii R. and Miyashita Y. (1975) Receptor mechanisms in fish chromatophores-I. Alpha nature of adrenoceptors mediating melanosome aggregation in guppy melanophores. Cor33p. B&hem. Physioi. 51C, 171-178. Fujii R. and Miyashita Y. (1976) Receptor mechanisms in fish chromatophores-III. Neurally controlled melanosome aggregation in a siluroid (Parnsilurus asotus) is strangely mediated by cholinoceptors. Camp. Biochem. Physiol. SSC, 4349.
Fujii R. and Gshima N. (1986) Control of chromatophore movements in teleost &&es. Zaul. Sci. 3, 13-47. Fujii R., Mi~ash~ta Y. and Fujii Y. (1982) Mu~rjnic cholinoceptors mediate neurally evoked pigment aggregation in glass catfish melanophores. J. Neural Transmission 54, 29-39.
Goldman I. P. and Hadley M. E. (1968) Acetylcholineinduced aggregation of melanin granules within epidermal (frog) melanocytes. 1. invesr. Dermutol. 50, 59-66. Greenwood P., Rosen A,, Weitzman I-I. and Myers G.
et ai.
(1966) Phyletic studies of teleostean fishes, with a provisional classification oFliving forms. Bull. Am. Mus. Nat. Hist. 131, 339456. Grove D. J. (1969) The effects of adrenergic drugs on melanophore~ of the minnow, P&oxinur g,&z&r~r (I_.). Camp. E&&em. Physiof. 28, 37-54. Healey E. G. and Ross D. M. (1966) The effects of drugs on the background colour responses of the minnow Phoxinus
phoxinus
L.
Comp.
Biochem.
Physiol.
19,
545-580. Iwata K. S. and Fukuda H. (1973) Central control of color changes in fish. In ,Respunses cf Fish to Environments Changes (Edited by CItavin W.), pp. 316341. C. C. Thomas, Springfield. Kasukawa H. and Fujii R. (1985) Receptor mechanisms in fish chromatophores-VII. Muscarinic cholinoceptors and alpha adrenoceptors, both mediating pigment aggregation, strangely coexist in Corydoras melanophores. Copnp. Biochem. Physbf. LXX, 21 l-21 5. Le Douarin N. M., Renaud D., Teillet M. A. and Le Douarin G. H. (1975) Cholinergic differentiation of presumptive adrenergic neuroblasts in interspeciiic chimeras after heterotopic transplantations. Proc. natn. Acad. Sci. U.S.A. 72, 728-732.
Moller H. and Lerner A. B. (1966) Melanocyte stimulating hormone inhibition by acetylcholine and noradrenaline in the frog skin bioassay. Acta endocrinol. 51, I49-I6@ Gshima N. and Fujii R. (1984) A precision photoekztric method for recording chromatophore responses in u&u. Zool Sci. I, 545.-552. Reed B. L. and Finnln B. C. (1972) Adrenergic innervation of melanophores in a teleost fish. In Pigmentation: Its Genesis and Biologic Control (Edited by Riley V.), pp. 285-294. Appleto~“~entury-Crofts, New York. __ Reichardt L. F. and Patterson P. H. (19771 > , Neurotransmitter synthesis and uptake by isolated sympathetjc neurones in microcultures. Nature, Lend. 270, 147-151. S&line R. R. (1963) Adrenergic mechanisms in fish: chromatophore pigment concentration in cuckoo wrasse Labrus ossifasus (I,,). Comp. Bin&em. Physiol. 9+ 215-227. Uvnas B, (1954) Sympathetic vasodilator outflow. Physiol. Rpt’. 34, 5088618.
Physid.
Vol. 8%
0306~4492/86 $3.00+ 0.00 Pergamon Journals Ltd
No. 1, pp. 115-120, 1986
Printedin Great Britain
A COMPARATIVE SURVEY OF THE TYPE OF SYMPATHETIC NEURO-MELANOPHORE TRANSMISSION IN CATFISHES HIROAKI KASUKAWA,NORIKO OSHIMAand RYOZO FUJII* Department of Biology, Faculty of Science, Toho University;Miyama, Funabashi, Chiba 274, Japan. Telephone: (0474) 72- 114 I (Received 13 January 1986)
Abstract-l. The types of innervation to integumental melanophores were surveyed comparatively within the order Siluriformes. 2. In fish of many families, pigment aggregation within melanophores was found to be under the control of adrenergic sympathetic postganglionic fibers as in other fish. 3. In silurid catfish, on the other hand, the cells were found to be regulated cholinergically, though the fibers concerned were sympathetic postganglionic. Muscarinic cholinoceptors mediate the melaninaggregating response. 4. In some catfish belonging to Bagridae, Pimelodidae and Callichthyidae, the melanophores strangely possessed cholinoceptors, notwithstanding the fact that they were under adrenergic nervous control. 5. These results were discussed in conjunction with the phylogeny of Siluriformes.
hope of finding some key for elucidating the phylogeny of this group of fish.
INTRODUffION It is now established that in many fish the rapid pigment-aggregating responses of integumental chromatophores are primarily controlled by sympathetic postganglionic fibers (Iwata and Fukuda, 1973; Fujii and Oshima, 1986). Peripheral transmission to the effector cells has naturally been proved to be adrenergic (Fujii, 1961; Scheline, 1963; Healey and Ross, 1966), and the receptor involved has now been characterized as being of an alpha adrenergic nature (Grove, 1969; Reed and Finnin, 1972; Fernando and Grove, 1974; Fujii and Miyashita, 1975). Meanwhile, Fujii and Miyashita (1976) discovered that in a siluroid catfish (Purusilurus mom) the neurotransmission to the melanophores was peculiarly cholinergic, though the nerve fibers concerned were sympathetic postganglionic as usual. Further pharmacological analyses enabled them to characterize the postsynaptic cholinoceptor mediating the pigment aggregation as of a muscarinic nature. The same type of melanophore control has lately been found in the translucent glass catfish Kryptopterus bicirrhi, a popular aquarium fish, which also belongs to the family Siluridae (Fujii et al., 1982). Recently, we found that, although they were controlled by adrenergic fibers, the melanophores of a mailed catfish (Corydorus paleatus; Callichthyidae) possess muscarinic chlolinoceptors of unknown physiological significance (Kasukawa and Fujii, 1985). To date, such unusual cases have not been found in any teleostean orders other than Siluriformes. We therefore decided to make a comparative survey of the type of peripheral neural mechanisms controlling melanophores among fish within the order with the *Author to whom correspondence should be addressed.
MATERIALSAND
METHODS
A number of fish categorized as so-called “catfish” in a broad sense or into the order Siluriformes were used. They are listed in Table 1. In this study, we adopted the taxonomy of Chardon (1968), wherein the family nomenclature and classification as proposed by Greenwood er al. (1966) were incorporated. All the fish, with the exception of the marine catfish (Plotosus lineatus), were purchased from local dealers and were maintained in freshwater aquaria. Specimens of PIotosus lineatus were collected near the Kominato Marine Biological Laboratory, Faculty of Science, Ciba University, Awa, Chiba Prefecture, and were reared in a seawater tank. When we used isolated scales as the skin preparation, they were taken from the dorsal surface of the trunk with a pair of finely pointed forceps. Split fin pieces were prepared according to a previously described method (Fujii, 1959). In mailed catfish, melanophores on the inner surface of an isolated scale were observed (Kasukawa and Fujii, 1985). In some scaleless species, pieces of the dorsal trunk skin were used (Fujii and Miyashita, 1976). In all cases, the skin specimens were prepared and irrigated in a perfusion chamber with a physiological saline solution of the following composition (mM): NaCl 128, KC1 2.7, CaCI, 1.8, MgCI, 1.8, o-glucose 5.6, Tris-HCl buffer 10.0 (PH 7.2). Experimental procedures were fundamentally the same as those described earlier (Fujii and Miyashita, 1975). We adopted an improved method of microscopic photometry, however, for recording quantitatively the motile responses of melanophores (Oshima and Fujii, 1984). For some measurements, the pigment-aggregating response of the melanophore was elicited via nervous stimulation. For this purpose, a modified saline containing an increased K+ concentration (50 mM) was employed (Fujii, 1959). When this was done, the same amount of Na+ was compensatorily withdrawn in order to keep the tonicity of the fluid constant. 115
116
HIROAKI KASUKAWA et
al.
Table I. Types of pigment-aggregating innervation and neurotransmitter receptors of melanophoresin catfishes studiedto date Melanosolre-aggregating receptor
We Family
of
Species
Ictaluridae
Ictatwm
Bagridae
Alpha Muscarinic 'nnervat'on adrenergic cholinergic
nebuZosw
A
Mystus vittatus
A
M&us
A
sp.
+ + + + + + + ?
A*
ICtaluruSplmotatuw
Lsiocassissimarsis
A
Pseudobagms aumtiacus
A
P8eudobagmS fu'k&iRzCO
A
Liobagm
?
North America North America
+ +
Xryptopterusbioirrhi~
C
~ptopte~E
c
A glass
C
+
Schilbeidae
Eutropiellussp.
A
Pangasiidae
PangasiuspoZyumnodon
A
Heteropneustidae
Heteropmustes mCcrops
A
Chacidae
chaca c??aca
A
Mochokidae
symodo?ltis nigriumtiis
A
Doradidae
Tmchycorystes galeatus
A
Plotosidae
Plotosus Zineatus
A
Neosilums gleocoensis
A
PimelodeZlapi&us
A
PimelodeZtasp.
A
Smebodus sp.
A
Corydmas pateatus3
A
corydorastriZZineatus
A
CoqjdorasjulZi
A
Corydorassp.
A
Dianema tongibarbis
A
Pamque sp.
A
QtoocincZus affSni8
A
catfish (unidentified)
Pimelodidae
Callichthyidae
Loricarfidae
+ f + + + + + + + + + + + + + + + +
Asia
Japan
c**
sp.
India Japan
Parasilurusa60tus1
Siluridae
India Southeast
? + + +
mini
Habitat
Japan
India
Japan India India
Africa Southeast Asia Southeast Asia India Africa South America Asia Australia
+ + + + + + +
South America South America South America South America South America South America South America South America South America South America
‘Adrenergic. l*ChoEnergic. Among the above species, three have already been described by us elsewhere: 1, Fujii and Miyashita (1976); 2, Fujii et al. (1982); 3, Kasukawa and Fujii (1985).
The drugs employed were norepinephrine hydrochloride (Sankyo, Tokyo), phentolamine mesylate (Ciba-Geigy, Basel). acetylcholine chloride (Daiichi Seiyaku, Tokyo), atropine sulfate (Tanabe Seiyaku, Tqkyo), scopolamine hydrobromide (Yamanouchi Pharmaceutical, Tokyo) and
melatonin (Nakarai Chemical, Kyoto). Experiments were performed at room (20-26°C).
temperature
RESULTS
The results obtained are summarized in Table 1. In the families Ictaluridae, Schilbeidae, Pangasiidae, Heteropneustidae, Chacidae, Mochokidae, Doradidae, Plotosidae and Loricariidae, all the fishes investigated were found to have melanophores quite orthodoxly regulated by adrenergic postganglionic fibers, although not many species were examined in each family. Typical examples of the responses of the melanophores of this type can be seen in Figs 1A and 2A,
which illustrate the recordings obtained on the marine catfish, Plotosus lineatus, belonging to Plotosidae. These pharmacological results clearly indicated the adrenergic nature of peripheral transmission to the effector cells. Of the four species of silurid catfish listed in Table 1, two, i.e. the Japanese common catfish Parasilurus usatus (Fujii and Miyashita, 1976), and the translucent catfish Kryptopterus bicirrhi (Fujii et nl., 1982), had already been examined for the nature of innervation to the melanophores. Figures 1B and 2B show a typical series of the responses of the melanophores of the Japanese catfish Parusilurus asotus, recorded again for this study. Confirming surely earlier observations, the present results indicate even more clearly that the melanophores are regulated through the mediation of cholinoceptors of a muscarinic nature. In addition, we employed another species of translucent glass catfish, which apparently belongs to the
Neuro-melanophore transmission in catfishes
117
Fig. 1. Photoelectric recordings showing the responses of melanophores of three catfish species: A, the marine catfish Plotosus lineatus; B, the Japanese common catfish Parasilurus asotus; and C, the peppered catfish Corydoras paleatus. Each fish is representative of those in which melanophores were found to be under one of three types of regulatory system, i.e. orthodox adrenergic (A), muscarinic cholinergic (B) and adrenergic, but in which the effector cells possess extra cholinoceptors (C). The pigment-aggregating effect of acetylcholine (ACh) or norepinephrine (NE) was first examined. If the effect was detected, its influence on an alpha adrenolytic agent, phentolamine (PA), or a muscarinic cholinolytic agent, atropine (AP), was examined. Melatonin (MT, 1O-5M) was finally employed to induce the full level of melanosome aggregation.
same genus Kryptopterus, and another larger glass catfish. Neither the genus nor species of the latter could be determined. Judging from its external form and gross morphology, however, it undoubtedly belongs to this family. The melanophores in these fish as well were proved pharmacologically to receive cholinergic innervation in the same manner as that of the above-mentioned identified species of silurids.
Fig. 2. Photoelectric recordings of the responses of melanophores of the marine catfish (A), the Japanese common catfish (B) and the peppered catfish (C). Melanosomeaggregating stimulation by K+ (50mM) was applied in order to examine whether phentolamine (PA), an alpha adrenolytic agent, or scopolamine @A), a muscarinic cholinolytic, might antagonize the neurally evoked response. Melatonin (MT, 10m5M) was used to induce the full level of pigment aggregation.
We have already shown that in the peppered catfish paleatus the melanophores possess muscarinic cholinoceptors of unknown significance, although they are under the control of adrenergic postganglionics as usual (Kasukawa and Fujii, 1985). In this study, therefore, we tried to broadly survey similar systems among varieties of catfish. As expected, we found that three Corydoras spp. in addition to C. paleatus possessed the same system. As typical examples of melanophores regulated in this manner, the responses to testing of pharmacological agents in Corydoras paleatus were examined again, and sections of the recordings are shown in Figs 1C and 2C. Another catfish of the genus Dianema (species unidentified) was then examined. Although this fish is included in the order Callichthyidae, to which Corydoras catfish also belong, its regulatory system for melanophores was found to be of an orthodox adrenergic type without extra cholinoceptors. We also found that all three South American pimelodid catfish tested had systems of the Corydorus type in which melanophores are adrenergically controlled but have extra muscarinic cholinoceptors. Furthermore, two Mystus spp. among the six species examined in this survey and which belong to four genera from the family Bagridae were also found to have the same regulatory mechanism for melanophore motility. Corydoras
DISCUSSION
In examining the present results, it can first be said that, even in the order Siluriformes, melanophores are usually under the regulation of the adrenergic postganglionic fibers of the sympathetic system. That is, the melanophores are quite orthodoxly regulated in the same way as they are in many of the tele-
118
H~ROAKI KASUKAWA et af.
osts examined and described to date (~5 Fujii and Oshima, 1986). As we have already shown, the melanophores of two silurids, i.e. the Japanese common catfish, Parasilurus astutus (Fujii and Miyashita, 1976) and the translucent glass catfish, Kryptopterus bicirrhi (Fujii et al., 1982), are strangely controlled by cholinergic fibers. In the present study, a Kryptopterus glass catfish (species unidentified), and another glass catfish, which, although even the genus has not been identified, apparently belongs to the same family, were added to this group of fish. To our knowledge, no further species have been studied within this family, but other silurids may possibly have an identical system of nervous control for melanophores. It should be emphasized here, however, that in families other than Siluridae no species have been found to possess such a mode of neurotransmission to the pigment cells, although there still remains the possibility that the melanophores of Amblycipitidae might also be regulated by cholinergic fibers. Unfortunately, to date, we have not been able to obtain materials for these species. Furthermore, we have as yet no info~ation regarding the presence of cholinergic nervous control of chromatophores in animals of other classes. Silurid catfish may thus be considered a very unique group among the vertebrate classes. We have also learned that the melanophores of the peppered catfish, Corydoras paleatus, possess cholin~eptors of ~usca~nic nature in addition to the common adrenergic nervous supply (Kasukawa and Fujii, 1985). In the present survey, a quite similar system was found in other mailed catfish (Corydoras spp.), and in other families rather sporadically, i.e. in
AFRICA
SOUTH
EURASIA I
7 Schifbeidae
I I
I
NORTH AMERICA
Doradidae
I ! Heteropneustidae
i
AMEZRICA
BAGROIDEI 1
I
Mochokidae
two bagrid catfish belonging to the genus Mystus and in all three pimelodid species studied so far. As near as we can guess, there are no catfish in which melanophores do not receive pigmentaggregating innervation, or in which the cells are controlled by a different kind of chemical transmitter. In Siluriformes species, therefore, there exist three types of peripheral neuronal systems for controlling melanophore movement: an orthodox adrenergic one, a cholinergic one known only in Siluridae, and a third type in which adrenergically controlled melanophores possess extra cholinoceptors. A number of opinions have been put forward to date regarding the taxonomy and phylogeny of teleost fishes. If we restrict ourselves to those concerning catfish, however, only a few proposals have been presented. Among these, Chardon’s concept (1968) seems to be currently the most popular, although it still remains rather controversial. Figure 3 illustrates an abstraction from his phylogenetical tree of Siluroformes with special attention to the families actually dealt with in this work. Siluriformes has generally been classified into three suborders. Chardon (1968) himself defined and designated these suborders as Siluroidei, Bagroidei and Loricarioidei, all of them being derived from the same prototype (Stock II in Fig. 3). Glancing over the tree, we find the family Siluridae, in which the unique cholinergic nervous system operates, situated close to the stock, or rather directly originating from the prototype of Siluriformes. Bagridae, Pimelodidae and ~alIichthyidae, in which at least in some forms muscarinic cholinoceptors of unknown functional significance coexist with the functioning alpha adrenoceptors, are also found not far from the root. The tree also indicates that
i,
Pangasiidae
I
i
Chacidae Plotosidae
1
/Ictaluridae
I 1 Pimelodidae**
i
kStock I -(Diplomystidae~
Fig. 3. A genealogical tree based essentially on the concepts of Chardon (1968) regarding the evolution of catfish within the order Siluriformes. The names of families with which the present work was concerned were selectively postered in this figure. In the families, whose names are without asterisks, the melanophores are adrenergically controlled as in most teleosts. *Melanophores are cholinergically controlled. **Melanophores are adrenergically controlled but have extra muscarinic cholinoceptors.
Nemo-melanophore transmission in catfishes Ictaluridae, Schilbeidae, Pangasiidae, Heteropneustidae, Chacidae, Mochokidae, Doradidae and Plotosidae, in which melanophores are controlled in the same way as in fish of other orders, have been derived from Pimelodidae or Bagridae. Based on the results from a few species of catfish, we have already speculated that the cholinergic controlling mechanism for melanophore motility may be of a more evolved type than the orthodox one, and that those cells having both adrenoceptors and cholinoceptors may be transitional forms (Fujii et al., 1982; Kasukawa and Fujii, 1985). After conducting the present wide-ranging survey of Siluriformes, we have found no reason to change our previous opinion. Actually, examples of “cholinalso exist in ergic” sympathetic postganglionics higher animals, i.e. the sympathetic innervation to sweat glands (Dale and Feldberg, 1934) or to blood vessels in some muscular tissues in mammals (Uvnas, 1954). Such fibers may safely be assumed to have derived from conventional adrenergic ones. In addition, Le Douarin et al. (1975) showed that intrinsically adrenergic avian sympathetic ganglion cells can be artificially differentiated into either adrenergic or cholinergic cells. Working on mammalian sympathetic ganglion cells in culture, Reichardt and Patterson (1977) have also recently demonstrated that adrenergic-to-cholinergic conversion of the neuronal output can take place rather easily by selecting a proper culture medium. The reverse conversion, however, has not been shown to be an easy process. Therefore, it seems to be quite reasonable to assume that cholinergic control of the pigment cells has been derived from adrenergic control, and that cholinergic control is a more evolved type of regulation. The first of two speculations which we wish to present in this article therefore is quite inconsistent with the conjecture presented by Chardon (1968), who put Siluridae, Pimelodidae and Bagridae near the stock of his genealogical tree. As has been indicated earlier, the transition of adrenergic fibers to chohnergic ones might not have been a very difficult task for fish during their long period of evolutionary history (Fujii et al., 1982). We may thus surmise that conversion may have occurred in an ancient silurid from which all recent ones derived, or, later, frequently and independently among many of the silurids already radiated adaptively. In any case, the induction or expression of muscarinic cholinoceptors on the postsynaptic membrane must have been an easier process for fish, since vertebrate cells are undoubtedly loaded with the gene of the receptor protein. Thus, the sporadic occurrence of cholinoceptors, being independent of phylogeny, may be rather easily explained. In this connection, we can point out here the finding that even outside the fish classes, cholinoceptors are present on the melanophore membrane. The described species was an anuran Rana #ens, although not all the specimens examined possessed the receptor (MBller and Lerner, 1966; Goldman and Hadley, 1968). All these observations seem to favor the view that the simpler cholinergic system may have been derived from the regular adrenergic one during the evolution of chromatophore controlling mechanisms. The second hypothesis on the phylogeny of Siluri-
119
formes which we would like to put forward here is in good conformity with that of Chardon (1968), or, in a manner of speaking, is essentially based on it. As pointed out above, Pimelodidae, Bagridae, Callichthyidae and Siluridae can be found near the stock of the genealogical tree shown in Fig. 3. Chardon (1968) further regarded the former two families to be ancestral groups within the suborder Bagroidei (Chardon, 1968). In his tree, therefore, the families in which all the species studied in this survey were shown to possess an orthodox adrenergic melanosome-aggregating nervous system, are located more peripherally, being derived from ancient forms of the abovementioned two families. In conclusion, it may be put forward that, on the adrenergically innervated melanophores of the common ancestral catfish (Stock II in Fig. 3), muscarinic cholinoceptors may have come into existence by chance or by mistake. Thereafter, the adrenergic-tochohnergic conversion may have taken place in the melanophore system of primitive silurids. Radiating adaptively, their descendants may have formed the several recent silurid species found in Eurasia. In a few recently discovered bagrids and pimelodids, on the other hand, the original regulatory system for the pigment cell movements may have survived until today. When coexisting with alpha adrenoceptors, such cholinoceptors can naturally be thought to be practically useless, as has been suggested earlier (Fujii et al., 1982; Kasukawa and Fujii, 1985). As a matter of course, they may have disappeared from the melanophore membrane in many families assumed by Chardon (1968) to have been derived from the abovementioned two families. In Loricarioidei, melanophores of the genus Corydoras (family: Callichthyidae) were found to possess muscarinic cholinoceptors, while those of Dianema (family: Callichthyidae) did not. Since only two families have been examined within this order, it may ‘not be appropriate at this point to discuss the phyletic status further. In order to strengthen the hypothesis of this line, we should explain why and how such an unusual system, in which both alpha adrenoceptors and muscarinic cholinoceptors coexist, appeared in Stock I or II. Although the above-mentioned observations of the sporadic presence of cholinoceptors in melanophores of a Rana frog may be of a help, available information is still very meager. Further comparative surveys are therefore required. In any case, we hope that our results on the types of innervation to effector cells of one sort, the chromatophores, will provide some clue to a better understanding of the phylogeny of not only catfish but also of teleostean orders, which may be closely related to the order dealt with in this article. Acknowledgements-We wish to express our gratitude to Dr R. Arai, Department of Zoology, National Science Museum, Tokyo, for classifying some materials employed in this study. Our thanks are also due to Dr Y. Miyashita for interest and providing us some information relevant to our present study, to Dr S. Kikuchi and the staff of the Kominato Marine Biological Laboratory, Faculty of Science, Chiba University, for the supply of marine catfish, and to .I. Igarashi for technical assistance. This work was
120
H~R~AKI
supported in part by grants from the Ministry Science and Culture of Japan.
KAS~JKAWA
ofEducation,
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