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The Metabolism of Biogenic Monoamines during Embryogenesis and Metamorphosis in Two Anuran Species
General and Comparative Endocrinology 106, 361–373 (1997) Article No. GC976885
The Metabolism of Biogenic Monoamines during Embryogenesis and Metamorphosis in Two Anuran Species Naokuni Takeda Department of Biotechnology, COSMO Research Institute, Research and Development Center, Satte, Saitama 340-01, Japan Accepted January 13, 1997
This study investigated the pathways to many monoamines and their metabolites in the central nervous system of the frog Rana nigromaculata and the toad Bufo bufo japonicus during embryonic development and metamorphosis. Metabolites were analyzed by three-dimensional HPLC. The two species provided evidence of similar pathways, with slightly different timetables for the development of their monoamine systems. During embryonic development, the main metabolic pathways in entire embryos were tyrosine (TYR) 8 [3,4-dihydroxyphenylalanine in Bufo] 8 3-hydroxytyramine 8 norepinephrine or epinine (EPIN) 8 epinephrine, TYR 8 tyramine 8 (octopamine in Rana) and TYR 8 3-O-methyldopa for catecholamines, and tryptophan 8 kynurenine and 5hydroxytryptamine (5-HT) 8 [5-hydroxyindoleacetic acid and N-methyl-5-hydroxytryptamine (N-MET) in Bufo]. The monoamine system in the brain was similar during metamorphosis to that during embryogenesis with a few exceptions. The most striking change was the development of the bufotenine (5-hydroxy-N,N-dimethyltryptamine) pathway from 5-HT via N-MET. EPIN and norepinephrine in Rana and octopamine in both species disappeared during metamorphosis. These results are discussed in relation to the roles of the various pathways in development. r 1997 Academic Press Biogenic monoamines are neurotransmitters that act as chemical mediators of intercellular communication via the activation of specific receptors and second 0016-6480/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
messengers in postsynaptic cells. Their presence and distribution in the nervous system have been analyzed from a phylogenetic perspective in the animal kingdom (e.g., Klemm, 1985; Takeda, 1996a). Preliminary ontogenetic analysis has also been performed in some species, such as Bombyx mori (Takeda et al., 1991) Xenopus laevis (Takeda and Takaoka, 1991), and Gallus domesticus (Takeda, 1992). In addition, immunohistochemical studies of the hypothalamo–hypophysis system have provided some information about the ontogeny of monoaminergic systems at late larval stages in some frogs (McKenna and Rosenbluth, 1975; Corio and Doerr-Schott, 1988). The life history in frogs and toads includes the dramatic changes associated with metamorphosis, which occurs when the larva is transformed into the adult form. Each larva undergoes a dramatic morphological, biochemical, and ecological transformation to become an adult. The hormonal and neuroendocrine mechanisms of metamorphosis are well established (e.g., Norris and Dent, 1989; Kikuyama et al., 1993; Denver, 1996; Kaltenbach, 1996). However, the monoaminergic systems that might play a very basic role in metamorphosis remain to be fully characterized. There are only a few studies on the changes of monoaminergic systems in the central nervous system of amphibians during their metamorphosis and development (Norris et al., 1992; Lowry et al., 1996; Takeda, 1996a). Developmental changes in levels of tyrosine
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hydroxylase and dopamine in the brain have been examined immunohistochemically in Xenopus laevis and Rana ridibunda (Gonza´lez et al., 1994). However, no studies have examined the quantitative relationship between monoaminergic systems and embryonic development or metamorphosis in frogs and toads. In the present report, the dynamics of monoaminergic systems in the embryo and the central nervous system of the frog and the toad are described for the first time, as part of our efforts to gain a better understanding of the development of this system with the progress of embryonic development and metamorphosis.
MATERIALS AND METHODS Animals Animals studied were the frog Rana nigromaculata (Ranidae) and the toad Bufo bufo japonicus (Bufonidae). Adult frogs of both species were collected in the field and reared under natural conditions. Egg masses were obtained from these species. The collection and analysis of samples were performed repeatedly from 1990 to 1994 to obtain representative values or tendencies because the analytical data sometimes exhibited large variations. Oviposition in both species was monitored visually. The developmental stages were determined by reference to the normal tables for Rana (Iwasawa, 1996) and Bufo (Ichikawa and Tahara, 1989). For convenience, we used the following stages for the analysis of whole embryos: stage I, cleavage period (from stage 1 to 6 in the normal table); stage II, gastrula period (from stage 12 to 14 in the normal table); stage III, neurula period (from stage 19 to 20 in the normal table); stage IV, tail bud period (stage 23 in the normal table); stage V, middle branchial bud stage during the external gill period (stage 26 in the normal table); and stage VI, external gill completion stage in the external gill period (stage 30 in the normal table). Furthermore, to examine the role of monoamines in early neurogenesis, we divided embryos at the tail bud stage into two parts, the dorsal part and the ventral part, and analyzed the monoamines in each part separately. The stages at which we analyzed the brain in
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Naokuni Takeda
tadpoles were as follows: stage VII, completion of the branchial mantle during the external gill period (stage 34 in the normal table); stage VIII, hindleg formation stage (stage 37 in the normal table); stage IX, hindleg completion stage (stage 41 in the normal table); stage X, foreleg appearance stage of metamorphosis (stage 42 in the normal table); stage XI, degradation of the caudal portion during metamorphosis (stage 43 in the normal table); and stage XII, completion of metamorphosis (stage 45 in the normal table). Tadpole larvae in breeding containers after stage VII were supplied with boiled spinach as food. Each embryo and each extirpated brain was put separately into a mixture of 300 µl of 0.4 N perchloric acid, 20 µl of 50 mM EDTA, and 10 µl of 0.2 N sodium hydrogensulfite and each sample was homogenized with a Physcotron (Nichi-On, Tokyo, Japan). The homogenates were centrifuged at 3000 rpm for 20 min. After filtration, 80 µl of sample was injected into the HPLC system.
HPLC with Electrochemical Detection The analytical apparatus used was a three-dimensional HPLC system (Coulochem Electrode Array System; ESA, Chelmsford, MA). The system consists of a gradient HPLC system and 16 high-sensitivity coulometric electrochemical detectors coupled with a compatible computer. The concept and inherent advantages of the multielectrode HPLC system have been described elsewhere (Matson et al., 1984). A reversephase C18 column (4.60 nm i.d. 3 150 m; NBS column, Niko Bioscience, Tokyo, Japan) was used. Two mobile phases, solvents A and B, were employed with both linear and step gradients. Solvent A consisted of 0.1 M sodium phosphate and 10 mg/ml sodium dodecyl sulfate at pH 3.35. Solvent B consisted of methanol and water (1:1, v/v) and 50 mg/liter of sodium dodecyl sulfate at pH 3.35. Another solution consisting of 50 mM sodium phosphate buffer that included 8.18% methanol and 1.82% acetonitrile, was sometimes also used. Our protocol permitted the simultaneous separation of about 30 compounds that were relevant to our analyses. A standard chromatogram including typical compounds is shown in Fig. 1, and various compounds, with their abbreviations and oxidation potentials, are shown in Table 1. The 16 serial electrodes were set in an incremental 60-mV array that ranged
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FIG. 1. Standard chromatogram showing profiles of elution of 28 compounds. Scale, 5 µm. Names of compounds are given in Table 1. Each number in the chromatogram corresponds to a number in Table 1.
from 0 to 900 mV. The column and electrodes were maintained at 34°. Sometimes, the temperature was lowered to 25° to obtain separation of peaks. Each compound was characterized not only by its retention times but also by the oxidation potential at which a maximal response was observed. Typically, each compound was detected at three electrodes, with an average ratio of peak heights between electrodes of 1:6:1. However, the exact ratio was specific to each compound and could be used to establish the purity of compounds present in peaks of unidentified material that eluted from the column with the same retention times as known standards. Unidentified peaks obtained after HPLC of the sample were matched with standards on the basis of both retention time and oxidation potential. A ratio accuracy of 100% means that the unknown compound detected at a certain retention time spread over the array of electrodes in exactly the same way as the standard and was, therefore, pure. In biological samples, there were always some other ions and trace metabolites that shifted this ratio slightly. In this system, compounds with a ratio accuracy above 70% were usually recognized as being present in a relatively pure form. Final concentrations were calculated by comparing the size of the dominant
peak of the standard to the dominant peak of the unknown compounds in the sample.
RESULTS The Metabolism of Monoamines in Whole Embryos during Early Development In general, the precursor amino acids, tyrosine (TYR) and tryptophan (TRP), first appeared in the embryo. As embryological development progressed, the metabolites were gradually detected in increasing amounts. In R. nigromaculata (Table 2), high levels of TYR, TRP, and many of their metabolites were detected in embryos just after oviposition. The levels of these amino acids, monoamines, and their metabolites increased gradually with the progress of development, with the exception of a somewhat temporary decrease at stage V. With respect to catecholamines, a characteristic result was the detection of TYRA and octopamine (OCT) at high levels throughout embryogenesis. 3-Hydroxytyramine (DA), 3-O-methyldopa (3-O-MD), 3-methoxytyramine (3-MT), and epinine (EPIN) were detected
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TABLE 1 Compounds Detected in the Analysis, with Abbreviations and Oxidation Potentials Oxidation potential (mV) Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Dihydroxyphenylethyleneglycol Vanillylmandelic acid Norepinephrine 3,4-Dihydroxyphenylalanine 3-Methoxy-4-hydroxyphenylethyleneglycol Octopamine Epinephrine Tyrosine 3,4-Dihydroxyphenylacetic acid Normetanephrine 3-O-Methyldopa 5-Hydroxyindoleacetic acid 3-Hydroxytyramine Metanephrine 5-Hydroxytryptophan Epinine N-Acetyl-5-hydroxytryptamine Hydroxyphenylacetic acid-4 Kynurenine Homovanillic acid Vanillactic acid Vanillic acid 5-Hydroxytryptamine 5-hydroxy-N,N-dimethyltryptamine N-Methyl-5-hydroxytryptamine 3-Methoxytyramine Melatonin Tryptophan
Abbreviation
First
Second
DOPEG VMA NE DOPA
240 300 180 150
— 600 — —
MHPG OCT E TYR DOPAC NMN 3-O-MD 5-HIAA DA MN 5-HTP EPIN N-ACT-5-HT HPAC-4 KYN HVA VLA VA 5-HT
450 620 180 650 150 480 450 180 150 480 300 120 180 650 800 450 360 480 180
— — — — — — — 750 — — 650 — 700 — — — — — 700
BUTN N-MET 3-MT MEL TRP
240 300 450 600 600
840 700 — — —
throughout embryogenesis but 3,4-dihydroxyphenylalanine (DOPA), found in Bufo, was not detected. Levels of 3,4-dihydroxyphenylacetic acid (DOPAC), at stages IV and V, and of vanillylmandelic acid (VMA) and homovanillic acid (HVA) were unstable in detection with a high degree of accuracy. Both norepinephrine (NE) and epinephrine (E) were found from stage II onward. With respect to indolealkylamines, kynurenine (KYN) and 5-hydroxytryptamine (5-HT) were detected throughout embryogenesis. These results suggested that the following main pathways were present: TYR = (DOPA) = DA or 3-O-MD; DA = NE or EPIN = E; DA = DOPAC or 3-MT = HVA; and TYR = TYRA = OCT for catecholamines; and TRP = KYN and 5-HT for idolealkylamines (Figs. 2 and 3).
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Although levels of 3-O-MD and 3-MT were relatively high, the levels of the final metabolite, HVA, were low. In B. japonicus (Table 3), high levels of TYR and TRP and of their metabolites, with the exception of DOPAC and EPIN, were detected in embryos just after oviposition. Levels of these amino acids and metabolites generally increased gradually with the progress of development, except at stage VI. DOPA, DA, 3-O-MD, 3-MT, and TYRA were detected at high levels throughout embryogenesis. E, EPIN, and NE were also detected. The final metabolites HVA and VMA were detected throughout embryogenesis. OCT, which we found in Rana, was not detected. KYN and 5-HT were detected throughout embryogenesis. At stage VI, we detected 5-hydroxyindoleacetic acid (5-HIAA), the final metabolite of 5-HT. In Bufo, unlike in Rana, the methylated derivative of 5-HT, N-methyl-5-hydroxytryptamine (N -MET), was newly detected. These results suggested that the following main pathways were present: TYR = DOPA = DA = NE or EPIN = E · · = VMA; DOPA = 3-O-MD; DA = DOPAC or 3-MT = HVA; and TYR = TYRA for catecholamines; and TRP = 5-HT = 5-HIAA; 5-HT = N-MET; and TRP = KYN for indolealkylamines (Figs. 2 and 3).
Localization of Monoamines in the Embryo at Tail Bud Period To monitor the appearance of the monoamines in the neural tube region, the embryos were divided at the tail bud period into two parts: the dorsal part, including the neural tube; and the ventral part, including the yolk. At the early tail bud stage in B. b. japonicus, for example, levels of monoamines were generally lower in the dorsal part than in the ventral part, except in the case of TYRA and 5-HT (Table 4, A). Levels of TYR, TRP, and KYN in the neural part were about half of those in the nonneural part. NE, E, and EPIN were not detected. Levels of TYRA and 5-HT were about three times higher than those in the nonneural part, although the ratio of accuracy was low for 5-HT. The high levels of monoamines, such as E, DA, 5-HT, N-MET, and 5-hydroxy-N,N-dimethyltryptamine (BUTN), which were found during metamorphosis, were not or not fully recognized at this period. By contrast, in R. nigromaculata at the late tail bud stage, for example, high levels of monoamines such as NE, E, and 5-HT were detected in the neural parts (Table 4, B).
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TABLE 2 Levels of Monoamines in Whole Embryos of the Frog Rana nigromaculata with the Progress of Embryonic Development
Note. See text for details of stages. pg/mg embryo; *, ng; —, nondetectable level; mean 6 SD, n 5 25.
As was also the case in Bufo, high levels of monoamines were not detected prior to this stage. These results suggest, therefore, that monoamines were introduced into the nervous system after completion of the neural tube.
The Metabolism of Brain Monoamines during Metamorphosis In R. nigromaculata (Table 5), high levels of TYR, TRP, DA, E, and some of their metabolites, such as 3-O-MD and 3-MT, were detected in whole brains throughout metamorphosis. The peak levels of TRP, TYR, DA, E, 3-MT, VA, TYRA, and KYN were found at stage VIII, the hindleg formation stage. With respect to catecholamines, EPIN and NE were not detected except at stage VII. TYRA was detected only at stages VIII and IX. Vanillic acid (VA) was detected for the first time at stage VIII. Final metabolites, such as HVA and VMA, were detected at all stages except at stage VII and just at stage VIII, respectively. With respect to indolealkylamines, KYN, 5-HT, and N-MET were detected through-
out metamorphosis. The peak of KYN was found at stage VIII. Conversion of 5-HT to 5-HIAA was evident from stage IX. High levels of N-MET were detected at the time of migration to land. These results suggested that the following metabolic pathways were present: TYR = (DOPA) = DA = NE or EPIN = E · · = VMA = VA; DA = DOPAC or 3-MT = HVA; and TYR = TYRA for catecholamines; and TRP = KYN; TRP = 5-HT = 5-HIAA; and 5-HT = N-MET for indolealkylamines (Figs. 2 and 3). The highest levels of monoamines were found at stage X. In B. b. japonicus (Table 6), high levels of TYR and TRP, as well as of monoamines and almost all of the metabolites examined, were detected in whole brains throughout metamorphosis. Furthermore, BUTN, a dimethylated derivative of 5-HT, found via N-MET, was first detected during metamorphosis. The peak levels of TYR, TRP, DA, E, TYRA, KYN, 5-HT, and BUTN were found at stage X, when the forelegs appeared. With respect to catecholamines, high levels of DA, E, TYRA, 3-O-MD, MN, and 3-MT were detected. Metanephrine (MN), a metabolite of E, was
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FIG. 2.
Main metabolic pathways of catecholamines in the frog Rana nigromaculata and the toad Bufo bufo japonicus.
detected at stages IX, X, and XI. VA was also detected. VMA was detected only at stage X. HVA was detected throughout metamorphosis. With respect to indolealkylamines, KYN, 5-HT, N-MET, and BUTN were detected. These results suggested that the following metabolic pathways were present: TYR = (DOPA) = DA = E = MN = · · = VMA = VA; DA = DOPAC or 3-MT = HVA; and TYR = 3-O-MD or TYRA for catecholamines; and TRP = KYN; TRP = 5-HT =
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5-HIAA; and 5-HT = N-MET = BUTN for indolealkylamines (Figs. 2 and 3). The highest levels of monoamines were found at stage VIII. Some compounds found during embryogenesis were not detected in brains during metamorphosis, namely DOPA in Bufo, OCT in Rana, and NE and EPIN (except stage VII) in Rana. These compounds might act mainly during embryogenesis and not during metamorphosis.
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FIG. 3.
Main metabolic pathways of indolealkylamines in the frog Rana nigromaculata and the toad Bufo bufo japonicus.
DISCUSSION Various changes in metabolic pathways from the precursor amino acids, TYR and TRP, to many monoamines and their metabolites were clearly demonstrated with the progress of embryogenesis and metamorphosis in the frog B. b. japonicus and the toad R. nigromaculata. Similar pathways were found in both species with slightly different timetables for the development of monoaminergic systems. In both species, even the embryos just after oviposition contained some metabolites of TYR and TRP. Since unfertilized eggs in the female abdomen contain no metabolites, fertilization might trigger the rapid induction of this aspect of metabolism by activating hydroxylases for precursor amino acids such as TYR and TRP. In sea urchins and chickens, monoamines, including
5-HT and NE, are synthesized and released by yolk granules after fertilization (Buznikov, 1984, 1991; Renaud et al., 1983; Emanuelsson et al., 1988). During embryonic development, the main metabolic pathways in whole embryos of the two species studied were TYR = (DOPA in Bufo) = DA = EPIN or NE = E, TYR = TYRA = (OCT in Rana) and TYR = 3-O-MD for catecholamines, and TRP = KYN and 5-HT = (5-HIAA and N-MET in Bufo) for indolealkylamines. Thus, the development of the monoaminergic systems appeared to be similar in both species during embryogenesis. In a report of preliminary data for X. laevis (Takeda, 1996a), the first metabolic pathways to develop were suggested to be TYR = (DOPA) = 3-O-MD = · · = HVA and then DA = 3-MT = HVA, DA = DOPAC = HVA and DA = E, in that order, for catecholamines; and TRP = 5-HT = 5-HIAA and
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TABLE 3 Levels of Monoamines in Whole Embryos of the Toad Bufo bufo japonicus with the Progress of Embryonic Development
Note. See text for details of stages. pg/mg embryo; *, ng; —, nondetectable level; mean 6 SD, n 5 25.
TYP = KYN at the same time and then 5-HT = N-MET for indolealkylamines. The present two species, Rana and Bufo, seem to resemble X. laevis. Although high levels of DOPA were detected by two-dimensional analysis, as reported by Rowe et al. (1993), the poor ratio accuracy prevented accurate quantitation of DOPA. The same was true for DA and DOPAC during early embryogenesis. In earlier phylogenetic analysis (Takeda, 1996a), monoamines were detected in primitive organisms, such as protozoa and porifera, and in embryos, including those of vertebrates, prior to the development of the nervous system. What role could these neurotransmitters play in early embryos that have no nervous system? With respect to the differentiation of embryonic neurons, Rowe et al. (1993) suggested that NE might bind to a-epinephrinergic receptors on neural plate cells and might be involved in the differentiation of nerve cells in X. laevis. Experimental evidence
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suggests that 5-HT and NE in the sea urchin and chicken might regulate the cleavage divisions of the blastula and gastrulation (e.g., Buznikov, 1984, 1991). These monoamines have also been suggested to regulate the morphogenetic movements of cells that are important for neurulation in amphibians and avians and for palatal morphogenesis in the mouse (e.g., Godin, 1986; Lauder, 1988, 1993). Thus, morphogenetic roles of monoamines during embryogenic development, which were adopted early in evolution, are still operative at early embryonic stages of ontogeny. Therefore, the so-called neurotransmitters seem to have evolved to their specialized roles from more general humoral functions in primitive organisms. Although the roles of KYN, TYRA, and OCT in embryogenesis are unknown, these are phylogenetically ancient monoamines (Takeda, 1996a). In the adult nervous system, monoamines, such as DA, NE, and E, are discrete intercellular signaling
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TABLE 4 Localization of Monoamines in the Embryos of the Frog Rana nigromaculata and the Toad Bufo bufo japonicus at Stage IV Compounds
Neural part
Nonneural part
A: Stage IV, early tail bud stage, in Bufo bufo japonicus TYR 3-O-MD DA DOPAC 3-MT HVA EPIN NE E TYRA
34.5 6 20.0* 266.2 6 147.3 2.9 6 1.7* 23.4 6 17.0 1.6 6 0.1* 133.3 6 88.6 — — — 6.1 6 3.4*
73.6 6 113.1* 816.7 6 988.3 3.0 6 4.2* 51.4 6 58.2 1.76 6 2.7* 97.1 6 148.2 20.4 6 27.2 34.7 6 43.1 267.1 6 350.7 1.1 6 1.8*
TRP 5-HT KYN
1.6 6 9.9* 6.6 6 3.8 256.2 6 117.9
30.0 6 46.5* 2.6 6 2.4 434.2 6 483.2
B: Stage IV, late tail bud stage, in Rana nigromaculata TYR DOPA 3-O-MD DA 3-MT EPIN NE E TYRA OCT
58.0 6 18.2* 302.7 6 358.6 1.4 6 0.2* 105.8 6 15.1 367.4 6 58.0 56.2 6 11.5 323.5 6 48.8 474.4 6 80.2 19.3 6 14.7* 0.8 6 0.3*
28.2 6 7.9* 134.2 6 31.8 0.7 6 0.1* 73.8 6 59.5 175.6 6 31.9 32.6 6 1.4 52.6 6 7.9 55.7 6 35.9 14.2 6 4.7* 1.0 6 0.4*
TRP 5-HT KYN
19.3 6 3.2* 80.7 6 31.9 507.8 6 282.0
12.2 6 2.1* 16.8 6 6.6 446.3 6 146.0
Note. —, nondetectable level; pg/mg embryo; *, ng; n 5 15.
compounds which mediate communication between nerve cells as neurotransmitters. This specialized role seems to have evolved from more primitive functions in lower organisms, in which these compounds serve as both intra- and intercellular signaling molecules. In general, the levels of monoamines in the neural tube area of these embryos were low at the early tail bud stage. However, levels gradually increased and became higher than those of others at the late tail bud stage. In Rana, high levels of DOPA and NE in the neural part might be important for embryogenesis because these compounds were not detected in the brains of tadpoles. In both Rana and Bufo, high levels of 5-HT and KYN in the neural part appeared to be important for the development of the nervous system
(Schwarcz et al., 1991). Developmental features of serotonergic neurons in the brain stem in the neural tube have been examined in X. laevis (van Mier et al., 1989): the first serotonergic neuron appeared in the rostral part of the brain stem and axonal outgrowth reached the rostral part of the spinal cord at stage IV. The growth and development of amphibians during metamorphosis are under the control of three hormones: prolactin, growth hormone, and thyroxine (e.g., Kikuyama et al., 1993; Denver, 1996; Kaltenbach, 1996). Prolactin stimulates the growth of larval organs and inhibits the growth of adult organs, while growth hormone stimulates only the growth of adult organs. Thyroxine is an important hormone that controls metamorphosis; while it acts on larval organs to inhibit growth and to stimulate degradation, it stimulates the growth of adult organs. How, then, are monoamines involved in the action of these hormones or in metamorphosis directly? Monoamines in the brain appear to function as neurotransmitters that are involved in controlling the hypothalamic hormone-secreting neurons that secrete releasing factors (Nakai et al., 1986; Pang and Schrebman, 1986). There is evidence that aminergic neurons make synaptic contacts with these neurons. While it can be argued that the systems are not fully characterized, there is sufficient information to allow us to make some general comments and to provide some illustrative examples. The hypothalamus is innervated by aminergic neurons that have been shown to contain DA, NE, E, and 5-HT (e.g., Nakai et al., 1986). Some of the dopaminergic neurons secrete dopamine into the hypophyseal portal blood through which it is transported to, and acts directly upon, the anterior pituitary to inhibit the tonic secretion of prolactin (McCann et al., 1984). NE and DA appear to stimulate secretion of thyroid-stimulating hormone by stimulating neurons that secrete thyrotropin-releasing hormone (TRH; e.g., Krulich et al., 1977) and growth hormone by stimulating neurons that secrete growth hormone-releasing hormone (Kakucoka and Makara, 1983). By contrast, 5-HT inhibits the release of TRH. From results of studies with DA and known agonists and antagonists (Seki and Kikuyama, 1979, 1982; Kikuyama and Seki, 1980), DA is thought to be the physiological inhibitor of the release of prolactin. Seki and Kikuyama (1982)
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TABLE 5 Levels of Monoamines in the Brain of the Frog Rana nigromaculata during Metamorphosis
Note. See text for details of stages. pg/mg brain; *, ng; —, nondetectable level; mean 6 SD, n 5 15.
showed that the release of prolactin was enhanced by the direct action of 5-HT on the pituitary gland, suggesting that 5-HT might be a prolactin-releasing neurohormone. In anuran larvae, the only aminergic nuclei that are active in the hypothalamus prior to metamorphosis are the paraventricular organ and the dorsal infundibular nucleus (Hanke, 1976). McKenna and Rosenbluth (1975) provided the first link between thyroid hormones and hypothalamic monoamines in the preoptic recess organ of metamorphosing tadpoles of Bufo marinus. The peptidergic hormones released from the posterior pituitary might also be under aminergic control. KYN can act as an excitatory neurotransmittor (Stone and Connick, 1983). Moreover, BUTN is capable of promoting the secretion of prolactin (Seeman and Brown, 1985) within a few minutes after peripheral injection in rats (Meltzer et al., 1978). During metamorphosis, the pathway 5-HT = N-MET = BUTN was shown to be operative. Many
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bioactive secretions have been detected in the amphibian integument. Since the presumptive areas of the epidermis and the nervous system have the same origin in the embryo, it is not unreasonable that we found these compounds in the adult nervous system. In Bufo, not only the parotid glands but also the dermal glands synthesize and secrete bioactive substances that include amines, peptides, alkaloids, and bufodienolides (Ersparmer, 1994). In B. b. japonicus, the brain, blood, and urine contain BUTN, and the presence of BUTN suggests a route for its metabolism from the brain to the urine (Takeda, 1994). BUTN detected in whole embryos of Bufo at stage VII might have originated from these presumptive areas. In Xenopus, the dermal glands also contain BUTN (Takeda, 1996a). By contrast, the integument and the brain of Rana do not contain BUTN. BUTN may play several roles in animals. The 5-HT = N-MET pathway has been demonstrated in the poison
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TABLE 6 Levels of Monoamines in the Brain of the Toad Bufo bufo japonicus during Metamorphosis
Note. See text for details of stages. pg/mg brain; *, ng; —, nondetectable level; mean 6 SD, n 5 15.
glands of insects and in some coelenterates, including Hydra (Takeda and Svendsen, 1991; Shimizu and Takeda, 1993). In humans, BUTN acts as a hallucinogenic compound. BUTN has been detected in the urine of some psychiatric patients and has been suggested to be a diagnostic marker for some psychiatric disorders (Takeda et al., 1995). Therefore, B. b. japonicus may be a useful model animal for neurochemical studies to psychiatric disorders. Several secretions, including the precursor of BUTN, in X. laevis induce highly stereotyped oralfacial movements in the predators such as anuran-eating snakes (Barthalmus, 1994). The stereotyped oralfacial movements in snakes affected by BUTN precursors may be acting through effects on the serotonergic system. In rat, BUTN was suggested to have an action on 5-HT neurons comparable to that of p-chloroamphetamine (Monroe et al., 1994). The role of BUTN remains unclear in amphibians, and more analytical research, for example, with antibodies against
BUTN (Takeda, 1996b) might provide some important clues to the possible role of this compound.
ACKNOWLEDGMENTS The author expresses his thanks to his son, Takuro Takeda, for collecting materials in the fields during a period of 3 years.
REFERENCES Barthalmus, G. T. (1994). Biological roles of amphibian skin secretions. In ‘‘Amphibian Biology I. The Integument’’ (H. Heatwole and G. T. Barthalmus, Eds.), pp. 382–410. Surrey Beatty & Sons, Chipping Norton, Australia. Buznikov, G. A. (1984). The action of neurotransmitters and related
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372 substances on early embryogenesis. In ‘‘Developmental Pharmacology’’ (J. Gy. Papp, Ed.), pp. 23–59. Pergamon, London. Buznikov, G. A. (1991). The biogenic monoamines as regulators of early (pre-nervous) embryogenesis: New data. In ‘‘Plasticity and Regeneration of the nervous system’’ (P. S. Timiras et al., Eds.), pp. 33–48. Plenum, New York. Corio, M., and Doerr-Schott, J. (1988). The monoaminergic system in the diencephalon of the newt tadpole, Triturus alpestris (Mert). A histofluorescence study. J. Hirnforsch. 29, 377–384. Denver, R. J. (1996). Neuroendocrine control of amphibian metamorphosis. In ‘‘Metamorphosis, Postembryonic Reprogramming of Gene Expression in Amphibian and Insect Cells’’ (L. I. Gilbert, J. R. Taka, and B. G. Atkinson, Eds.), pp. 433–464. Academic Press, San Diego. Emanuelsson, H., Carlberg, M., and Lowkvist, B. (1988). Presence of serotonin in early chick embryos. Cell Differ. 24, 191–200. Erspamer, V. (1994). Bioactive secretions of the amphibian integument. In ‘‘Amphibian Biology I. The Integument’’ (H. Heatwole and G. T. Barthalmus, Eds.), pp. 178–350. Surrey Beatty & Sons, Chipping Norton, Australia. Godin, I. (1986). Explanted and implanted notocord of amphibian anuran embryos. Histofluorescence study on the ability to synthesize catecholamines. Anat. Embryol. 173, 393–399. Gonza´lez, A., Marı´n, O., Tuinhof, R., and Smeets, W. J. A. J. (1994). Ontogeny of catecholamine systems in the central nervous system of anuran amphibians: An immunohistochemical study with antibodies against tyrosine hydroxylase and dopamine. J. Comp. Neurol. 346, 63–79. Hanke, W. (1976). Neuroendocrinology. In ‘‘Frog Neurobiology’’ (R. Linas and W. Frecht, Eds.), pp. 975–1020. Springer–Verlag, Berlin. Ichikawa, M., and Tahara, Y. (1989). Normal table of a toad. In ‘‘Development of Vertebrates’’ (T. S. Okada, Ed.), pp. 357–373. Baihukan, Tokyo [In Japanese]. Iwasawa, H. (1996). Normal table of Rana porosa porosa. In ‘‘Atlas of Animal Developmental Stages’’ (K. Ishihara, Ed.), pp. 307–316. Kyoritsu Shuppan, Tokyo [In Japanese]. Kaltenbach, J. C. (1996). Endocrinology of amphibian metamorphosis. In ‘‘Metamorphosis, Postembryonic Reprogramming of Gene Expression in Amphibian and Insect Cells’’ (L. I. Gilbert, J. R. Tata, and B. G. Atkinson, Eds.), pp. 403–431. Academic Press, San Diego. Kakucoka, I., and Makara, G. B. (1983). Various putative neurotransmitters affect growth hormone (GH) release in rats with anterolateral hypothalamic deafferentiation of the medical hypothalamus: Evidence for mediation by a GH-releasing factor. Endocrinology 113, 318–323. Kikuyama, S., and Seki, T. (1980). Possible involvement of dopamine in the release of prolactin-like hormone from the bullfrog pituitary gland. Gen. Comp. Endocrinol. 41, 173–179. Kikuyama, S., Kawamura, K., Tanaka, S., and Yamamoto, K. (1993). Aspects of amphibian metamorphopsis: Hormonal control. Int. Rev. Cytol. 145, 105–148. Klemm, N. (1985). The distribution of biogenic monoamines in invertebrates. In ‘‘Neurobiology’’ (R. Gilles and J. Balthazart, Eds.), pp. 280–296. Springer–Verlag, Berlin.
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Naokuni Takeda Krulich, L., Giachetti, A., Marchlewsaka-Koj, A., Hefco, E., and Jameson, H. E. (1977). On the role of the central noradrenergic and dopaminergic systems in the regulation of TSH secretion in the rat. Endocrinology 100, 496–505. Lauder, J. M. (1988). Neurotransmitters as morphogens. Prog. Brain Res. 73, 365–387. Lauder, J. M. (1993). Neurotransmitters as growth-regulatory signals: role of receptors and second messengers. Trends Neurosci. 16, 233–240. Lowry, C. A., Renner, K. J., and Moore, F. L. (1996). Catecholamines and indoleamines in the central nervous system of a urodela amphibia: A microdissection study with emphasis on the distribution of epinephrine. Brain Behav. Evol. 48, 70–93. Matson, W. R., Langlais, P., Volicer, L., Gamache, P. H., Bird, E. D., and Mark, K. A. (1984). n-Electrode three-dimensional liquid chromatography with electrochemical detection for the determination of neuro-transmitters. Clin. Chem. 30, 1477–1488. McCann, S. M., Lumpkin, M. D., Mizunuma, H., Khorram, O., Ottleecz, A., and Samson, W. K. (1984). Peptidergic and dopaminergic control of prolactin release. Trends Neurosci. 7, 127–131. McKenna, O. C., and Rosenbluth, J. (1975). Ontogenic studies of a catecholamine-containing nucleus of the toad hypothalamus in relation to metamorphosis. Exp. Neurol. 46, 496–505. Meltzer, H. Y., Fessler, R. G., Simonovic, M., and Fang, V. S. (1978). Stimulation of rat prolactin secretion by indolealkylamine hallucinogens. Psychopharmacology 56, 255–259. Monroe, P. J., Smith, D. L., Williams, G. M., and Smith, D. J. (1994). Bufotenine has a parachloroamphetamine-like action on the storage and release of serotonin in rat spinal cord synaptosomes. Biogenic Amines 10, 273–284. Nakai, Y., Shioda, S., Ochiai, H., and Kozasa, K. (1986). Catecholamine–peptide interactions in the hypothalamus. In ‘‘Morphology of the Hypothalamus and its Connections’’ (D. Ganten and D. Pfaff, Eds.), pp. 135–160. Springer–Verlag, Berlin. Norris, D. O., and Dent, J. N. (1989). Neuroendocrine aspects of amphibian metamorphosis. In ‘‘Development, Maturation, and Senescence of Neuroendocrine Systems: A Comparative Approach’’ (M. P. Schreibman and C. G. Scanes, Eds.), pp. 63–90. Academic Press, San Diego. Norris, D. O., Carr, J. A., Desan, P. H., Smock, T. K., and Norman, M. F. (1992). Monoamines and their metabolites in the amphibian (Ambystoma tigrinum) brain: Quantitative changes during metamorphosis and captivity. Comp. Biochem. Physiol. 103A, 279–283. Pang, P. K. T., and Schreibman, M. P. (1986). ‘‘Vertebrate Endocrinology: Fundamentals and Biomedical Implications. 1. Morphological Considerations,’’ Academic Press, London/New York. Renaud, F., Parisi, E., Capasso, A., and De Prisco, E. P. (1983). On the role of serotonin and 5-methoxytryptamine in the regulation of cell division in sea urchin eggs. Dev. Biol. 98, 37–46. Rowe, S. J., Messenger, N. J., and Warner, A. E. (1993). The role of noradrenaline in the differentiation of amphibian embryonic neurons. Development 119, 1343–1357. Schwarcz, R., Young, S. N., and Brown, R. R. (Eds.) (1991). ‘‘Kynurenine and Serotonin Pathways,’’ Plenum, New York/London.
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Seeman, G., and Brown, G. M. (1985). Indolealkylamine and prolactin secretion. Neuropharmacology 24, 1195–1200. Seki, T., and Kikuyama, S. (1979). Effects of ergocornine and reserpine on metamorphosis in Bufo bufo japonicus tadpoles. Endocrinol. Japon 26, 675–678. Seki, T., and Kikuyama, S. (1982). In vitro studies on the regulation of prolactin secretion in the bullfrog pituitary gland. Gen. Comp. Endocrinol. 46, 473–479. Shimizu, T., and Takeda, N. (1993). Aromatic amino acids in the venom of the Braconid parasitoid Apanteles kariyai. Z. Naturforsch. 48C: 108–109. Stone, T. W., and Connick, J. H. (1985). Quinonic acid and other kynurenines in the central nervous system. Neuroscience 15, 597– 617. Takeda, N., and Svendsen, C. N. (1991). Monoamine concentrations in Hydra magnipapillata. J. Hydrobiol. 216/217, 549–554. Takeda, N., Takaoka, H., Shimizu, T., Yazawa, M., and Yagi, S. (1991). Biogenic monoamine levels in the central nervous system and hemolymph of the silkworm, Bombyx mori. Comp. Biochem. Physiol. 99C, 588–593. Takeda, N., and Takaoka, H. (1991). Dynamics of neurotransmitter
373 metabolism in the central nervous system of frogs. Soc. Neurosci. 17, 745. Takeda, N. (1992). Development of neurotransmitter metabolism in the brain of the chick, Gallus gallus domesticus. Soc. Neurosci. 18, 1472. Takeda, N. (1994). Serotonin-degradative pathways in the toad (Bufo bufo japonicus) brain: Clues to the pharmacological analysis of human psychiatric disorders. Comp. Biochem. Physiol. 107C, 275–281. Takeda, N., Ikeda, R., Ohba, K., and Kondo, M. (1995). Bufotenine reconsidered as a diagnostic indicator of psychiatric disorders. Neuro Report 6, 2378–2380. Takeda, N. (1996). Antibodies to bufotenine. Soc. Neurosci. 18, 604. Takeda, N. (1997). A phylogenetic analysis of biogenic monoamines in the central nervous system of various organisms and applications to the field of medical chemistry. In ‘‘Progress in HPLC, Multielectrode EC Array; Applications to Trace Analysis of Neurotransmitters, Pharmaceuticals and Environmental Markers’’ (S. Naoi, H. Parvez, I. N. Acworth, S. Parvez, and W. Matson, Eds.), pp. 169–211. VSP International Press, the Netherlands. Van Mier, P., Joosten, H. W. J., Van Rheden, R., and Ten Donkelaar, H. J. (1989). The development of serotonergic raphespinal projections in Xenopus laevis. Int. J. Dev. Neurosci. 4, 465–475.
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The Metabolism of Biogenic Monoamines during Embryogenesis and Metamorphosis in Two Anuran Species Naokuni Takeda Department of Biotechnology, COSMO Research Institute, Research and Development Center, Satte, Saitama 340-01, Japan Accepted January 13, 1997
This study investigated the pathways to many monoamines and their metabolites in the central nervous system of the frog Rana nigromaculata and the toad Bufo bufo japonicus during embryonic development and metamorphosis. Metabolites were analyzed by three-dimensional HPLC. The two species provided evidence of similar pathways, with slightly different timetables for the development of their monoamine systems. During embryonic development, the main metabolic pathways in entire embryos were tyrosine (TYR) 8 [3,4-dihydroxyphenylalanine in Bufo] 8 3-hydroxytyramine 8 norepinephrine or epinine (EPIN) 8 epinephrine, TYR 8 tyramine 8 (octopamine in Rana) and TYR 8 3-O-methyldopa for catecholamines, and tryptophan 8 kynurenine and 5hydroxytryptamine (5-HT) 8 [5-hydroxyindoleacetic acid and N-methyl-5-hydroxytryptamine (N-MET) in Bufo]. The monoamine system in the brain was similar during metamorphosis to that during embryogenesis with a few exceptions. The most striking change was the development of the bufotenine (5-hydroxy-N,N-dimethyltryptamine) pathway from 5-HT via N-MET. EPIN and norepinephrine in Rana and octopamine in both species disappeared during metamorphosis. These results are discussed in relation to the roles of the various pathways in development. r 1997 Academic Press Biogenic monoamines are neurotransmitters that act as chemical mediators of intercellular communication via the activation of specific receptors and second 0016-6480/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
messengers in postsynaptic cells. Their presence and distribution in the nervous system have been analyzed from a phylogenetic perspective in the animal kingdom (e.g., Klemm, 1985; Takeda, 1996a). Preliminary ontogenetic analysis has also been performed in some species, such as Bombyx mori (Takeda et al., 1991) Xenopus laevis (Takeda and Takaoka, 1991), and Gallus domesticus (Takeda, 1992). In addition, immunohistochemical studies of the hypothalamo–hypophysis system have provided some information about the ontogeny of monoaminergic systems at late larval stages in some frogs (McKenna and Rosenbluth, 1975; Corio and Doerr-Schott, 1988). The life history in frogs and toads includes the dramatic changes associated with metamorphosis, which occurs when the larva is transformed into the adult form. Each larva undergoes a dramatic morphological, biochemical, and ecological transformation to become an adult. The hormonal and neuroendocrine mechanisms of metamorphosis are well established (e.g., Norris and Dent, 1989; Kikuyama et al., 1993; Denver, 1996; Kaltenbach, 1996). However, the monoaminergic systems that might play a very basic role in metamorphosis remain to be fully characterized. There are only a few studies on the changes of monoaminergic systems in the central nervous system of amphibians during their metamorphosis and development (Norris et al., 1992; Lowry et al., 1996; Takeda, 1996a). Developmental changes in levels of tyrosine
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hydroxylase and dopamine in the brain have been examined immunohistochemically in Xenopus laevis and Rana ridibunda (Gonza´lez et al., 1994). However, no studies have examined the quantitative relationship between monoaminergic systems and embryonic development or metamorphosis in frogs and toads. In the present report, the dynamics of monoaminergic systems in the embryo and the central nervous system of the frog and the toad are described for the first time, as part of our efforts to gain a better understanding of the development of this system with the progress of embryonic development and metamorphosis.
MATERIALS AND METHODS Animals Animals studied were the frog Rana nigromaculata (Ranidae) and the toad Bufo bufo japonicus (Bufonidae). Adult frogs of both species were collected in the field and reared under natural conditions. Egg masses were obtained from these species. The collection and analysis of samples were performed repeatedly from 1990 to 1994 to obtain representative values or tendencies because the analytical data sometimes exhibited large variations. Oviposition in both species was monitored visually. The developmental stages were determined by reference to the normal tables for Rana (Iwasawa, 1996) and Bufo (Ichikawa and Tahara, 1989). For convenience, we used the following stages for the analysis of whole embryos: stage I, cleavage period (from stage 1 to 6 in the normal table); stage II, gastrula period (from stage 12 to 14 in the normal table); stage III, neurula period (from stage 19 to 20 in the normal table); stage IV, tail bud period (stage 23 in the normal table); stage V, middle branchial bud stage during the external gill period (stage 26 in the normal table); and stage VI, external gill completion stage in the external gill period (stage 30 in the normal table). Furthermore, to examine the role of monoamines in early neurogenesis, we divided embryos at the tail bud stage into two parts, the dorsal part and the ventral part, and analyzed the monoamines in each part separately. The stages at which we analyzed the brain in
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Naokuni Takeda
tadpoles were as follows: stage VII, completion of the branchial mantle during the external gill period (stage 34 in the normal table); stage VIII, hindleg formation stage (stage 37 in the normal table); stage IX, hindleg completion stage (stage 41 in the normal table); stage X, foreleg appearance stage of metamorphosis (stage 42 in the normal table); stage XI, degradation of the caudal portion during metamorphosis (stage 43 in the normal table); and stage XII, completion of metamorphosis (stage 45 in the normal table). Tadpole larvae in breeding containers after stage VII were supplied with boiled spinach as food. Each embryo and each extirpated brain was put separately into a mixture of 300 µl of 0.4 N perchloric acid, 20 µl of 50 mM EDTA, and 10 µl of 0.2 N sodium hydrogensulfite and each sample was homogenized with a Physcotron (Nichi-On, Tokyo, Japan). The homogenates were centrifuged at 3000 rpm for 20 min. After filtration, 80 µl of sample was injected into the HPLC system.
HPLC with Electrochemical Detection The analytical apparatus used was a three-dimensional HPLC system (Coulochem Electrode Array System; ESA, Chelmsford, MA). The system consists of a gradient HPLC system and 16 high-sensitivity coulometric electrochemical detectors coupled with a compatible computer. The concept and inherent advantages of the multielectrode HPLC system have been described elsewhere (Matson et al., 1984). A reversephase C18 column (4.60 nm i.d. 3 150 m; NBS column, Niko Bioscience, Tokyo, Japan) was used. Two mobile phases, solvents A and B, were employed with both linear and step gradients. Solvent A consisted of 0.1 M sodium phosphate and 10 mg/ml sodium dodecyl sulfate at pH 3.35. Solvent B consisted of methanol and water (1:1, v/v) and 50 mg/liter of sodium dodecyl sulfate at pH 3.35. Another solution consisting of 50 mM sodium phosphate buffer that included 8.18% methanol and 1.82% acetonitrile, was sometimes also used. Our protocol permitted the simultaneous separation of about 30 compounds that were relevant to our analyses. A standard chromatogram including typical compounds is shown in Fig. 1, and various compounds, with their abbreviations and oxidation potentials, are shown in Table 1. The 16 serial electrodes were set in an incremental 60-mV array that ranged
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FIG. 1. Standard chromatogram showing profiles of elution of 28 compounds. Scale, 5 µm. Names of compounds are given in Table 1. Each number in the chromatogram corresponds to a number in Table 1.
from 0 to 900 mV. The column and electrodes were maintained at 34°. Sometimes, the temperature was lowered to 25° to obtain separation of peaks. Each compound was characterized not only by its retention times but also by the oxidation potential at which a maximal response was observed. Typically, each compound was detected at three electrodes, with an average ratio of peak heights between electrodes of 1:6:1. However, the exact ratio was specific to each compound and could be used to establish the purity of compounds present in peaks of unidentified material that eluted from the column with the same retention times as known standards. Unidentified peaks obtained after HPLC of the sample were matched with standards on the basis of both retention time and oxidation potential. A ratio accuracy of 100% means that the unknown compound detected at a certain retention time spread over the array of electrodes in exactly the same way as the standard and was, therefore, pure. In biological samples, there were always some other ions and trace metabolites that shifted this ratio slightly. In this system, compounds with a ratio accuracy above 70% were usually recognized as being present in a relatively pure form. Final concentrations were calculated by comparing the size of the dominant
peak of the standard to the dominant peak of the unknown compounds in the sample.
RESULTS The Metabolism of Monoamines in Whole Embryos during Early Development In general, the precursor amino acids, tyrosine (TYR) and tryptophan (TRP), first appeared in the embryo. As embryological development progressed, the metabolites were gradually detected in increasing amounts. In R. nigromaculata (Table 2), high levels of TYR, TRP, and many of their metabolites were detected in embryos just after oviposition. The levels of these amino acids, monoamines, and their metabolites increased gradually with the progress of development, with the exception of a somewhat temporary decrease at stage V. With respect to catecholamines, a characteristic result was the detection of TYRA and octopamine (OCT) at high levels throughout embryogenesis. 3-Hydroxytyramine (DA), 3-O-methyldopa (3-O-MD), 3-methoxytyramine (3-MT), and epinine (EPIN) were detected
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TABLE 1 Compounds Detected in the Analysis, with Abbreviations and Oxidation Potentials Oxidation potential (mV) Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Dihydroxyphenylethyleneglycol Vanillylmandelic acid Norepinephrine 3,4-Dihydroxyphenylalanine 3-Methoxy-4-hydroxyphenylethyleneglycol Octopamine Epinephrine Tyrosine 3,4-Dihydroxyphenylacetic acid Normetanephrine 3-O-Methyldopa 5-Hydroxyindoleacetic acid 3-Hydroxytyramine Metanephrine 5-Hydroxytryptophan Epinine N-Acetyl-5-hydroxytryptamine Hydroxyphenylacetic acid-4 Kynurenine Homovanillic acid Vanillactic acid Vanillic acid 5-Hydroxytryptamine 5-hydroxy-N,N-dimethyltryptamine N-Methyl-5-hydroxytryptamine 3-Methoxytyramine Melatonin Tryptophan
Abbreviation
First
Second
DOPEG VMA NE DOPA
240 300 180 150
— 600 — —
MHPG OCT E TYR DOPAC NMN 3-O-MD 5-HIAA DA MN 5-HTP EPIN N-ACT-5-HT HPAC-4 KYN HVA VLA VA 5-HT
450 620 180 650 150 480 450 180 150 480 300 120 180 650 800 450 360 480 180
— — — — — — — 750 — — 650 — 700 — — — — — 700
BUTN N-MET 3-MT MEL TRP
240 300 450 600 600
840 700 — — —
throughout embryogenesis but 3,4-dihydroxyphenylalanine (DOPA), found in Bufo, was not detected. Levels of 3,4-dihydroxyphenylacetic acid (DOPAC), at stages IV and V, and of vanillylmandelic acid (VMA) and homovanillic acid (HVA) were unstable in detection with a high degree of accuracy. Both norepinephrine (NE) and epinephrine (E) were found from stage II onward. With respect to indolealkylamines, kynurenine (KYN) and 5-hydroxytryptamine (5-HT) were detected throughout embryogenesis. These results suggested that the following main pathways were present: TYR = (DOPA) = DA or 3-O-MD; DA = NE or EPIN = E; DA = DOPAC or 3-MT = HVA; and TYR = TYRA = OCT for catecholamines; and TRP = KYN and 5-HT for idolealkylamines (Figs. 2 and 3).
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Although levels of 3-O-MD and 3-MT were relatively high, the levels of the final metabolite, HVA, were low. In B. japonicus (Table 3), high levels of TYR and TRP and of their metabolites, with the exception of DOPAC and EPIN, were detected in embryos just after oviposition. Levels of these amino acids and metabolites generally increased gradually with the progress of development, except at stage VI. DOPA, DA, 3-O-MD, 3-MT, and TYRA were detected at high levels throughout embryogenesis. E, EPIN, and NE were also detected. The final metabolites HVA and VMA were detected throughout embryogenesis. OCT, which we found in Rana, was not detected. KYN and 5-HT were detected throughout embryogenesis. At stage VI, we detected 5-hydroxyindoleacetic acid (5-HIAA), the final metabolite of 5-HT. In Bufo, unlike in Rana, the methylated derivative of 5-HT, N-methyl-5-hydroxytryptamine (N -MET), was newly detected. These results suggested that the following main pathways were present: TYR = DOPA = DA = NE or EPIN = E · · = VMA; DOPA = 3-O-MD; DA = DOPAC or 3-MT = HVA; and TYR = TYRA for catecholamines; and TRP = 5-HT = 5-HIAA; 5-HT = N-MET; and TRP = KYN for indolealkylamines (Figs. 2 and 3).
Localization of Monoamines in the Embryo at Tail Bud Period To monitor the appearance of the monoamines in the neural tube region, the embryos were divided at the tail bud period into two parts: the dorsal part, including the neural tube; and the ventral part, including the yolk. At the early tail bud stage in B. b. japonicus, for example, levels of monoamines were generally lower in the dorsal part than in the ventral part, except in the case of TYRA and 5-HT (Table 4, A). Levels of TYR, TRP, and KYN in the neural part were about half of those in the nonneural part. NE, E, and EPIN were not detected. Levels of TYRA and 5-HT were about three times higher than those in the nonneural part, although the ratio of accuracy was low for 5-HT. The high levels of monoamines, such as E, DA, 5-HT, N-MET, and 5-hydroxy-N,N-dimethyltryptamine (BUTN), which were found during metamorphosis, were not or not fully recognized at this period. By contrast, in R. nigromaculata at the late tail bud stage, for example, high levels of monoamines such as NE, E, and 5-HT were detected in the neural parts (Table 4, B).
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TABLE 2 Levels of Monoamines in Whole Embryos of the Frog Rana nigromaculata with the Progress of Embryonic Development
Note. See text for details of stages. pg/mg embryo; *, ng; —, nondetectable level; mean 6 SD, n 5 25.
As was also the case in Bufo, high levels of monoamines were not detected prior to this stage. These results suggest, therefore, that monoamines were introduced into the nervous system after completion of the neural tube.
The Metabolism of Brain Monoamines during Metamorphosis In R. nigromaculata (Table 5), high levels of TYR, TRP, DA, E, and some of their metabolites, such as 3-O-MD and 3-MT, were detected in whole brains throughout metamorphosis. The peak levels of TRP, TYR, DA, E, 3-MT, VA, TYRA, and KYN were found at stage VIII, the hindleg formation stage. With respect to catecholamines, EPIN and NE were not detected except at stage VII. TYRA was detected only at stages VIII and IX. Vanillic acid (VA) was detected for the first time at stage VIII. Final metabolites, such as HVA and VMA, were detected at all stages except at stage VII and just at stage VIII, respectively. With respect to indolealkylamines, KYN, 5-HT, and N-MET were detected through-
out metamorphosis. The peak of KYN was found at stage VIII. Conversion of 5-HT to 5-HIAA was evident from stage IX. High levels of N-MET were detected at the time of migration to land. These results suggested that the following metabolic pathways were present: TYR = (DOPA) = DA = NE or EPIN = E · · = VMA = VA; DA = DOPAC or 3-MT = HVA; and TYR = TYRA for catecholamines; and TRP = KYN; TRP = 5-HT = 5-HIAA; and 5-HT = N-MET for indolealkylamines (Figs. 2 and 3). The highest levels of monoamines were found at stage X. In B. b. japonicus (Table 6), high levels of TYR and TRP, as well as of monoamines and almost all of the metabolites examined, were detected in whole brains throughout metamorphosis. Furthermore, BUTN, a dimethylated derivative of 5-HT, found via N-MET, was first detected during metamorphosis. The peak levels of TYR, TRP, DA, E, TYRA, KYN, 5-HT, and BUTN were found at stage X, when the forelegs appeared. With respect to catecholamines, high levels of DA, E, TYRA, 3-O-MD, MN, and 3-MT were detected. Metanephrine (MN), a metabolite of E, was
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FIG. 2.
Main metabolic pathways of catecholamines in the frog Rana nigromaculata and the toad Bufo bufo japonicus.
detected at stages IX, X, and XI. VA was also detected. VMA was detected only at stage X. HVA was detected throughout metamorphosis. With respect to indolealkylamines, KYN, 5-HT, N-MET, and BUTN were detected. These results suggested that the following metabolic pathways were present: TYR = (DOPA) = DA = E = MN = · · = VMA = VA; DA = DOPAC or 3-MT = HVA; and TYR = 3-O-MD or TYRA for catecholamines; and TRP = KYN; TRP = 5-HT =
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5-HIAA; and 5-HT = N-MET = BUTN for indolealkylamines (Figs. 2 and 3). The highest levels of monoamines were found at stage VIII. Some compounds found during embryogenesis were not detected in brains during metamorphosis, namely DOPA in Bufo, OCT in Rana, and NE and EPIN (except stage VII) in Rana. These compounds might act mainly during embryogenesis and not during metamorphosis.
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FIG. 3.
Main metabolic pathways of indolealkylamines in the frog Rana nigromaculata and the toad Bufo bufo japonicus.
DISCUSSION Various changes in metabolic pathways from the precursor amino acids, TYR and TRP, to many monoamines and their metabolites were clearly demonstrated with the progress of embryogenesis and metamorphosis in the frog B. b. japonicus and the toad R. nigromaculata. Similar pathways were found in both species with slightly different timetables for the development of monoaminergic systems. In both species, even the embryos just after oviposition contained some metabolites of TYR and TRP. Since unfertilized eggs in the female abdomen contain no metabolites, fertilization might trigger the rapid induction of this aspect of metabolism by activating hydroxylases for precursor amino acids such as TYR and TRP. In sea urchins and chickens, monoamines, including
5-HT and NE, are synthesized and released by yolk granules after fertilization (Buznikov, 1984, 1991; Renaud et al., 1983; Emanuelsson et al., 1988). During embryonic development, the main metabolic pathways in whole embryos of the two species studied were TYR = (DOPA in Bufo) = DA = EPIN or NE = E, TYR = TYRA = (OCT in Rana) and TYR = 3-O-MD for catecholamines, and TRP = KYN and 5-HT = (5-HIAA and N-MET in Bufo) for indolealkylamines. Thus, the development of the monoaminergic systems appeared to be similar in both species during embryogenesis. In a report of preliminary data for X. laevis (Takeda, 1996a), the first metabolic pathways to develop were suggested to be TYR = (DOPA) = 3-O-MD = · · = HVA and then DA = 3-MT = HVA, DA = DOPAC = HVA and DA = E, in that order, for catecholamines; and TRP = 5-HT = 5-HIAA and
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TABLE 3 Levels of Monoamines in Whole Embryos of the Toad Bufo bufo japonicus with the Progress of Embryonic Development
Note. See text for details of stages. pg/mg embryo; *, ng; —, nondetectable level; mean 6 SD, n 5 25.
TYP = KYN at the same time and then 5-HT = N-MET for indolealkylamines. The present two species, Rana and Bufo, seem to resemble X. laevis. Although high levels of DOPA were detected by two-dimensional analysis, as reported by Rowe et al. (1993), the poor ratio accuracy prevented accurate quantitation of DOPA. The same was true for DA and DOPAC during early embryogenesis. In earlier phylogenetic analysis (Takeda, 1996a), monoamines were detected in primitive organisms, such as protozoa and porifera, and in embryos, including those of vertebrates, prior to the development of the nervous system. What role could these neurotransmitters play in early embryos that have no nervous system? With respect to the differentiation of embryonic neurons, Rowe et al. (1993) suggested that NE might bind to a-epinephrinergic receptors on neural plate cells and might be involved in the differentiation of nerve cells in X. laevis. Experimental evidence
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suggests that 5-HT and NE in the sea urchin and chicken might regulate the cleavage divisions of the blastula and gastrulation (e.g., Buznikov, 1984, 1991). These monoamines have also been suggested to regulate the morphogenetic movements of cells that are important for neurulation in amphibians and avians and for palatal morphogenesis in the mouse (e.g., Godin, 1986; Lauder, 1988, 1993). Thus, morphogenetic roles of monoamines during embryogenic development, which were adopted early in evolution, are still operative at early embryonic stages of ontogeny. Therefore, the so-called neurotransmitters seem to have evolved to their specialized roles from more general humoral functions in primitive organisms. Although the roles of KYN, TYRA, and OCT in embryogenesis are unknown, these are phylogenetically ancient monoamines (Takeda, 1996a). In the adult nervous system, monoamines, such as DA, NE, and E, are discrete intercellular signaling
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TABLE 4 Localization of Monoamines in the Embryos of the Frog Rana nigromaculata and the Toad Bufo bufo japonicus at Stage IV Compounds
Neural part
Nonneural part
A: Stage IV, early tail bud stage, in Bufo bufo japonicus TYR 3-O-MD DA DOPAC 3-MT HVA EPIN NE E TYRA
34.5 6 20.0* 266.2 6 147.3 2.9 6 1.7* 23.4 6 17.0 1.6 6 0.1* 133.3 6 88.6 — — — 6.1 6 3.4*
73.6 6 113.1* 816.7 6 988.3 3.0 6 4.2* 51.4 6 58.2 1.76 6 2.7* 97.1 6 148.2 20.4 6 27.2 34.7 6 43.1 267.1 6 350.7 1.1 6 1.8*
TRP 5-HT KYN
1.6 6 9.9* 6.6 6 3.8 256.2 6 117.9
30.0 6 46.5* 2.6 6 2.4 434.2 6 483.2
B: Stage IV, late tail bud stage, in Rana nigromaculata TYR DOPA 3-O-MD DA 3-MT EPIN NE E TYRA OCT
58.0 6 18.2* 302.7 6 358.6 1.4 6 0.2* 105.8 6 15.1 367.4 6 58.0 56.2 6 11.5 323.5 6 48.8 474.4 6 80.2 19.3 6 14.7* 0.8 6 0.3*
28.2 6 7.9* 134.2 6 31.8 0.7 6 0.1* 73.8 6 59.5 175.6 6 31.9 32.6 6 1.4 52.6 6 7.9 55.7 6 35.9 14.2 6 4.7* 1.0 6 0.4*
TRP 5-HT KYN
19.3 6 3.2* 80.7 6 31.9 507.8 6 282.0
12.2 6 2.1* 16.8 6 6.6 446.3 6 146.0
Note. —, nondetectable level; pg/mg embryo; *, ng; n 5 15.
compounds which mediate communication between nerve cells as neurotransmitters. This specialized role seems to have evolved from more primitive functions in lower organisms, in which these compounds serve as both intra- and intercellular signaling molecules. In general, the levels of monoamines in the neural tube area of these embryos were low at the early tail bud stage. However, levels gradually increased and became higher than those of others at the late tail bud stage. In Rana, high levels of DOPA and NE in the neural part might be important for embryogenesis because these compounds were not detected in the brains of tadpoles. In both Rana and Bufo, high levels of 5-HT and KYN in the neural part appeared to be important for the development of the nervous system
(Schwarcz et al., 1991). Developmental features of serotonergic neurons in the brain stem in the neural tube have been examined in X. laevis (van Mier et al., 1989): the first serotonergic neuron appeared in the rostral part of the brain stem and axonal outgrowth reached the rostral part of the spinal cord at stage IV. The growth and development of amphibians during metamorphosis are under the control of three hormones: prolactin, growth hormone, and thyroxine (e.g., Kikuyama et al., 1993; Denver, 1996; Kaltenbach, 1996). Prolactin stimulates the growth of larval organs and inhibits the growth of adult organs, while growth hormone stimulates only the growth of adult organs. Thyroxine is an important hormone that controls metamorphosis; while it acts on larval organs to inhibit growth and to stimulate degradation, it stimulates the growth of adult organs. How, then, are monoamines involved in the action of these hormones or in metamorphosis directly? Monoamines in the brain appear to function as neurotransmitters that are involved in controlling the hypothalamic hormone-secreting neurons that secrete releasing factors (Nakai et al., 1986; Pang and Schrebman, 1986). There is evidence that aminergic neurons make synaptic contacts with these neurons. While it can be argued that the systems are not fully characterized, there is sufficient information to allow us to make some general comments and to provide some illustrative examples. The hypothalamus is innervated by aminergic neurons that have been shown to contain DA, NE, E, and 5-HT (e.g., Nakai et al., 1986). Some of the dopaminergic neurons secrete dopamine into the hypophyseal portal blood through which it is transported to, and acts directly upon, the anterior pituitary to inhibit the tonic secretion of prolactin (McCann et al., 1984). NE and DA appear to stimulate secretion of thyroid-stimulating hormone by stimulating neurons that secrete thyrotropin-releasing hormone (TRH; e.g., Krulich et al., 1977) and growth hormone by stimulating neurons that secrete growth hormone-releasing hormone (Kakucoka and Makara, 1983). By contrast, 5-HT inhibits the release of TRH. From results of studies with DA and known agonists and antagonists (Seki and Kikuyama, 1979, 1982; Kikuyama and Seki, 1980), DA is thought to be the physiological inhibitor of the release of prolactin. Seki and Kikuyama (1982)
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370
Naokuni Takeda
TABLE 5 Levels of Monoamines in the Brain of the Frog Rana nigromaculata during Metamorphosis
Note. See text for details of stages. pg/mg brain; *, ng; —, nondetectable level; mean 6 SD, n 5 15.
showed that the release of prolactin was enhanced by the direct action of 5-HT on the pituitary gland, suggesting that 5-HT might be a prolactin-releasing neurohormone. In anuran larvae, the only aminergic nuclei that are active in the hypothalamus prior to metamorphosis are the paraventricular organ and the dorsal infundibular nucleus (Hanke, 1976). McKenna and Rosenbluth (1975) provided the first link between thyroid hormones and hypothalamic monoamines in the preoptic recess organ of metamorphosing tadpoles of Bufo marinus. The peptidergic hormones released from the posterior pituitary might also be under aminergic control. KYN can act as an excitatory neurotransmittor (Stone and Connick, 1983). Moreover, BUTN is capable of promoting the secretion of prolactin (Seeman and Brown, 1985) within a few minutes after peripheral injection in rats (Meltzer et al., 1978). During metamorphosis, the pathway 5-HT = N-MET = BUTN was shown to be operative. Many
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bioactive secretions have been detected in the amphibian integument. Since the presumptive areas of the epidermis and the nervous system have the same origin in the embryo, it is not unreasonable that we found these compounds in the adult nervous system. In Bufo, not only the parotid glands but also the dermal glands synthesize and secrete bioactive substances that include amines, peptides, alkaloids, and bufodienolides (Ersparmer, 1994). In B. b. japonicus, the brain, blood, and urine contain BUTN, and the presence of BUTN suggests a route for its metabolism from the brain to the urine (Takeda, 1994). BUTN detected in whole embryos of Bufo at stage VII might have originated from these presumptive areas. In Xenopus, the dermal glands also contain BUTN (Takeda, 1996a). By contrast, the integument and the brain of Rana do not contain BUTN. BUTN may play several roles in animals. The 5-HT = N-MET pathway has been demonstrated in the poison
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Metabolism of Biogenic Monoamines
TABLE 6 Levels of Monoamines in the Brain of the Toad Bufo bufo japonicus during Metamorphosis
Note. See text for details of stages. pg/mg brain; *, ng; —, nondetectable level; mean 6 SD, n 5 15.
glands of insects and in some coelenterates, including Hydra (Takeda and Svendsen, 1991; Shimizu and Takeda, 1993). In humans, BUTN acts as a hallucinogenic compound. BUTN has been detected in the urine of some psychiatric patients and has been suggested to be a diagnostic marker for some psychiatric disorders (Takeda et al., 1995). Therefore, B. b. japonicus may be a useful model animal for neurochemical studies to psychiatric disorders. Several secretions, including the precursor of BUTN, in X. laevis induce highly stereotyped oralfacial movements in the predators such as anuran-eating snakes (Barthalmus, 1994). The stereotyped oralfacial movements in snakes affected by BUTN precursors may be acting through effects on the serotonergic system. In rat, BUTN was suggested to have an action on 5-HT neurons comparable to that of p-chloroamphetamine (Monroe et al., 1994). The role of BUTN remains unclear in amphibians, and more analytical research, for example, with antibodies against
BUTN (Takeda, 1996b) might provide some important clues to the possible role of this compound.
ACKNOWLEDGMENTS The author expresses his thanks to his son, Takuro Takeda, for collecting materials in the fields during a period of 3 years.
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