Serotonin metabolism in directly developing frog embryos during paternal care

Neuroscience Letters 388 (2005) 100–105

Serotonin metabolism in directly developing frog embryos during paternal care Gary R. Ten Eyck a,∗ , Patrick J. Ronan b,c , Kenneth J. Renner d , Cliff H. Summers d b

a Department of Psychology, Biopsychology Area, The University of Michigan, Ann Arbor, MI 48109, USA Avera Research Institute, Medical Research Service, Sioux Falls VA Medical Center, Sioux Falls, SD 57105, USA c Neuroscience Group, The University of South Dakota, Vermillion, SD 57069, USA d Department of Biology and Neuroscience Group, Division of Basic Biomedical Sciences, School of Medicine, The University of South Dakota, Vermillion, SD 57069, USA

Received 25 March 2005; received in revised form 20 June 2005; accepted 22 June 2005

Abstract Central serotonin (5-HT) metabolism during embryogenesis and a 3-day post-hatching period was analyzed using high performance liquid chromatography in the directly developing frog, Eleutherodactylus coqui. This anuran bypasses the free-swimming larval stage and embryos hatch as miniature frogs in the adult phenotype. During embryogenesis and for a short time immediately after hatching, male E. coqui provide paternal care by brooding and guarding eggs/embryos to prevent desiccation and predation. Serotonin and its catabolite, 5-HIAA, were measured from whole brain during embryogenesis and at 3 days post-hatch to identify critical periods in 5-HT development and to determine the relationship between 5-HT and life history events such as hatching and frog dispersal from the nest site. Serotonergic activity was highest during the early-mid embryonic stages as indicated by the ratio of 5-HIAA/5-HT, a general indicator of turnover and metabolism. There were significant increases in tissue concentrations of 5-HT during the latest or terminal embryonic stage, just prior to hatching, and also at 3 days post-hatch, shortly before neonates disperse into the rainforest. These two increases probably represent different functional requirements during development. The first may occur as a result of the surge of development in the 5-HT system during late embryogenesis that occurs in E. coqui and the second may be from the increase demand in sensory and motor neural development required before dispersal from the nest site. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: 5-HT; Development; Amphibian; Directly developing frogs; Paternal care

Serotonergic (5-HT) systems are involved in an array of behavioral and physiological functions in animals, including movement, feeding, aggression, stress, and developmental regulation [8,14,20,43]. Investigations of mammals [1,19,21,56], birds [30,35,55], amphibians [10,49,50,54,58], and fish [4,5,11,12,23] have found that the serotonergic system develops early and is typically functional by the completion of embryogenesis [1,21,23,41,58]. Furthermore, the 5-HT system appears to have an important functional role during the transition from embryo to neonate, especially events ∗ Corresponding author at: Department of Biological Sciences, Idaho State University, 638 E. Dunn Street, Campus Box 8007, Pocatello, ID 832098007, USA. Tel.: +1 208 282 4410; fax: +1 208 282 4570. E-mail address: [email protected] (G.R. Ten Eyck).

0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.06.039

associated with hatching. For example, it has been demonstrated in the chick that the 5-HT system is activated during hatching [45] and that this activation is important for normal hatching [42]. If embryos are subjected to disruption of normal 5-HT2 receptor signaling, with either 5-HT2 receptor antagonists or agonists during late embryonic development, normal hatching behavior is disrupted [42]. Most anuran amphibians (frogs) hatch from eggs relatively early during development and subsequently enter a larval period. This larval stage is typically a free-swimming tadpole, which is terminated by metamorphosis, a dramatic change in the physiology, morphology, behavior, and ecology of the frog [31,59]. Investigations have found that patterns of 5-HT development appear specific to life history changes such as metamorphosis [29,46], which suggests that 5-HT

G.R. Ten Eyck et al. / Neuroscience Letters 388 (2005) 100–105

metabolism may play a critical role in larval development and metamorphosis. In the salamander, Ambystoma tigrinum, 5-HT levels are the highest during mid-metamorphosis [29] and in the metamorphic frogs, Bufo bufo and Rana nigromaculata, a gradual rise in 5-HT levels occurs throughout embryonic development that peaks during metamorphic climax [46]. The Puerto Rican coqu´ı frog, Eleutherodactylus coqui, is a directly developing frog that lacks the free-swimming larval stage and hatches as a miniature froglet in the terrestrial environment. This frog develops inside a gelatinous egg capsule and possesses both larval (fleshy tail, thyroid hormone-dependent developmental period [9]) and non-larval (absent larval olfactory organ [15], modified peripheral nervous system and skull development [36,13]) characters. An additional derived characteristic of this frog includes paternal care, whereby males remain with the eggs after oviposition by the female. Paternal males perform two major parental activities, brooding of eggs and the defense of the nest site from potential predators. Following hatching, froglets and the paternal male can remain at the nest site for up to 5 days [52]. All of the behaviors described above are also observed in captive breeding colonies [26]. The objective of this study was to determine the development of the serotonergic system in the brain of E. coqui by measuring 5-HT and 5-HIAA, which are reflections of 5-HT synthetic capacity and system activity. We will quantify 5HT and 5-HIAA during embryogenesis and the post-hatching period which correlates to the temporal period throughout which paternal care occurs. Our purpose was also to compare these results with developmental changes in the serotonergic system in metamorphic frogs and other vertebrates. Embryos of E. coqui were obtained from spontaneous mating between wild-caught adults that were captured in the rainforest of northeastern Puerto Rico, under permits issued by the Departmento de Recursos Naturales y Ambientales of Puerto Rico. Adult frogs were maintained as a laboratory-breeding colony in the Department of Biology at The University of South Dakota. Care of animals followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the University of South Dakota IACUC. Animals were kept on a 12:12 h photoperiod, fed live crickets (dusted with vitamin and mineral powder) three times a week, and maintained at 30/21 ◦ C (day/night) in humid terraria. Embryos were staged according to the table of Townsend and Stewart [51] that defines a total of 15 embryonic stages from oviposition to hatching (TS 1–15). Measurements of whole brain concentrations of 5-HT and 5-HIAA were done using high performance liquid chromatography (HPLC) with electrochemical detection [34]. Embryos (TS 5–15) and hatchlings (immediately hatched and 3 days post-hatched) had their brains (∼1.0–3.0 mm in length) dissected rapidly from the cranium on a dry ice/ice mixture and then stored at −80 ◦ C. Brains were expelled into 60 ␮l of sodium acetate buffer (pH 5) containing 1 × 10−7 alpha methyl DA (Sigma Chemical Co.; internal standard),

101

freeze–thawed, and then were exposed to very brief bursts or pulses of sonic homogenization. Prior to centrifugation (15,000 × g for 2 min), 2 ␮l of a 1 mg/10 ml H2 O ascorbate oxidase solution (Boehringer Mannheim) was added to each sample [22]. The supernatant was removed and 45 ␮l was injected into a Waters chromatography system (Waters Associates, Milford, MA) and analyzed electrochemically with a LC-4B potentiostat and a glassy carbon electrode (Bioanalytical Systems Inc., West Lafayette, IN). The electrode potential was set at +0.65 V with respect to an Ag/AgCl reference electrode. Separation was accomplished using a 4 ␮m C-18 radial compression cartridge (Waters Associates, Milford, MA). The mobile phase consisted of 11 g citric acid, 8.6 g sodium acetate, 110 mg octylsulfonic acid (Eastman Kodak), 250 mg EDTA and 100 ml methanol in 1 liter of water. The tissue pellets were dissolved in 0.3N NaOH and analyzed for protein content [6]. Samples were quantified by comparison with standard solutions (5-HIAA and 5-HT, Sigma) of known concentration and corrected for recovery of the internal standard and volume using a Waters 745 integrator. Amine levels are expressed as pg amine/␮g protein. Significance between means of neurotransmitter and metabolite concentrations for the different stages was performed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range tests for posthoc analyses (α < 0.05). The estimate of turnover or activity is reported as the ratio of catabolite to transmitter (5-HIAA/5-HT). Interpretation of data: Although neurotransmitter levels, expressed as pg amine/␮g protein, are apparently uncomplicated, understanding relative changes in activity of monoamine systems often requires some interpretation. The data may be presented as ratios of catabolite to transmitter (i.e., 5-HIAA/5-HT), which is an estimate of monoaminergic turnover. As such, the activity of a given monoamine system can be said to increase as the ratio increases. This kind of monoaminergic activity, approximated by the ratio of the catabolite to transmitter, presumes that transmitter levels (i.e., 5-HT) decrease (or remain constant) as they are secreted and converted to catabolite (5-HIAA) and thereby catabolite concentrations increase. However, the relative progression of synthesis of the transmitter is critical for interpretation of results, especially in developmental studies. Changes in the catabolite are often seen along with changes in the ratio [57]. When this is so, as during the early stages of development (TS stages 5–8), the ratio is often a more direct index of monoaminergic activity than catabolite levels per se, because variance related to tissue sampling, and to total levels of transmitter and catabolite, are reduced [37]. Accessible transmitter concentration is often greater than demand, as appears to be the case for the middle to late stages of development (TS 9 to hatching); hence monoamine levels often remain unchanged. Constant or growing transmitter concentrations may also occur when synthesis is rapidly elevated in response to stimulus or development, as appears to be the case in later stages of E. coqui development. When production offsets release, the ratio (5-HIAA/5-HT) becomes meaningless

102

G.R. Ten Eyck et al. / Neuroscience Letters 388 (2005) 100–105

as a measure of monoaminergic activity. If rapid synthesis or reuptake of transmitter coincides with extensive release and catabolism, both monoamine transmitter and catabolite concentrations are likely to rise, as is evident in posthatching froglets. While this clearly indicates an increase in system activity, the ratio remains unchanged and is not valuable for determining monoaminergic activity. There was a gradual and significant increase (F12,57 = 40.6, P < 0.0001, n = 70, mean sample size = 5.3 ± 0.76) in 5-HT levels in the brain throughout embryogenesis (Fig. 1a and b). However, two considerably significant increases in 5-HT concentration occurred beyond the gradual developmental rise in 5-HT (Fig. 1a and b). The first significant increase occurred at TS 15 and the second in 3-day-old neonates. Levels of 5-HT catabolite 5-HIAA were variable but higher, relative to other 5-HIAA points, during embryogenesis and post-embryonic development, showing one dramatically significant increase (F12,61 = 7.5, P < 0.0001, n = 74, mean sample size = 5.6 ± 0.57), which occurred in 3-day post-hatched neonates (Fig. 1c and d). Overall 5-HT activity can be divided into two periods: (1) early high turnover (estimated by 5-HIAA/5-HT ratio), which gradually decreases during embryonic development, after significant elevation (F12,54 = 29.0, P < 0.0001, n = 67, mean sample size = 5.8 ± 0.81) from TS 5 through 7 (Fig. 1e and f), predominantly caused by high 5-HIAA concentrations, relative to lower 5-HT concentrations, and (2) increased capacity later in development, during which progressively higher 5-HT levels result in reduced 5-HIAA/5-HT ratios. Concentrations of 5-HT gradually increase in the brain of E. coqui throughout embryogenesis, a pattern of change which also occurs in metamorphic frogs [46], until the first significant rise at the termination of embryogenesis. A second significant rise occurs immediately prior to neonate dispersal into the forest. Interestingly, these two significant increases could be a consequence of different life history demands. The first increase could be related to the surge of development that occurs during the late stages of embryogenesis. This period is denoted by dynamic morphological development. This includes skeletal development [13,28] and 5-HT has been shown to regulate morphogenesis, particularly craniofacial development [3,27,40]. Also during this later period, substantial development occurs in the peripheral nervous system and associated neuromuscular connections [36]. Furthermore, during mid-late to late embryogenesis E. coqui undergoes a thyroid hormone dependence period [9] that is reminiscent of frogs undergoing metamorphosis [38,47]; treatment with the goitrogen, methimazole, drastically inhibits normal morphogenesis in E. coqui [9]. At this time, an up-regulation also occurs in the thyroid hormone receptor message, TR␤ mRNA [9]. In metamorphic frogs, the quantity of 5-HT is highest during the climax of metamorphosis [46] and in E. coqui whole brain concentrations of 5-HT during embryogenesis were highest in the latter stages of development. Both increases are concurrent with the rise in thyroid hormone and hormone action. This rise in central 5-HT could partially be a function

of intense embryonic development, which may depend upon 5-HT for developmental regulation [8,18]. The two increases could be indicative of a metabolic connection between the thyroid axis and the serotonergic system [2,39], especially between thyroid hormone and the 5-HT1A receptor function [17,48,53]. Furthermore, increasing thyroid hormone levels result in increasing serotonin transmission [2] and diminished levels of thyroid hormone during neural development result in the disruption of the normal upsurge of 5-HT metabolism in the rat brain [33]. The later embryonic stages in E. coqui, which are characterized by high thyroid metabolism, are also denoted with intense morphological development of the 5-HT system. An increase in serotonergic-immunoreactive neurons occurs in the diencephalon and hindbrain regions, and prominent increases in immunoreactive varicose fibers develop throughout the brain [49]. Lastly, if E. coqui embryos are treated with the goitrogen, methimazole, during embryogenesis, morphological development of the 5-HT system is altered (unpublished data). Serotonergic activity (estimated by 5-HIAA/5-HT ratio) is highest during the early-mid embryonic stages (TS 5–8), with a significant increase at TS 5 and 6, followed by a gradual decrease throughout embryogenesis. These ratios are particularly high and high turnover ratios are indicative of increased 5-HT metabolism [16,37,44,57], as evidenced by high 5-HIAA concentrations measured at this time (Fig. 1c and d). High 5-HT turnover during these early stages would be biologically reasonable. At TS 6, embryos are beginning movement in egg capsules, and by TS 7, tail thrashing occurs [51]. This early movement coupled with high 5-HT metabolism during embryonic development is consistent with 5-HT enhancing the development and modulation of locomotion [7,24,25] but also with the role of 5-HT stimulating neural development [3,8,18]. Interestingly, in mice, another direct developing organism, serotonin-induced motor activity also begins at an early embryonic stage [1]. In E. coqui, during this same temporal period, immunocytochemistry reveals developing 5-HT cells and fibers in the hindbrain and diencephalon [49]. Additionally, in tadpoles and fish larvae it has been established that 5-HT activity occurs early and is correlated with early movement [23,58]. Thus, increased or intense 5-HT turnover is likely to modulate both the locomotor/arousal and developmental demands during early to mid embryogenesis. The second significant rise in 5-HT occurs 3 days after hatching which coincides with the time period during which the terrestrial froglets are still at the nest site and guarded by the paternal male. This increase is the largest and is also correlated with a significant increase of 5-HT catabolite 5-HIAA. Several factors may attribute to this rise. First, during this non-dispersal period, the 5-HT system, and presumably other neurochemical systems, may be undergoing further maturation and refinement in sensory and motor systems. Post-embryonic development and maturation are seen in fish [11], birds [30], and mammals [1,32], including marsupials [21]. In the opossum, Monodelphis domestica, at birth,

G.R. Ten Eyck et al. / Neuroscience Letters 388 (2005) 100–105

103

Fig. 1. Mean concentrations (±S.E.M.) of (a and b) 5-HT, (c and d) 5-HIAA, and (e and f) serotonergic activity (estimated by 5-HIAA/5-HT ratio) in the brain of E. coqui during embryonic stages TS 5–15 and post-embryonic periods immediately after hatching (JH) and 3 days post-hatched (PH). Serotonin levels gradually increase throughout embryogenesis by stage (a) with significant increases marked by means that share no common superscript letters. This gradual increase in 5-HT is also apparent when analyzed by day of development (b) where ‘*’ denotes significantly more 5-HT than on day 6; ‘**’, significantly more 5-HT than day 10; ‘***’, significantly more than day 19.5; ‘****’, significantly more 5-HT than day 21.5. Brain levels of 5-HIAA fluctuate during embryogenesis, where significant changes are denoted by (c) developmental stage means without common superscript letters, or (d) where ‘*’ denotes significantly elevated 5-HIAA concentrations compared to days 10, 15 and 22; ‘**’, significantly more 5-HIAA than all other days. (e and f) Serotonergic activity, as measured by the ratio of 5-HIAA (serotonin catabolite) to 5-HT, turnover is highest at earlier embryonic stages (TS 5–7), and decreases as embryogenesis progresses. However, this measure of serotonergic activity, the ratio 5-HIAA/5-HT, is not meaningful when both 5-HT and 5-HIAA concentrations are increasing or decreasing.

104

G.R. Ten Eyck et al. / Neuroscience Letters 388 (2005) 100–105

the serotonergic system is well developed and comparable to a 14-day-old mouse. Interestingly, similar to E. coqui, substantial postnatal serotonergic development occurs during the period of parental care [21]. The up-regulation of 5-HT during these developmental periods may reflect functional changes linked to successful dispersal, which presumably requires increased motor and sensory function to negotiate the environment (i.e., avoiding predators, preventing desiccation), in which 5-HT is required [1,23,24]. Finally, there may have been selection for 5-HT increases, maturation, and/or adjustments during this non-dispersal period since froglets are typically not required to negotiate, move, or forage outside the nest site since they still contain a large amount of yolk for energy [51] and the paternal male continues to provide vigilance and protection from predators [52].

Acknowledgements We are grateful to the Departmento de Recursos Naturales y Ambientales of Puerto Rico for issuing collecting permits to obtain the adult Coqu´ı for the breeding colony. A special thanks goes to the staff at the El Verde Field Station, Puerto Rico for providing support and use of their facilities, especially Drs. Alonso Ram´ırez (Director of El Verde Field Station), Jess Zimmerman, Jill Thompson, and Ms. Hilda Hugo. The field station is part of the Long Term Ecological Research (LTRE) project funded by the National Science Foundation and The University of Puerto Rico. Financial support of this research was made possible by the Department of Biology at The University of South Dakota, the National Science Foundation OSR-9452894 (CHS), the National Institutes of Health P20 RR15567 (CHS), and the Office of the Vice President of Research at The University of Michigan (GTE).

References [1] B. Ballion, P. Branchereau, J. Chapron, D. Viala, Ontogeny of descending serotonergic innervation and evidence for intraspinal 5HT neurons in the mouse spinal cord, Brain Res. Dev. Brain Res. 137 (2002) 81–88. [2] M. Bauer, A. Heinz, P.C. Whybrow, Thyroid hormones, serotonin and mood: of synergy and significance in the adult brain, Mol. Psychiatry 7 (2002) 140–156. [3] N. Bhasin, A.S. LaMantia, J.M. Lauder, Opposing regulation of cell proliferation by retinoic acid and the serotonin (2B) receptor in the mouse frontonasal mass, Anat. Embryol. 208 (2004) 135–143. [4] V. Bolliet, M.A. Ali, Immunohistochemical study of the development of serotoninergic neurons in the brain of the brook trout, Salvelinus fontinalis, Brain Behav. Evol. 40 (1992) 234–249. [5] V. Bolliet, S. Perreault, M.A. Ali, Development of serotonergic neurons in the brain of the mackerel, Scomber scombrus. An immunohistochemical study, J. Fish Biol. 44 (1994) 241–253. [6] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254.

[7] E. Brustein, M. Chong, B. Holmqvist, P. Drapeau, Serotonin patterns locomotor network activity in the developing zebrafish by modulating quiescent periods, J. Neurobiol. 57 (2003) 303–322. [8] G.A. Buznikov, H.W. Lambert, J.M. Lauder, Serotonin and serotoninlike substances as regulators of early embryogenesis and morphogenesis, Cell Tissue Res. 305 (2001) 177–186. [9] E.M. Callery, R.P. Elinson, Thyroid hormone-dependent metamorphosis in a direct developing frog, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 2615–2620. [10] P. Clairambault, N. Christophe, C. Pairault, M. Herbin, R. Ward, J. Reperant, Organization of the serotoninergic system in the brain of two amphibian species, Ambystoma mexicanum (Urodela) and Typhlonectes compressicauda (Gymnophiona), Anat. Embryol. 190 (1994) 87–99. [11] P. Ekstr¨om, Developmental changes in the brain-stem serotonergic nuclei of teleost fish and neural plasticity, Cell. Mol. Neurobiol. 14 (1994) 381–393. [12] P. Ekstr¨om, L. Nyberg, T. van Veen, Ontogenetic development of serotonergic neurons in the brain of a teleost, the three-spined stickleback. An immunohistochemical analysis, Brain Res. Dev. Brain Res. 17 (1985) 209–224. [13] J. Hanken, M.W. Klymkowsky, C.H. Summers, D.W. Seufert, N. Ingebrigtsen, Cranial ontogeny in the direct-developing frog, Eleutherodactylus coqui (Anura: Leptodactylidae), analyzed using whole-mount immunohistochemistry, J. Morphol. 211 (1992) 95–118. [14] B.L. Jacobs, C.A. Fornal, Activity of serotonergic neurons in behaving animals, Neuropsychopharmacology 21 (2 Suppl.) (1999) 9S–15S. [15] W.J. Jermakowicz III, D.A. Dorsey, A.L. Brown, K. Wojciechowski, C.L. Giscombe, B.M. Graves, C.H. Summers, G.R. Ten Eyck, Development of the nasal chemosensory organs in two terrestrial anurans: the directly developing frog, Eleutherodactylus coqui (Anura: Leptodactylidae), and the Metamorphosing Toad, Bufo americanus (Anura: Bufonidae), J. Morphol. 261 (2004) 225–248. [16] W.J. Korzan, C.H. Summers, Serotonergic response to social stress and artificial social sign stimuli during paired interactions between male Anolis carolinensis, Neuroscience 123 (2004) 835–845. [17] A.V. Kulikov, R. Jeanningro, The effects of hypothyroidism on 5HT1A and 5-HT 2A receptors and the serotonin transporter protein in the rat brain, Neurosci. Behav. Physiol. 31 (2001) 445–449. [18] J.M. Lauder, Neurotransmitters as growth regulatory signals: role of receptors and second messengers, TINS 16 (1993) 233–240. [19] H.G.W. Lidov, M.E. Molliver, Immunohistochemical study of the development of serotonergic neurons in the rat CNS, Brain Res. Bull. 9 (1982) 559–604. [20] I. Lucki, The spectrum of behaviors influenced by serotonin, Biol. Psychiatry 44 (1998) 151–162. [21] J.M. Luque, V. Biou, J.G. Nicholls, Three-dimensional visualization of the distribution, growth, and regeneration of monoaminergic neurons in whole mounts of immature mammalian CNS, J. Comp. Neurol. 390 (1998) 427–438. [22] L. McKay, C. Bradberry, A. Oke, Ascorbic acid oxidase speeds up analysis for catecholamines, indoleamines and their metabolites in brain tissue using HPLC-EC, J. Chromatogr. 311 (1985) 167–169. [23] D.L. McLean, J.R. Fetcho, Ontogeny and innervation patterns of dopaminergic, noradrenergic, and serotonergic neurons in larval zebrafish, J. Comp. Neurol. 480 (2004) 38–56. [24] D.L. McLean, S.D. Merrywest, K.T. Sillar, The development of neuromodulatory systems and the maturation of motor patterns in amphibian tadpoles, Brain Res. Bull. 53 (2000) 595–603. [25] D.L. McLean, K.T. Sillar, Divergent actions of serotonin receptor activation during fictive swimming in frog embryos, J. Comp. Physiol. A 190 (2004) 391–402. [26] S.F. Michael, Captive breeding of two species of Eleutherodactylus (anura: Leptodactylidae) from Puerto Rico, with notes on behavior in captivity, Herpetol. Rev. 26 (1995) 27–29.

G.R. Ten Eyck et al. / Neuroscience Letters 388 (2005) 100–105 [27] J.R.D. Moiseiwitsch, The role of serotonin and neurotransmitters during craniofacial development, Crit. Rev. Oral Biol. Med. 11 (2000) 230–239. [28] J.R.D. Moiseiwitsch, J.M. Lauder, Serotonin regulates mouse cranial neural crest migration, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 7182–7186. [29] D.O. Norris, J.A. Carr, P.H. Desan, T.K. Smock, M.F. Norman, Monoamines and their metabolites in the amphibian (Ambystoma tigrinum) brain: quantitative changes during metamorphosis and captivity, Comp. Biochem. Physiol. 103A (1992) 279–283. [30] N. Okado, H. Sako, S. Homma, K. Ishikawa, Development of serotoninergic system in the brain and spinal cord of the chick, Prog. Neurobiol. 38 (1992) 93–123. [31] F.H. Pough, R.M. Andrews, J.E. Cadle, M.L. Crump, A.H. Savitzky, K.D. Wells, Herpetology, Prentice Hall, Upper Saddle River, NJ, 1998, 570 pp. [32] N. Rajaofetra, F. Sandillon, M. Geffard, A. Privat, Pre- and postnatal ontogeny of serotonergic projections to the rat spinal cord, J. Neurosci. Res. 22 (1989) 305–321. [33] R.B. Rastogi, R.L. Singhal, The effect of thyroid hormone on serotonergic neurons: depletion of serotonin in discrete brain areas of developing hypothyroid rats, Naunyn Schmiedebergs Arch. Pharmacol. 304 (1978) 9–13. [34] K.J. Renner, V. Luine, Analysis of temporal and dose-dependent effects of estrogen on monoamines in brain nuclei, Brain Res. 366 (1986) 64–71. [35] H. Sako, T. Kojima, N. Okado, Immunohistochemical study on the development of serotonergic neurons in the chick: I. Distribution of cell bodies and fibers in the brain, J. Comp. Neurol. 253 (1986) 61–78. [36] G. Schlosser, G. Roth, Evolution of nerve development in frogs: II. Modified development of the peripheral nervous system in the directdeveloping frog, Eleutherodactylus coqui (Leptodactylidae), Brain Behav. Evol. 50 (1997) 94–128. [37] N.J. Shannon, J.W. Gunnet, K.E. Moore, A comparison of biochemical indices of 5-hydroxytryptaminergic neuronal activity following electrical stimulation of the dorsal raphe nucleus, J. Neurochem. 47 (1986) 958–965. [38] Y.-B. Shi, Thyroid hormone-regulated early and late genes during amphibian metamorphosis, in: L.I. Gilbert, J.R. Tata, B.G. Atkinson (Eds.), Metamorphosis: post-embryonic reprogramming of gene expression in amphibian and insect cells, Academic Press, San Diego, 1996, pp. 505–533. [39] Z.X. Shi, W. Xu, M.K. Selmanoff, J.F. Wilber, Serotonin (5HT) stimulates thyrotropin-releasing hormone (TRH) gene transcription in rat embryonic cardiomyocytes, Endocrine 6 (1997) 153– 158. [40] D.L. Shuey, T.W. Sadler, J.M. Lauder, Serotonin as a regulator of craniofacial morphogenesis: site specific malformations following exposure to serotonin uptake inhibitors, Teratology 46 (1992) 367–378. [41] K.T. Sillar, C.A. Reith, J.R. McDearmid, Development and aminergic neuromodulation of a spinal locomotor network controlling swimming in Xenopus larvae, Ann. N. Y. Acad. Sci. 860 (2002) 318–332. [42] S.B. Sparber, A. Rizzo, B. Berra, Excessive stimulation of serotonin 2 (5-HT2 ) receptors during late development of chicken embryos causes decreased embryonic motility, interferes with hatching, and

[43] [44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

105

induces herniated umbilici, Pharmacol. Biochem. Behav. 53 (1996) 603–611. C.H. Summers, Social interaction over time: implications for stress responsiveness, Integr. Comp. Biol. 42 (2002) 591–599. C.H. Summers, T.R. Summers, M.C. Moore, W.J. Korzan, S.K. Woodley, P.J. Ronan, E. H¨oglund, M.J. Watt, N. Greenberg, Temporal patterns of limbic monoamine and plasma corticosterone response during social stress, Neuroscience 116 (2003) 553–563. T.R. Summers, C.H. Summers, P.H. Desan, T.K. Smock, Activation of the serotonergic system in chick spinal cord during hatching, J. Exp. Zool. 257 (1991) 330–335. N. Takeda, The metabolism of biogenic monoamines during embryogenesis and metamorphosis in two anuran species, Gen. Comp. Endocrinol. 106 (1997) 361–373. J.R. Tata, Hormonal interplay and thyroid hormone receptor expression during amphibian metamorphosis, in: L.I. Gilbert, J.R. Tata, B.G. Atkinson (Eds.), Metamorphosis: Postembryonic Reprogramming of Gene Expression in Amphibian and Insect Cells, Academic Press, San Diego, 1996, pp. 466–503. S. Tejani-Butt, J. Yang, A. Kaviani, Time course of altered thyroid states on 5-HT1A receptors and 5-HT uptake sties in rat brain: an autoradiographic analysis, Neuroendocrinology 57 (1993) 1011–1018. G.R. Ten Eyck, W.J. Jermakowicz, A.F. Chinn, C.H. Summers, Ontogeny of central serotonergic neurons in the directly developing frog, Eleutherodactylus coqui, Anat. Embryol. (in press). M. Terlou, H.W.M. van Straaten, P.G.W.J. van Oordt, The development of hypothalamic monoaminergic neurons in Xenopus laevis tadpoles in relation to background-dependent colour change, Int. J. Dev. Neurosci. 4 (1972) 465–475. D.S. Townsend, M.M. Steward, Direct development in Eleutherodactylus coqui (anura: Leptodactylidae): a staging table, Copeia (1985) 423–436. D.S. Townsend, M.M. Stewart, F.H. Pough, Male paternal care and its adaptive significance in a neotropical frog, Anim. Behav. 32 (1984) 421–431. V.A. Vaidya, M.E. Castro, Q. Pei, M.E. Sprakes, D.G. GrahameSmith, Influence of thyroid hormone on 5-HT1A and 5-HT2A receptor-mediated regulation of hippocampal BDNF mRNA expression, Neuropharmacology 40 (2001) 48–56. P. van Mier, H.W.J. Joosten, R. van Rheden, H.J. ten Donkelaar, The development of serotonergic raphespinal projections in Xenopus laevis, Int. J. Dev. Neurosci. 4 (1986) 465–475. J.A. Wallace, An immunocytochemical study of the development of central serotonergic neurons in the chick embryo, J. Comp. Neurol. 236 (1985) 443–453. J.A. Wallace, J.M. Lauder, Development of the serotonergic system in the rat embryo: an immunocytochemical study, Brain Res. Bull. 10 (1983) 459–479. S. Winberg, G.E. Nilsson, Roles of brain monoamine transmitters in agonistic behaviour and stress reactions, with particular reference to fish, Comp. Biochem. Physiol. 106C (1993) 597–614. A.-M. Woolston, J.F.S. Wedderburn, K.T. Sillar, Descending serotonergic spinal projections and modulation of locomotor rhythmicity in Rana temporaria embryos, Proc. R. Soc. Lond. B Biol. Sci. 255 (1994) 73–79. G.R. Zug, Herpetology: An Introductory Biology of Amphibians and Reptiles, Academic Press, San Diego, 1993, 527 pp.