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Hypothalamic and telencephalic catecholamine content in the brain of the teleost fish, Pygocentrus notatus, during the annual reproductive cycle
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
80,
257-263 (1%)
Hypothalamic and Telencephalic Catechoiamine Content in the Brain of the Teleost Fish, Pygocentrus notatus, during the Annual Reproductive Cycle HILDA Y. GUERRERO,GISELA CACERES,CARMEN
L. PAIVA, AND DAYSSI MARCANO’
Department of Physiology, “J. M. Vargas” Medical School, and Institute for Experimental Medicine, Universidad Central de Venezuela, Apartado 47633, Caracas 1041-A, Venezuela Accepted November 1, 1989 The catecholamines noradrenaline (NA), dopamine (DA), and adrenaline (A) were measured in hypothalamic and telencephalic extracts of the Venezuelan freshwater fish “caribe colorado,” Pygocentrus notatus, at different stages of the reproductive cycle. The concentration of NA was found to be significantly higher in the telencephalon than in the hypothalamus, but that of DA was higher in the hypothalamus than in the telencephalon. Fluctuations depending upon the reproductive stage and environmental conditions occurred in both hypothalamus and telencephalon. In the hypothalamus, DA content was highest during the prespawning period (June) as compared to other periods of the cycle. Although the NA concentration was reduced during spawning there was no significant variation during any other period. DA concentrations in both telencephalon and hypothalamus showed a similar pattern of changes. In the telencephalon, NA levels increased between preparatory and prespawning periods but decreased sharply during spawning. No sex differences were observed in either area at any stage of reproduction. 8 IWOAcademic RUSS, hc.
The “caribe colorado,” Pygocentrus notutus, is a seasonal breeder, widely distributed in rivers and lagoons of the Venezuelan plains. Under natural conditions this fish shows an annual gonadal cycle, which can be divided into four periods: preparatory (February-April), prespawning (MayJune), spawning (July-August), and postspawning (September-January) (Gentile, 1983). The pattern of gonadal maturation of this species seems to be closely related to changes in the environmental conditions of the Venezuelan plains (Mago-Leccia, 1970), which are characterized by a dry season (December-April) followed by a rainy period (May-November). Catecholaminergic neuronal systems are widespread in the animal kingdom, and ’ To whom correspondence should be addressed.
and reprint requests
nerve fibers of this type have been described in the brain of different species of fish (Kotrschal and Adam, 1983; Parent, 1984). In teleosts the aminergic cells seem to be highly concentrated in the hypothalamus, particularly in the paraventricular organ from where projections extend to the midbrain, telencephalon, and pituitary gland (Dube and Parent, 1982; Fryer et al., 1985; Kah et al., 1987; Parent and Northoutt, 1982; Peute et al., 1987). Recently Corio et al. (1985) showed the presence of immunoreactive dopamine (DA) neurons in the nucleus ventromedialis, the nucleus posterioris periventricularis, and the lateral extensions of the nucleus lateralis tuberis in the African catfish. In the goldfish, DA fibers from the hypothalamus innervate the different lobes of the pituitary gland, and DA terminals are often observed in direct contact with the gonadotrophs (Kah et al., 1986). On the basis of brain-
257 0016~648OBo $1.50 Copyright 0 1990 by Academic Press, Inc. AU rights of reproduction in any form reserved.
258
GUERRERO ET AL.
lesion studies and immunohistochemistry, Kah et al. (1987) demonstrated the existence of a dopaminergic preoptic-hypophyseal pathway in the goldfish. The involvement of catecholamines in the neuroendocrine regulation of gonadotrophin release in teleosts was first demonstrated, in the goldfish, by Chang et al. (1983). There is evidence that noradrenaline (NA) stimulates gonadotrophin secretion, and it has been shown that DA acts as an inhibitory factor on gonadotrophin secretion, to directly inhibit spontaneous gonadotrophin release and modulate gonadotrophin releasing hormone (GnRH) action in the pituitary gland (De Leeuw et al., 1986; Peter et al., 1986; Peute et al., 1987). As catecholamines appear to play an important role in the regulation of gonadotrophin secretion in teleosts, and hence intluence their reproductive cycles, the purpose of this study was to evaluate the changes in NA, DA, and adrenaline (A) levels in the hypothalamus and telencephalon of P. notutus, in relation to the gonadal maturation. MATERIALS
AND METHODS
Adult male and female P. notarus (Lutken, 1874) (Teleostei-Characidae) were collected monthly during 1987 from lagoons and ponds of the Guarico River and its tributaries (Guarico and Apure states) and transported in aerated tanks to the laboratory. The animals were sacrificed by decapitation, then the brains were immediately removed and dissected, and the tissues were weighed and maintained at -40°C until amine extraction. At the same time, the gonads were extirpated and weighed, and the gonadosomatic index (GSI) was calculated. Tissue dissection and extraction procedures. Immediately following removal, the brains were dissected. Both the whole hypothalamus (H) and remaining forebrain comprising the telencephalon and olfactory bulb (TEL) were homogenized (at a final tissue concentration of 160 mg/ml) in ice-cold 0.4 M perchloric acid (PCA) containing 40 mM sodium metabisulfite and 10 r&ml of 3,4-dihydroxybenzilamine (DHBA) as an internal standard. After centrifugation at 20,OOOgthe supematants were filtered through a 0.45urn pore diameter filter (Millipore S.A.) and aliquots (S-100 ul) of this were directly analyzed by HPLC. The measurement was performed by comparing the peak heights of
the samples with those of a standard run, and correcting according to the internal standard. The HPLC system used was a Model 6000A solvent delivery pump coupled to a U6K injector (Waters Associates, Milford, MA). Separation of catecholamines was achieved on MBondapak Cl8 IO-pm Radial-Pak cartridges (10 cm x 5 mm I.D.) from Waters Associates. The detection system consisted of an electrochemical detector (Bioanalytical Systems, Model LC 4B) fitted with a glassy carbon electrode (Model TL.5) and a recorder-integrator (Data Module 730, Waters Associates). A slight modification of the working conditions reported by Moyer and Jiang (1978) was established: the operating potential was set at +0.55 V against the Ag/AgCl, reference electrode. The mobile phase consisted of 0.075 M sodium phosphatecitric acid buffer containing 0.1 m&f EDTA (free acid) and 2.5 mM sodium octyl sulfate, pH 4.0, with an aqueous and methanol ratio of 92:8 (v/v). The flow rate was set at 1.5 ml/min. Noradrenaline, dopamine, and adrenaline were supplied by Sigma Chemical Co. (St. Louis, MO). The stock solutions were prepared in PCA and the working solutions were made fresh every day. Concentrations of biogenic amines were expressed as pg/mg wet tissue weight. Statistical analysis. Although it is not shown in the paper, the statistical comparison between male and female brain catecholamine contents during the stages of the reproductive cycle studied did not indicate sexual dimorphism; therefore, both sexes were considered together for the purposes of statistical analysis. Data for all parameters were expressed as $he means f SEM and statistical comparisons were made using the Mann-Whitney test and Kruskal-Wallis test.
RESULTS HPLC Chromatography
Figure 1 shows a typical chromatogram of standards and tissue extracts. Linear relations for catecholamine concentration were found between 10 and 500 pg/pl and the lower limit of detection was 20 pg. The complete run took 6 min at a 1.5 ml/min flow-rate. Gonudosomutic Index (GSZ)
The GSI of female and male caribe colorado are shown in Table 1. The fish underwent gonadal maturation throughout the preparatory to prespawning periods, and spawning or spermiation occurred between July and August. The GSI for females in-
CATECHOLAMINES
A)
IN TELEOST
259
BRAIN
TABLE 1 GONOSOMATIC~NDEX (GSI) OFMALEAND FEMALE Pygocentrus notatus SAMPLED PERIODICALLY DURINGAREPRODUCTNECYCLE
B)
GM (%) Date January March April May June July
1
0.02 nA
IN* J t 0;4;,
MINUTES
MINUTES
FIG. 1. HPLC-ED traces showing the separation of adrenaline (A), noradrenaline (NA), and dopamine (DA) in (A) a mixed standard containing 200 pg of each amine, and (B) hypothalamic extracts. In both cases, 5 (~1was injected at a flow of 1 mYmin.
creased from 0.35 + 0.04% in January to 7.91 + 0.27% in April, and remained relatively high during May and June. By the end of July, all fish captured had spawned (Table 1). In males, the changes in the GSI were less marked (Table 1). Seasonal Changes in Brain Catecholamine Content Significant differences in hypothalamus and telencephalon concentrations of A, NA, and DA were seen during the reproductive cycle (Tables 2 and 3). The content of DA showed a well-defined seasonal increase in both hypothalamus and telencephalon. It can be seen that levels of DA in the hypothalamus were significantly higher than those in the telencephalon in April, June, and July, while there were no differences in March and May. In contrast, the NA content of the telencephalon was higher than the hypothalamus in May, June, and July and there was a linear in-
Males 29 17 28 27 30 29
0.076 0.131 0.476 0.586 0.548 0.106
‘* k f f f
0.01 0.01 0.03 0.03 0.03 0.01
Females (25) (18) (20) (18) (25) (10)
0.347 1.914 7.907 5.624 5.202 0.214
+ + + + + +
0.04 0.41 0.27 0.41 0.98 0.02
(32) (12) (17) (7) (7) (3)
Note. Values are expressed as means ? SEM of the number of animals in parentheses. The statistical differences were analyzed using the Kruskal-Wallis test at P < 0.05. Males: January compared with March, April, May, June, and July; March compared with April, May, June, and July; April compared with May and July; May compared with July; June compared with July. Females: January compared with March, April, May, and June; March compared with April, May, June, and July; April compared with May, June, and July; May compared with July; June compared with July.
crease of NA levels from April to June, but these declined in July. It was also observed that in the hypothalamus, the DA!NA ratio in April (7.56%) and May (12.9%) was lower than in June (27.6%) and July (64.4%), (Table 4). The telencephalon also showed similar changes; the DA/NA ratio increased from April (2.4%) to July (8.9%), (Table 4). The content of A in the telencephalon did not change significantly throughout the reproductive cycle (Table 3). In contrast, A was not detected in the hypothalamus in March, June, and July. However, there was a significant difference between A content in April compared to that in May (48.9 + 7.5 and 4.75 it 0.9 pglmg wet tissue, respectively). Concentrations of A, NA, and DA in the brain regions studied showed no significant sex differences. DISCUSSION
Previous studies have shown the presence of catecholamines in fish brain (Baum-
260
GUERRERO ET AL. TABLE CONCENTRATIONS
A
Month March April May June
2
OF ADRENALINE, NORADRENALINE, AND D~PAMINE IN THE HYPOTHALAMUS Fygocentrus nottlt~s DURING THE ANNUAL REPRODUCTIVE CYCLE
ND 48.9 f 7.5 4.7 " 0.9
ND ND
July
NA (10) (10)
DA
590 458 608 230
(12) (15)
(11)
+ f f 2
OF
(10)
72 28 25 47
(12) (15)
(11)
1.9 + 0.5 (10) (10)
42.8 + 2.4 59.5 2 10.5 200.0 + 39.3 94.2 +- 11.0
(12) (15)
(11)
Note. Adrenaline (A), noradrenaline (NA), and dopamine (DA) were simultaneously measured using HPLCED. The values are expressed as pg/mg wet tissue f SEM; number of animals is shown in parentheses. ND, not detectable; -, not assayed. The statistical significance of differences was determined using the Mann-Whitney test and Kruskal-Wallis test. Significant differences were found between groups at P < 0.005. A: April compared with May. NA: April compared with July; May compared with June and July; June compared with July. DA: March compared with April, May, June, and July; April compared with June and July; May compared with June and July; June compared with July.
and Braak, 1967; Juorio, 1973; Yoshida et al., 1983). However, the methods used for measurement of these biogenic amines were complex and the specificity was variable. HPLC associated with electrochemical detection has allowed greatly increased sensitivity for catecholamine analysis. This method has been used to measure the levels of biogenic amines in brain regions of various fish species (Sloley and Rehnberg, 1988), and in the whole brain of Anguilla anguilla (Caroff et al., 1986). In the present study we have demonstrated, using HPLC with EC detection, changes in NA, DA, and A content in hypogarten
TABLE CONCENTRATIONS
Month
May
June July
3
OF ADRENALINE,
Pygocentrus
March April
thalamus and telencephalon throughout the annual reproductive cycle of P. notatus. A dual neurosecretory mechanism for the control of adenohypophyseal cells was first suggested by Knowles and Vollrath (1966). The most important neurotransmitters in this respect are the monoamines NA, DA, and 5-hydroxytryptamine (5HT). In teleosfs, the source of most of the amine& nerves supplying the pars distalis and pars intermedia seems to be the hypothalamic nucleus recessus posterioris and the nucleus lateralis tuberis (Corio et al., 1985; Kah et al., 1987; Peute et al., 1987; Yoshida et al., 1983).
NORADRENALINE, AND DOPAMINE IN THE TELENCEPHALON notatus DURING THE ANNUAL F&PRODUCTIVE CYCLE
A 2 1.1 1.4 ? 0.5 ND ND 3.0 + 1.0
2.9
OF
NA (10) (16) (12) (12) (11)
DA
383 f 709 f
1.7 f 0.4 38 58
(16) (12)
1005 f 134 (12) 513 * 56 (11)
8.9 37.2 76.2 44.4
+ " + f
1.4 7.1 5.0 4.1
(10) (16) (12) (12)
(11)
Note, Adrenaline (A), noradrenaline (NA), and dopamine (DA) were simultaneously measured using HPLCED. The values are expressed as pg/mg wet tissue 2 SEM; number of animals is shown in parentheses. ND, not detectable; -, not assayed. The statistical significance of differences was determined using the Mann-Whitney test and Kruskal-Wallis test. Significant differences were found between groups at P < 0.05. A: No signi&ance. NA: April compared with May and June; May compared with July; June compared with July. DA: March compared with April, May, June, and July; April compared with May, June, and July; May compared with June; June compared with July.
CATECHOLAMINES TABLE
4
D~PAMINE AND NORADRENALINE RATIO (DA/NA) IN THE HYPOTHALAMUS AND THE TELENCEPHALON OF Pygocentrus notatus DURING THE ANNUAL REPRODUCTIVE CYCLE
DA/NA (%) Month April May June July
Hypothalamus 7.6 12.9 27.6 64.4
+ 1.0 (10) * 1.9 (12) ” 2.7 (15) f 16.7 (11)
Telencephalon 2.4 5.8 8.9 9.9
f 0.4 (16) f 1.1 (12) * 1.0 (12) k 1.6 (11)
Note. The values are expressed as percentage * SEM, number of samples are shown in parentheses. The statistical significance of differences was determined using the Mann-Whitney test and KruskalWallis test. Significant differences were found between groups at P < 0.05. Hypothalamus: April compared with May, June, and July; May compared with June and July. Telencephalon: April compared with May, June, and July.
Stimulation of DA receptors could inhibit the gonadotrophin-releasing ability of GnRH, as well as noradrenaline-stimulated GnRH release. In addition, the DA inhibition of gonadotrophin secretion was blocked by administration of drugs which block dopaminergic D2-like receptors (Chang ef al., 1984; Omeljaniuk et al., 1987; Peter et al., 1986). Although turnover was not measured in this study, it would be interesting to relate the present findings to functional neural activity in catecholamine neurons by making a tentative assumption that decreased amine content generally reflects an increase in neurotransmission, while increased amine levels indicate a decreased neuronal activity. Thus, the increases in DA content observed in the hypothalamus during prespawning may indicate decreased dopaminergic transmission at this stage. In contrast, NA content did not show significant changes through the preparatory to prespawning period, but declined during spawning. The content of DA in the hypothalamus was 2.5fold greater than in the telencephalon in specimens captured in June (prespawning). This observation is in
IN TELEOST
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261
agreement with that of Juorio (1973) who reported high concentrations of DA in the goldfish hypothalamus compared to other brain areas. This difference could possibly be due to seasonal fluctuations of catecholamine content. The exact timing of the gonadotrophin surge in P. notatus is unknown. However, we have recently shown that the content of immunoreactive GnRH in the hypothalamus in this species was greatest a month before the maximal GSI was attained (Gentile et al., 1986), which suggests that the gonadotrophin surge may occur just before spawning. This rise in hypothalamic GnRH content during the preparatory period, together with a decreased neural DA and increased NA activity, could contribute to the increase of the gonadotrophin-releasing pool at the pituitary gland and may be part of the mechanism that regulates the ovulatory gonadotrophin surge in some teleosts. This would be consistent with pharmacological studies that suggest an inhibitory role for DA in gonadotrophin secretion (Peter et al., 1986). The annual changes of NA and DA in the telencephalon are particulary interesting because the telencephalon has been implicated in the control of sexual behavior, nest building, spawning, and sperm release (Davis et al., 1976; Schwagmeyer et al., 1977), and recent lesion studies have shown that destruction of the medial nuclei of the ventral telencephalon results in impairment of spawning behavior in male goldfish (Kyle and Peter, 1982). Furthermore, NA and DA cells have been found in various telencephalic areas (Kotrschal and Adam, 1983; Yoshida et al., 1983). In the normal in vivo situation, removal of the DA inhibition and an increase of GnRH stimulation of gonadotrophin secretion are probably both required to allow the rapid increase in circulating gonadotrophin levels that trigger ovulation. The variations in DA/NA ratio in the hypothalamus during the reproductive cycle
262
GUERRERO ET AL.
may reflect changes in dopaminergic and REFERENCES noradrenergic activity that could be respon- Baumgarten, H. G., and Braak, H. (1967). Catecholsible for the maintenance of relatively low amine im hypothalamus vom Goldlisch (Cnrascirculating gonadotrophin levels in the sius auratus). Z. Zellforsch A80, 246-263. phase of increases in the readily releasable Callard, G. V., Petro, Z., Ryan, K. J., and Claibome, J. B. (1981). Estrogen synthesis in vitro and in gonadotrophin pool as the fish enters the vivo in the brain of a marine teleost (Myoxocereproductive season and/or gonadotrophin phalus). Gen. Comp. Endocrinol. 43, 243-255. surge before ovulation. Caroff, J., Barth&l&my, L., and Seber, Ph. (1986). One striking result from the present inBrain and plasma biogenic amines analysis by the vestigation was the high concentration of A EC-HPLC technique: Application to fish. Comp. Biochem. Physiol. 84C, 151-153. in the hypothalamus during the preparatory Chang, J. P., Cook, A. F., and Peter, R. E. (1983). period, while low levels were observed in Influence of catecholamines on gonadotropin sethe telencephalon throughout the cycle and cretion in goldfish, Carussius auratus. Gen. A was not detected in hypothalamic exComp. Endocrinol. 49, 22-31. tracts from animals captured at the pre- Chang, J. P., Peter R. E., Nahomiak, C. S., and Sokolowska, M. (1984). Effects of catecholaminspawning-spawning stage which could reergic agonists and antagonists on serum gonadoflect an increased activity of A neural pathtropin concentrations and ovulation in goldfish: ways at this time. Evidence for specificity of dopamine inhibition of Regions which show changes in catecholgonadotropin secretion. Gen. Comp. Endocrinol. amine content dependent on the GSI are 55, 35 l-366. known to be involved in teleostean repro- Corio, M., Peute, J., Steinbusch, H. W. M., and Doerr-Schott, J. (1985). Immunoreactive dopamiduction and are target tissues for steroid nergic neurons in the hypothalamus of a fish, hormones (Callard et al., 1981; Kim et al., Clarias lazera, and an amphibian, Xenopus laevis. 1978, 1979; Morrell et al., 1975). It would Neurosci. Left. (Suppl.) 22, 110. seem that in white rats, steroid hormones Davis, R. E., Kassel, J., and Schwagmeyer, P. (1976). Telencephalic lesions and behavior in the teleost, may exert their actions by altering hypothaMacropodus opercularis: Reproduction startle relamic catecholaminergic transmission. In action and operant behavior in the male. Behav. particular, estrogen appears to reduce doBiol. 18, 165-177. paminergic activity and to augment norad- De Leeuw, R., Goos, H. J. Th., and Van Oordt, renergic activity in the hypothalamus beP. G. W. J. (1986). The dopaminergic inhibition fore ovulation. of the gonadotropin-releasing hormone-induced gonadotropin release: An in vitro study with fragThe results suggest several functional anments and cell suspensions from pituitaries of the atomical correlations which are likely to be African catfish, Clarias gariepinus (Burchell). involved in reproductive physiology and Gen. Comp. Endocrinol. 63, 171-177. behavior of P. notatus. Synchronization of Dube, L., and Parent, A. (1982). The organization of environmental signals with gonadal develmonoamine-containing neurons in the brain of the salamander, Necturus maculosus. J. Comp. Neuopment and sexual behavior is probably rol. 211, 21-30. regulated by steroid modulation of cateFryer, J. N., Boudreault-Chateauvert, C., and Kirby, cholaminergic activity. R. P. (1985). Pituitary afferents originating in the ACKNOWLEDGMENTS This work was supported by grants from CONICIT Sl-1623 and C.D.C.H. M.09.19.85. We are grateful to Dr. Neil Lynch for revision of the manuscript. We also thank Msc. Miguel Fontaine, Msc. Otto Castillo, and the other members of FONAIAP for their valuable help in the fish collection.
paraventricular organ (PVO) of the goldfish hypothalamus. J. Comp. Neurol. 242, 475-484. Gentile. F. (1983). “Distribution de la hormona liberadora de las gonadotropinas (GnRH, LHRH) en el encefalo de1 caribe Colorado, Pygocentrus notarus, (Lutken, 1874), (Teleostei-Characidae).” Undergraduate Thesis, Universidad Central de Venezuela, Caracas, Venezuela. Gentile, F., Lira, O., and Marcano-de Cotte, D. (1986). Relationship between brain gonadotro-
CATECHOLAMINES
phin-releasing hormone (GnRH) and seasonal reproductive cycle of “caribe colorado,” fygocentrus
notatus.
Gen.
Comp.
Endocrinol.
64, 239-
245. Juorio, A. V. (1973). The distribution of catecholamines in the hypothalamus and other brain areas of some lower vertebrates, J. Neurochem. 20, 641-645. Kah, O., Dubourg, P., Onteniente, B., Geffard, M., and Calas, A. (1986). The dopaminergic innervation of the goldfish pituitary: An immunocytochemical study at the electron microscopy level using antibodies to dopamine. Cell Tissue Rex 244, 577-582. Kah, O., Dulka, J. G., Dubourg, P., Thibault, J., and Peter, R. E. (1987). Neuroanatomical substrate for the inhibition of gonadotrophin secretion in goldfish: Existence of a dopaminergic preoptichypophyseal pathway, Neuroendocrinology 45, 451458. Kim, Y. S., Stumpf, W. E., and Sar, M. (1978). Topography of estrogen target cells in the forebrain of goldfish Carassius aura&s. J. Comp. Neural. 182, 61 l-620. Kim, Y. S., Stumpf, W. E., and Sar, M. (1979). Topographical distribution of estrogen target cells in the forebrain of platyiish, Xiphophorus maculatus, studied by autoradiography. Brain Res. 170, 43-59. Knowles, S. F., and Vollrath, L. (1966). Changes in the pituitary of the migrating European eel during its journey from river to the sea. Z. Zellforsch. 75, 3 17-327. Kotrschal, K., and Adam, H. (1983). The amine&c system in the brain of Blennius incognitus (Bath 1%8) (Teleostei, Perciformes). Cell Tissue Res. 229,403-409. Kyle, A. L., and Peter, R. E. (1982). Effects of forebrain lesions on spawning behavior in the male goldfish. Physiol. Behav. 28, 1103-l 109. Mago-Leccia, F. (1970). Estudios preliminares sobre la ecologia de 10s peces de 10s llanos de Venezuela. Acta Biol. Venez. 7, 71-102. Morrell, J. I., Kelley D. B., and Pfaff, D. W. (1975). Sex steroid binding in the brains of vertebrates. In “Brain-Endocrine Interaction II,” (K. M. Knigge, D. E. Scott, H. Kobayashi, and S. Ishii, Eds.), pp. 230-256. Karger, Basel.
IN
TELEOST
BRAIN
263
Moyer, T. P., and Jiang, N. S. (1978). Optimized isocratic conditions for analysis of catecholamines by high-performance reversed-phase paired-ion chromatography with amperometric detection. J. Chromatogr.
153, 365-372.
Omeljaniuk, R. J., Shih, S. H., and Peter, R. E. (1987). In-vivo evaluation of dopamine receptormediated inhibition of gonadotrophin secretion from the pituitary gland of the goldfish, Curassius aura&s. J. Endocrinol. 114, 449-458. Parent, A. (1984). Functional anatomy and evolution of monoaminergic systems. Amer. Zool. 24, 7837%. Parent, A., and Northcutt, R. G. (1982). The monoamine-containing neurons in the brain of the garfish, Lepisosteus osseus. Brain Res. Bull. 9, 189-204. Peter, R. E., Chang, J. P., Nahomiak, C. S., Omeljaniuk, R. J., Sokolowska, M., Shih, S. H., and Billard, R. (1986). Interactions of catecholamines and GnRH in regulation of gonadotropin secretion in teleosts fish. In “Recent Progress in Hormone Research” (R. 0. Greep, Ed.), Vol. 42, pp. 513548. Academic Press, New York. Peute, J., Schild, R. G., Schild, V. A., Buijs, R. M., van Asselt, L. A. C., and von Oordt, P. G. W. J. (1987). Immunocytochemical evidence for peptidergic (GNRH) and dopaminergic innervation of the gonadotropic cells in the pituitary of the African catfish, Clarias gariepinus. Gen. Comp. Endocrinol. 67, 303-310. Schwagmeyer, P., Davis, R. E., and Kassel, J. (1977). Telencephalic lesions and behavior in the teleost Macropodus opercularis (L): Effects of telencephalon and olfactory bulb ablation on spawning and foamnest building. Behavioral Biol. 20, 463470. Sloley, B. D., and Rehnberg, B. G. (1988). Noradrenaline, dopamine, 5-hydroxytryptamine and tryptophan concentrations in the brains of four cohabiting species of fish. Comp. Biochem. Physiol. 89, 197-199. Yoshida, M., Nagatsu, I., Kawakami-Kondo, Y., Karasawa, M., Spatz, M., and Nagatsu, T. (1983). Monoaminergic neurons in the brain of goldfish as observed by immunohistochemical techniques. Experientia 39, 1171-l 174.
AND
COMPARATIVE
ENDOCRINOLOGY
80,
257-263 (1%)
Hypothalamic and Telencephalic Catechoiamine Content in the Brain of the Teleost Fish, Pygocentrus notatus, during the Annual Reproductive Cycle HILDA Y. GUERRERO,GISELA CACERES,CARMEN
L. PAIVA, AND DAYSSI MARCANO’
Department of Physiology, “J. M. Vargas” Medical School, and Institute for Experimental Medicine, Universidad Central de Venezuela, Apartado 47633, Caracas 1041-A, Venezuela Accepted November 1, 1989 The catecholamines noradrenaline (NA), dopamine (DA), and adrenaline (A) were measured in hypothalamic and telencephalic extracts of the Venezuelan freshwater fish “caribe colorado,” Pygocentrus notatus, at different stages of the reproductive cycle. The concentration of NA was found to be significantly higher in the telencephalon than in the hypothalamus, but that of DA was higher in the hypothalamus than in the telencephalon. Fluctuations depending upon the reproductive stage and environmental conditions occurred in both hypothalamus and telencephalon. In the hypothalamus, DA content was highest during the prespawning period (June) as compared to other periods of the cycle. Although the NA concentration was reduced during spawning there was no significant variation during any other period. DA concentrations in both telencephalon and hypothalamus showed a similar pattern of changes. In the telencephalon, NA levels increased between preparatory and prespawning periods but decreased sharply during spawning. No sex differences were observed in either area at any stage of reproduction. 8 IWOAcademic RUSS, hc.
The “caribe colorado,” Pygocentrus notutus, is a seasonal breeder, widely distributed in rivers and lagoons of the Venezuelan plains. Under natural conditions this fish shows an annual gonadal cycle, which can be divided into four periods: preparatory (February-April), prespawning (MayJune), spawning (July-August), and postspawning (September-January) (Gentile, 1983). The pattern of gonadal maturation of this species seems to be closely related to changes in the environmental conditions of the Venezuelan plains (Mago-Leccia, 1970), which are characterized by a dry season (December-April) followed by a rainy period (May-November). Catecholaminergic neuronal systems are widespread in the animal kingdom, and ’ To whom correspondence should be addressed.
and reprint requests
nerve fibers of this type have been described in the brain of different species of fish (Kotrschal and Adam, 1983; Parent, 1984). In teleosts the aminergic cells seem to be highly concentrated in the hypothalamus, particularly in the paraventricular organ from where projections extend to the midbrain, telencephalon, and pituitary gland (Dube and Parent, 1982; Fryer et al., 1985; Kah et al., 1987; Parent and Northoutt, 1982; Peute et al., 1987). Recently Corio et al. (1985) showed the presence of immunoreactive dopamine (DA) neurons in the nucleus ventromedialis, the nucleus posterioris periventricularis, and the lateral extensions of the nucleus lateralis tuberis in the African catfish. In the goldfish, DA fibers from the hypothalamus innervate the different lobes of the pituitary gland, and DA terminals are often observed in direct contact with the gonadotrophs (Kah et al., 1986). On the basis of brain-
257 0016~648OBo $1.50 Copyright 0 1990 by Academic Press, Inc. AU rights of reproduction in any form reserved.
258
GUERRERO ET AL.
lesion studies and immunohistochemistry, Kah et al. (1987) demonstrated the existence of a dopaminergic preoptic-hypophyseal pathway in the goldfish. The involvement of catecholamines in the neuroendocrine regulation of gonadotrophin release in teleosts was first demonstrated, in the goldfish, by Chang et al. (1983). There is evidence that noradrenaline (NA) stimulates gonadotrophin secretion, and it has been shown that DA acts as an inhibitory factor on gonadotrophin secretion, to directly inhibit spontaneous gonadotrophin release and modulate gonadotrophin releasing hormone (GnRH) action in the pituitary gland (De Leeuw et al., 1986; Peter et al., 1986; Peute et al., 1987). As catecholamines appear to play an important role in the regulation of gonadotrophin secretion in teleosts, and hence intluence their reproductive cycles, the purpose of this study was to evaluate the changes in NA, DA, and adrenaline (A) levels in the hypothalamus and telencephalon of P. notutus, in relation to the gonadal maturation. MATERIALS
AND METHODS
Adult male and female P. notarus (Lutken, 1874) (Teleostei-Characidae) were collected monthly during 1987 from lagoons and ponds of the Guarico River and its tributaries (Guarico and Apure states) and transported in aerated tanks to the laboratory. The animals were sacrificed by decapitation, then the brains were immediately removed and dissected, and the tissues were weighed and maintained at -40°C until amine extraction. At the same time, the gonads were extirpated and weighed, and the gonadosomatic index (GSI) was calculated. Tissue dissection and extraction procedures. Immediately following removal, the brains were dissected. Both the whole hypothalamus (H) and remaining forebrain comprising the telencephalon and olfactory bulb (TEL) were homogenized (at a final tissue concentration of 160 mg/ml) in ice-cold 0.4 M perchloric acid (PCA) containing 40 mM sodium metabisulfite and 10 r&ml of 3,4-dihydroxybenzilamine (DHBA) as an internal standard. After centrifugation at 20,OOOgthe supematants were filtered through a 0.45urn pore diameter filter (Millipore S.A.) and aliquots (S-100 ul) of this were directly analyzed by HPLC. The measurement was performed by comparing the peak heights of
the samples with those of a standard run, and correcting according to the internal standard. The HPLC system used was a Model 6000A solvent delivery pump coupled to a U6K injector (Waters Associates, Milford, MA). Separation of catecholamines was achieved on MBondapak Cl8 IO-pm Radial-Pak cartridges (10 cm x 5 mm I.D.) from Waters Associates. The detection system consisted of an electrochemical detector (Bioanalytical Systems, Model LC 4B) fitted with a glassy carbon electrode (Model TL.5) and a recorder-integrator (Data Module 730, Waters Associates). A slight modification of the working conditions reported by Moyer and Jiang (1978) was established: the operating potential was set at +0.55 V against the Ag/AgCl, reference electrode. The mobile phase consisted of 0.075 M sodium phosphatecitric acid buffer containing 0.1 m&f EDTA (free acid) and 2.5 mM sodium octyl sulfate, pH 4.0, with an aqueous and methanol ratio of 92:8 (v/v). The flow rate was set at 1.5 ml/min. Noradrenaline, dopamine, and adrenaline were supplied by Sigma Chemical Co. (St. Louis, MO). The stock solutions were prepared in PCA and the working solutions were made fresh every day. Concentrations of biogenic amines were expressed as pg/mg wet tissue weight. Statistical analysis. Although it is not shown in the paper, the statistical comparison between male and female brain catecholamine contents during the stages of the reproductive cycle studied did not indicate sexual dimorphism; therefore, both sexes were considered together for the purposes of statistical analysis. Data for all parameters were expressed as $he means f SEM and statistical comparisons were made using the Mann-Whitney test and Kruskal-Wallis test.
RESULTS HPLC Chromatography
Figure 1 shows a typical chromatogram of standards and tissue extracts. Linear relations for catecholamine concentration were found between 10 and 500 pg/pl and the lower limit of detection was 20 pg. The complete run took 6 min at a 1.5 ml/min flow-rate. Gonudosomutic Index (GSZ)
The GSI of female and male caribe colorado are shown in Table 1. The fish underwent gonadal maturation throughout the preparatory to prespawning periods, and spawning or spermiation occurred between July and August. The GSI for females in-
CATECHOLAMINES
A)
IN TELEOST
259
BRAIN
TABLE 1 GONOSOMATIC~NDEX (GSI) OFMALEAND FEMALE Pygocentrus notatus SAMPLED PERIODICALLY DURINGAREPRODUCTNECYCLE
B)
GM (%) Date January March April May June July
1
0.02 nA
IN* J t 0;4;,
MINUTES
MINUTES
FIG. 1. HPLC-ED traces showing the separation of adrenaline (A), noradrenaline (NA), and dopamine (DA) in (A) a mixed standard containing 200 pg of each amine, and (B) hypothalamic extracts. In both cases, 5 (~1was injected at a flow of 1 mYmin.
creased from 0.35 + 0.04% in January to 7.91 + 0.27% in April, and remained relatively high during May and June. By the end of July, all fish captured had spawned (Table 1). In males, the changes in the GSI were less marked (Table 1). Seasonal Changes in Brain Catecholamine Content Significant differences in hypothalamus and telencephalon concentrations of A, NA, and DA were seen during the reproductive cycle (Tables 2 and 3). The content of DA showed a well-defined seasonal increase in both hypothalamus and telencephalon. It can be seen that levels of DA in the hypothalamus were significantly higher than those in the telencephalon in April, June, and July, while there were no differences in March and May. In contrast, the NA content of the telencephalon was higher than the hypothalamus in May, June, and July and there was a linear in-
Males 29 17 28 27 30 29
0.076 0.131 0.476 0.586 0.548 0.106
‘* k f f f
0.01 0.01 0.03 0.03 0.03 0.01
Females (25) (18) (20) (18) (25) (10)
0.347 1.914 7.907 5.624 5.202 0.214
+ + + + + +
0.04 0.41 0.27 0.41 0.98 0.02
(32) (12) (17) (7) (7) (3)
Note. Values are expressed as means ? SEM of the number of animals in parentheses. The statistical differences were analyzed using the Kruskal-Wallis test at P < 0.05. Males: January compared with March, April, May, June, and July; March compared with April, May, June, and July; April compared with May and July; May compared with July; June compared with July. Females: January compared with March, April, May, and June; March compared with April, May, June, and July; April compared with May, June, and July; May compared with July; June compared with July.
crease of NA levels from April to June, but these declined in July. It was also observed that in the hypothalamus, the DA!NA ratio in April (7.56%) and May (12.9%) was lower than in June (27.6%) and July (64.4%), (Table 4). The telencephalon also showed similar changes; the DA/NA ratio increased from April (2.4%) to July (8.9%), (Table 4). The content of A in the telencephalon did not change significantly throughout the reproductive cycle (Table 3). In contrast, A was not detected in the hypothalamus in March, June, and July. However, there was a significant difference between A content in April compared to that in May (48.9 + 7.5 and 4.75 it 0.9 pglmg wet tissue, respectively). Concentrations of A, NA, and DA in the brain regions studied showed no significant sex differences. DISCUSSION
Previous studies have shown the presence of catecholamines in fish brain (Baum-
260
GUERRERO ET AL. TABLE CONCENTRATIONS
A
Month March April May June
2
OF ADRENALINE, NORADRENALINE, AND D~PAMINE IN THE HYPOTHALAMUS Fygocentrus nottlt~s DURING THE ANNUAL REPRODUCTIVE CYCLE
ND 48.9 f 7.5 4.7 " 0.9
ND ND
July
NA (10) (10)
DA
590 458 608 230
(12) (15)
(11)
+ f f 2
OF
(10)
72 28 25 47
(12) (15)
(11)
1.9 + 0.5 (10) (10)
42.8 + 2.4 59.5 2 10.5 200.0 + 39.3 94.2 +- 11.0
(12) (15)
(11)
Note. Adrenaline (A), noradrenaline (NA), and dopamine (DA) were simultaneously measured using HPLCED. The values are expressed as pg/mg wet tissue f SEM; number of animals is shown in parentheses. ND, not detectable; -, not assayed. The statistical significance of differences was determined using the Mann-Whitney test and Kruskal-Wallis test. Significant differences were found between groups at P < 0.005. A: April compared with May. NA: April compared with July; May compared with June and July; June compared with July. DA: March compared with April, May, June, and July; April compared with June and July; May compared with June and July; June compared with July.
and Braak, 1967; Juorio, 1973; Yoshida et al., 1983). However, the methods used for measurement of these biogenic amines were complex and the specificity was variable. HPLC associated with electrochemical detection has allowed greatly increased sensitivity for catecholamine analysis. This method has been used to measure the levels of biogenic amines in brain regions of various fish species (Sloley and Rehnberg, 1988), and in the whole brain of Anguilla anguilla (Caroff et al., 1986). In the present study we have demonstrated, using HPLC with EC detection, changes in NA, DA, and A content in hypogarten
TABLE CONCENTRATIONS
Month
May
June July
3
OF ADRENALINE,
Pygocentrus
March April
thalamus and telencephalon throughout the annual reproductive cycle of P. notatus. A dual neurosecretory mechanism for the control of adenohypophyseal cells was first suggested by Knowles and Vollrath (1966). The most important neurotransmitters in this respect are the monoamines NA, DA, and 5-hydroxytryptamine (5HT). In teleosfs, the source of most of the amine& nerves supplying the pars distalis and pars intermedia seems to be the hypothalamic nucleus recessus posterioris and the nucleus lateralis tuberis (Corio et al., 1985; Kah et al., 1987; Peute et al., 1987; Yoshida et al., 1983).
NORADRENALINE, AND DOPAMINE IN THE TELENCEPHALON notatus DURING THE ANNUAL F&PRODUCTIVE CYCLE
A 2 1.1 1.4 ? 0.5 ND ND 3.0 + 1.0
2.9
OF
NA (10) (16) (12) (12) (11)
DA
383 f 709 f
1.7 f 0.4 38 58
(16) (12)
1005 f 134 (12) 513 * 56 (11)
8.9 37.2 76.2 44.4
+ " + f
1.4 7.1 5.0 4.1
(10) (16) (12) (12)
(11)
Note, Adrenaline (A), noradrenaline (NA), and dopamine (DA) were simultaneously measured using HPLCED. The values are expressed as pg/mg wet tissue 2 SEM; number of animals is shown in parentheses. ND, not detectable; -, not assayed. The statistical significance of differences was determined using the Mann-Whitney test and Kruskal-Wallis test. Significant differences were found between groups at P < 0.05. A: No signi&ance. NA: April compared with May and June; May compared with July; June compared with July. DA: March compared with April, May, June, and July; April compared with May, June, and July; May compared with June; June compared with July.
CATECHOLAMINES TABLE
4
D~PAMINE AND NORADRENALINE RATIO (DA/NA) IN THE HYPOTHALAMUS AND THE TELENCEPHALON OF Pygocentrus notatus DURING THE ANNUAL REPRODUCTIVE CYCLE
DA/NA (%) Month April May June July
Hypothalamus 7.6 12.9 27.6 64.4
+ 1.0 (10) * 1.9 (12) ” 2.7 (15) f 16.7 (11)
Telencephalon 2.4 5.8 8.9 9.9
f 0.4 (16) f 1.1 (12) * 1.0 (12) k 1.6 (11)
Note. The values are expressed as percentage * SEM, number of samples are shown in parentheses. The statistical significance of differences was determined using the Mann-Whitney test and KruskalWallis test. Significant differences were found between groups at P < 0.05. Hypothalamus: April compared with May, June, and July; May compared with June and July. Telencephalon: April compared with May, June, and July.
Stimulation of DA receptors could inhibit the gonadotrophin-releasing ability of GnRH, as well as noradrenaline-stimulated GnRH release. In addition, the DA inhibition of gonadotrophin secretion was blocked by administration of drugs which block dopaminergic D2-like receptors (Chang ef al., 1984; Omeljaniuk et al., 1987; Peter et al., 1986). Although turnover was not measured in this study, it would be interesting to relate the present findings to functional neural activity in catecholamine neurons by making a tentative assumption that decreased amine content generally reflects an increase in neurotransmission, while increased amine levels indicate a decreased neuronal activity. Thus, the increases in DA content observed in the hypothalamus during prespawning may indicate decreased dopaminergic transmission at this stage. In contrast, NA content did not show significant changes through the preparatory to prespawning period, but declined during spawning. The content of DA in the hypothalamus was 2.5fold greater than in the telencephalon in specimens captured in June (prespawning). This observation is in
IN TELEOST
BRAIN
261
agreement with that of Juorio (1973) who reported high concentrations of DA in the goldfish hypothalamus compared to other brain areas. This difference could possibly be due to seasonal fluctuations of catecholamine content. The exact timing of the gonadotrophin surge in P. notatus is unknown. However, we have recently shown that the content of immunoreactive GnRH in the hypothalamus in this species was greatest a month before the maximal GSI was attained (Gentile et al., 1986), which suggests that the gonadotrophin surge may occur just before spawning. This rise in hypothalamic GnRH content during the preparatory period, together with a decreased neural DA and increased NA activity, could contribute to the increase of the gonadotrophin-releasing pool at the pituitary gland and may be part of the mechanism that regulates the ovulatory gonadotrophin surge in some teleosts. This would be consistent with pharmacological studies that suggest an inhibitory role for DA in gonadotrophin secretion (Peter et al., 1986). The annual changes of NA and DA in the telencephalon are particulary interesting because the telencephalon has been implicated in the control of sexual behavior, nest building, spawning, and sperm release (Davis et al., 1976; Schwagmeyer et al., 1977), and recent lesion studies have shown that destruction of the medial nuclei of the ventral telencephalon results in impairment of spawning behavior in male goldfish (Kyle and Peter, 1982). Furthermore, NA and DA cells have been found in various telencephalic areas (Kotrschal and Adam, 1983; Yoshida et al., 1983). In the normal in vivo situation, removal of the DA inhibition and an increase of GnRH stimulation of gonadotrophin secretion are probably both required to allow the rapid increase in circulating gonadotrophin levels that trigger ovulation. The variations in DA/NA ratio in the hypothalamus during the reproductive cycle
262
GUERRERO ET AL.
may reflect changes in dopaminergic and REFERENCES noradrenergic activity that could be respon- Baumgarten, H. G., and Braak, H. (1967). Catecholsible for the maintenance of relatively low amine im hypothalamus vom Goldlisch (Cnrascirculating gonadotrophin levels in the sius auratus). Z. Zellforsch A80, 246-263. phase of increases in the readily releasable Callard, G. V., Petro, Z., Ryan, K. J., and Claibome, J. B. (1981). Estrogen synthesis in vitro and in gonadotrophin pool as the fish enters the vivo in the brain of a marine teleost (Myoxocereproductive season and/or gonadotrophin phalus). Gen. Comp. Endocrinol. 43, 243-255. surge before ovulation. Caroff, J., Barth&l&my, L., and Seber, Ph. (1986). One striking result from the present inBrain and plasma biogenic amines analysis by the vestigation was the high concentration of A EC-HPLC technique: Application to fish. Comp. Biochem. Physiol. 84C, 151-153. in the hypothalamus during the preparatory Chang, J. P., Cook, A. F., and Peter, R. E. (1983). period, while low levels were observed in Influence of catecholamines on gonadotropin sethe telencephalon throughout the cycle and cretion in goldfish, Carussius auratus. Gen. A was not detected in hypothalamic exComp. Endocrinol. 49, 22-31. tracts from animals captured at the pre- Chang, J. P., Peter R. E., Nahomiak, C. S., and Sokolowska, M. (1984). Effects of catecholaminspawning-spawning stage which could reergic agonists and antagonists on serum gonadoflect an increased activity of A neural pathtropin concentrations and ovulation in goldfish: ways at this time. Evidence for specificity of dopamine inhibition of Regions which show changes in catecholgonadotropin secretion. Gen. Comp. Endocrinol. amine content dependent on the GSI are 55, 35 l-366. known to be involved in teleostean repro- Corio, M., Peute, J., Steinbusch, H. W. M., and Doerr-Schott, J. (1985). Immunoreactive dopamiduction and are target tissues for steroid nergic neurons in the hypothalamus of a fish, hormones (Callard et al., 1981; Kim et al., Clarias lazera, and an amphibian, Xenopus laevis. 1978, 1979; Morrell et al., 1975). It would Neurosci. Left. (Suppl.) 22, 110. seem that in white rats, steroid hormones Davis, R. E., Kassel, J., and Schwagmeyer, P. (1976). Telencephalic lesions and behavior in the teleost, may exert their actions by altering hypothaMacropodus opercularis: Reproduction startle relamic catecholaminergic transmission. In action and operant behavior in the male. Behav. particular, estrogen appears to reduce doBiol. 18, 165-177. paminergic activity and to augment norad- De Leeuw, R., Goos, H. J. Th., and Van Oordt, renergic activity in the hypothalamus beP. G. W. J. (1986). The dopaminergic inhibition fore ovulation. of the gonadotropin-releasing hormone-induced gonadotropin release: An in vitro study with fragThe results suggest several functional anments and cell suspensions from pituitaries of the atomical correlations which are likely to be African catfish, Clarias gariepinus (Burchell). involved in reproductive physiology and Gen. Comp. Endocrinol. 63, 171-177. behavior of P. notatus. Synchronization of Dube, L., and Parent, A. (1982). The organization of environmental signals with gonadal develmonoamine-containing neurons in the brain of the salamander, Necturus maculosus. J. Comp. Neuopment and sexual behavior is probably rol. 211, 21-30. regulated by steroid modulation of cateFryer, J. N., Boudreault-Chateauvert, C., and Kirby, cholaminergic activity. R. P. (1985). Pituitary afferents originating in the ACKNOWLEDGMENTS This work was supported by grants from CONICIT Sl-1623 and C.D.C.H. M.09.19.85. We are grateful to Dr. Neil Lynch for revision of the manuscript. We also thank Msc. Miguel Fontaine, Msc. Otto Castillo, and the other members of FONAIAP for their valuable help in the fish collection.
paraventricular organ (PVO) of the goldfish hypothalamus. J. Comp. Neurol. 242, 475-484. Gentile. F. (1983). “Distribution de la hormona liberadora de las gonadotropinas (GnRH, LHRH) en el encefalo de1 caribe Colorado, Pygocentrus notarus, (Lutken, 1874), (Teleostei-Characidae).” Undergraduate Thesis, Universidad Central de Venezuela, Caracas, Venezuela. Gentile, F., Lira, O., and Marcano-de Cotte, D. (1986). Relationship between brain gonadotro-
CATECHOLAMINES
phin-releasing hormone (GnRH) and seasonal reproductive cycle of “caribe colorado,” fygocentrus
notatus.
Gen.
Comp.
Endocrinol.
64, 239-
245. Juorio, A. V. (1973). The distribution of catecholamines in the hypothalamus and other brain areas of some lower vertebrates, J. Neurochem. 20, 641-645. Kah, O., Dubourg, P., Onteniente, B., Geffard, M., and Calas, A. (1986). The dopaminergic innervation of the goldfish pituitary: An immunocytochemical study at the electron microscopy level using antibodies to dopamine. Cell Tissue Rex 244, 577-582. Kah, O., Dulka, J. G., Dubourg, P., Thibault, J., and Peter, R. E. (1987). Neuroanatomical substrate for the inhibition of gonadotrophin secretion in goldfish: Existence of a dopaminergic preoptichypophyseal pathway, Neuroendocrinology 45, 451458. Kim, Y. S., Stumpf, W. E., and Sar, M. (1978). Topography of estrogen target cells in the forebrain of goldfish Carassius aura&s. J. Comp. Neural. 182, 61 l-620. Kim, Y. S., Stumpf, W. E., and Sar, M. (1979). Topographical distribution of estrogen target cells in the forebrain of platyiish, Xiphophorus maculatus, studied by autoradiography. Brain Res. 170, 43-59. Knowles, S. F., and Vollrath, L. (1966). Changes in the pituitary of the migrating European eel during its journey from river to the sea. Z. Zellforsch. 75, 3 17-327. Kotrschal, K., and Adam, H. (1983). The amine&c system in the brain of Blennius incognitus (Bath 1%8) (Teleostei, Perciformes). Cell Tissue Res. 229,403-409. Kyle, A. L., and Peter, R. E. (1982). Effects of forebrain lesions on spawning behavior in the male goldfish. Physiol. Behav. 28, 1103-l 109. Mago-Leccia, F. (1970). Estudios preliminares sobre la ecologia de 10s peces de 10s llanos de Venezuela. Acta Biol. Venez. 7, 71-102. Morrell, J. I., Kelley D. B., and Pfaff, D. W. (1975). Sex steroid binding in the brains of vertebrates. In “Brain-Endocrine Interaction II,” (K. M. Knigge, D. E. Scott, H. Kobayashi, and S. Ishii, Eds.), pp. 230-256. Karger, Basel.
IN
TELEOST
BRAIN
263
Moyer, T. P., and Jiang, N. S. (1978). Optimized isocratic conditions for analysis of catecholamines by high-performance reversed-phase paired-ion chromatography with amperometric detection. J. Chromatogr.
153, 365-372.
Omeljaniuk, R. J., Shih, S. H., and Peter, R. E. (1987). In-vivo evaluation of dopamine receptormediated inhibition of gonadotrophin secretion from the pituitary gland of the goldfish, Curassius aura&s. J. Endocrinol. 114, 449-458. Parent, A. (1984). Functional anatomy and evolution of monoaminergic systems. Amer. Zool. 24, 7837%. Parent, A., and Northcutt, R. G. (1982). The monoamine-containing neurons in the brain of the garfish, Lepisosteus osseus. Brain Res. Bull. 9, 189-204. Peter, R. E., Chang, J. P., Nahomiak, C. S., Omeljaniuk, R. J., Sokolowska, M., Shih, S. H., and Billard, R. (1986). Interactions of catecholamines and GnRH in regulation of gonadotropin secretion in teleosts fish. In “Recent Progress in Hormone Research” (R. 0. Greep, Ed.), Vol. 42, pp. 513548. Academic Press, New York. Peute, J., Schild, R. G., Schild, V. A., Buijs, R. M., van Asselt, L. A. C., and von Oordt, P. G. W. J. (1987). Immunocytochemical evidence for peptidergic (GNRH) and dopaminergic innervation of the gonadotropic cells in the pituitary of the African catfish, Clarias gariepinus. Gen. Comp. Endocrinol. 67, 303-310. Schwagmeyer, P., Davis, R. E., and Kassel, J. (1977). Telencephalic lesions and behavior in the teleost Macropodus opercularis (L): Effects of telencephalon and olfactory bulb ablation on spawning and foamnest building. Behavioral Biol. 20, 463470. Sloley, B. D., and Rehnberg, B. G. (1988). Noradrenaline, dopamine, 5-hydroxytryptamine and tryptophan concentrations in the brains of four cohabiting species of fish. Comp. Biochem. Physiol. 89, 197-199. Yoshida, M., Nagatsu, I., Kawakami-Kondo, Y., Karasawa, M., Spatz, M., and Nagatsu, T. (1983). Monoaminergic neurons in the brain of goldfish as observed by immunohistochemical techniques. Experientia 39, 1171-l 174.