4 pp 291-297 (1990) ... When the fish were exposed to long photoperiod, with or without altering ... during the preparatory (February-March) phase of the annual ...
Fish Physiology and Biochemistry vol. 8 no. 4 pp 291-297 (1990) Kugler Publications, Amsterdam/Berkeley
Differential effects of photoperiod and temperature on hypothalamic monoaminergic activity in the teleost Channa punctatus (Bloch) I.A. Khan and K.P. Joy Centre of Advanced Study in Zoology, BanarasHindu University, Varanasi-221 005, India Keywords: photoperiod, temperature, hypothalamus, norepinephrine, dopamine, 5-hydroxytryptamine, MAO
Abstract In Channapunctatus maintained under ambient photothermal conditions (10.5L:13.5D; 13 + 2C) in the postspawning season, hypothalamic dopamine (DA) and norepinephrine (NE) content varied diurnally, but serotonin (5-HT) and monoamine oxidase (MAO) did not. Exposure of the fish to high temperature (25 + 2°C), with or without altering the photopheriod, increased the NE and DA content, and NE turnover but decreased DA turnover. Increasing the photoperiod (16L:8D) alone had no significant effect on catecholaminergic (CA) content or activity. When the fish were exposed to long photoperiod, with or without altering the temperature, a day-night variation in 5-HT content and activity was induced in the postspawning season. An increase in temperature alone had no significant effect on 5-HT content or activity. Hypothalamic MAO activity was elevated in fish exposed to high temperature alone, or in combination with long photoperiod, but was not affected significantly by long photoperiod treatment. These results indicate that CA activity is influenced largely by ambient high temperature, whereas serotonergic activity is controlled primarily by photoperiod, with high temperature having an additive effect. MAO activity seems to be influenced by both temperature and photoperiod in this species.
Introduction Although the role of environmental factors, such as photoperiod and temperature on different aspects of teleost physiology, including annual reproductive cycle, is well known (Bromage et al. 1982; Duston and Bromage 1986), the mechanisms by which they synchronize and modulate various functions are not clearly understood. The hypothalamohypophyseal system is regarded as a possible mediator of the environmental information received via the pineal organ or the lateral eyes, transducing it into appropriate neuroendocrine signals to produce
metabolic, physiological and behavioral alterations. In higher vertebrates, hypothalamic monoamines are implicated in the control of endocrine rhythms (Kordon et al. 1978; Scapagnini et al. 1978). Circadian activity of hypothalamic serotonin (5-HT) is related to transmission of day length information in ferrets (Yates and Herbert 1976) and a serotonergic mechanism is suggested to be involved in photosexual responses in mammals (Meyer and Quay 1976) and birds (Elhalawani et al. 1978). In teleosts, the hypothalamic monoaminergic system is known to play a central role in the reg-
Address for correspondence: K.P. Joy, Centre of Advanced Study in Zoology, Banaras Hindu University, Varanasi-221 005, India.
292 ulation of the secretion of pituitary hormones by acting directly on the adenohypophyseal cells, or by modulating the secretions of releasing and inhibiting hormones in the hypothalamus (Ball 1981; Peter and Fryer 1983; Peter et al. 1986; DeLeeuw et al. 1987). Hypothalamic monoaminergic activity is reported to be influenced by environmental photothermal conditions in goldfish (Olcese and DeVlaming 1979, 1980; Olcese et al. 1979, 1980) and in C. punctatus (Khan and Joy 1987, 1988a) suggesting the possibility that environmental factors modulate monoaminergic activity to influence various physiological activities of teleosts, including endocrine functions. Furthermore, in C. punctatus we have reported diurnal and seasonal variations in hypothalamic monoamines and monoamine oxidase (MAO) (Khan and Joy 1988b), and that the diurnal variation in 5-HT and MAO levels occur exclusively during the preparatory (February-March) phase of the annual reproductive cycle. The present study was undertaken to determine the effects of photoperiod and temperature on hypothalamic monoamines and MAO activity during the postspawning season (December) when the hypothalamo-hypophyseal-gonadal system is normally photorefractory as reported in Heteropneustes fossilis (Sundararaj and Vasal 1976) which coexists with C. punctatus.
Materials and methods Freshly caught C. punctatus were purchased from local fish market at Varanasi in late November (postspawning phase of the reproductive cycle) and were acclimated to laboratory conditions for 10 days prior to initiation of experiments. Female adult fish weighing 65-70 g were divided into four groups of 40 fish each and were maintained under the following photothermal conditions: Group 1. Ambient photoperiod and temperature (10.5L:13.5D; 13 + 2C); Group 2. Long photoperiod and ambient temperature (16L:8D; 13 + 2°C); Group 3. Ambient photoperiod and elevated temperature (10.5L:13.5D; 25 2C); Group 4. Long photoperiod and elevated temperature (16L:8D; 25 2C).
After one month, five fish were sacrificed by decapitation at mid-light and mid-dark phase from each of the 4 groups. The brain of each fish was removed quickly and frozen at -20°C. Hypothalami were dissected, and processed for the estimation of MAO activity by the radioisotopic method of Parvez and Parvez (1973), as described previously (Khan and Joy 1987). The remaining fish were further divided into three batches of ten each. In the first batch, the fish were injected with pargyline (75 mg/kg body weight), an irreversible MAO inhibitor, 6 h prior to sacrificing as described by Olcese et al. (1979). The second batch of the fish was given ae-methyl ptyrosine (a-MPT; 250 mg/kg body weight), a known tyrosine hydroxylase inhibitor, 1 h before sacrificing, as described by Advis et al. (1978), while the third batch served as saline (0.6% NaCI)injected control. Five fish each were sacrificed at mid-light and mid-dark phase for the estimation of 5-HT from batch 1 and 3, and of norepinephrine (NE) and dopamine (DA) contents from batch 2 and 3 by the spectrophotofluorometric method of Cox and Perhach (1973). The details of the technique have been described earlier (Khan and Joy 1988a). The percentage recovery was found to be 77%o for 5-HT, 64% for NA and 61.5%o for DA and final values were adjusted accordingly. The turnover index of NE and DA was calculated as the percentage decrease of NE and DA content, respectively due to a-MPT as compared to saline-injected controls. Similarly, the turnover index of 5-HT was calculated as the percentage increase in 5-HT content due to pargyline injection. Statistical analysis involved two-way analysis of variance for daily variations and photothermal conditions followed by Newman Keuls' multiple range test for comparison of group means. Significance was tested at the p < 0.05 level.
Results NE content and turnover index There was a significant day-night difference in hypothalamic NE content, but not in turnover in-
293 dex (group 1), in the fish sampled during the postspawning phase of the reproductive cycle and held under the ambient photothermal conditions (Fig. 1, Table 1). Long photoperiod (group 2) alone failed to alter the NE content or turnover index. Elevated temperature (group 3), however, significantly increased the NE content and turnover index during both the photophase and the scotophase without altering the daily pattern. Similar changes were observed in fish exposed to both long photoperiod and elevated temperature (group 4). DA content and turnover index Hypothalamic DA content showed a significant day-night difference under the ambient photothermal conditions. Increasing the photoperiod alone failed to cause any significant change in DA content or turnover index. Exposure of the fish to elevated temperature increased the DA content and decreased the turnover index significantly. 5-HT content and turnover index Hypothalamic 5-HT content and turnover index showed no day-night difference under the ambient photothermal conditions (group 1). When the fish were exposed to long photoperiod alone (group 2), the midphotophase value of 5-HT content decreased resulting in a significant day-night difference (Fig. 1). In the same group, serotonergic activity (turnover index) also exhibited a significant daynight difference with a rise in mid-photophase value (Table 1). Increasing the temperature alone failed to bring about any significant change in 5-HT content or activity. A combination of long photoperiod and elevated temperature (group 4) induced a further increase in 5-HT turnover index over group 2 values and a significant decrease in its midphotophase content as seen in group 2, resulting in significant diurnal variation. MAO activity There was no significant day-night variation in hypothalamic MAO activity in fish held under the
ambient photothermal conditions (Fig. 1). Long photoperiod alone could not influence hypothalamic MAO activity significantly. However, elevated temperature alone, as well as in combination with long photoperiod induced a significant elevation of MAO activity without changing the daily pattern.
Discussion The present study shows that hypothalamic catecholamines (CA), 5-HT and MAO of C. punctatus responded differentially to long photoperiod and elevated temperature, alone or in combination. Although circadian and seasonal variations in the brain/hypothalamic NE and DA have been reported in goldfish (Sauerbier and Meyer 1977), eel (Le Bras, 1984) and C. punctatus (Khan and Joy 1988b), investigations on the effects of altered environmental photothermal conditions on hypothalamic CA activity in teleosts are meagre. Previously, we have demonstrated that exposure of C. punctatus to long photoperiod and high temperature (16L:8D; 22 ± I°C) in the preparatory phase increased both NE and DA content of the hypothalamus without altering the diurnal variations (Khan and Joy 1988a). However, the differential effects of photoperiod and temperature on the CA systems was not clear from that study. In the present investigation, increasing the temperature, regardless of photoperiod increased noradrenergic, and concomitantly decreased dopaminergic activities. Increasing the photoperiod alone had no significant effect on the CA systems. Our results show that the hypothalamic CA system is responsive primarily to changes in environmental temperature. In the postspawning season, when ambient photoperiod and temperature are low, hypothalamic DA activity predominates over NE activity as shown previously (Khan and Joy 1988b) and in the present (group 1) study. In the breeding phase, when the ambient photoperiod and temperature are high, hypothalamic NE level is increased over DA level (Khan and Joy 1988b), lending support to the view that an increase in environmental photoperiod and temperature favours NE activity. It has been reported that
id-light phase
L D c y c l e 10-5:13'5
W a t e r t e m p . 1322'C
-
id dark phase @pargyline ord-MPT
16:8 10-5713.5 1 3 2 2 'C
25+2'C
Fig. 1. Hypothalamic monoamine levels and M A 0 activity in C. punctatus maintained under different photothermal conditions. *Significant day-night variation, a-significantly different from 10.5L:13.5D; 13 k 2°C (G,) regime, b-Significantly different from 16L:8D; 13 k 2'C (G2) regime, c-significantly different from 10.5L:13.5D; 25 k 2°C (G3) regime.
Table 1. Effects of photoperiod and temperature on turnover index of NE, DA and 5-HT in C.punctatus. ~~~~~
Groups
Treatments
G1 G2 G3 G4
10.5L:13.5D;13+2°C 16L:8D;13k2OC 10.5L:13.5D;25 f2OC 16L:8D;25 k2"C
Turnover Index (To)*
20.5k4.3 21.6k5.0 38.1 k4.4a.b 43.2*4.5a*b
28.6k4.9 27.2k5.1 46.4k4.8a.b 49.8k4.7a.b
39.8k6.2 42.3k4.4 20.1 f4.7a.b 18.9f 4.4a*b
52.6+6.8 50.7f 4.8 29.2k 5.3a,b 29.5f5.3aJJ
67.3 t 4.5 129.6f 6.2a 78.2t 5.6 159.4+_8.7a.b+
* Mean * SEM of 5 samples each; ** Significant day-night variation; a Significantly different from G1 values; from G2 values; Significantly different from G3 values.
75.8k5.1 71.9+5.7** 70.3 _+ 4.5 84.2+3.8**
Significantly different
295 NE acts at the pituitary or the hypothalamic level to stimulate gonadotropin release in goldfish with regressed ovaries or ovaries in early stages of recrudescence (Chang and Peter 1984). On the other hand, DA is inhibitory to gonadotropin secretion in goldfish (Peter et al. 1986) and the African catfish (DeLeeuw et al. 1987). In subtropical species, e.g. C. punctatus, the gonads grow and mature under the influence of gradually increasing photoperiod and temperature. In this species, the 16L:8D; 22 + 1 C regime accelerated ovarian growth in the preparatory phase (unpublished data), indicating that long photoperiod and high temperature are stimulatory to gonadal recrudescence, as has been demonstrated in H. fossilis (Sundararaj and Vasal 1976). It is, therefore, suggested that the effect of long photoperiod and high temperature on the gonads may be indirectly modulated through the hypothalamic monoaminergic mechanisms. However, further studies are required to relate the stimulation of noradrenergic system to gonadotropin secretion in this species. The demonstration that hypothalamic 5-HT exhibited a significant diurnal variation, both in its content and activity when the fish were exposed to either a 16L:8D; 13 + 2C or 16L:8D; 25 + 2°C regime is significant in that such a trend is normally absent in the postspawning phase (group 1). In a previous study (Khan and Joy 1988b), we have reported that the diurnal variation in hypothalamic 5-HT and MAO is restricted to the preparatory phase (February and March) only, and is not observed in other seasons (prespawning, spawning and postspawning; April-January). An increase in temperature from 13 2 0C to 25 + 2C alone was ineffective in generating day-night differences in 5-HT content or turnover. However, the combination of altered day length and temperature had a cumulative effect on 5-HT content and activity. Thus, it is primarily the increase in photoperiod which is responsible for the generation of diurnal variation in 5-HT; temperature may be having an additive effect. In goldfish, the only other teleost investigated, Olcese et al. (1980) reported that day-night difference in hypothalamic serotonergic activity was photoperiod-dependent; transfer of the fish from a short photoperiod (8L: 16D; 20°C) to a long photo-
period (16L:8D; 20°C) regime produced day-night difference in 5-HT content within 72 h and in its activity after 2 weeks. Goldfish maintained in short photoperiod did not exhibit a diurnal variation in 5-HT content or activity. However, this study does not refer to season or the seasonal effects of photoperiod. In C. punctatus, in the preparatory phase (March), long photoperiod and high temperature caused a 180 ° shift in the diurnal pattern of hypothalamic 5-HT content with the mid-photophase value remaining unchanged (Khan and Joy 1988a). In the postspawning season also, a similar 180 ° shifted day-night pattern was observed in the 16L:8D groups, but was achieved differently. In both the groups (2 and 4), the mid-photophase content of 5-HT decreased significantly compared to the control values, resulting in the appearance of a diurnal variation. Likewise, the day-night difference in 5-HT activity was produced by elevation of the mid-photophase values over the control values. Apparently, the mechanism by which long photoperiod influences 5-HT diurnal variation seems to be season-dependent. Hypothalamic 5-HT activity has been related to photosexual responses in mammals (Meyer and Quay 1976) and birds (Elhalawani et al. 1978), the control of endocrine rhythms (Kordon et al. 1978; Scapagnini et al. 1978) and to the transmittance of day-night information in ferrets (Yates and Herbert 1976). A hypothalamic serotonergic system is implicated in the regulation of the secretion of prolactin (Olivereau 1978a; Olcese et al. 1979; James and Wigham 1984), MSH (Olivereau 1978b) and gonadotropin (Groves 1984) from the pituitary of teleosts; all of which are influenced by environmental light regimes. In the reproductive cycle of C. punctatus, the occurrence of 5-HT diurnal variation is significant in that it coincides with the beginning of the breeding phase (preparatory phase; February-March) when the hypothalamo-hypophyseal-gonadal system becomes responsive to the gradually increasing photoperiod and temperature (Khan and Joy 1988b). It may be hypothesized that the appearance of 5-HT day-night variation may signal the initiation of the photosensitivity of the hypothalamo-hypophyseal system. Furthermore, the elevation of temperature alone or in combina-
296 tion with long photoperiod had significantly enhanced hypothalamic MAO activity in this species. Unlike 5-HT, neither treatments nor photoperiod alone could generate a significant day-night difference in hypothalamic MAO activity in the postspawning season. It is not clear whether the influence of altered photothermal conditions is obscured by seasonal factors or by relatively large individual variations. Most of the studies on hypothalamic monoaminergic system of teleosts are concerned with histochemical/immunocytochemical localization of monoamines in the hypothalamo-hypophyseal system (Terlou et al. 1978; Batten 1979; Kah and Chambolle 1983; Ekstr6m and van Veen 1984; Peute et al. 1987). In teleosts, the nucleus recessus lateralis and the nucleus recessus posterioris, homologues of paraventricular organ of higher vertebrates contain monoaminergic neurons innervating the nucleus preopticus, the nucleus lateralis tuberis and the hypophysis (Terlou et al. 1978). Although such studies have not yet been carried out in C. punctatus, the hypothalamus used in this experiment includes all these nuclei. However, functional studies correlating changes in content and turnover of monoamines in individual aminergic nuclei with specific functions of the hypothalamo-hypophyseal system in teleosts will be fruitful. Acknowledgements The first author is grateful to the University Grants Commission of India for a Senior Research Fellowship. References cited Advis, J.P., Simpkins, J.W., Chen, H.T. and Meites, J. 1978. Relation of biogenic amines to onset of puberty in the female rat. Endocrinology 103: 11-16. Ball, J.N. 1981. Hypothalamic control of the pars distalis in fishes, amphibians and reptiles. Gen. Comp. Endocrinol. 44: 135-170. Batten, T.F.C., Ingleton, P.M. and Ball, J.N. 1979. Ultrastructural and formaldehyde-fluorescence studies on the hypothalamus of Poecilia latipinna (Teleostei: Cyprinodontiformes). Gen. Comp. Endocrinol. 39: 87-109.
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