The Effect of Stress and Starvation on Brain Serotonin Utilization in Arctic Charr (Salvelinus Alpinus)

In a recent study we found that social rank in Arctic charr (Salvelinus alpinus) was inversely correlated with the concentrations of 5-hydroxyindoleacetic acid (5-HIAA), a major serotonin (5-hydroxytryptamine, 5-HT) metabolite, present in the telencephalon and the brain stem (Winberg et al. 1991). Thus, subordinate individuals showed much higher brain 5-HIAA concentrations than dominant fish, indicating elevated serotonergic activity in subordinate fish. Since the 5-HT levels remained unchanged, the 5-HIAA/5-HT ratios, an index of serotonin utilization (Fuller, 1985), were also increased in low-ranking fish.

In mammals, serotonergic neurones are thought to be involved in stress reactions in the brain (Culman et al. 1984). The effect of stress on brain serotonergic activity in rodents has been the subject of several studies (Bliss et al. 1972; Curzon et al. 1972; Morgan et al. 1975; Morgan and Rudeen, 1976; Adell et al. 1988; Mitchell and Thomas, 1988). In general, these studies have reported that stress increases brain levels of 5-HIAA without affecting 5-HT concentrations.

In fish, a low position in a dominance hierarchy has been associated with physiological signs of increased stress (Noakes and Leatherland, 1977; Ejike and Schreck, 1980; Peters et al. 1980; Scott and Currie, 1980), implying that the observed increase in 5-HIAA levels in subordinate fish (Winberg et al. 1991) could be stress-mediated. However, to our knowledge, there have been no studies on the effect of stress on brain serotonergic activity in fish.

There is also another possible explanation for the increased 5-HIAA levels displayed by subordinate Arctic charr. In a dominance hierarchy, subordinate fish have reduced access to food, since their feeding attempts often induce attacks from the dominant fish. Indeed, fish occupying low positions in a dominance hierarchy show much lower weight gains than dominant individuals (Jobling and Wandsvik, 1983; Abbott et al. 1985; Abbott and Dill, 1989), indicating malnutrition in subordinates. In mammals, food deprivation for 24-72 h has been found to elevate brain 5-HIAA levels (Curzon et al. 1972; Kantak et al. 1978; Fuenmayor and Garcia, 1984). Thus, it is possible that the increase in 5-HIAA levels seen in subordinate Arctic charr (Winberg et al. 1991) is an effect of food deprivation.

The aim of the present study was to investigate the effect of stress and starvation on brain levels of 5-HT and 5-HIAA in Arctic charr. In our previous study (Winberg et al. 1991) 2-11 weeks of subordinate experience increased brain 5-HIAA levels and 5-HIAA/5-HT ratios. In this study we tried to imitate the chronic stress experienced by a subordinate individual by subjecting the fish to repeated daily stress during a four-week period. The effect of stress was studied both in fish given food and in fish deprived of food during the experiment.

The fish were 2-year-old offspring of Arctic charr (Salvelinus alpinus L.) caught in Lake Torron, Jamtland, Sweden. The fish were kept indoors at our department for more than one year before the experiment. The holding tank was continuously supplied with aerated Uppsala tap water (8-11 °C) and the photoperiod was kept at 10h:14h L:D (light between 08:00h and 18:00h). The fish were fed daily with commercial trout pellets (EWOS ST40, Astra-EWOS Sweden) at 1-2 % of the body weight.

All experiments were performed in four glass aquaria (1000 mm × 500mm × 500mm), each divided into four compartments by black plastic walls, and continuously supplied with aerated Uppsala tap water (0.801 min ^{−1, 8 −} 10°C).

The water level was maintained at 400 mm by a standpipe. To minimize disturbance of the fish, a fine nylon-mesh screen was attached to the front of the aquaria while black plastic covered the remaining sides. Light was provided by fluorescent tubes (2 × 20 W, warm white), placed 100 mm above the water surface. The photoperiod was 12h:12h L:D with light on between 07:00 h and 19:00 h. The bottom of each compartment was covered with a plastic net that could be lifted by lines. Stress was induced, three times a day (each day), by elevating this net bottom so that the dorsal fin of the fish was above the water surface; the fish was left in the elevated position for 15 min. While lifted, the fish was given three pinches (with fingers) to the caudal fin. During the stress experiments, control fish were kept in identical aquaria but the plastic nets were never lifted.

The fish, initially weighing 46±12g (mean±s.D., N=16), were kept isolated in individual compartments and daily given commercial trout pellets at 2-4 % of the body weight. Stress was induced as described above during a four-week period (22 May to 12 June).

The fish, initially weighing 54±18g (mean±s.D., N=16), were kept isolated in individual compartments. Experimental fish were starved for a four-week period (14 June to 12 July) while controls were fed daily with trout pellets (2-4 % of body weight).

This experiment was performed as for experiment 1 but neither stressed fish nor controls were fed during the experiment. At the start of the experiment the mean mass of the fish was 141±26g (mean±s.D., 7V=16). The experiment was run during the period 22 September to 15 October.

At the end of each experimental period, fish were decapitated, between 17:00 and 18:00 h. The brain (excluding the olfactory bulbs and the pituitary gland) was rapidly removed and divided into two parts: the telencephalon (weighing ll±3mg) and the remaining parts of the brain (weighing 156±38mg), here called the brain stem. The brain samples were wrapped in aluminium foil, frozen in liquid nitrogen (within 2 min of decapitation), and kept at -80°C. Each fish was weighed after tissue sampling.

The frozen brain samples were homogenized in 4% (w/v) ice-cold perchloric acid (PCA) containing 0.2% EDTA, 0.05% sodium bisulphite and 40 ng ml⁻ epinine (deoxyepinephrine, the internal standard), using a Potter-Elvehjem homogenizer (brain stem) or an MSE 100 W ultrasonic disintegrator (telencephalon).

5-HT and 5-HIAA were quantified using high performance liquid chromatography with electrochemical detection as described by Nilsson (1989). As a measure of serotonergic activity, the 5-HIAA/5-HT ratio was calculated for each individual (Fuller, 1985; Winberg et al. 1991).

Stressed fish, whether given food (expt 1) or starved (expt 3), showed significantly higher 5-HIAA concentrations in both the telencephalon and the brain stem (Fig. 1). The 5-HIAA/5-HT ratio was also higher in stressed fish (Fig. 2) and a significant increase in the 5-HIAA/5-HT ratio was found in the telencephalon as well as in the brain stem of stressed fish deprived of food during the experiment (expt 3, Fig. 2). Furthermore, an increase in the 5-HIAA/5-HT ratio was seen in stressed fish given food during the experiment (expt 1), although, in this experiment, the increase in the 5-HIAA/5-HT ratio was only significant in the telencephalon (Fig. 2).

There was no effect of starvation on 5-HIAA concentrations in either telencephalon or brain stem (Fig. 1) and starvation did not significantly affect 5-HIAA/5-HT ratios (Fig. 2).

The 5-HT concentration was significantly decreased in the telencephalon of starved fish (expt 2, Fig. 3). However, in brain stem no significant differences in 5-HT concentrations were seen between starved fish and controls (expt 2, Fig. 3).

There were no significant differences in brain 5-HT concentrations between stressed fish and controls when both groups were given food (expt 1, Fig. 3).

Where both groups were deprived of food during the experiment (expt 3), a small non-significant (P=0.094) decrease in 5-HT levels in the telencephalon occurred in the stressed individuals (Fig. 3).

In expt 1, where both controls and stressed fish were given food, the stressed fish showed a significant weight loss during the experiment compared with their starting weight. However, in this experiment, the difference in weight change between stressed fish and controls was not significant (Fig. 4).

As expected, starvation per se (expt 2) resulted in a significant weight loss. There was a significant difference in weight change between starved fish and controls (Fig. 4).

In expt 3, in which controls as well as stressed fish were deprived of food during the experiment, significant weight losses were seen in both groups. The weight loss was, however, significantly higher in stressed fish (Fig. 4).

During the first days of the stress experiments the fish made furious efforts to escape when pinched on the caudal fin. As the experiments continued, these flight attempts gradually decreased. Stressed fish were inactive at the end of the experiment, spending most of their time close to the bottom of the aquarium. Strikingly, stressed fish made little effort to escape when netted at the end of the experiment. In contrast, control fish and fish subjected to starvation were active and moved around in their compartments throughout the experiment.

No mortality occurred in any of the groups during the experiments.

The results from the present study clearly demonstrate that stress increases brain levels of 5-HIAA, the main 5-HT metabolite, without affecting 5-HT concentrations in Arctic charr. Thus, the 5-HIAA/5-HT ratio, which can been used as an index of serotonergic activity (Fuller, 1985; Winberg et al. 1991), was elevated in stressed fish, indicating that stress induces an increase in the activity of the central serotonergic system in this species. Starvation, in contrast, had no effect on either 5-HIAA concentrations or 5-HIAA/5-HT ratios.

The stress-induced increase in 5-HT activity was present in both the telencephalon and the brain stem, although it was somewhat more pronounced in the telencephalon. Thus, stress seems to have a general effect on 5-HT utilization in the brain of Arctic charr. It should be mentioned that stress in mammals has been reported to increase the concentration of tryptophan, the precursor of 5-HT, in blood as well as in brain (Curzon et al. 1972; Neckers and Sze, 1975; Dunn, 1988). The activity of tryptophan hydroxylase, the enzyme catalyzing the rate-limiting step in 5-HT synthesis, is normally restricted by substrate (i.e. tryptophan) availability. Consequently, increased brain tryptophan concentrations are likely to increase 5-HT synthesis and 5-HT turnover (Boadle-Biber, 1982). However, this mechanism could hardly explain the increased 5-HIAA levels seen in stressed fish, since the 5-HT concentration remained unchanged or even showed a tendency to decrease during stress. Hence, it is more likely that the increased 5-HIAA/5-HT ratios during stress were caused by an increased use (release) of 5-HT.

Interestingly, Dunn (1988) observed a decrease in the 5-HT concentration in prefrontal cortex and hypothalamus in mice after 15 min of footshock, although plasma as well as brain levels of tryptophan increased progressively during the stress treatment. The decrease in 5-HT concentration was, however, reversed after a further 15 min of footshock. The 5-HIAA/5-HT ratio was elevated in both these brain regions after 15 and 30 min of footshock. Dunn (1988) suggests that the transient decrease in 5-HT reflects a stress-induced increase in 5-HT release, depleting existing 5-HT stores. Using in vivo voltametry in rats, Joseph and Kennett (1983) found that hippocampal 5-HIAA levels increased immediately after initiation of immobilization stress, suggesting stress-induced increases in 5-HT release into the extracellular space. Tryptophan administration did not increase brain 5-HIAA release, as determined by in vivo voltametry, but increased tissue 5-HIAA levels determined by post mortem neurochemical analysis (De Simoni et al. 1987). Furthermore, Lookingland et al. (1986) showed that exogenous tryptophan, given to rats, produced a dose-dependent increase in both 5-HT and 5-HIAA concentrations in several hypothalamic regions, without affecting 5-HIAA/5-HT ratios in these regions. Thus, it seems that both tryptophan administration and stress can increase 5-HT turnover but only the latter increases 5-HT release.

The central serotonergic system is thought to be involved in the regulation of locomotor activity in fish (Fingerman, 1976; Genot et al. 1984). During the present stress experiments, the attempts of the fish to escape when pinched on the caudal fin gradually disappeared. Indeed, the locomotor activity of the stressed fish showed a general decrease during the experimental period. Similarly, subordinate Arctic charr in a dominance hierarchy show lower locomotor activity compared to dominant fish (Winberg et al. 1991). The observation that subordinate fish show physiological stress symptoms (Noakes and Leatherland, 1977; Ejike and Schreck, 1980; Peters et al. 1980; Scott and Currie, 1980) as well as elevated brain 5-HIAA/5-HT ratios (Winberg et al. 1991) makes it tempting to suggest a causal relationship between stress, increased serotonergic activity and reduced locomotor activity in these individuals.

Food deprivation has been reported to increase brain 5-HIAA levels in mammals (Curzon et al. 1972; Kantak et al. 1978; Fuenmayor and Garcia, 1984). In the present study, starvation had no effect on brain 5-HIAA concentrations in Arctic charr. Starvation did, however, significantly decrease the 5-HT concentration in the telencephalon. In the mammalian experiments, the animals were only deprived of food for 24-72 h, which may explain the discrepancy between these studies and our results. As tryptophan is an essential amino acid, the four-week starvation period used in our experiment may very well have reduced tryptophan availability, and thus 5-HT synthesis. Phylogenetic factors are, of course, also likely to explain these discrepancies.

As expected, fish subjected to four weeks of starvation lost weight. A weight loss was also seen in stressed fish given food during the experiment. The latter result could be related to a decreased food intake due to the stress. Indeed, the stressed fish often paid little attention to the food. However, most interestingly, starved stressed fish lost more weight than starved controls, even though they showed less locomotor activity. This could be related to a stress-induced increase in metabolic rate. Abbott and Dill (1989) showed that the dominant individual in a size-matched pair of steelhead trout (Oncorhynchus mykiss) had a higher growth rate than the subordinate, even when both fish were fed equal amounts. As subordinates were less active, Abbott and Dill (1989) suggested that the reduced growth in subordinate fish correlated with increased metabolic rate caused by stress.

Brain concentrations of 5-HT and 5-HIAA varied between experiments. This variation is likely to reflect seasonal variations in brain monoamine levels. Seasonal and daily variations in brain monoamine concentrations are well known and have previously been shown to occur in fish (Fingerman, 1976; Sauerbier and Meyer, 1977; Popek, 1983; Ehrenstrom and Johansson, 1987; Khan and Joy, 1988). Furthermore, there was an increase in the size of fish used in later experiments and it is possible that this growth affected brain 5-HT and 5-HIAA concentrations.

However, since a control group was included in each experiment, the significant changes seen within an experiment must reflect changes in serotonergic function that are responses to the experimental treatment, that is short-term changes that occur in addition to seasonal variations in serotonergic function. Indeed, our experiments clearly suggest that the seasonal and weight changes seen in the present study did not interfere with the main conclusion -that stress increases serotonin utilization in Arctic charr. Thus, in the first two experiments, which were run close together (May-July, fish weighing approximately 50 g), we show that stress in fish given food (expt 1) increases serotonergic activity and that this is not due to food deprivation (expt 2). In the third experiment, we show that stress also has this effect in September when on average the fish weighed 141 g.

In conclusion, the present results show that stress significantly increases brain 5-HIAA levels without affecting 5-HT concentrations in Arctic charr. This pattern of change appears to be identical to that displayed by subordinate Arctic charr in a dominance hierarchy (Winberg et al. 1991). Thus, increased stress, which subordinate individuals are likely to experience, could very well be the cause of the increased 5-HIAA/5-HT ratios seen in these fish. The stress regime used here could be regarded as a number of acute stresses. Apart from the acute stress caused by each attack by dominant fish, a subordinate fish is probably also exposed to a chronic challenge by the constant presence of the dominant fish. Such a constant stress is probably impossible to imitate experimentally. In spite of this difficulty, the experimental stress regime used caused changes in serotonergic activity that were very similar to those in a subordinate fish. In contrast, the food deprivation experienced by subordinate Arctic charr can probably be ruled out as a factor causing the increased 5-HIAA/5-HT ratios observed in low-ranking fish.

This work was supported by The Swedish Council for Forestry and Agricultural Research (no. 0890/89 V 88 and 48.0594/91 to G. E. Nilsson), the Helge Ax:son Johnson Foundation and the Royal Swedish Academy of Sciences (the Hierta-Retzius and the Regnell Founds).