Behavioural Brain Research 292 (2015) 283–287
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Chronic social isolation affects thigmotaxis and whole-brain serotonin levels in adult zebrafish Soaleha Shams a , Diptendu Chatterjee b , Robert Gerlai a,b,∗ a b
Department of Cell & Systems Biology, University of Toronto, Toronto, Ontario, Canada Department of Psychology, University of Toronto Mississauga, Mississauga, Ontario, Canada
h i g h l i g h t s • • • • •
Zebrafish were socially isolated for 90 days. Isolated fish showed reduced thigmotaxis but unaltered activity compared to control. Isolated fish showed reduced serotonin levels but unaltered dopamine levels. Social isolation appears to have anxiolytic effects in zebrafish. Standard high density social housing may be stressful for zebrafish.
a r t i c l e
i n f o
Article history: Received 7 March 2015 Received in revised form 24 May 2015 Accepted 29 May 2015 Available online 25 June 2015 Keywords: Social isolation Zebrafish Anxiety Thigmotaxis Serotonin
a b s t r a c t The popularity of the zebrafish has been growing in behavioral brain research. Previously utilized mainly in developmental biology and genetics, the zebrafish has turned out to possess a complex behavioral repertoire. For example, it is a highly social species, and individuals form tight groups, a behavior called shoaling. Social isolation induced changes in brain function and behavior have been demonstrated in a variety of laboratory organisms. However, despite its highly social nature, the zebrafish has rarely been utilized in this research area. Here, we investigate the effects of chronic social isolation (lasting 90 days) on locomotor activity and anxiety-related behaviors in an open tank. We also examine the effect of chronic social isolation on levels of whole-brain serotonin and dopamine and their metabolites. We found that long-term social deprivation surprisingly decreased anxiety-related behavious during open-tank testing but had no effect on locomotor activity. We also found that serotonin levels, decreased significantly in socially isolated fish, but levels of dopamine and metabolites of these neurotransmitters 5HIAA and DOPAC, respectively, remained unchanged. Our results imply that the standard high density housing employed in most zebrafish laboratories may not be the optimal way to keep these fish, and open a new avenue towards the analysis of the biological mechanisms of social behavior and of social deprivation induced changes in brain function using this simple vertebrate model organism. © 2015 Elsevier B.V. All rights reserved.
The zebrafish is a social species, widely used in genetics, pharmacology, and neuroscience research. Social interaction is an important and complex aspect of zebrafish behavior that has started to be analyzed [1,2]; however, neural mechanisms regulating zebrafish social behavior are not well-understood. Similarly, effects of social environment on behavior and physiological indices also remain largely unknown in this species. Social deprivation can
∗ Corresponding author at: Department of Psychology, University of Toronto, Mississauga, 3559 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada. Fax: +1 905 569 4326. E-mail address: robert [email protected] (R. Gerlai). http://dx.doi.org/10.1016/j.bbr.2015.05.061 0166-4328/© 2015 Elsevier B.V. All rights reserved.
be used as a tool to study regulation and consequences of social interaction and the dependence of social behaviors on presence of social stimuli. The effects of social isolation have been investigated in various model species including insects, birds, rodents, and non-human primates, where a wide range of behavioral and physiological effects have been reported [3,4]. Social isolation slows growth of sensory and learning areas of the brain in bees [5], while in fruit flies, social isolation decreases lifespan [6]. Socially-isolated chickens show higher anxiety in the open-field [7] and isolated male song birds have increased dendritic spines [8]. In rodents, social isolation affects locomotor activity and anxiety-related behavior, global epigenetic changes, wound healing, and reactivity of the Hypothalamic–Pituitary–Adrenocortical
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axis (for review, see [9,10]). Furthermore, social isolation was reported to alter dopamine and serotonin neurotransmission in rats [11,12] and to reduce serotonin levels in monkeys [13]. Social isolation has been found to increase locomotor activity in angelfish [14], to alter androgen and cortisol levels in male cichlids and swordtails [15], and to reduce the level of serotonin metabolite 5HIAA, while increasing cell proliferation in rainbow trouts [16]. In zebrafish, the behavior of isolated fish has been described as unpredictable and variable [1], with reduction in shoaling, locomotor activity, anxiety and stress reactivity [17,18]. Physiological indices showed that fish isolated prior to and/or during testing had reduced cortisol [4], decreased proliferating cells in sensory areas of the brain [19] and reduced serotonin in response to novelty [2,20]. For dopamine neurotransmission, conflicting reports exist. Some report no change in dopamine and its metabolite DOPAC [2,20] while others show reduction of dopamine and DOPAC in isolated fish [21]. Despite the wide ranging effects of social isolation reported in the literature, zebrafish are often kept isolated prior to or during experimental manipulation as it allows the experimenters to individually recognize, monitor, or manipulate the otherwise uniform looking subjects [21,22]. Furthermore, some reports may lack details about social environment prior to or during experimental trials, or use isolated fish as a control group [18,20]. Behavioral results, obtained in particular with the use of social interaction tasks or tests of anxiety and locomotor activity may be difficult to interpret if prior social experience and social environment of the testing or stimulus fish are not taken into consideration. In contrast to extensive social isolation literature in other model species, literature on social deprivation in zebrafish is scarce and conflicting. Even harder to find are studies that purposefully and systemically study social isolation and not as part of other experimental manipulation. To address this, in the current study, we investigated the effects of chronic social isolation in adult zebrafish on locomotion and anxiety-related behaviors in an open-tank. Isolation-induced hyperactivity has been previously reported in socially deprived monkeys and rats [9]. Similar to rats, zebrafish also shows thigmotaxis or wall-hugging behavior, and this response has been utilized as a measure of anxiety [23]. In the current study, we quantify the distance of the fish from the center of their open-tank. We also investigate the effects of social deprivation on neurotransmitter systems by quantifying whole-brain serotonin level, its metabolite 5-hydroxyindoleacetic acid (5HIAA) level and calculating the ratio of 5HIAA to serotonin. Similarly, we also measured levels of dopamine, its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), and calculated the ratio of DOPAC to dopamine. Adult zebrafish (Danio rerio) of AB strain were used in this study (50–50% male–female). These fish were bred and kept at the University of Toronto Mississauga vivarium and were fifth generation descendant of the progenitor fish from the Zebrafish International Research Center (ZIRC, Eugene, OR). Fish were housed in transparent acrylic tanks on a high-density rack system (Aquaneering Inc., San Diego, USA). Details about this system, feeding and housing conditions can be found elsewhere [24]. All fish were kept in groups under identical conditions throughout their development, until experimental manipulation started around 12–14 months of age. All experimental procedures used were in accordance with the ethical guidelines provided by the Canadian Council on Animal Care and were approved by the Local Animal Care Committee at the University of Toronto Mississauga. Experimental fish were randomly assigned to either the isolated or the social group. Isolated fish were kept singly for 3 months (90 days) in 1.4 l tanks with opaque corrugated plastic dividers between the tanks, which maintained visual isolation from conspecifics. Social fish were kept in groups of five in 9.5 l tanks for the same period with the same opaque corru-
gated plastic divider between the tanks to provide a similar visual environment. After 90 days, behavioral tests were conducted in an opentank whose linear dimensions were 20 times of the body length of the experimental fish (70 cm × 70 cm, width × length, filled to 20 cm water depth). An over-head digital video-camera (Sony HDR-XR550, Sony Corporation, Japan) allowed us to monitor the movements of the experimental fish. Each fish was tested individually for 20 min in the open-tank. The recordings were transferred to a hard-drive and later analyzed using EthoVision XT 8.5 (Noldus Information Technology, Wageningen, The Netherlands). The analysis included the quantification of locomotor activity (total distance traveled), and anxiety-related behaviors such as thigmotaxis (distance to the center of the tank) as well as freezing (duration of immobility). Immediately after behavioral testing, each fish was decapitated and their heads were stored at −80 ◦ C. Subsequently, brains were dissected and processed using high precision liquid chromatography (HPLC) as previously described [24]. Briefly, levels of whole brain serotonin, 5-HIAA (serotonin metabolite), dopamine, and DOPAC (dopamine metabolite) from experimental zebrafish were quantified in a single run using BAS 460 MICRO-BORE-HPLC system (Bio-analytical Systems Inc., West Lafayette, IN) with a Uniget C-18 reverse phase microbore column as the stationary phase (BASi, Catalogue# 8949). Chemically pure serotonin, 5HIAA, dopamine, and DOPAC standards (Sigma) were used to calibrate and identify peaks for experimental samples from the isolated and control zebrafish. All neurochemical levels detected were normalized to total brain protein weight for each fish to account for potential differences in brain size. Data were analyzed using statistical software SPSS 17.0 (SPSS, Inc.) for Windows. Hypothesis tests were completed using p < 0.05 as the criterion for significance. For behavioral analysis, data were analyzed using repeated measures ANOVA with social experience (isolated vs social control) as a between-subject factor and time (1-min intervals) as the within-subject factor. Neurochemical data were analyzed using one-way ANOVA with social isolation as the between-subject factor. The statistical analysis showed that chronic social isolation had no significant effect on locomotor activity (Fig. 1a). There was no significant difference between social or isolated fish in the timecourses of their activity or in the overall total distance traveled in the open-tank (p > 0.5). However, isolated fish appeared to swim closer to the center of the open-tank (Fig. 1b), indicating reduced thigmotaxis. Average distance to center point was significantly decreased in isolated fish, F (1,16) = 5.277, p = 0.035. Also, there was a significant effect of time, F (1,16) = 5.338, p < 0.001, revealing behavioral changes across the 20 min, which may be interpreted as a time-dependent increase in active avoidance. There was no group × time interaction suggesting that the group differences in thigmotaxis are independent of time. However, analysis of variance is known to be insensitive to detect interaction between main effects [25], and our figure suggests that the distance from center increased faster in social fish compared to isolated fish, a result that suggests interaction between the main factors group and time. Isolated fish also appeared to spend reduced amount of time freezing as compared to control non-isolated fish (data not shown), but this difference failed to reach statistical significance (F (1,16) = 3.530, p = 0.077). Levels of neurotransmitters serotonin and dopamine and their metabolites are depicted in Fig. 2. We found chronic social isolation to significantly decrease whole-brain serotonin level (see Fig. 2a) (F (1,16) = 7.803, p = 0.013), without altering levels of serotonin metabolite 5HIAA (p > 0.5; Fig. 2b). Ratio of metabolite to serotonin (5HIAA:5HT) was significantly increased in isolated fish as compared to the social fish (F (1,16) = 8.369, p = 0.011; Fig. 2c).
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Fig. 1. Temporal change of behaviour (mean ± S.E.M.) during open-tank exploration in fish socially isolated for 90 days (open circles) and in fish that were group housed (black filled circles). (a) Locomotor activity, measured as distance traveled, did not significantly differ between control (social) and socially isolated (isolated) fish, across the 1-min intervals. Total distance moved during the entire 20-min testing period is also shown (inset). (b) Thigmotaxis, measured as distance to center point, significantly decreased in isolated fish. The line graph shows the temporal change of this behavior across 1-min intervals. The average performance over the 20-min period is also shown (inset). n = 10 for isolated fish and n = 8 for social control fish.
Fig. 2. Mean (± S.E.M.) level of whole-brain (a) serotonin, 5HT, (b) serotonin metabolite 5HIAA and (c) their ratio (5HIAA:5HT), and (d) dopamine, (e) its metabolite DOPAC and (f) the ratio of DOPAC:dopamine. Serotonin levels significantly decreased following chronic social isolation for 90 days but no change was seen in levels of serotonin’s metabolite 5HIAA, or in dopamine and DOPAC (metabolite). Neurochemical levels are expressed as weight of neurochemical (ng) per weight of total brain protein (mg). n = 10 for isolated fish and n = 8 for social control fish.
The metabolite:neurotransmitter ratio is usually interpreted as a measure of turn-over of the given neurotransmitter due to neurotransmitter release and metabolism. Thus, the increased 5HIAA:5HT ratio found here may indicate elevated mono-amine oxidase (MAO)-dependent metabolism of serotonin in isolated fish. Notably, the reduced serotonin amount detected from whole brain extracts suggests compensation for reduced serotonin production by increased release and metabolism of this neurotransmitter in the isolated zebrafish. In contrast, we found no significant effect of social isolation on the levels of dopamine, its metabolite DOPAC, and the ratio between them (p > 0.5 for all).
In summary, chronic social isolation did not affect locomotor activity but it reduced thigmotaxis, and it also reduced serotonin levels in the brain of adult zebrafish. Previously, hyperactivity has been noted in socially isolated rats and monkeys [10]. Isolated zebrafish in this study, however, did not show any locomotor hyperactivity in the open-tank. Nevertheless, our results are comparable to those of Pagnussat et al. [1], who found no difference in zebrafish tested in isolation or in groups in an exploratory behavior task. In contrast, Kerr [17] reported hypoactivity in zebrafish isolated for 6-months, longer isolation period than what was employed in our current study. Our results are also inconsistent
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with previous reports of hyperactivity in angelfish after 4 and 10 days of isolation [14]. The above inconsistencies may be due to different isolation, housing and experimental procedures used or also to species-specific characteristics. Nevertheless, these inconsistencies demonstrate that isolation induced changes may be highly dependent on experimental procedure, a problem that may be viewed as a source of information on the factors and mechanisms influencing social behavior. Isolated zebrafish in this experiment exhibited decreased thigmotaxis and an apparent reduction of freezing, changes that may signal reduction of anxiety. It is possible that testing socially housed fish singly in the experimental tank contributed to the novel nature of the test environment, whereas testing singly housed fish alone in the experimental tank did not and the putatively enhanced novel nature of the test tank was the reason why socially housed fish were more anxious. Although plausible, findings in the literature support another possibility, namely that social housing as employed with zebrafish is actually stressful. Previously, Parker et al. [18] reported a decrease in anxiety in zebrafish isolated for 14-days. Giacomini et al. [4] found similar changes after 15-days or 30-days of isolation. Together, these findings, along with reports of decrease or no difference in cortisol levels in isolated fish [18,19] suggest that social isolation has anxiolytic effects in zebrafish. In contrast, isolation in social mammals (rodents, primates, and humans) has been reported as anxiogenic [3,12]. This is a notable inconsistency. Zebrafish have been shown to exhibit strong preference towards conspecifics [2]. Furthermore, the sight of conspecifics has been found to be a positive reinforcer [22]. Finding social isolation to have anxiolytic effects conflicts with these results. A possible explanation for these inconsistencies is that the social housing employed in the current and previous zebrafish studies may not be optimal. Being confined to the small plastic tank of the standard high density rack system with a large number of conspecifics as employed in this study and also in many other laboratories may induce anxiety and stress in zebrafish. For example, it may increase competition for food, limit the ability of subordinates to move away from aggressive dominant subjects, and it may also increase physiological stress due to possibly reduced oxygen levels and/or increased organic waste products. Irrespective of what contributed to the differences between isolated and socially housed fish, the mechanisms underlying social isolation induced behavioral changes are not well understood. We found social isolation to decrease levels of whole-brain serotonin without affecting levels of dopamine. Similar findings have been reported where, reduction in only serotonin, and not dopamine, were seen after overnight or 7-day long isolation in zebrafish [2,20]. Decreased cortical and plasma serotonin has also been reported in isolated rats and monkeys [11,13], and the dysfunction of serotonergic system has been associated with fear, anxiety, and aggression in other mammals, and with antisocial behavior in humans [3]. Given the possibility that social housing as usually employed in high density zebrafish racks is stressful to the fish, it is too early to draw mechanistic conclusions about the similarities and differences in social isolation induced functional changes in the brain across multiple species. Our results suggests that systematic analysis of social factors (isolation, crowding, sex-ratio, etc.) and also importantly non-social environmental factors too (holding tank size, oxygen levels, organic waste, etc.) are needed before proper comparison may be made across species. Such studies can help determine how these various factors modulate anxiety and stress individually and in combination. In conclusion, we report here that unlike in social mammals, chronic social isolation in zebrafish can reduce anxiety and serotonin levels in this species. These effects may have important implications for the high density housing employed in most zebrafish laboratories all over the world. While this method of
housing may be acceptable for studies focusing on features of the embryo, analysis of adult fish, and especially analysis of the behavior and brain function of adult fish may be significantly impacted by the potentially stressful aspect of high density housing. Irrespective of this, however, our results demonstrate that analysis of brain function and behavior and manipulation of the social environment of the zebrafish may be utilized as a tool to study both the environmental conditions that affect as well as the biological mechanisms that underlie social behavior, a line of research that may in the future also enable the investigator to model social deficits and anxiety-related disorders using this simple vertebrate.
Acknowledgements We would like to thank Ms. Diane Seguin and Mr. Steven Tran for their technical help, and our volunteers and vivarium staff at the University of Toronto Mississauga for animal care and monitoring. Supported by NSERC (311637).
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Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Chronic social isolation affects thigmotaxis and whole-brain serotonin levels in adult zebrafish Soaleha Shams a , Diptendu Chatterjee b , Robert Gerlai a,b,∗ a b
Department of Cell & Systems Biology, University of Toronto, Toronto, Ontario, Canada Department of Psychology, University of Toronto Mississauga, Mississauga, Ontario, Canada
h i g h l i g h t s • • • • •
Zebrafish were socially isolated for 90 days. Isolated fish showed reduced thigmotaxis but unaltered activity compared to control. Isolated fish showed reduced serotonin levels but unaltered dopamine levels. Social isolation appears to have anxiolytic effects in zebrafish. Standard high density social housing may be stressful for zebrafish.
a r t i c l e
i n f o
Article history: Received 7 March 2015 Received in revised form 24 May 2015 Accepted 29 May 2015 Available online 25 June 2015 Keywords: Social isolation Zebrafish Anxiety Thigmotaxis Serotonin
a b s t r a c t The popularity of the zebrafish has been growing in behavioral brain research. Previously utilized mainly in developmental biology and genetics, the zebrafish has turned out to possess a complex behavioral repertoire. For example, it is a highly social species, and individuals form tight groups, a behavior called shoaling. Social isolation induced changes in brain function and behavior have been demonstrated in a variety of laboratory organisms. However, despite its highly social nature, the zebrafish has rarely been utilized in this research area. Here, we investigate the effects of chronic social isolation (lasting 90 days) on locomotor activity and anxiety-related behaviors in an open tank. We also examine the effect of chronic social isolation on levels of whole-brain serotonin and dopamine and their metabolites. We found that long-term social deprivation surprisingly decreased anxiety-related behavious during open-tank testing but had no effect on locomotor activity. We also found that serotonin levels, decreased significantly in socially isolated fish, but levels of dopamine and metabolites of these neurotransmitters 5HIAA and DOPAC, respectively, remained unchanged. Our results imply that the standard high density housing employed in most zebrafish laboratories may not be the optimal way to keep these fish, and open a new avenue towards the analysis of the biological mechanisms of social behavior and of social deprivation induced changes in brain function using this simple vertebrate model organism. © 2015 Elsevier B.V. All rights reserved.
The zebrafish is a social species, widely used in genetics, pharmacology, and neuroscience research. Social interaction is an important and complex aspect of zebrafish behavior that has started to be analyzed [1,2]; however, neural mechanisms regulating zebrafish social behavior are not well-understood. Similarly, effects of social environment on behavior and physiological indices also remain largely unknown in this species. Social deprivation can
∗ Corresponding author at: Department of Psychology, University of Toronto, Mississauga, 3559 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada. Fax: +1 905 569 4326. E-mail address: robert [email protected] (R. Gerlai). http://dx.doi.org/10.1016/j.bbr.2015.05.061 0166-4328/© 2015 Elsevier B.V. All rights reserved.
be used as a tool to study regulation and consequences of social interaction and the dependence of social behaviors on presence of social stimuli. The effects of social isolation have been investigated in various model species including insects, birds, rodents, and non-human primates, where a wide range of behavioral and physiological effects have been reported [3,4]. Social isolation slows growth of sensory and learning areas of the brain in bees [5], while in fruit flies, social isolation decreases lifespan [6]. Socially-isolated chickens show higher anxiety in the open-field [7] and isolated male song birds have increased dendritic spines [8]. In rodents, social isolation affects locomotor activity and anxiety-related behavior, global epigenetic changes, wound healing, and reactivity of the Hypothalamic–Pituitary–Adrenocortical
284
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axis (for review, see [9,10]). Furthermore, social isolation was reported to alter dopamine and serotonin neurotransmission in rats [11,12] and to reduce serotonin levels in monkeys [13]. Social isolation has been found to increase locomotor activity in angelfish [14], to alter androgen and cortisol levels in male cichlids and swordtails [15], and to reduce the level of serotonin metabolite 5HIAA, while increasing cell proliferation in rainbow trouts [16]. In zebrafish, the behavior of isolated fish has been described as unpredictable and variable [1], with reduction in shoaling, locomotor activity, anxiety and stress reactivity [17,18]. Physiological indices showed that fish isolated prior to and/or during testing had reduced cortisol [4], decreased proliferating cells in sensory areas of the brain [19] and reduced serotonin in response to novelty [2,20]. For dopamine neurotransmission, conflicting reports exist. Some report no change in dopamine and its metabolite DOPAC [2,20] while others show reduction of dopamine and DOPAC in isolated fish [21]. Despite the wide ranging effects of social isolation reported in the literature, zebrafish are often kept isolated prior to or during experimental manipulation as it allows the experimenters to individually recognize, monitor, or manipulate the otherwise uniform looking subjects [21,22]. Furthermore, some reports may lack details about social environment prior to or during experimental trials, or use isolated fish as a control group [18,20]. Behavioral results, obtained in particular with the use of social interaction tasks or tests of anxiety and locomotor activity may be difficult to interpret if prior social experience and social environment of the testing or stimulus fish are not taken into consideration. In contrast to extensive social isolation literature in other model species, literature on social deprivation in zebrafish is scarce and conflicting. Even harder to find are studies that purposefully and systemically study social isolation and not as part of other experimental manipulation. To address this, in the current study, we investigated the effects of chronic social isolation in adult zebrafish on locomotion and anxiety-related behaviors in an open-tank. Isolation-induced hyperactivity has been previously reported in socially deprived monkeys and rats [9]. Similar to rats, zebrafish also shows thigmotaxis or wall-hugging behavior, and this response has been utilized as a measure of anxiety [23]. In the current study, we quantify the distance of the fish from the center of their open-tank. We also investigate the effects of social deprivation on neurotransmitter systems by quantifying whole-brain serotonin level, its metabolite 5-hydroxyindoleacetic acid (5HIAA) level and calculating the ratio of 5HIAA to serotonin. Similarly, we also measured levels of dopamine, its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), and calculated the ratio of DOPAC to dopamine. Adult zebrafish (Danio rerio) of AB strain were used in this study (50–50% male–female). These fish were bred and kept at the University of Toronto Mississauga vivarium and were fifth generation descendant of the progenitor fish from the Zebrafish International Research Center (ZIRC, Eugene, OR). Fish were housed in transparent acrylic tanks on a high-density rack system (Aquaneering Inc., San Diego, USA). Details about this system, feeding and housing conditions can be found elsewhere [24]. All fish were kept in groups under identical conditions throughout their development, until experimental manipulation started around 12–14 months of age. All experimental procedures used were in accordance with the ethical guidelines provided by the Canadian Council on Animal Care and were approved by the Local Animal Care Committee at the University of Toronto Mississauga. Experimental fish were randomly assigned to either the isolated or the social group. Isolated fish were kept singly for 3 months (90 days) in 1.4 l tanks with opaque corrugated plastic dividers between the tanks, which maintained visual isolation from conspecifics. Social fish were kept in groups of five in 9.5 l tanks for the same period with the same opaque corru-
gated plastic divider between the tanks to provide a similar visual environment. After 90 days, behavioral tests were conducted in an opentank whose linear dimensions were 20 times of the body length of the experimental fish (70 cm × 70 cm, width × length, filled to 20 cm water depth). An over-head digital video-camera (Sony HDR-XR550, Sony Corporation, Japan) allowed us to monitor the movements of the experimental fish. Each fish was tested individually for 20 min in the open-tank. The recordings were transferred to a hard-drive and later analyzed using EthoVision XT 8.5 (Noldus Information Technology, Wageningen, The Netherlands). The analysis included the quantification of locomotor activity (total distance traveled), and anxiety-related behaviors such as thigmotaxis (distance to the center of the tank) as well as freezing (duration of immobility). Immediately after behavioral testing, each fish was decapitated and their heads were stored at −80 ◦ C. Subsequently, brains were dissected and processed using high precision liquid chromatography (HPLC) as previously described [24]. Briefly, levels of whole brain serotonin, 5-HIAA (serotonin metabolite), dopamine, and DOPAC (dopamine metabolite) from experimental zebrafish were quantified in a single run using BAS 460 MICRO-BORE-HPLC system (Bio-analytical Systems Inc., West Lafayette, IN) with a Uniget C-18 reverse phase microbore column as the stationary phase (BASi, Catalogue# 8949). Chemically pure serotonin, 5HIAA, dopamine, and DOPAC standards (Sigma) were used to calibrate and identify peaks for experimental samples from the isolated and control zebrafish. All neurochemical levels detected were normalized to total brain protein weight for each fish to account for potential differences in brain size. Data were analyzed using statistical software SPSS 17.0 (SPSS, Inc.) for Windows. Hypothesis tests were completed using p < 0.05 as the criterion for significance. For behavioral analysis, data were analyzed using repeated measures ANOVA with social experience (isolated vs social control) as a between-subject factor and time (1-min intervals) as the within-subject factor. Neurochemical data were analyzed using one-way ANOVA with social isolation as the between-subject factor. The statistical analysis showed that chronic social isolation had no significant effect on locomotor activity (Fig. 1a). There was no significant difference between social or isolated fish in the timecourses of their activity or in the overall total distance traveled in the open-tank (p > 0.5). However, isolated fish appeared to swim closer to the center of the open-tank (Fig. 1b), indicating reduced thigmotaxis. Average distance to center point was significantly decreased in isolated fish, F (1,16) = 5.277, p = 0.035. Also, there was a significant effect of time, F (1,16) = 5.338, p < 0.001, revealing behavioral changes across the 20 min, which may be interpreted as a time-dependent increase in active avoidance. There was no group × time interaction suggesting that the group differences in thigmotaxis are independent of time. However, analysis of variance is known to be insensitive to detect interaction between main effects [25], and our figure suggests that the distance from center increased faster in social fish compared to isolated fish, a result that suggests interaction between the main factors group and time. Isolated fish also appeared to spend reduced amount of time freezing as compared to control non-isolated fish (data not shown), but this difference failed to reach statistical significance (F (1,16) = 3.530, p = 0.077). Levels of neurotransmitters serotonin and dopamine and their metabolites are depicted in Fig. 2. We found chronic social isolation to significantly decrease whole-brain serotonin level (see Fig. 2a) (F (1,16) = 7.803, p = 0.013), without altering levels of serotonin metabolite 5HIAA (p > 0.5; Fig. 2b). Ratio of metabolite to serotonin (5HIAA:5HT) was significantly increased in isolated fish as compared to the social fish (F (1,16) = 8.369, p = 0.011; Fig. 2c).
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Fig. 1. Temporal change of behaviour (mean ± S.E.M.) during open-tank exploration in fish socially isolated for 90 days (open circles) and in fish that were group housed (black filled circles). (a) Locomotor activity, measured as distance traveled, did not significantly differ between control (social) and socially isolated (isolated) fish, across the 1-min intervals. Total distance moved during the entire 20-min testing period is also shown (inset). (b) Thigmotaxis, measured as distance to center point, significantly decreased in isolated fish. The line graph shows the temporal change of this behavior across 1-min intervals. The average performance over the 20-min period is also shown (inset). n = 10 for isolated fish and n = 8 for social control fish.
Fig. 2. Mean (± S.E.M.) level of whole-brain (a) serotonin, 5HT, (b) serotonin metabolite 5HIAA and (c) their ratio (5HIAA:5HT), and (d) dopamine, (e) its metabolite DOPAC and (f) the ratio of DOPAC:dopamine. Serotonin levels significantly decreased following chronic social isolation for 90 days but no change was seen in levels of serotonin’s metabolite 5HIAA, or in dopamine and DOPAC (metabolite). Neurochemical levels are expressed as weight of neurochemical (ng) per weight of total brain protein (mg). n = 10 for isolated fish and n = 8 for social control fish.
The metabolite:neurotransmitter ratio is usually interpreted as a measure of turn-over of the given neurotransmitter due to neurotransmitter release and metabolism. Thus, the increased 5HIAA:5HT ratio found here may indicate elevated mono-amine oxidase (MAO)-dependent metabolism of serotonin in isolated fish. Notably, the reduced serotonin amount detected from whole brain extracts suggests compensation for reduced serotonin production by increased release and metabolism of this neurotransmitter in the isolated zebrafish. In contrast, we found no significant effect of social isolation on the levels of dopamine, its metabolite DOPAC, and the ratio between them (p > 0.5 for all).
In summary, chronic social isolation did not affect locomotor activity but it reduced thigmotaxis, and it also reduced serotonin levels in the brain of adult zebrafish. Previously, hyperactivity has been noted in socially isolated rats and monkeys [10]. Isolated zebrafish in this study, however, did not show any locomotor hyperactivity in the open-tank. Nevertheless, our results are comparable to those of Pagnussat et al. [1], who found no difference in zebrafish tested in isolation or in groups in an exploratory behavior task. In contrast, Kerr [17] reported hypoactivity in zebrafish isolated for 6-months, longer isolation period than what was employed in our current study. Our results are also inconsistent
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with previous reports of hyperactivity in angelfish after 4 and 10 days of isolation [14]. The above inconsistencies may be due to different isolation, housing and experimental procedures used or also to species-specific characteristics. Nevertheless, these inconsistencies demonstrate that isolation induced changes may be highly dependent on experimental procedure, a problem that may be viewed as a source of information on the factors and mechanisms influencing social behavior. Isolated zebrafish in this experiment exhibited decreased thigmotaxis and an apparent reduction of freezing, changes that may signal reduction of anxiety. It is possible that testing socially housed fish singly in the experimental tank contributed to the novel nature of the test environment, whereas testing singly housed fish alone in the experimental tank did not and the putatively enhanced novel nature of the test tank was the reason why socially housed fish were more anxious. Although plausible, findings in the literature support another possibility, namely that social housing as employed with zebrafish is actually stressful. Previously, Parker et al. [18] reported a decrease in anxiety in zebrafish isolated for 14-days. Giacomini et al. [4] found similar changes after 15-days or 30-days of isolation. Together, these findings, along with reports of decrease or no difference in cortisol levels in isolated fish [18,19] suggest that social isolation has anxiolytic effects in zebrafish. In contrast, isolation in social mammals (rodents, primates, and humans) has been reported as anxiogenic [3,12]. This is a notable inconsistency. Zebrafish have been shown to exhibit strong preference towards conspecifics [2]. Furthermore, the sight of conspecifics has been found to be a positive reinforcer [22]. Finding social isolation to have anxiolytic effects conflicts with these results. A possible explanation for these inconsistencies is that the social housing employed in the current and previous zebrafish studies may not be optimal. Being confined to the small plastic tank of the standard high density rack system with a large number of conspecifics as employed in this study and also in many other laboratories may induce anxiety and stress in zebrafish. For example, it may increase competition for food, limit the ability of subordinates to move away from aggressive dominant subjects, and it may also increase physiological stress due to possibly reduced oxygen levels and/or increased organic waste products. Irrespective of what contributed to the differences between isolated and socially housed fish, the mechanisms underlying social isolation induced behavioral changes are not well understood. We found social isolation to decrease levels of whole-brain serotonin without affecting levels of dopamine. Similar findings have been reported where, reduction in only serotonin, and not dopamine, were seen after overnight or 7-day long isolation in zebrafish [2,20]. Decreased cortical and plasma serotonin has also been reported in isolated rats and monkeys [11,13], and the dysfunction of serotonergic system has been associated with fear, anxiety, and aggression in other mammals, and with antisocial behavior in humans [3]. Given the possibility that social housing as usually employed in high density zebrafish racks is stressful to the fish, it is too early to draw mechanistic conclusions about the similarities and differences in social isolation induced functional changes in the brain across multiple species. Our results suggests that systematic analysis of social factors (isolation, crowding, sex-ratio, etc.) and also importantly non-social environmental factors too (holding tank size, oxygen levels, organic waste, etc.) are needed before proper comparison may be made across species. Such studies can help determine how these various factors modulate anxiety and stress individually and in combination. In conclusion, we report here that unlike in social mammals, chronic social isolation in zebrafish can reduce anxiety and serotonin levels in this species. These effects may have important implications for the high density housing employed in most zebrafish laboratories all over the world. While this method of
housing may be acceptable for studies focusing on features of the embryo, analysis of adult fish, and especially analysis of the behavior and brain function of adult fish may be significantly impacted by the potentially stressful aspect of high density housing. Irrespective of this, however, our results demonstrate that analysis of brain function and behavior and manipulation of the social environment of the zebrafish may be utilized as a tool to study both the environmental conditions that affect as well as the biological mechanisms that underlie social behavior, a line of research that may in the future also enable the investigator to model social deficits and anxiety-related disorders using this simple vertebrate.
Acknowledgements We would like to thank Ms. Diane Seguin and Mr. Steven Tran for their technical help, and our volunteers and vivarium staff at the University of Toronto Mississauga for animal care and monitoring. Supported by NSERC (311637).
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