Evidence that acute serotonergic activation potentiates the locomotor-stimulating effects of corticotropin-releasing hormone in juvenile chinook salmon (Oncorhynchus tshawytscha)☆

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Evidence that acute serotonergic activation potentiates the locomotorstimulating effects of corticotropin-releasing hormone in juvenile chinook salmon (Oncorhynchus tshawytscha)夞 Shaun Clements,a,* Frank L. Moore,b and Carl B. Schrecka a

Oregon Cooperative Fish and Wildlife Research Unit,1 Department of Fisheries and Wildlife, Oregon State University, and U.S.G.S., Corvallis, OR 97331-3803, USA, b Department of Zoology, Oregon State University, Corvallis, OR 97331-3803, USA Received 1 March 2002; revised 1 October 2002; accepted 14 October 2002

Abstract The present study investigated whether the serotonergic system is involved in mediating the behavioral effects of corticotropin-releasing hormone (CRH) in juvenile spring chinook salmon, Oncorhynchus tshawytscha. An intracerebroventricular (ICV) injection of CRH induced hyperactivity. The effect of CRH was potentiated in a dose-dependent manner by the concurrent administration of the serotonin (5-HT) selective reuptake inhibitor fluoxetine. However, administration of fluoxetine alone had no effect on locomotor activity, suggesting that the locomotor-stimulating effect of CRH is mediated by the activation of the serotonergic system. Conversely, ICV injections of the 5-HT1A receptor antagonist NAN-190 attenuated the effect of CRH on locomotor activity when given in combination with CRH but had no effect when administered alone. These results provide the first evidence to support the hypothesis that the effect of CRH on locomotor activity in teleosts is mediated by activating the serotonergic system. © 2003 Elsevier Science (USA). All rights reserved. Keywords: CRH; CRF; Serotonin; Locomotor; Teleost

Introduction Corticotropin-releasing hormone (CRH) has recently been characterized in salmonids (Oncorhynchus nerka) (Ando et al., 1999). Pohl et al. (2001) have also reported the existence of two CRH receptors in chum salmon (Oncorhynchus keta). In mammals CRH is the primary regulator of the hypothalamic–pituitary–adrenal/interrenal axis. CRH is also involved in the regulation of many behavioral and physiological responses, particularly those involved with stress (Dunn and Berridge, 1987; Moore et al., 1984;

夞 Oregon Agricultural Experimental Station Technical Report 11928. * Corresponding author. 104 Nash Hall, Department of Fisheries and Wildlife, Oregon State University, Corvallis, OR 97331. Fax: ⫹1-541-7373590. E-mail address: [email protected] (S. Clements). 1 Supported cooperatively by the U.S.G.S., Oregon State University, and the Oregon Department of Fish and Wildlife.

Sutton et al., 1982). Conversely, in teleosts Urotensin 1, not CRH, appears to be most potent at stimulating the secretion of ACTH (Fryer et al., 1983; Tran et al., 1990); however, little is known about the behavioral effects of CRH. Administration of CRH in mammals often results in increased activity (Sutton et al., 1982). Lowry et al. (1990) have also demonstrated that CRH causes hyperactivity in roughskin newts (Taricha granulosa) and does so independently of the pituitary gland (Moore et al., 1984). More recently, we have shown that intracerebroventricular (ICV) administration of CRH to juvenile chinook salmon, Oncorhynchus tshawytscha, can lead to behavioral changes such as increased locomotor activity (Clements et al., 2001). These results suggest that the involvement of CRH in the control of locomotor activity is highly conserved across all vertebrates. However, CRH operates within a complex system of neurohormones and neurotransmitters; therefore interactions with other substances are likely to be important in determining behavioral output. Both in vitro and in vivo

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studies suggest that CRH is an important regulator of serotonergic activity. Lowry et al. (2000) showed that CRH increases the firing rates of a specific subpopulation of serotonergic neurons in the rat brain. Similarly, the administration of exogenous CRH can lead to the alteration of serotonin (5-HT) metabolism and neurotransmission (Singh et al., 1992). This functional link is further supported by topographical studies showing that serotonergic centers within the brain express CRH receptors, and are innervated by CRH fibers (Price et al., 1998). The concentration of 5-HT within the brain has also been correlated with changes in locomotor activity in goldfish, Carassius auratus (Fenwick, 1970), and the Texas killifish, Fundulus grandis (Fingerman, 1976). Winberg et al. (1993) demonstrated that inhibition of brain serotonergic activity caused a significant increase in the activity levels of arctic charr, Salvelinus alpinus. In contrast, Genot et al. (1984) reported that inhibiting the synthesis of the serotonin precursor, 5-hydroxytryptophan (5-HTP), caused a significant decline in the activity of eels, Anguilla anguilla, and that the effect could be reversed by treatment with 5-HTP. Based on these findings we hypothesized that the increase in locomotor activity following administration of CRH was due to the activation of serotonergic mechanisms. Using a behavioral assay we investigated whether the changes observed following CRH administration were affected by changes in serotonergic activity.

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standard teleost Ringer’s solution (0.20% NaHCO3 in 0.6% NaCl solution). At the beginning of each experiment aliquots of the appropriate concentrations were made by serial dilution of the stock in Ringer’s solution. A fresh aliquot was used on each day of testing. Both the stock solutions and aliquots were stored frozen (⫺20°C) when not in use. To test the effects of the DMSO vehicle on control behavior, in Experiment 4 a modified Ringer’s solution was made by diluting DMSO 1:10 in 1.1⫻ Ringer’s solution. In addition, CRH (500 ng) was dissolved in this modified Ringer’s solution to test the effect of DMSO on induced hyperactivity. Administration procedure Fish were netted from the holding tank and anesthetized (5–7 min in 50 mg L⫺1 tricaine methanesulfonate buffered with 125 mg L⫺1 NaHCO3). Chemicals were administered following a procedure described previously (Clements et al., 2001). In summary, injections were performed midline, immediately behind the pineal gland into the third ventricle. The needle was inserted to a depth sufficient to penetrate the cartilaginous brain casing and enter the third ventricle. The total injection volume was 1 ␮l. A preliminary trial established the accuracy of this procedure as approximately 85%. Behavioral testing

General methods Fish Spring chinook salmon parr (8 –10 months old and 109.8 ⫾ 1.4 mm in length) (Willamette stock) of mixed sex were held under ambient photoperiod in a 336-L circular tank at Oregon State University’s Fish Performance and Genetics Laboratory. Flow through water (12°C) was supplied from a well. Fish were fed twice daily with semi-moist pellet (BioOregon). All experiments were conducted between August and October 2000. Animals were treated in accordance with the principles and procedures of the Laboratory Animal Resource Center at OSU. All manipulations in this manuscript were approved by the LARC prior to experimentation. Chemicals Ovine CRH, fluoxetine (5-HT selective reuptake inhibitor), and NAN-190 (5-HT1A receptor antagonist) were obtained from Sigma Chemical Co. (St. Louis, MO). Due to the insoluble nature of NAN-190 it was first dissolved in a 1:10 solution of DMSO and then further diluted 1:10 in 1.1⫻ teleost Ringer’s solution (0.22% NaHCO3 in 0.66% NaCl solution) to maintain the appropriate molality of the final solution. The remaining chemicals were dissolved in

Fish were injected and tested individually. The assignment of treatments and the arena for testing were random. Following the injection, each fish was transferred to the testing arena and placed in a dark perforated plastic container for recovery. Fifteen minutes later the container was removed and the fish was able to swim freely in the arena. The testing arena consisted of a light blue fiberglass tank with inside dimensions of 965 ⫻ 965 ⫻ 609 mm. Two identical arenas were used. Each arena was filled with well water to a depth of 10 cm. The water was replaced every hour to maintain the temperature within 0.5°C of the holding tanks. Each arena was lit by two 100-W incandescent bulbs mounted 2.28 m above the water surface. During the tests, activity was monitored from above by 8-mm videocameras for a 10-min period beginning at the time of release. During analysis of locomotor activity the tank was divided into 36 equal segments by superimposing a grid onto the recorded image. Activity was then quantified by counting the number of line crossings during the 10-min period. Line crossings were recorded when the fish was actively swimming/gliding in a forward direction. Stereotyped circling behavior was not recorded as activity. Statistical analysis All data were analyzed using nonparametric methods because of unequal variances between treatments. Group

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differences were analyzed using a Kruskal–Wallis test. Treatment differences were analyzed using Dunn’s multiple range post test. Only planned comparisons were considered in the post tests. Differences between days within a treatment and differences between the saline and CRH treatments and their respective DMSO controls in Experiment 4 were analyzed using the Mann–Whitney U test.

Specific methods and results Experiment 1: dose–response study: the effect of acute treatment with fluoxetine To determine if enhancing endogenous serotonergic activity increases locomotor activity, fish were given an ICV injection of either saline or fluoxetine (10, 100, or 1000 ng). Fifteen fish were injected in each treatment group. The experiment was conducted over 2 days. There was no difference in activity levels within any of the treatment groups over the 2 days; therefore, we combined data within each treatment. ICV injections of fluoxetine had no effect on locomotor activity at any of the three doses. Mean levels of locomotor activity (⫾ SEM) were 129 ⫾ 39, 119 ⫾ 39, 146 ⫾ 33, and 72 ⫾ 18 for the saline controls, 10, 100, and 1000 ng treatments, respectively. Experiment 2: interaction between CRH and fluoxetine To determine if the effect of CRH on locomotor activity was facilitated by serotonergic activity, fish were injected ICV with saline, CRH (500 ng), or CRH (500 ng) and fluoxetine (10 or 100 ng) concurrently. Fifteen fish were injected in each treatment group. The experiment was conducted over 2 days. There were no differences in activity levels within any of the treatment groups over the 2 days; therefore, we combined data within each treatment. In agreement with previous results an ICV injection of CRH stimulated locomotor activity (P ⬍ 0.05) (Fig. 1). The concurrent administration of fluoxetine (100 ng) with CRH significantly potentiated the effect compared to CRH alone (P ⬍ 0.001). Experiment 3: dose–response study: the effect of acute treatment with NAN-190 To further investigate the role of serotonergic activity in the control of locomotor activity, fish were injected ICV with either saline or the 5-HT1A receptor antagonist NAN190 (10, 100, or 1000 ng). Fifteen fish were injected in each treatment group. The experiment was conducted on a single day. ICV injections of NAN-190 had no effect on locomotor activity at any of the three doses. Mean levels of locomotor activity (⫾ SEM) were 41 ⫾ 9, 39 ⫾ 7, 40 ⫾ 7, and 47 ⫾ 13 for the saline controls, 10, 100, and 1000 ng treatments, respectively.

Fig. 1. Locomotor activity (means ⫾ 1 SEM) in juvenile chinook salmon following ICV injections of saline, CRH (500 ng) or a combination of CRH (500 ng) and fluoxetine (10 or 100 ng). Locomotor activity was quantified by placing the fish into a square tank that was divided into 36 equal quadrants. Each column represents the mean number of line crossings over a 10 min period starting 15 min post-injection. Columns that share a common superscript are not different (P ⬎ 0.05, Dunn’s multiple range test). n ⫽ 15 for all treatments.

Experiment 4: interaction between CRH and NAN-190 To determine the effect of partially inhibiting serotonergic activity on CRH-induced locomotor activity, fish were injected ICV with saline, CRH (500 ng), or CRH (500 ng) and NAN-190 (10, 100, or 500 ng) concurrently. The effect of the DMSO vehicle on locomotor behavior was also evaluated. Saline controls were compared to fish injected ICV with the modified Ringer’s solution containing DMSO. In addition, the effect of DMSO on induced hyperactivity was determined by comparing the treatment group given CRH dissolved in standard Ringer’s solution to a second group given CRH dissolved in the modified Ringer’s solution. Fifteen fish were injected in each treatment group. The experiment was conducted over 2 days. There was no difference in activity levels within any of the treatment groups over the 2 days; therefore we combined data in each treatment. DMSO had no effect on control or CRH-induced levels of activity. Therefore, all further comparisons were made to the original saline and CRH treatment groups that did not receive DMSO. ICV injections of CRH stimulated locomotor activity (P ⬍ 0.001) (Fig. 2). The concurrent administration of NAN190 with CRH tended to attenuate the effect of CRH alone, but the reduction was only significant at the highest dose (500 ng NAN-190) (P ⬍ 0.001).

Discussion Previously we have shown that the central administration of CRH causes increased locomotor activity in teleosts

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Fig. 2. Locomotor activity (means ⫾ 1 SEM) in juvenile chinook salmon following ICV injections of saline, a modified Ringer’s solution containing DMSO (DMSO), CRH (500 ng), CRH dissolved in the modified Ringer’s solution (CRH 500 ng/DMSO), or a combination of CRH (500 ng) and NAN-190 (10, 100, or 500 ng). Locomotor activity was measured as described in Fig. 1. Saline and CRH (500 ng) were compared to their respective treatments with DMSO. Columns that share a common symbol are not different (P ⬎ 0.05, Mann–Whitney U test). Saline, CRH (500 ng), and CRH/NAN-190 (10, 100, or 500 ng) were compared. Columns that share a common superscript are not different (P ⬎ 0.05, Dunn’s multiple range test). n ⫽ 15 for all treatments.

(Clements et al., 2001). The current study provides the first evidence that this action may be mediated by the activation of the serotonergic system. Acute administration of fluoxetine into the CNS clearly potentiated the effect of exogenous CRH on locomotor activity. This response is robust and repeatable, having been observed during three independent experiments between 1999 and 2001. In vitro experiments suggest that serotonin stimulates the release of CRH (Jones et al., 1976; Tizabi and Calogero, 1992). Therefore, one hypothesis is that the increase in locomotor activity is due to the release of CRH following stimulation by serotonin. However, if this were the case, injections of fluoxetine alone should cause an increase in locomotor activity. By itself fluoxetine had no significant effect on locomotor compared to control fish; therefore, a more likely explanation is that the increased activity is due to the stimulation of the serotonergic system by CRH. This mechanism has been proposed for other vertebrates by Lowry et al. (2000), who suggest that during stress serotonergic neurotransmission in the mesolimbocortical system is increased by the direct action of CRH on specific subpopulations of serotonergic neurons within the caudal dorsal raphe nucleus, a region that is densely innervated by CRH neurons (Kirby et al., 2000). Kirby et al. (2000) also suggest that the topographical organization of the CRH fibers in the dorsal raphe nucleus (DRN) would allow specific stimuli to differentially alter 5-HT release in the DRN by activation of selective CRH afferents. This is supported by earlier work in T. granulosa showing that injections of CRH cause a rapid increase in the firing rate of neurons in the raphe and adjacent reticular

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region of the rostral medulla (Lowry et al., 1996). However, both Price et al. (1998) and Kirby et al. (2000) found that CRH has predominately inhibitory effects on serotonergic activity in the DRN. Again, however, this may relate to differences in the population of cells that were examined in the three studies, as suggested by Lowry et al. (2000), who hypothesize that mesolimbocortical and mesostriatal serotonergic systems are differentially regulated by CRH. In agreement with this are the results of Isogawa et al. (2000), who reported that intraperitoneal (IP) administration of a CRH 1 receptor antagonist reduced 5-HT concentrations in the hippocampus, but not the frontal cortex. More recently, Lowry et al. (2001) reported that peripheral injections of corticosterone resulted in an increase in 5-HT concentrations in the dorsomedial hypothalamus. The authors suggest that the increase may be due to innervation from serotonergic cells located in the median raphe nucleus and the DRN. Interestingly, though, central administration of CRH did not alter levels of 5-HT in any of the regions measured. The link between CRH and serotonin in telelosts was previously examined by DePedro et al. (1998) in a study examining feeding behavior. Their results suggest that CRH mediates, in part, the inhibitory effect of serotonin on feeding behavior in goldfish (C. auratus). Based partly on the finding that administration of the CRH antagonist results in increased hypothalamic content of 5-HT but has no effect on hypothalamic 5-HIAA, the authors hypothesize that 5-HT activates CRH neurons which then inhibit serotonergic neurotransmission. Although this mechanism differs from the one proposed for locomotor control, most likely due to the different populations of neurons involved, it does demonstrate that CRH and 5-HT interact to control a variety of behaviors in teleosts as well as mammals. Jacobs and Fornal (1999) proposed that the actions of 5-HT are most closely linked to motor activity and autonomic regulation. In this regard our current results are in agreement, showing that 5-HT is involved in the central control of locomotor activity. Jacobs and Fornal (1999) further suggest that serotonergic activity is a general correlate of motor activity, but is not significantly perturbed in response to stressful stimuli. Our current results do not support this hypothesis, as one would expect a general increase in activity following fluoxetine treatment if this were the case. Given that the addition of CRH is required to elicit the locomotor-stimulating effect, and the well-known involvement of CRH in the stress response, our results align more closely with the hypothesis of Lowry et al. (2000) that there are topographically organized subpopulations of serotonergic neurons that are dedicated to specific functions associated with the stress response. Furthermore, the results of Overli et al. (2001) suggest that selection for stress responsiveness in rainbow trout also leads to changes in the brain serotonergic systems. This finding lends further support to the notion that specific populations of serotonergic neurons are involved in output during the stress response. The differences in interpretation may lie in the absence of

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discrimination between specific subpopulations of DRN serotonergic cells in earlier studies (Jacobs and Fornal, 1995; Jacobs et al., 1999). Behavioral investigations into the role of 5-HT in locomotor activity are often contradictory in vertebrates, with either excitatory or inhibitory effects being reported. For example, Ferrer and Artigas (1994) reported that an IP injection of the monoamine oxidase inhibitor tranylcypromine significantly increased locomotor activity as well as increasing concentrations of 5-HT in the DRN and prefrontal cortex (PFC). Similarly, Mignon and Wolf (2002) recently showed that treatment with a 5-HT agonist significantly enhanced locomotor activity. In teleosts the literature is also divided; Fingerman (1976) and Winberg et al. (1993) suggested that increased serotonergic activity is associated with reduced locomotor activity in fish. Similarly, Overli et al. (1998) found that subordinate fish had increased serotonergic activity but decreased locomotor output as measured by swimming speed. Despite the fact that these studies do not correspond with our current results, it does demonstrate that serotonin and locomotor activity change concurrently, and again, the differences are most likely due to the brain regions examined. For example, Overli et al. (1998) determined tissue concentrations of 5-HT and 5-HIAA in the hypothalamus and brain stem and so would not distinguish changes in the specific population of cells within the DRN that is the proposed mechanism for action in the current study. In contrast, our results and those of Genot (1984) suggest that serotonin can also facilitate increases in activity. Interestingly, Overli et al. (2002) recently reported that rainbow trout (Oncorhynchus mykiss) selected for high stress responsiveness (HR) had significantly higher levels of locomotor activity when challenged with a conspecific intruder compared to trout selected for low stress responsiveness (LR). Earlier Overli et al. (2001) also showed that HR fish had increased 5-HT concentrations in the brain stem relative to LR fish. In light of this it would be interesting to compare the responses of these two lines to various CRH/5-HT treatments to determine the mechanics of this difference. The apparent contradiction for both teleosts and mammals may arise due to experimental differences in the manipulation of serotonergic activity. Depletion of serotonin within the CNS is often associated with increased levels of motor activity in mammals (Bradford, 1986), suggesting that serotonin is likely to be inhibitory at a macroscopic level with respect to locomotor behavior. However, based on recent studies it is unlikely that such gross scale manipulation would reveal the true nature of the involvement of 5-HT in locomotor activity control. Increases in synaptic levels of serotonin, or stimulation of postsynaptic serotonin receptors, can induce hyperactivity in rats and mice (GrahameSmith, 1971). This is in agreement with our results that demonstrated that acute treatment with fluoxetine potentiated the effect of CRH. Other studies have also shown marked differences in the behavioral response to microin-

jections of specific brain regions. For example, injection of a 5-HT1A agonist into the median raphe induced hyperactivity, whereas injection into the dorsal raphe produced dose-dependent decreases in motor activity (Hillegart and Hjorth, 1989; Hillegart et al., 1989). This result is also in agreement with our finding that injection of a 5-HT1A antagonist blocks the stimulatory effect of CRH. Takahashi et al. (2000) investigated the effect of various 5-HT manipulations within the striatum, hippocampus, and PFC cortex on locomotor activity in rats. Their results suggest that only the hippocampus and PFC are involved in the locomotorstimulating effects of 5-HT. However, even within these two areas there were differences in the nature of the hyperactivity. The results of this study are, however, in agreement with the hypothesis that the serotonergic neurons in the median raphe are involved in the control of locomotor activity. Fluoxetine was used in our study to enhance serotonergic activity; however, the mechanism by which acute treatment with fluoxetine exerts its serotonergic effect is not clear. Baxter et al. (2001) argue that acute administration of fluoxetine would decrease forebrain flux of 5-HT. In contrast Malagie et al. (1995) have shown that the acute administration of fluoxetine increased extracellular levels of 5-HT in the raphe nucleus of rats. These seemingly contradictory theories may be reconciled when examined in light of the work by Bel and Artigas (1992), who showed that another SSRI increased extracellular 5-HT in the raphe but not the frontal cortex. Furthermore, the work of de Montigny and Aghajanian (1978) and Blier et al. (1988) suggests that an acute increase in 5-HT in the terminal area might lead to “overactivation” of somatodendritic and/or terminal autoreceptors, which in turn might lead to compensatory reduced 5-HT neuronal firing. More recently, Belzung et al. (2001) examined the mechanism by which acute treatment with fluoxetine exerts its effect in a “free exploration” test. Their results suggest that dopaminergic mechanisms may underlie, at least in part, the behavioral effects (anxiogenic-like, locomotor impairment) of fluoxetine, whereas 5-HT1A and 5-HT2 may not be involved primarily in these effects. In contrast Bagdy et al. (2001) demonstrated that the 5-HT1A receptor was involved in facilitating the effect of fluoxetine on locomotor activity. In the latter paper, fluoxetine resulted in decreased activity. Although these findings and our own are not in complete agreement, it is important to note the methodological differences between the experiments, particularly given the known biphasic effects of many neuroactive substances in familiar vs unfamiliar environments and the species-specific effects of fluoxetine on locomotor activity (Brocco et al., 2002). In the current study we hypothesize that fluoxetine is most likely working within the DRN to enhance 5-HT levels, although we cannot rule out the possibility that it works directly on forebrain structures. The possible involvement of the 5-HT1A receptor in the mechanism is also an avenue for future investigation. Our results do suggest that the stimulatory effect of CRH

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may be mediated by a 5-HT1A type receptor in teleost fish. Injections of the 5-HT1A antagonist NAN-190 significantly attenuated the CRH-induced increase in locomotor activity in juvenile chinook. Similarly, we have found that longterm treatment with fluoxetine attenuates the locomotor response of control animals and prevents the increase in locomotor activity observed following the central administration of CRH (Clements et al., in preparation). This may be due to the depletion of 5-HT within the brain; however, it may also be due to an effect on the 5-HT receptors. Previous studies have shown that chronic fluoxetine treatment results in the desensitization of the 5-HT1A receptor (Berlin et al., 1998; Raap et al., 1999). The involvement of the 5-HT1A receptor in controlling locomotor activity has been proposed previously for mammals (Mignon and Wolf, 2002; O’Neill and Sanger, 1999), and amphibians (Wedderburn and Sillar, 1994) and may be due to stimulation of noradrenaline (Suwabe et al., 2000). Alternately, there is evidence that the 5-HT1A receptor may be involved in behavioral sensitization during stress (Grahn et al., 1999). Lowry et al. (2000) have suggested that CRH may contribute to this sensitization, which may explain the exaggerated locomotor response in animals that are given exogenous CRH. In the current study fish were taken from a rearing tank, anesthetized, and injected, a procedure that has been shown previously to be stressful, but does not alter the behavior of fish relative to uninjected or unanesthetized fish (Clements et al., 2001). It is not clear whether the arenas that the fish are tested in represents a novel or unfamiliar environment, and this represents an area for future research to determine whether the response to CRH varies with the environment as in mammals (Britton et al., 1982; Koob et al., 1993). The effect of prior stressors on the physiological/behavioral responses to CRH/5-HT suggests that this system may be important in behavioral adaptation and conditioned fear responses. Lowry et al. (2000) showed that repeated restraint stress and isolation housing for 5 days enhanced the serotonergic response to CRH. Similarly Pelton et al. (1997) showed that repeated stress enhanced the effect of CRH to subsequent stressors. Conversely, Price et al. (2002) report that prior exposure to a stressor reduced the ability of CRH to modify serotonergic activity within the lateral septum, and further, prior stressors attenuated the discharge of neurons in the dorsal raphe. In the current experiments it is unlikely that the handling prior to testing was sufficient to observe any sensitization; however, it is possible that this may explain some of the differences in absolute levels of locomotor activity in control animals between experiments. All fish in an experiment were taken from a single tank so prior differences in the history of tanks may result in sensitization of fish in some tanks but not others and thus an enhanced response. Based on these results we speculate that the increases in CRH activity during stress (Chappell et al., 1996; Lederis et al., 1994; Rivier and Plotsky, 1986) are responsible, in part,

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for the changes in serotonergic activity observed following a stressor (Winberg et al., 1993; Winberg and Nilsson, 1993). This link was proposed by Lowry et al. (2000) and is supported by neuroanatomical (Cummings et al., 1983), behavioral (Lazosky and Britten, 1991), and physiological studies (Singh et al., 1992). In fish CRH and serotonin may regulate both locomotor behavior and habitat choice in response to a stressor. Such a system would allow for coordinated physiological and behavioral responses. It would now be most interesting to determine whether CRH and serotonin interact via a direct neuronal mechanism. In summary, the current study provides evidence to support the hypothesis that the stimulatory effect of CRH on locomotor activity is due, in part at least, to interactions with the serotonergic system.

Acknowledgments We thank Rob Chitwood for his technical assistance. The Animal Care and Use Committee at OSU approved all manipulations in this paper.

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