Corticotropin releasing factor induces anxiogenic locomotion in trout and alters serotonergic and dopaminergic activity

Abstract

Corticotropin releasing factor (CRF) and serotonin (5-HT) are strongly linked to stress and anxiety in vertebrates. As a neuromodulator in the brain, CRF has anxiogenic properties often characterized by increased locomotion and stereotyped behavior in familiar environments. We hypothesized that expression of anxiogenic behavior in response to CRF will also be exhibited in a teleost fish. Rainbow trout were treated with intracerebroventricular (icv) injections of artificial cerebrospinal fluid (aCSF), 500 or 2000 ng ovine CRF, or not injected. Treatment with either dose of CRF elicited greater locomotion and pronounced head shaking behavior but did not influence water column position. Locomotor and head shaking behaviors may be analogous to the increased stereotypy evoked by icv CRF in rats and may reflect the expression of stress/anxiety behavior. Injection with either aCSF or CRF produced significant increases in plasma cortisol. The absence of behavioral changes in aCSF-injected fish suggests that the behavioral responses following CRF were not due to cortisol. Treatment with 2000 ng CRF significantly increased serotonin, 5-HIAA and dopamine concentrations in the subpallium and raphé and increased 5-HIAA in the preoptic hypothalamus (POA). Concurrent effects of CRF on central monoamines, locomotion and head shaking in trout suggest that anxiogenic properties of CRF are evolutionarily conserved. In addition, positive linear correlations between locomotion and serotonergic and dopaminergic function in the subpallium, POA and raphé nuclei suggest a locomotory function for these monoamines.

Figure 1

Photomicrographs of A sagittal view of the trout brain indicating the third ventricle injection site (arrows) for icv administration of CRF or aCSF; B coronal view (TEL = telencephalon, OT = optic tectum, HYP = hypothalamus, IIIv = third ventricle, CER = cerebellum, MED = medulla). C Mean plasma cortisol (F ± SEM) concentrations are influenced by icv injections of CRF or aCSF. Trout injected icv with 500 or 2000 ng CRF (hatched bars) had significantly elevated plasma F (* indicate significance; P < 0.05) concentrations, as did trout injected with artificial cerebrospinal fluid (aCSF, gray bars), as compared with uninjected controls (clear bars).

Figure 2

Time spent moving forward (in seconds ± SEM) before (clear bars) and after treatment (hatched bars) during the five minute observation periods. There were no differences in locomotion between groups prior to injection, and those groups that were not injected or injected icv with artificial cerebrospinal fluid (aCSF) did not change their forward movement significantly. However, trout inject icv with 500 or 2000 ng CRF had significantly elevated forward locomotion (* indicate significance; P < 0.05) compared with pretreatment measurements and with injected and uninjected controls.

Figure 3

Mean (± SEM) A serotonin (5-HT) and B serotonin catabolite 5-HIAA concentrations in the commissural (Vc) nuclei of the ventral area of the subpallial region of the telencephalon. The Vc nuclei constitute putative amygdalar/striatal regions in the teleost. Trout injected icv with 2000 ng CRF (hatched bars) had significantly more 5-HT (* indicates significance; P < 0.05) than those given 500 ng CRF, artificial cerebrospinal fluid (aCSF, gray bars) or uninjected controls (clear bars). Dose of CRF injected icv (0 = uninjected + aCSF controls, 500 ng, or 2000 ng) is positively correlated with C 5-HT (linear regression: r² = 0.38, P < 0.001) and D 5-HIAA (linear regression: r² = 0.17, P < 0.029) in the subpallium. No correlations between E 5-HT (linear regression: r² = 0.00, P > 0.95) or F 5-HIAA (linear regression: r² = 0.08, P > 0.18) concentrations in subpallium and locomotion exist in rainbow trout.

Figure 4

Mean A dopamine (DA ± SEM) concentrations in subpallium. Trout injected icv with 2000 ng CRF (hatched bars) had significantly more DA (* indicates significance; P < 0.05) than those given 500 ng CRF, artificial cerebrospinal fluid (aCSF, gray bars) or uninjected controls (clear bars). Dose of CRF injected icv (0 = uninjected + aCSF controls, 500 ng, or 2000 ng) is positively correlated with B DA (linear regression: r² = 0.26, P < 0.006) in the subpallium. In contrast to serotonergic parameters, a positive correlation between C DA (linear regression: r² = 0.26, P < 0.013) concentrations in Vc and locomotion in rainbow trout suggests that DA in the subpallium is involved in locomotion.

Figure 5

Mean A serotonin catabolite 5-hydroxyindoleacetic acid (5-HIAA ± SEM) concentrations in the hypothalamic preoptic area (POA). Trout injected icv with 2000 ng CRF (hatched bars) had significantly more 5-HIAA (* indicates significance; P < 0.05) than those given 500 ng CRF, artificial cerebrospinal fluid (aCSF, gray bars) or uninjected controls (clear bars). Dose of CRF injected icv (0 = uninjected + aCSF controls, 500 ng, or 2000 ng) is positively correlated with B 5-HIAA (linear regression: r² = 0.31, P < 0.002) concentration in the POA. A positive correlation also exists between C 5-HIAA (linear regression: r² = 0.24, P < 0.011) concentrations in POA and locomotion in rainbow trout.

Figure 6

Mean (± SEM) A serotonin (5-HT) and B 5-HIAA concentrations in the raphé nucleus of the rainbow trout brain. Trout injected icv with 2000 ng CRF (hatched bars) had significantly more 5-HT (* indicate significance; P < 0.05) than those given 500 ng CRF, artificial cerebrospinal fluid (aCSF, gray bars) or uninjected controls (clear bars). Dose of CRF injected icv (0 = uninjected + aCSF controls, 500 ng, or 2000 ng) is positively correlated with C 5-HT (linear regression: r² = 0.50, P < 0.0001) and D 5-HIAA (linear regression: r² = 0.45, P < 0.0007) in the trout subpallium. In addition, E 5-HT (linear regression: r² = 0.50, P < 0.0002) and F 5-HIAA (linear regression: r² = 0.55, P < 0.0001) concentrations in subpallium were positively correlated with locomotion in rainbow trout.

Figure 7

Mean A dopamine (DA ± SEM) concentrations in raphé. Trout injected icv with 2000 ng CRF (hatched bars) had significantly more DA (* indicates significance; P < 0.05) than those given 500 ng CRF, artificial cerebrospinal fluid (aCSF, gray bars) or uninjected controls (clear bars). Dose of CRF injected icv (0 = uninjected + aCSF controls, 500 ng, or 2000 ng) is positively correlated with B DA (linear regression: r² = 0.35, P < 0.001) concentration in the raphé. A positive correlation also exists between C DA (linear regression: r² = 0.26, P < 0.008) concentrations in raphé and locomotion in rainbow trout.