The effect of lateral septum corticotropin-releasing factor receptor 2 activation on anxiety is modulated by stress

Abstract

Corticotropin-releasing factor (CRF), a 41 amino acid peptide, mediates endocrine, autonomic, and behavioral responses to stress. Whereas the CRF1 receptor appears to contribute to anxiety associated with stress, the role of the CRF2 receptor remains unclear and may depend on drug dose, brain location, or testing environment. Results involving treatments with selective CRF2 receptor agonists or antagonists and the behavior of CRF2 receptor knock-out mice suggest both anxiogenic and anxiolytic effects of CRF2 receptor activation. The present study tested the hypothesis that the effect of CRF2 receptor activation on anxiety depends on the stress level of the animal. The selective CRF2 receptor agonist urocortin 2 was infused into the lateral septum of mice under low- or high-stress (30 min of immobilization) testing conditions, and then behavior in the light-dark box, open-field, and novel-object tests was assessed. In the low-stress environment, 240 pmol of septal urocortin 2 increased anxiety, but lower doses (0.48, 4.8, and 48 pmol) did not have consistent effects. However, in the high-stress condition, 48 pmol of septal urocortin 2 significantly increased anxiety compared with control in wild-type but not CRF2 receptor knock-out mice in the light-dark box. Septal administration of the relatively selective CRF2 antagonist astressin-2B, but not the CRF1-selective antagonist antalarmin, blocked the anxiogenic effects of urocortin 2. Urocortin 2 infusion into the medial septum or lateral ventricle did not affect anxiety measures. These results indicate that the effect of septal CRF2 receptor activation on anxiety is dependent on stress level.

Figure 1.

Histological verification of the cannula injector placements. Cannula placement was verified after each experiment with the infusion of methylene blue dye as shown in the lateral (b) and medial (c) septum. Scale bars, 500 μm. a is an illustration of the septum showing successful cannula placements in the intermediate lateral septum (filled circles) and placements excluded from data analyses (open circles). d illustrates the central amygdala region targeted for antalarmin infusion, with an example section shown in e. LS, Lateral septum; MS, medial septum; LV, lateral ventricle; cc, corpus callosum; aca, anterior commissure, IC internal capsule; OP, optic tract; CEA, central nucleus of the amygdala; BLA, basolateral amygdala.

Figure 2.

Septal urocortin 2 increased anxiety. Effect of bilateral septal infusion of urocortin 2 (vehicle or 0.48, 4.4, 48, or 240 pmol) into ICR mice on the following: a, latency to enter the light side of light–dark box (seconds); b, total time spent in the light side of light–dark box (seconds); c, locomotion in open field (total square entries); d, percentage of time in the center of the open field; e, locomotion in the open field with a novel object (total square entries); and f, percentage of time spent in center with novel object. Transitions in the light–dark box and percentage of center entries in the open-field and novel-object tests not shown. The high dose of 240 pmol increased anxiety-like behavior in the light–dark box and novel-object tests compared with vehicle, with a trend toward increased anxiety-like behavior in the open-field test, but did not alter locomotion. Lower doses of 0.48 and 48 pmol did not significantly alter anxiety. Urocortin 2 at 4.8 pmol increased anxiety-like behavior in the novel-object test but had no effect in the light–dark box or open-field tests. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01.

Figure 3.

Intracerebroventricular urocortin 2 decreased motor activity at high doses. Effect of urocortin 2 infusion into the lateral ventricle of ICR mice (vehicle or 24, 120, 240, or 1200 pmol) on the following: a, latency to enter the light side of the light–dark box (seconds); b, total time spent in the light side of the light–dark box (seconds); c, locomotion in the open field (total square entries); d, percentage time in the center of the open field; e, locomotion in the open field with a novel object (total square entries); and f, percentage time spent in the center with a novel object. Transitions in the light–dark box and percentage center entries in open-field and novel-object tests are not shown. The highest dose of intracerebroventricular urocortin 2 (1200 pmol) significantly reduced locomotion in the open-field and novel-object tests compared with vehicle, but the peptide had no effect on anxiety or locomotion at lower doses. Data are presented as mean ± SEM. *p < 0.05.

Figure 4.

Effect of septal urocortin 2 on anxiety is dependent on stress. Effect of septal urocortin 2 (48 pmol) in ICR mice treated with no stress or 30 min of immobilization stress on the following: a, latency to enter the light side of the light–dark box (seconds); b, total time spent in the light side of the light–dark box (seconds); c, locomotion in the open field (total square entries); d, percentage time in the center of the open field; e, locomotion in the open field with a novel object (total square entries; and f, percentage time spent in the center with a novel object. Transitions in the light–dark box and percentage center entries in open-field and novel-object tests are not shown. This dose of urocortin 2 had no effect on anxiety-like behavior in the low-stress condition but significantly increased anxiety-like behavior compared with vehicle in the light–dark box, open-field, and novel-object tests when combined with 30 min of immobilization stress. There was no difference in locomotion between vehicle- and peptide-treated mice in the stress condition. Data are presented as mean ± SEM. **p < 0.01;***p < 0.001.

Figure 5.

Septal urocortin 2 increased anxiety when combined with stress in CRF₂ receptor wild-type mice on a mixed C57BL/6 × 129 background. Effect of septal urocortin 2 (vehicle or 0.48, 4.8, or 48 pmol) in CRF₂ receptor wild-type mice treated with no stress or 30 min of immobilization stress on the following: a, latency to enter the light side of the light–dark box (seconds); b, total time spent in the light side of the light–dark box (seconds); c, locomotion in the open field (total square entries); d, percentage of time in the center of the open field; e, locomotion in the open field with a novel object (total square entries); and f, percentage of time spent in the center with a novel object. Transitions in the light–dark box and percentage of center entries in the open-field and novel-object tests are not shown. Urocortin 2 did not affect anxiety-like behavior in the no-stress condition at any dose tested. In the stress condition, 48 pmol of urocortin 2 increased anxiety-like behavior in the light–dark box and novel-object tests; the 4.8 pmol dose increased anxiety in the novel-object test in the stress condition but not the no-stress condition. Data are presented as mean ± SEM. ***p < 0.001.

Figure 6.

No effect of septal urocortin 2 in CRF₂ receptor knock-out mice. Effect of septal urocortin 2 (48 pmol) in CRF₂ receptor wild-type (WT) and knock-out (KO) mice treated with no stress or 30 min of immobilization stress on the following: a, latency to enter the light side of the light–dark box (seconds); b, total time spent in the light side of the light–dark box (seconds); c, locomotion in the open field (total square entries); d, percentage of time in the center of the open field; e, locomotion in the open field with a novel object (total square entries); and f, percentage of time spent in the center with a novel object. Transitions in the light–dark box and percentage of center entries in the open-field and novel-object tests are not shown. In wild-type mice, urocortin 2 had no effect on anxiety-related behavior in the no-stress condition but increased anxiety-related behavior in the light–dark box, open-field, and novel-object tests when combined with immobilization stress. Urocortin 2 had no effect in CRF₂ receptor knock-out mice in the light–dark box (a, b). CRF₂ receptor knock-out mice showed increased anxiety compared with wild-type, spending less time in the center of the open-field (d) and novel-object (f) tests, regardless of drug treatment. Knock-out mice treated with vehicle also exhibited greater latency to enter the light side of the light–dark box compared with vehicle-treated wild-type mice (a). Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7.

CRF₂ antagonist astressin-2B blocked the effect of urocortin 2 and stress on anxiety. Effect of septal CRF₂ receptor antagonist astressin-2B (24, 96, or 192 pmol) administered 10 min before urocortin 2 (48 pmol) in CRF₂ receptor wild-type mice treated with no stress or 30 min of immobilization stress on the following: a, latency to enter the light side of the light–dark box (seconds); b, total time spent in the light side of the light–dark box (seconds); c, locomotion in the open field (total square entries); d, percentage of time in the center of the open field; e, locomotion in the open field with a novel object (total square entries); and f, percentage of time spent in the center with a novel object. Transitions in the light–dark box and percentage of center entries in the open-field and novel-object tests are not shown. Astressin-2B decreased anxiety-like behavior in the light–dark box, open-field, and novel-object tests in the stress condition but had little effect in the no-stress condition. In the light–dark box, 24 and 96 pmol of astressin-2B decreased the anxiogenic effect of urocortin 2 in the stress condition but had no effect in vehicle-treated mice, whereas the 192 pmol dose reduced anxiety in all stressed mice, decreasing the latency to enter the light side of the light–dark box (a) and increasing time spent in the light side (b). In the open field, 24 pmol of astressin-2B decreased the anxiogenic effect of urocortin 2 but had no effect in vehicle-treated mice in the stress condition (d). In the novel-object test, 24 and 96 pmol of the antagonist inhibited the anxiogenic effect of urocortin 2 but had no significant effect in vehicle-treated mice (f), whereas 192 pmol decreased anxiety in both groups. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 8.

Septal administration of the CRF₁ antagonist antalarmin does not block the effect of urocortin 2. Effect of septal administration of the CRF₁ receptor antagonist antalarmin (vehicle, 264 pmol/100 ng, of 792 pmol/300 ng) on the anxiogenic effect of septal urocortin 2 (48 pmol) on the following: a, latency to enter the light side of the light–dark box (seconds); b, total time spent in the light side of the light–dark box (seconds); c, locomotion in the open field (total square entries); d, percentage of time in the center of the open field; e, locomotion in the open field with a novel object (total square entries); and f, percentage of time spent in the center with a novel object. Transitions in the light–dark box and percentage of center entries in the open-field and novel-object tests are not shown. Urocortin 2 significantly increased anxiety-like behavior in the light–dark box (a, b), open-field (d), and novel-object (f) tests in immobilization-stressed mice pretreated with septal antalarmin or vehicle. Antalarmin did not block the anxiogenic effect of urocortin 2.

Figure 9.

Administration of the CRF₁ antagonist antalarmin into the central amygdala reduced the anxiogenic effect of immobilization stress. Effect of administration of the CRF₁ receptor antagonist antalarmin into central amygdala (vehicle or 264 or 792 pmol) on the anxiogenic effect of 30 min of immobilization stress on the following: a, latency to enter the light side of the light–dark box (seconds); b, total time spent in the light side of the light–dark box (seconds); c, locomotion in the open field (total square entries); d, percentage time in the center of open field; e, locomotion in the open field with a novel object (total square entries); and f, percentage time spent in the center with a novel object. Transitions in the light–dark box and percentage center entries in the open-field and novel-object tests are not shown. Antalarmin at 792 pmol reduced the anxiogenic effect of immobilization stress in the light–dark box (a, b) and open field (d), with a trend toward decreased anxiety in the novel-object test (f).The lower dose of 264 pmol did not have a significant effect. Data are presented as mean ± SEM. **p < 0.01; ***p < 0.001.