Importance of CRF receptor-mediated mechanisms of the bed nucleus of the stria terminalis in the processing of anxiety and pain

Abstract

Corticotropin-releasing factor (CRF)-mediated mechanisms in the bed nucleus of the stria terminalis (BNST) have a pivotal role in stress-induced anxiety and hyperalgesia. Although CRF is known to activate two receptor subtypes, CRF1 and CRF2, attempts to delineate the specific role of each subtype in modulating anxiety and nociception have been inconsistent. Here we test the hypothesis that CRF1 and CRF2 receptor activation in the anteriolateral BNST (BNSTAL) facilitates divergent mechanisms modulating comorbid anxiety and hyperalgesia. Microinfusions of the specific antagonists CP376395 and Astressin2B into the BNSTAL were used to investigate CRF1 and CRF2 receptor functions, respectively. We found that CRF1 and CRF2 receptors in the BNSTAL had opposing effects on exploratory behavior in the elevated plus-maze, somatic mechanical threshold, and the autonomic and endocrine response to stress. However, CRF1 or CRF2 receptor antagonism in the BNSTAL revealed complementary roles in facilitating the acoustic startle and visceromotor reflexes. Our results suggest that the net effect of CRF1 and CRF2 receptor activation in the BNSTAL is pathway-dependent and provides important insight into the CRF receptor-associated circuitry that likely underpins stress-induced pathologies.

Figure 1

Infusions are restricted to the anteriolateral bed nucleus of the stria terminalis (BNST_AL). Before being killed, animals were infused with horseradish peroxidase (HRP) to assess diffusion radius. (a) Post-mortem tissue slices were stained with cresyl violet to determine viability (left) and show minimal damage to the BNST. Negative control (middle), and the addition of DAB substrate (right), show that infusions were restricted to the BNST_AL and minor outlying areas. (b) The BNST receives inputs from regions associated with sensory and affect processing. To verify infusion accuracy, sections were collected from the regions that project to the BNST including the paraventricular nucleus (PVN), the central amygdala (CeA), medial nucleus (MDN), the periaqueductal grey (PAG), and nucleus accumbens (NTS). Positive staining was revealed in the CeA, PVN, and NTS. Minimal staining was seen in the MDN, and not visible in the PAG. (c) Brain atlas sections for coordinates (from top) bregma −0.12, −0.24, −0.36, respectively, depicting the localization of each cannula placement.

Figure 2

Repeated water avoidance stress (WAS) increases corticotropin-releasing factor (CRF) and CRF receptor expression. CRF expression was assessed using qRT-PCR, whereas CRF₁ and CRF₂ receptors were quantified using western blot analysis. Compared with animals that received SHAM stress, animals that were exposed to chronic WAS expressed higher levels of (a) CRF, (b) CRF₁ receptors, and (c) CRF₂ receptors in the anteriolateral bed nucleus of the stria terminalis (BNST_AL). (d) The ratio between CRF1 and CRF2 was higher following WAS compared with SHAM-treated animals. (e) The target location of the micropunches is illustrated. Data represent mean±SEM; *P<0.05, **P<0.01, and ***P<0.001 by Student's unpaired t-test; n=6/group.

Figure 3

Effect of corticotropin-releasing factor (CRF) receptor antagonists on baseline behavior. Animals implanted with bilateral cannulas localized to the anteriolateral bed nucleus of the stria terminalis (BNST_AL) received infusions of either vehicle (VEH; normal saline; n=5), the CRF₁ receptor antagonist CP376395 (10 mg/ml; n=6), or the CRF₂ receptor antagonist Astressin₂B (100 μM; n=6). Compared with VEH infusions, treatment with CP376395 had no effect on (a) the percent time spent exploring the open arms, (b) total distance traveled, or (c) somatic mechanical threshold. In contrast, infusion with Astressin₂B decreased open arm exploration without changing total distance traveled, and decreased the somatic mechanical threshold. (d) Both antagonists decreased the visceromotor reflex (VMR) relative to VEH treatment. Data represent mean±SEM; **P<0.01 and ***P<0.001 by one-way analysis of variance (ANOVA) or two-way ANOVA with repeated measures (ANOVA-RM) with Bonferonni post hoc analysis.

Figure 4

Effect of corticotropin-releasing factor (CRF) receptor antagonist following water avoidance stress (WAS). Animals were implanted with bilateral cannulas localized to the anteriolateral bed nucleus of the stria terminalis (BNST_AL) and exposed to the WAS protocol. Before behavioral assessment, the animals received infusions of either vehicle (VEH; normal saline; n=5), the CRF₁ receptor antagonist CP376395 (10 mg/ml; n=6), or the CRF₂ receptor antagonist Astressin₂B (100 μM; n=6). Compared with WAS-treated animals with VEH infusions, (a) antagonizing CRF₁ receptors increased percent time spent on the open arm. Blocking CRF₂ had no effect on the time spent exploring the open arm. (b) Neither CRF₁ nor CRF₂ receptor antagonists changed the total distance traveled compared with VEH control. (c) Following WAS, inhibiting CRF₁ receptors in the BNST_AL attenuated the decrease in somatic mechanical threshold induced by WAS compared with VEH infusions, but blocking CRF₂ receptors had no effect on threshold. (d) Both CRF₁ and CRF₂ decreased the number of abdominal contractions in response to colorectal distension. At the highest distension pressure, CRF₂ receptor was no longer significantly different from VEH. (e) Serum ACTH and (f) corticosteroid (CORT) output was quantified following 30 min of novel environment stress. Animals exposed to WAS and infused with CP376395 had decreased plasma ACTH and CORT compared with animals exposed to the WAS protocol and infused with VEH or Astressin₂B. Data represent mean±SEM; *P<0.05, **P<0.01, and ***P<0.001 by one-way analysis of variance (ANOVA) or two-way ANOVA with repeated measures (ANOVA-RM) with Bonferonni post hoc analysis.

Figure 5

Effect of water avoidance stress (WAS) on corticotropin-releasing factor (CRF) receptor antagonism. The data for animals exposed to the SHAM protocol and animals exposed to the WAS protocol were compared. Overall, there were significant effects of infusion, stress protocol, and significant interaction between the two for anxiety-like behavior on the (a) percent of time spent exploring the open arm of the EPM, (b) somatic mechanical threshold, and (c) the visceromotor reflex (VMR) to colorectal distension (CRD) analyzed at 60 mm Hg (see text for details). Data represent mean±SEM, *P<0.05, and ***P<0.001 compared with SHAM+vehicle (VEH) infusion, and ⁺P<0.05 and ⁺⁺⁺P<0.001 compared with WAS+indicated infusion by two-way analysis of variance (ANOVA) with Tukey's post hoc analysis.

Figure 6

Effect of corticotropin-releasing factor (CRF) receptor antagonists on acoustic startle reflex (ASR) and prepulse inhibition (PPI). Animals were implanted with bilateral cannulas localized to the anteriolateral bed nucleus of the stria terminalis (BNST_AL) and exposed to the water avoidance stress (WAS) protocol. Before behavioral assessment, the animals received infusions of either vehicle (VEH; normal saline; n=5), the CRF₁ receptor antagonist CP376395 (10 mg/ml; n=5), or the CRF₂ receptor antagonist Astressin₂B (100 μM; n=5). Following WAS, animals infused with CRF₁ receptor antagonists had (a) a decrease in startle amplitude and (b) increased prepulse inhibition compared with animals infused with VEH. Animals infused with CRF₂ receptor antagonists also had a decrease in startle amplitude, but to a lesser extent, and no change in prepulse inhibition. (c) Neither treatment had any effect on percent habituation. Data represent mean±SEM, *P<0.05, **P<0.01, and ***P<0.001 by one-way analysis of variance (ANOVA) or two-way ANOVA with repeated measures (ANOVA-RM) with Bonferonni post hoc analysis.