Corticotropin-Releasing Factor Receptors Modulate Oxytocin Release in the Dorsolateral Bed Nucleus of the Stria Terminalis (BNST) in Male Rats

Abstract

The neuropeptide oxytocin (OT) plays an important role in the regulation of social and anxiety-like behavior. Our previous studies have shown that OT neurons send projections from the hypothalamus to the dorsolateral bed nucleus of the stria terminalis (BNST_dl), a forebrain region critically involved in the modulation of anxiety-like behavior. Importantly, these OT terminals in the BNST_dl express presynaptic corticotropin releasing factor (CRF) receptor type 2 (CRFR2). This suggests that CRFR2 might be involved in the modulation of OT release. To test this hypothesis, we measured OT content in microdialysates collected from the BNST_dl of freely-moving male Sprague-Dawley rats following the administration of a selective CRFR2 agonist (Urocortin 3) or antagonist (Astressin 2B, As2B). To determine if type 1 CRF receptors (CRFR1) are also involved, we used selective CRFR1 antagonist (NBI35965) as well as CRF, a putative ligand of both CRFR1 and CRFR2. All compounds were delivered directly into the BNST_dl via reverse dialysis. OT content in the microdialysates was measured with highly sensitive and selective radioimmunoassay. Blocking CRFR2 with As2B caused an increase in OT content in BNST_dl microdialysates, whereas CRFR2 activation by Urocortin 3 did not have an effect. The As2B-induced increase in OT release was blocked by application of the CRFR1 antagonist demonstrating that the effect was dependent on CRFR1 transmission. Interestingly, CRF alone caused a delayed increase in OT content in BNST_dl microdialysates, which was dependent on CRF2 but not CRF1 receptors. Our results suggest that members of the CRF peptide family modulate OT release in the BNST_dl via a fine-tuned mechanism that involves both CRFR1 and CRFR2. Further exploration of mechanisms by which endogenous OT system is modulated by CRF peptide family is needed to better understand the role of these neuropeptides in the regulation of anxiety and the stress response.

Keywords: BNST; CRF; bed nucleus of the stria terminalis; microdialysis; oxytocin; release; urocortin.

Figure 1

Experimental timeline (upper panel) and representative brain sections with a track of microdialysis probe targeting BNST_dl (bottom panel). Upon completion of the microdialysis experiments the probes were perfused with Chicago Sky Blue 6B dye. All extracted brains were sliced and all BNST sections were photographed to confirm proper placement of the probe. (A) Only rats with cannula tip located in the BNST_dl (Bregma + 0.10 mm to Bregma − 0.36 mm) have been included in the analysis. Modified from Paxinos and Watson (2009). (B) An example of confirmed cannula location in the BNST_dl, which met the following criteria: above the anterior commissure (ac), below the lateral ventricle and medially to the internal capsule as indicated by the arrow (included in the analysis). (C) An example of misplaced cannula location with cannula track too lateral to the BNST_dl as indicated by the arrow (excluded from the analysis). (D) An example of unconfirmed cannula location due to inability to see the tip of the cannula as indicated by the arrow (excluded from the analysis).

Figure 2

CRFR2 blockade increases OT content in BNST_dl microdialysates. OT content in BNST_dl microdialysates is expressed as MEAN ± SEM of OT (pg) in 100 μl microdialysis samples for ACSF (A) and As2B (B) treated groups. Baseline samples were collected in 30 min intervals before a drug delivery. (A) Perfusion of ACSF alone did not affect OT content in BNST_dl microdialysates. (B) There was a significant main treatment effect in rats perfused with As2B (one-way ANOVA), and post-hoc analysis showed significantly increased OT content in the first sample collected after drug infusion (30 min, ^*P < 0.05) in comparison to pre-treatment OT content (Bonferroni's multiple comparisons).

Figure 3

Manipulation of CRFR2 affects OT content in BNST_dl microdialysates. OT content in the microdialysates collected at 30 min intervals is expressed as percent change from the baseline. Data is expressed as mean ± SEM. Although CRFR2 activation by Ucn3 did not significantly affect OT release (n = 7, olive, closed square), an increase in OT release at 30 and 90 min was induced by intra-BNST_dl application of the CRFR2 antagonist, As2B (n = 14, purple, closed circle) in comparison to vehicle (ACSF) group (n = 19, black, closed triangle, P < 0.01). ^**P < 0.01, ^*P < 0.05, two-way ANOVA with repeated measures; Bonferroni's multiple comparisons test.

Figure 4

The effect of CRFR2 antagonist (As2B) on OT content in BNST_dl microdialysates is modulated by CRFR1 manipulation. The effect of As2B on OT release at 30 min was occluded by intra-BNST_dl application of CRFR1 antagonist NBI 35965 (n = 6, teal, open circle, P < 0.05) as well as application of CRF (n = 7, blue diamond, P < 0.01). The delayed stimulatory effect of As2B at 90 min was not affected by CRF or NBI 35965 application (P > 0.05). ^**P < 0.01, ^*P < 0.05, two-way ANOVA with repeated measures.

Figure 5

OT content in BNST_dl microdialysates is modulated by CRF. CRF caused a significant increase in OT release at 90 min (n = 9, red triangle, P < 0.001 in comparison to ACSF group). CRFR1 blockade by NBI 35965 did not significantly affect the effect of CRF on OT release (n = 8, cyan, open square, P > 0.05). However, blocking CRFR2 with As2B abolished the effect of CRF on OT release (n = 7, blue diamond, P < 0.01). ^***P < 0.001, ^**P < 0.01 two-way ANOVA with repeated measures.