Bradycardic effects of microinjections of urocortin 3 into the nucleus ambiguus of the rat

Abstract

The presence of urocortin 3 (UCN3) and CRF2 receptors (CRF2R) has been demonstrated in brain tissue. Nucleus ambiguus (nAmb) is the predominant brain area providing parasympathetic innervation to the heart. On the basis of these reports, it was hypothesized that activation of CRF2Rs in the nAmb may elicit cardiac effects. Experiments were carried out in urethane-anesthetized, artificially ventilated, and adult male Wistar rats. Microinjections of l-glutamate (l-GLU, 5 mM) were used to identify the nAmb. Different concentrations of UCN3 (0.031, 0.062, 0.125, 0.25, and 0.5 mM) microinjected into the nAmb elicited decreases in heart rate (HR) (5.3 ± 1, 22 ± 3.3, 38 ± 4.9, 45.7 ± 2.7, and 27.3 ± 2.3 bpm, respectively). The volume of all microinjections was 30 nl. Blood pressure changes concomitant with decreases in HR were not observed. Bradycardia elicited by microinjections of UCN3 (0.25 mM; maximally effective concentration) into the nAmb was significantly (P < 0.05) attenuated by microinjections of selective CRF2R antagonists (K41498, 0.5 mM, and astressin 2B, 0.25 mM) at the same site. Bilateral vagotomy abolished the bradycardic responses to UCN3. These results indicated that activation of CRF2Rs in the nAmb by UCN3 elicited bradycardia, which was vagally mediated. UCNs have been reported to exert cardioprotective effects in heart failure and ischemia/reperfusion injury. In this situation, centrally induced bradycardia by UCN3 would be beneficial. The results of the present investigation provide a platform for future studies on the role of CRF2Rs in the nAmb in pathological states such as heart failure.

Fig. 1.

Concentration-response for urocortin 3 (UCN3) microinjections into the nAmb. The decreases in heart rate (HR) in response to microinjections of different concentrations of UCN3 into the nAmb elicited bradycardic responses. Maximum bradycardic responses were elicited by microinjections of 0.25 mM of UCN3. The volume of all microinjections was 30 nl. *P < 0.01; **P < 0.001.

Fig. 2.

Bar diagram showing the effect of bilateral vagotomy on l-glutamate (l-GLU) and UCN3 responses. Bilateral vagotomy (Vag-X) significantly (**P < 0.001) attenuated the bradycardic responses elicited by microinjections of l-GLU and UCN3 into the nAmb.

Fig. 3.

Tracing showing blockade of UCN3 responses in the nAmb by K41498. A: bradycardic responses to microinjection of l-GLU (5 mM). B: after allowing an interval of 5 min, the vehicle (artificial cerebrospinal fluid, aCSF) was microinjected at the same site; no significant changes were observed. C: UCN3 (0.25 mM) was microinjected at the same site; bradycardic responses were elicited. D: after an interval of 40 min to avoid tachyphylaxis to UCN3 responses, K41498 (a CRF2 receptor antagonist; 0.5 mM) was microinjected at the same site; the antagonist elicited no significant responses. E: a 2-min interval was allowed for the diffusion of the antagonist into the nAmb and UCN3 (0.25 mM) was again microinjected at the same site; the responses to UCN3 were blocked. F: responses to l-GLU (5 mM) were unaltered by K41498. PAP, pulsatile arterial pressure; MAP, mean arterial pressure; HR, heart rate.

Fig. 4.

Tracing showing blockade of UCN3 responses in the nAmb by astressin 2B. A: bradycardic responses to microinjection of l-GLU (5 mM). B: after an interval of 5 min, the vehicle (aCSF) was microinjected at the same site; no significant changes were observed. C: UCN3 (0.25 mM) was microinjected at the same site; bradycardic responses were elicited. D: after an interval of 40 min, astressin 2B (a CRF2R antagonist; 0.25 mM) was microinjected at the same site; the antagonist elicited no significant responses. E: UCN3 (0.25 mM) was again microinjected at the same site; after a 2-min interval; the responses to UCN3 were blocked. F: responses to l-GLU (5 mM) were unaltered by astressin 2B.

Fig. 5.

The effect of blockade of GABA, glycine, and ionotropic glutamate receptors in the nAmb on UCN3 responses. The bradycardic responses elicited by microinjections of UCN3 (0.25 mM) into the nAmb were significantly attenuated by microinjections of gabazine (0.01 mM) (A), strychnine (0.5 mM) (B), and combined microinjections of d-AP7 (5 mM) and NBQX (2 mM) into the nAmb (C). **P < 0.001. ***P < 0.0001.

Fig. 6.

Identification of cells immunoreactive for CRF2Rs and UCN3. A: cells immunoreactive for CRF2Rs in the nAmb area (white arrows). B: control in which the primary antibody for CRF2R was omitted from the protocol. C: control in which the primary antibody for CRF2R was preincubated with CRF2R peptide. D: UCN3-immunoreactive cells in the nAmb (white arrows). E: control in which the primary antibody for UCN3 was omitted from the protocol. F: control in which the primary antibody for UCN3 was preincubated with UCN3 control peptide. Calibration bar in each panel = 50 μm.

Fig. 7.

Histological verification of microinjection sites in nAmb. A: typical nAmb site marked with Lumafluor (30 nl); the site was located 0.24 rostral to the calamus scriptorius, 2.0 mm lateral to the midline, 2.2 mm deep from the medullary surface (arrow). B–H: drawings of coronal sections at levels 0.64 rostral (B) to 0.12 caudal (H) to the calamus scriptorius showing microinjection sites (solid circles). Each solid circle represents a microinjection site in the nAmb in one rat (n = 30). AP, area postrema; CC, central canal; nAmb, nucleus ambiguus; py, pyramid; 10N, dorsal motor nucleus of vagus; 12N, hypoglossal nucleus.