Pituitary adenylate cyclase-activating polypeptide: Postnatal development in multiple brain stem respiratory-related nuclei in the rat

Abstract

The pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in anterior pituitary hormone secretion, neurotransmission, and the control of breathing. Mice lacking PACAP die suddenly mainly in the 2^nd postnatal week, coinciding temporally with a critical period of respiratory development uncovered by our laboratory in the rat. The goal of the current study was to test our hypothesis that PACAP expression is reduced during the critical period in normal rats. We undertook immunohistochemistry and optical densitometry of PACAP (specifically PACAP38) in several brain stem respiratory-related nuclei of postnatal days P2-21 rats, and found that PACAP immunoreactivity was significantly reduced at P12 in the pre-Bötzinger complex, nucleus ambiguus, hypoglossal nucleus, and the ventrolateral subnucleus of the nucleus tractus solitarius. No changes were observed in the control, non-respiratory cuneate nucleus at P12. Results imply that the down-regulation of PACAP during normal postnatal development may contribute to the critical period of vulnerability, when the animals' response to hypoxia is at its weakest.

Keywords: Critical period; Hypoglossal nucleus; Immunohistochemistry; Nucleus ambiguus; PACAP; Pre-Bötzinger complex.

Figure 1.

PACAP-ir neurons and neuropil in the PBC (A), Amb (B), XII (C), NTS_VL (D), and CN (E) at P2 (A1 – E1), P10 (A2 – E2), P12 (A3 – E3), and P21 (A4 – E4). In the PBC, XII, and NTS_VL, PACAP immunoreactivity was relatively low at P2, markedly increased at P10, then significantly reduced at P12, followed by a rise and a plateau at P21. The level of immunoreactivity in Amb was relatively high from P2 to P11, but was significantly reduced at P12, followed by a rise and a plateau at P21. PACAP expression in the CN was relatively low at P2, followed by a gradual climb to a peak level at P10, then remained constant until P21. In about 5 – 10% of labeled neurons, the plasma membrane was also clearly labeled at P2 (not shown) and at P10 (arrows in A2 to E2, with higher magnifications shown in respective insets), but was less distinct thereafter. The insets at the upper left corners of A1 -E1 are schematic diagrams of the location of each of the five brain stem nuclei. Scale bar in A1: 20 μm for all low magnifications, and 8.33 μm for all high magnifications in insets.

Figure 2.

Optical densitometric measurements of PACAP immunoreaction product in individual neurons of the PBC (A), Amb (B), XII (C), NTS_VL (D), and CN (E) from P2 to P21. Data points represent the mean ± SEM. ANOVA revealed significant differences among the ages in the PBC, Amb, XII, and NTS_VL (P < 0.01), but not in the CN. Tukey’s Studentized tests showed a significant drop in immunoreactivity from P11 to P12 in the PBC and Amb, and from P10 to P12 in the XII and NTS_VL. Other significant differences are indicated in the text. *P < 0.05.