Mitogen-activated protein kinase contributes to lipopolysaccharide-induced activation of corticotropin-releasing hormone synthesizing neurons in the hypothalamic paraventricular nucleus

Abstract

To determine whether the p44/p42 MAPK (ERK1/2) signaling pathway is involved in the activation of CRH-containing neurons in the hypothalamic paraventricular nucleus (PVN) after bacterial lipopolysaccharide (LPS) administration, Sprague Dawley rats were injected with LPS, and studied after 2, 6, 9, and 12 h. In saline-treated controls, isolated weak phosphorylated (phospho)ERK1/2 immunoreactive neurons were observed in the PVN. However, a dramatic increase in phospho-ERK1/2 immunoreactivity was apparent in the PVN 2 h after LPS administration, and gradually declined to baseline levels 9-12 h after injection. By double-labeling immunofluorescence, all CRH-containing neurons in the PVN contained phospho-ERK1/2 2 h after LPS. Intracerebroventricular administration of the MAPK inhibitor, PD98059, prevented LPS-induced ERK1/2 phosphorylation, c-fos activation, and the increase of CRH gene expression in the PVN but had no effect on c-fos activation in brainstem A2-C1/C2 regions. We conclude that LPS rapidly increases the phospho-ERK1/2 in CRH-containing neurons in the PVN and that activation of MAPKs is necessary for LPS-induced activation of the hypothalamic-pituitary-adrenal axis.

Figure 1

Photomicrographs showing the effect of LPS administration on phospho-ERK1/2 immunoreactivity in the PVN of saline-treated control animals (A), and 2 (B), 6 (C), 9 (D), and 12 h (E) after LPS. Note maximal increase in phospho-ERK1/2 immunoreactivity 2 h after LPS administration. F, Semiquantitative image analysis of the phospho-ERK1/2 immunoreactivity (OD) in the PVN. *, P < 0.001 compared with control, 6-, 9-, and 12-h LPS. #, P < 0.05 compared with control, 9- and 12-h LPS. Scale bar, 200 μm. III, Third ventricle.

Figure 2

CRH immunofluorescence in the PVN of saline-treated controls (A) and LPS-treated animals (B). C, Doubly labeled phospho-ERK1/2- (red) and CRH-(green) immunoreactive neurons are not detected in the PVN of saline-treated control animals. D, Two hours after LPS administration, all CRH neurons in the PVN co-contain phospho-ERK1/2 and appear yellow due to color mixing (arrows). Scale bar, 200 μm. III, Third ventricle.

Figure 3

Effect of the MAPK inhibitor, PD98059, on LPS-induced ERK1/2 activation in the PVN. Veh (15% DMSO)-treated animals (A), LPS-treated animals (B), and PD98059-treated animals before LPS administration (C). Note attenuated LPS response in the PVN in C. D, Semiquantitative image analysis of the phospho-ERK1/2 immunoreactivity (optical density) in the PVN. *, P < 0.001 compared with Veh and PD98059/LPS groups. Scale bar, 200 μm. III, Third ventricle.

Figure 4

Effect of MAPK inhibitor, PD98059, on LPS-induced c-fos expression in the PVN. Veh (15% DMSO)-treated animals (A), LPS-treated animals (B), and PD98059-treated animals before LPS administration (C). Note profound reduction in LPS-induced c-fos immunoreactivity in the PVN in C. D, Semiquantitative image analysis of the number of c-fos immunoreactive cells in the PVN. *, P < 0.001 compared with Veh/Saline and PD98059/LPS groups. Scale bar, 200 μm. III, Third ventricle.

Figure 5

Effect of MAPK inhibitor, PD98059, on LPS-induced c-fos expression in the A2 (A–F) and C1 (G–I) cell groups in the lower brainstem. Veh (15% DMSO)-treated animals (A, D, and G), LPS-treated animals (B, E, and H), and PD98059-treated animals before LPS administration (C, F, and I). D–F, High-magnification photomicrographs of the area marked with a rectangle in A–C, respectively. MAPK inhibition has no effect on LPS-induced c-fos expression in the C1 and A2 regions. Scale bar, 200 μm.

Figure 6

Dark-field photomicrographs showing CRH mRNA signal in the PVN in icv Veh (15% DMSO)-treated animals receiving an ip injection of saline (Sal) (A) or LPS (B). The icv administration of the MAPK inhibitor, PD98059, has no effect on CRH mRNA expression in saline-treated control animals (C) but diminishes the CRH mRNA response after LPS administration (D). E, Semiquantitative image analysis of integrated density units representing CRH mRNA signal in the PVN. *, P < 0.05 compared with Veh/saline, PD98059/saline, and PD98059/LPS groups. Scale bar, 200 μm. III, Third ventricle.