Pituitary adenylate cyclase-activating polypeptide (PACAP) mimics neuroendocrine and behavioral manifestations of stress: Evidence for PKA-mediated expression of the corticotropin-releasing hormone (CRH) gene

Abstract

The physiologic response to stress is highly dependent on the activation of corticotropin-releasing hormone (CRH) neurons by various neurotransmitters. A particularly rich innervation of hypophysiotropic CRH neurons has been detected by nerve fibers containing the neuropeptide PACAP, a potent activator of the cAMP-protein kinase A (PKA) system. Intracerebroventricular (icv) injections of PACAP also elevate steady-state CRH mRNA levels in the paraventricular nucleus (PVN), but it is not known whether PACAP effects can be associated with acute stress responses. Likewise, in cell culture studies, pharmacologic activation of the PKA system has stimulated CRH gene promoter activity through an identified cAMP response element (CRE); however, a direct link between PACAP and CRH promoter activity has not been established. In our present study, icv injection of 150 or 300 pmol PACAP resulted in robust phosphorylation of the transcription factor CREB in the majority of PVN CRH neurons at 15 to 30 min post-injection and induced nuclear Fos labeling at 90 min. Simultaneously, plasma corticosterone concentrations were elevated in PACAP-injected animals, and significant increases were observed in face washing, body grooming, rearing and wet-dog shakes behaviors. We investigated the effect of PACAP on human CRH promoter activity in alphaT3-1 cells, a PACAP-receptor expressing cell line. Cells were transiently transfected with a chloramphenicol acetyltransferase (CAT) reporter vector containing region - 663/+124 of the human CRH gene promoter then treated for with PACAP (100 nM) or with the adenylate cyclase activating agent, forskolin (2.5 muM). Both PACAP and forskolin significantly increased wild-type hCRH promoter activity relative to vehicle controls. The PACAP response was abolished in the CRE-mutant construct. Pretreatment of transfected cells with the PKA blocker, H-89, completely prevented both PACAP- and forskolin-induced increases in CRH promoter activity. Furthermore, CREB overexpression strongly enhanced PACAP-mediated stimulation of hCRH promoter activity, an effect which was also lost with mutation of the CRE. Thus, we demonstrate that icv PACAP administration to rats under non-stressed handling conditions leads to cellular, hormonal and behavioral responses recapitulating manifestations of the acute stress response. Both in vivo and in vitro data point to the importance of PACAP-mediated activation of the cAMP/PKA signaling pathway for stimulation of CRH gene transcription, likely via the CRE.

Fig. 1

General pattern of nuclear PCREB immunolabeling in the PVN at 30 min following icv PACAP injection. (A) Control aCSF-injected PVN; (B) PACAP 150 pmol C PACAP 300 pmol. 3V = third cerebral ventricle; LM lateral magnocellular subdivision; dp, dorsal; mp, medial; vp, ventral; pv, periventricular parvocellular subdivisions of PVN. Scale bar, 200 μm.

Fig. 2

Time course of PCREB induction in the PVN following icv injection of PACAP. (A) Control 15 min; (B) PACAP 150 pmol 15 min; (C) PACAP 300 pmol 15 min; (D) control 30 min; (E) PACAP 150 pmol 30 min; (F) PACAP 300 pmol 30 min; (G) control 90 min; (H) PACAP 150 pmol 90 min; (I) PACAP 300 pmol 90 min. Scale bar, 100 μm.

Fig. 3

Representative images of PCREB-CRH dual immunolabeling at 30 min post-injection time point. High-power magnification light microscopic images show simultaneous immunolabeling for CRH neurons (brown cytoplasm) and PCREB positive cell nuclei (black nuclear label) in the medial parvocellular subdivision of the PVN. (A) Control and (B) 300 pmol PACAP-treated animals. High numbers of CRH positive neurons contain PCREB-labeled nuclei after icv infusion of PACAP (examples indicated by arrows). Scale bar, 20 μm.

Fig. 4

Percentages of PCREB-CRH double labeled neurons in the PVN following icv aCSF vehicle injection (Control) or PACAP (150 or 300 pmol) at 15, 30 and 90 min post-injection. Note robust elevations in the proportion of CRH neurons containing nuclear PCREB following icv PACAP injection. *P < 0.01 versus control; ^#P < 0.01 versus 150 pmol. n = numbers of animals in experimental groups.

Fig. 5

Nuclear Fos-immunoreactivity following icv PACAP injection in the PVN. (A) Control at 30 min; (B) control at 90 min; (C) PACAP 300 pmol at 30 min; (D) PACAP 300 pmol at 90 min. Scale bar in panel (D), 100 μm.

Fig. 6

Simultaneous Fos and CRH immunolabeling in the medial parvocellular subdivision of the PVN. (A) Control at 90 min. (B) PACAP 300 pmol at 90 min. Note the presence of several double-labeled neurons 90 min following PACAP injection, containing black nuclear Fos and brown cytoplasmic CRH immunoreactivity (some examples indicated by arrows). Scale bar, 50 μm.

Fig. 7

Quantitative scores of icv PACAP-induced behavioral manifestations during the first 10-min period following aCSF vehicle (Control) and 150 or 300 pmol doses of PACAP (PACAP150, PACAP300). Face = face washing and grooming. Asterisks indicate significant changes relative to control (in A, P < 0001; B, P < 0.005; C, P < 0.005; D, P < 0.001). n = numbers of animals in experimental groups.

Fig. 8

(A) PACAP stimulates hCRH gene promoter activity via the PKA system. αT3-1 gonadotrope cells were transiently transfected with a construct containing region −663/+124 of the human CRH gene promoter linked to a CAT reporter vector. Cells were cotransfected with an RSV-β-galactosidase expression vector. Cells were treated with PACAP38 (100 nM), forskolin (2.5 mM) or vehicle for 12–14 h prior to harvest. Where indicated, cells were pretreated for 1 h with the PKA specific inhibitor, H-89 (10 mM). CAT activity was normalized to β-galactosidase activity and promoter activity expressed as fold-change relative to expression in vehicle-treated control wells. Results are shown as the mean ± SEM. *P < 0.005 versus control; ^#P < 0.02 versus forskolin or PACAP alone. (B) Loss of PACAP effect with mutation of the cAMP response element (CRE) in the hCRH gene promoter. αT3-1 cells were transiently transfected with a CAT reporter vector containing region 663/+124 of the hCRH gene promoter present as the wild-type sequence (CRH-CAT, upper panel) or with deletion of the previously identified CRE at position −221 (ΔCRE-CAT, lower panel). Cells were treated with vehicle or PACAP (100 nM × 12–14 h) starting 36 h after transfection. *P < 0.005 versus control. (C) CREB augments PACAP-induced activation of the CRH gene. αT3-1 cells were transiently transfected with a CAT reporter vector containing region −663/+124 of the hCRH gene promoter present as the wild-type sequence (CRH-CAT, upper panel) or with deletion of the previously identified CRE (ΔCRE-CAT, lower panel). Cells were cotransfected with an RSV-driven expression vector encoding CREB or with the empty expression vector. Cells were treated with PACAP (100 nM × 12–14 h), and results calculated relative to vehicle treated wells that received the empty expression vector. Results are shown as the mean ± SEM. *P < 0.005 versus control.