Pituitary Adenylate Cyclase-activating Polypeptide mRNAs Research Articles

Expression Patterns of PACAP and PAC1R Genes and Anorexigenic Action of PACAP1 and PACAP2 in Zebrafish.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with potent suppressive effects on feeding behavior in rodents, chicken, and goldfish. Teleost fish express two PACAPs (PACAP1, encoded by the adcyap1a gene, and PACAP2, encoded by the adcyap1b gene) and two PACAP receptors (PAC1Rs; PAC1Ra, encoded by the adcyap1r1a gene, and PAC1Rb, encoded by the adcyap1r1b gene). However, the mRNA expression patterns of the two PACAPs and PAC1Rs, and the influence and relationship of the two PACAPs on feeding behavior in teleost fish remains unclear. Therefore, we first examined mRNA expression patterns of PACAP and PAC1R in tissue and brain. All PACAP and PAC1Rs mRNAs were dominantly expressed in the zebrafish brain. However, adcyap1a mRNA was also detected in the gut and testis. In the brain, adcyap1b and adcyap1r1a mRNA levels were greater than that of adcyap1a and adcyap1r1b, respectively. Moreover, adcyap1b and adcyap1r1a mRNA were dominantly expressed in telencephalon and diencephalon. The highest adcyap1a mRNA levels were detected in the brain stem and diencephalon, while the highest levels of adcyap1r1b were detected in the cerebellum. To clarify the relationship between PACAP and feeding behavior in the zebrafish, the effects of zebrafish (zf) PACAP1 or zfPACAP2 intracerebroventricular (ICV) injection were examined on food intake, and changes in PACAP mRNA levels were assessed against feeding status. Food intake was significantly decreased by ICV injection of zfPACAP1 (2 pmol/g body weight), zfPACAP2 (2 or 20 pmol/g body weight), or mammalian PACAP (2 or 20 pmol/g). Meanwhile, the PACAP injection group did not change locomotor activity. Real-time PCR showed adcyap1 mRNA levels were significantly increased at 2 and 3 h after feeding compared with the pre-feeding level, but adcyap1b, adcyap1r1a, and adcyap1r1b mRNA levels did not change after feeding. These results suggest that the expression levels and distribution of duplicated PACAP and PAC1R genes are different in zebrafish, but the anorexigenic effects of PACAP are similar to those seen in other vertebrates.

Distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) in the human testis and in testicular germ cell tumors

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide expressed in the central nervous system and peripheral organs. Previous studies revealed the role and distribution of PACAP in the rodent testis, however, its presence in the human testis and in testicular germ cell tumors is not known. We used RT-PCR and immunohistological observations to investigate whether human testicular tissue and testicular germ cell tumors contain PACAP. The mRNAs for PACAP and its receptors were detected in total RNA extracted from human testes. PACAP immunoreactivity was observed in spermatogonia and spermatids from normal testes. In contrast, diffuse PACAP immunopositivity was observed in seminoma tumor cells, while only faint immunoreactivity was observed in embryonal carcinoma cells. Our data suggest that PACAP may play a role in human spermatogenesis and in testicular germ cell tumor development.

PACAP Neurons in the Hypothalamic Ventromedial Nucleus Are Targets of Central Leptin Signaling

The adipose-derived hormone, leptin, was discovered over 10 years ago, but only now are we unmasking its downstream pathways which lead to reduced energy intake (feeding) and increased energy expenditure (thermogenesis). Recent transgenic models have challenged the long-standing supposition that the hypothalamic arcuate nucleus (Arc) is omnipotent in the central response to leptin, and research focus is beginning to shift to examine roles of extra-arcuate sites. Dhillon et al. (2006) demonstrated that targeted knock out of the signaling form of the leptin receptor (lepr-B) in steroidogenic factor 1 (SF-1) cells of the hypothalamic ventromedial nucleus (VMN) produces obesity of a similar magnitude to the pro-opiomelanocortin (POMC)-driven lepr-B deleted mouse, via a functionally distinct mechanism. These findings reveal that SF-1 cells of the VMN could be equally as important as POMC cells in mediating leptin's anti-obesity effects. However, the identification of molecular and cellular correlates of this relationship remains tantalizingly unknown. Here, we have shown that mRNA expression of the VMN-expressed neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is regulated according to energy status and that it exerts catabolic effects when administered centrally to mice. Furthermore, we have shown that SF-1 and PACAP mRNAs are colocalized in the VMN, and that leptin signaling via lepr-B is required for normal PACAP expression in these cells. Finally, blocking endogenous central PACAP signaling with the antagonist PACAP(6-38) markedly attenuates leptin-induced hypophagia and hyperthermia in vivo. Thus, it appears that PACAP is an important mediator of central leptin effects on energy balance.

Anorexigenic action of pituitary adenylate cyclase-activating polypeptide (PACAP) in the goldfish: Feeding-induced changes in the expression of mRNAs for PACAP and its receptors in the brain, and locomotor response to central injection

Our recent research has indicated that intracerebroventricular (ICV) administration of pituitary adenylate cyclase-activating polypeptide (PACAP) suppresses food intake in the goldfish. We therefore examined feeding- and fasting-induced changes in the expression of mRNAs for PACAP and its receptors in the goldfish brain, and the effect of ICV administration of synthetic PACAP on locomotor activity in the goldfish. Semiquantitative analysis revealed that the expression of mRNAs for PACAP and the PAC 1 receptor was significantly increased by excessive feeding (daily food supply at ≥5% of the body weight [BW]) for 7 days. ICV administration of PACAP at 20 pmol/g BW induced a significant decrease in locomotor activity during the 60-min post-treatment observation period. These results suggest that PACAP may have an anorexigenic action via hypomotility in goldfish.

Expression of pituitary adenylate cyclase activating polypeptide and its type I receptor mRNAs in human placenta

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and was known to stimulate the release of growth factor in various cells. Recently, we reported the cellular localization of PACAP and its type I (PAC1) receptor in rat placenta during pregnancy. Placenta is a critical organ that synthesizes several growth factors and angiogenic factors for the fetal development and its own growth. However, there is little information regarding the cellular localization of PACAP and its receptor in human placenta at various gestations. The aim of the present study was to define the expression and distribution of PACAP and PAC1 receptor mRNAs in the human placenta during the pregnancy period. PACAP and PAC1 receptor mRNAs were expressed in stroma cells of stem villi and terminal villi. At the early stage, on 7 and 14 weeks, PACAP and PAC1 receptor genes were moderately expressed in stroma cells surrounding the blood vessels within stem villi. These genes were strongly expressed in stroma cells of stem villi and terminal villi on 24 and 38 weeks. The expression of these genes was increased as gestation advanced, and localized in the same areas. Localization of PACAP and PAC1 receptor demonstrate the evidence that PACAP may play an important role, as an autoregulator or pararegulator via its PAC1 receptor. In conclusion, our findings strongly suggest that PACAP may have a critical role in physiological function of the placenta for gestational maintenance and fetal growth.

Differential expression of mRNAs for PACAP and its receptors during neural differentiation of embryonic stem cells

The expressions of mRNAs for pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and their receptors (PAC 1, VPAC 1 and VPAC 2) were examined in the five steps of the in vitro neuronal culture model of embryonic stem (ES) cell differentiation. mRNAs for PACAP, VIP, PAC 1 receptor, and VPAC 2 receptor were moderately expressed in neural stem cell-enriched cultures, while VPAC 1 receptor mRNA was most prominently expressed in embryoid bodies (EBs). The expression of PAC 1 receptor mRNA was further upregulated after terminal differentiation into neurons. In contrast, the expressions of PAC 1 receptor and PACAP mRNAs were markedly decreased after glial differentiation. These results suggest that this in vitro neuronal culture system will be a useful model for future studies on the functional role of the PACAPergic system during different stages of neuronal development.

Expression of mRNAs for PACAP and its receptor in human neuroblastomas and their relationship to catecholamine synthesis

Purpose: Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal peptide family, induces the expression of catecholamine-synthesizing enzymes in adrenal medullary cells. In addition, PACAP and its receptor have been detected in human neuroblastoma tissues and cell lines, though it is not yet known whether PACAP enhances the expression of genes encoding catecholamine-synthesizing enzymes. To address this question, we analyzed PACAP, PACAP receptor and tyrosine hydroxylase (TH) mRNAs in neuroblastomas. Methods: The levels of mRNA for PACAP and vasoactive intestinal peptide (VIP), as well as their receptors and the mRNA for TH were measured by RT-PCR or real-time PCR analysis. Results: VPAC1R mRNA was detected in all of 16 tissues and 3 cell lines that were examined, while VPAC2R mRNA was detected in 5 of 16 (31%) tissue and 2 of 3 cell lines. PAC1R mRNA was detected in 6 out of 16 (38%) tissues and none of 3 cell lines. mRNA expression of PACAP and TH were detected in many tissues (10/16 and 16/16, respectively). However, neither in tissues nor cell lines did PACAP mRNA expression correlate with TH mRNA expression. Conclusion: Our findings suggest that PACAP is not involved in the regulation of expression of TH in neuroblastomas.

The regulatory mechanism for neuron specific expression of PACAP gene

Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, is present abundantly in the central nervous system. In the 5'-flanking region of the PACAP gene, we found and characterized two negative regulatory elements, which are homologous to the neural-restrictive silencer element (NRSE). Their sequence and position were significantly conserved among mouse, human, and rat PACAP genes. NRSE is a crucial negative-acting DNA regulatory element for neuron-specific gene expression. NRSE acts through the transcription factor known as neural-restrictive silencer factor (NRSF). In non-neuronal cells, NRSF suppresses the expression of neuron-specific genes. On the other hand, in neuronal cells, NRnV, a NRSF truncated form, repress their expressions in a dominant negative manner. The electrophoretic mobility shift assay with 3T3 cells extract demonstrated the identical complexes among NRSLE-1, NRSLE2, and the NRSE of rat type II sodium channel gene. In the luciferase reporter assay, NRSLEs suppressed SV40 promoter activity in 3T3 cells, but not in PC12 cells. RT-PCR analysis revealed that PACAP and NRnV mRNAs are expressed in neuronal cells (differentiated PC12), but not in non-neuronal cells (3T3 or C6). These results suggested that the NRSE-NRSF system might be involved in the regulatory mechanism of neuron-specific expression of the PACAP gene.

Expression patterns of pituitary adenylate cyclase activating polypeptide and its type I receptor mRNAs in the rat placenta.

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and is known to act as a tropic factor in various cells. Recent report revealed the expression of PACAP and the PACAP type I (PAC(1)) receptor in human and rat placentas at term. Placenta is a critical organ that synthesizes several growth and angiogenic factors for its own growth as well as fetal development. However, there is little information regarding the expression pattern and cellular localization of PACAP and PAC(1) during pregnancy. The aim of this study was to define the expression and distribution of PACAP and PAC(1) receptor mRNAs in the rat placenta during pregnancy. PACAP and PAC(1) receptor mRNAs were expressed in decidual cells, chorionic vessels, and stromal cells of the chorionic villi. Interestingly, the expression of these genes varied with the day of gestation. For example, PACAP and PAC(1) receptor mRNAs expressed in decidual cells on day 13.5 and 15.5, their expression was strong in chorionic vessels and stromal cells of the chorionic villi within the labyrinth zone on day 17.5, 19.5, and 21.5. In fact, as gestation advanced, the expression of PACAP and PAC(1) receptor mRNAs in the decidua cells disappeared, as their high expression became evident in the chorionic vessels and stromal cells of the chorionic villi. Our finding that PACAP and the PAC(1) receptor are co-localized and their genes seemingly co-regulated within specific placental areas, strongly suggest that this peptide may play an important role, as an autoregulator or pararegulator via its PAC(1) receptor, in physiological functioning of the placenta for gestational maintenance.

Regulation of Pituitary Adenylate Cyclase-activating Polypeptide Gene Transcription by TTF-1, a Homeodomain-containing Transcription Factor

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an important hypophysiotrophic factor as well as a regulator for immune, reproductive, and neural tissues. We recently found that TTF-1, a homeodomain-containing transcription factor essential for the development of the fetal diencephalon, is postnatally expressed in the hypothalamic area and plays a transcription regulatory role for certain neurohormones. Based on the similarity of synthesis sites between PACAP and TTF-1 and, moreover, on the presence of conserved core TTF-1 binding motifs in the 5'-flanking region of the PACAP gene, we sought to uncover a regulatory role of TTF-1 in PACAP gene transcription. The TTF-1 homeodomain binds to six of the seven putative binding domains observed in the 5'-flanking region of the PACAP gene. In the C6 glioma cell-line, TTF-1 activates the PACAP promoter in a dose-dependent manner. This transactivation of PACAP by TTF-1 was totally removed when the core TTF-1 binding motif at -369 was deleted. RNase protection assays showed that TTF-1 and PACAP mRNAs have daily fluctuations in the rat hypothalamus. They both were at low levels during the day and high levels during the night. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased the PACAP mRNA level as well as TTF-1 protein content in the rat hypothalamus, suggesting that TTF-1 also regulates PACAP transcription in vivo. Moreover, the TTF-1 promoter was inhibited by molecular oscillators of CLOCK and BMAL-1. Taken together, these data suggest that TTF-1 plays an important regulatory role in the gene transcription for PACAP, which may be important for the generation of a daily rhythm of hypothalamic PACAP gene expression.

Mouse pituitary adenylate cyclase-activating polypeptide (PACAP): gene, expression and novel splicing

PACAP is a conserved neuropeptide present in all vertebrates. In the mouse, the PACAP gene and various mRNAs have been isolated. To further characterize the mouse PACAP gene ( Adcyap1), we have confirmed its sequence, identified its chromosomal location on distal chromosome 17 and used RT-PCR and 5′RACE in various tissues to identify eight PACAP mRNAs, four of which are novel. Three of these novel transcripts have alternate 5′UTRs, whereas the fourth is altered within the coding region. This is the first report of alternate splicing within the coding region of the PACAP gene. The expression pattern of PACAP in the mouse during embryonic development and adulthood is known. Here, expression of PACAP in the mouse at four postnatal stages in 12 tissues is assessed. We identify continuous expression of PACAP in the brain and eye and stage-specific expression in the gonads and thymus. The complex splicing of PACAP transcripts may regulate the tissue-and stage-specific expression.

Expression of pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP type I A receptor mRNAs in granulosa cells of preovulatory follicles of the rat ovary

Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalamus and known to stimulate the production of cAMP in anterior pituitary cells. In the recent report, the expression of PACAP was detected in preovulatory follicles, and treatment with PACAP stimulated the production of progesterone and prostaglandin E2 through the action of AC and PLC pathways in the ovary. PACAP binds to three type receptors. Type I A receptor is coupled to adenylate cyclase (AC) and phospholipase C (PLC) pathways, while type I B and type II receptors are only coupled to AC. Thus, the present study aimed to evaluate the temporal expression of PACAP and its type I A receptor mRNAs in the rat ovary after treatment with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG). Northern blot analysis showed that PACAP transcripts were transiently expressed from 3–9 hr after hCG treatment, reaching a maximum at 6 hr. During these time points, PACAP mRNAs were specifically and strongly expressed in granulosa cells and cumulus cells of large preovulatory follicles and interstitial glandular cells. Type I A receptor mRNAs were also transiently expressed in granulosa cells of large preovulatory follicles from 3–9 hr after hCG treatment. PACAP and its type I A receptor mRNAs were expressed in the same preovulatory follicles. These results demonstrate that PACAP acts as an autoregulator or pararegulator through type I A receptor in granulosa cells and cumulus cells of large preovulatory follicles. Thus, we suggest that PACAP may have a critical role in granulosa cells of preovulatory follicles for the preparation of ovulation. Mol. Reprod. Dev. 55:379–386, 2000. © 2000 Wiley-Liss, Inc.

Expression of pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP type I A receptor mRNAs in granulosa cells of preovulatory follicles of the rat ovary.

Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalamus and known to stimulate the production of cAMP in anterior pituitary cells. In the recent report, the expression of PACAP was detected in preovulatory follicles, and treatment with PACAP stimulated the production of progesterone and prostaglandin E(2) through the action of AC and PLC pathways in the ovary. PACAP binds to three type receptors. Type I A receptor is coupled to adenylate cyclase (AC) and phospholipase C (PLC) pathways, while type I B and type II receptors are only coupled to AC. Thus, the present study aimed to evaluate the temporal expression of PACAP and its type I A receptor mRNAs in the rat ovary after treatment with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG). Northern blot analysis showed that PACAP transcripts were transiently expressed from 3-9 hr after hCG treatment, reaching a maximum at 6 hr. During these time points, PACAP mRNAs were specifically and strongly expressed in granulosa cells and cumulus cells of large preovulatory follicles and interstitial glandular cells. Type I A receptor mRNAs were also transiently expressed in granulosa cells of large preovulatory follicles from 3-9 hr after hCG treatment. PACAP and its type I A receptor mRNAs were expressed in the same preovulatory follicles. These results demonstrate that PACAP acts as an autoregulator or pararegulator through type I A receptor in granulosa cells and cumulus cells of large preovulatory follicles. Thus, we suggest that PACAP may have a critical role in granulosa cells of preovulatory follicles for the preparation of ovulation.

Role of Progesterone Receptor Activation in Pituitary Adenylate Cyclase Activating Polypeptide Gene Expression in Rat Ovary1

It is well known that the pituitary gonadotropin surge induces progesterone receptor (PR) gene expression in luteinizing granulosa cells and that PR activation is critical for successful ovulation. To further understand the molecular mechanism(s) by which PR plays a role critical for granulosa cell functions, we wanted to identify progesterone-induced genes in granulosa cells. We employed a PCR-based subtraction cloning strategy to screen for genes expressed differentially in granulosa cells that were challenged with forskolin in the presence of progesterone or ZK98299. One such differentially expressed clone was identified as the pituitary adenylate cyclase activating polypeptide (PACAP). To begin to understand the relationship between PR activation and PACAP gene expression in luteinizing granulosa cells, we examined whether PR and PACAP messenger RNA (mRNA) expression is temporally correlated. In cultured granulosa cells, both human CG and forskolin induced PR and PACAP mRNA levels in a dose-dependent manner, as determined by semi-quantitative RT-PCR assays. However, the peak expression for PR and PACAP mRNAs was observed at 3 h and 6 h after hormone treatment, respectively. This time difference in cAMP-responsive expression of the PR and PACAP genes is due, at least in part, to the requirement of ongoing protein synthesis for PACAP expression, as demonstrated by the inhibitory effect of cycloheximide on cAMP-induced PACAP, but not PR, mRNA levels. To determine whether PR synthesis is prerequisite for PACAP expression, we examined the effect of ZK98299, a specific PR antagonist, on cAMP-induced PACAP mRNA expression. This compound blocked cAMP-induced PACAP mRNA expression in a dose-dependent manner, indicating that PR activation is required for PACAP gene expression in granulosa cells. We then compared cellular localization and hormonal regulation of ovarian PR and PACAP gene expression in immature rats treated with gonadotropins as well as in adult rats during the preovulatory period by using in situ hybridization and semiquantitative RT-PCR assays. Results show that both PR and PACAP mRNAs are induced in granulosa cells of preovulatory follicles by human CG, but that the PR gene is expressed before the PACAP gene. Taken together, these results demonstrate that PRs mediate the LH-induced PACAP gene expression in rat granulosa cells.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and Vasoactive Intestinal Peptide (VIP) Stimulate Interleukin-6 Production through the Third Subtype of PACAP/VIP Receptor in Rat Bone Marrow-Derived Stromal Cells

Regulation of Interleukin-6 (IL-6) production in bone marrow (BM)-derived stromal cells by neuropeptides, pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP), was examined. Both forms of PACAP, PACAP-27 and PACAP-38, as well as VIP significantly increased IL-6 production by rat BM-derived stromal cells at physiological concentrations ranging from 10−10–10−8m. The three related peptides (PACAP-27, -38, and VIP) stimulated the production of both cAMP and inositol 1,4,5-trisphosphate (IP3) in rat BM-derived stromal cells with similar 50% effective concentrations. The stimulatory potency of the three related peptides for the production of IL-6, cAMP, and IP3 was almost consistent, suggesting that the dual signaling transduction pathways may be involved in PACAP/VIP-induced IL-6 production in rat BM-derived stromal cells. The messenger RNA (mRNA) for the third subtype of PACAP receptor (PVR3) was found to be abundantly expressed in both BM-derived stromal cells and the BM tissue, whereas little of the mRNA for type 1 (PVR1) nor type 2 (PVR2) was detected. Furthermore, the mRNAs for PACAP and VIP were detected in the BM tissue, suggesting that both PACAP/VIP and PVR3 are synthesized in vivo in the BM. The results shown in this paper suggest that PACAP/VIP and their receptor play an important role in the IL-6 production and perhaps in the hematopoiesis in the BM.

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) stimulate interleukin-6 production through the third subtype of PACAP/VIP receptor in rat bone marrow-derived stromal cells.

Regulation of Interleukin-6 (IL-6) production in bone marrow (BM)-derived stromal cells by neuropeptides, pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP), was examined. Both forms of PACAP, PACAP-27 and PACAP-38, as well as VIP significantly increased IL-6 production by rat BM-derived stromal cells at physiological concentrations ranging from 10(-10)-10(-8) M. The three related peptides (PACAP-27, -38, and VIP) stimulated the production of both cAMP and inositol 1,4,5-trisphosphate (IP3) in rat BM-derived stromal cells with similar 50% effective concentrations. The stimulatory potency of the three related peptides for the production of IL-6, cAMP, and IP3 was almost consistent, suggesting that the dual signaling transduction pathways may be involved in PACAP/VIP-induced IL-6 production in rat BM-derived stromal cells. The messenger RNA (mRNA) for the third subtype of PACAP receptor (PVR3) was found to be abundantly expressed in both BM-derived stromal cells and the BM tissue, whereas little of the mRNA for type 1 (PVR1) nor type 2 (PVR2) was detected. Furthermore, the mRNAs for PACAP and VIP were detected in the BM tissue, suggesting that both PACAP/VIP and PVR3 are synthesized in vivo in the BM. The results shown in this paper suggest that PACAP/VIP and their receptor play an important role in the IL-6 production and perhaps in the hematopoiesis in the BM.

Ontogeny of pituitary adenylate cyclase activating polypeptide and its receptor mRNA in the mouse brain

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide which was first isolated from ovine hypothalamic tissue by screening for pituitary adenylate cyclase stimulating activity. Our previous data showed that radioimmunoassayable PACAP and PACAP-binding sites were detected in the whole rat brain as early as embryonic day 14 (E14). In order to understand more precisely the developmental pattern of the synthesis of PACAP and its receptors in the brain, we studied the expression of PACAP and its receptor genes in the prenatal and postnatal mouse brain using RNase protection assay. The mRNAs for both PACAP and its receptor were detected as early as 9.5 days of gestation (E9.5) in the whole head of mouse embryos. The levels of PACAP mRNA in the brain increased during the prenatal period peaking at postnatal day 0 (P0). On the other hand, the levels of PACAP receptor mRNA gradually increased after E9.5. The levels sharply increased at P6 (479.0 ± 82.5% of P0 levels), and then fell to the P3 levels at P10. These data together with our previous study on the ontogeny of PACAP immunoreactivity and its binding sites in the rat brain support the view that PACAP plays an important regulatory role in the development of brain.