Pituitary adenylate cyclase-activating polypeptide receptors during development: expression in the rat embryo at primitive streak stage

doi:10.1016/S0306-4522(99)00108-6

Neuroscience

Volume 93, Issue 1, June 1999, Pages 375-391

https://doi.org/10.1016/S0306-4522(99)00108-6 Get rights and content

Abstract

The distribution and localization of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor [the PAC₁ receptor (previously called the type 1 PACAP receptor or PVR1), which binds PACAP, but not vasoactive intestinal peptide, with high affinity] were first investigated in rats with in situ hybridization for its messenger RNA, and with immunohistochemical methods during prenatal and postnatal development. The expression of PACAP receptor messenger RNA was first detected in the rat embryo at the primitive streak stage as early as embryonic day 9, and it was intensely expressed in the neural plate. PACAP receptor messenger RNA was also intensely expressed in the neuroepithelia of the mesencephalon and rhombencephalon at embryonic day 11, and expressed in the basal telencephalon, hippocampal formation neuroepithelium, cortical neuroepithelium and cerebellar neuroepithelium after embryonic day 13. It was also expressed in the olfactory bulb neuroepithelium after embryonic day 16, and in mature regions of the older embryos. In postnatal developing brains, PACAP receptor messenger RNA was intensely expressed in the olfactory bulb, hippocampal formation, cerebellum and other scattered regions. The localization of PACAP receptor-like immunoreactivity coincided well with that of the gene transcripts. We also used reverse transcription–polymerase chain reaction methods to determine the expression of the splice variants of the PACAP receptor gene. At each ontogenetic stage of the rat from embryonic day 9 to postnatal day 60, two major products were detected with reverse transcription–polymerase chain reaction, a thick band (303 base pairs) corresponding to the short splice variant of the receptor that lacks both the “hip” and “hop” cassettes, and a thin band (387 base pairs) corresponding to the splice variant that contains one cassette of “hop” or “hip”. There was no evidence for the other larger splice variants. Some of the amplified products were sequenced and found to have the exact sequences of “PACAP receptor” and “PACAP receptor-hop1”, which are coupled to different signal transduction pathways.

These results indicate that the PACAP receptor is actively expressed in different neuroepithelia from early developmental stages and expressed in various brain regions during prenatal and postnatal development, and that the major splice variants are “PACAP receptor” and “PACAP receptor-hop1”. The initial mapping of ontogenetic localization of the PACAP receptor provides the basis for a better understanding of the functions of PACAP and its receptors during the development of the brain.

Section snippets

Animals and tissue preparation

Timed-pregnant Sprague–Dawley rats (Saitama Experimental Animal Center, Saitama, Japan) were deeply anesthetized with pentobarbital (50 mg/kg) and embryos were collected on E9–E20. The day on which a vaginal plug was observed was designated as E1. Whole embryos at E9, E9.5, E10, E11, E12, E13 and E14, the heads of E16 rats, and the brains from E18 and E20 rats were frozen with liquid nitrogen and stored at −70°C. Postnatal brains (the day of birth was designated as P0) were also collected on P0,

Authenticity of the experiments for the PACAP receptor

The antisense oligonucleotide probe for in situ hybridization showed tissue-specific hybridization at different developmental stages (Figs 1, 2). At each stage, three to six animals were analysed with consistently reproducible results. Moreover, the sense probe showed no specific signals at any time-point. The authenticity of the hybridization signals was confirmed by RT–PCR. Using the primers for PGK as an internal control generated the expected 182-bp band during each PAC₁ receptor cDNA PCR,

Expression of PACAP receptor in the early embryo

The present study is the first to demonstrate that the PAC₁ receptor is intensely expressed in the neuroepithelium of embryos at the primitive streak stage using in situ hybridization. It further demonstrates that at least two splice variants of the PAC₁ receptor are expressed during this developmental stage by RT–PCR, and that the main splice variants of the PAC₁ receptor are “PACAP-R” and “PACAP-R-hop1” by sequence analysis. It also shows that intense PAC₁ receptor-like immunoreactivities are

Conclusions

The PAC₁ receptor was first detected in the neuroepithelium at the primitive streak stage. During prenatal and postnatal development of the rat brain, the tissue-specific distributions of PAC₁ receptor were first determined, and two splice variants of the PAC₁ receptor were observed. These were strongly expressed in the developing olfactory bulb, hippocampal formation and cerebellum, which showed cell type-specific or transient patterns of expression. These results strongly suggest that PACAP

Acknowledgements

We thank Dr Jerome L. Maderdrut (Tulane University Medical Center, New Orleans, LA, U.S.A.) for a critical review of the manuscript, and Dr Syuri Kaneyama (Showa University School of Pharmaceutical Sciences, Tokyo, Japan) for assistance with this study. This research was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan (to S.S., Y.N. and S.K.), the High-Technology Research Center Project from the Ministry of Education, Science, Sports and Culture

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Cerebellum is one of the brain regions with continuous neurogenesis after birth. High concentrations of PACAP binding sites and PAC1 receptors were found in the external granule cell layer of the rodent cerebellum during the first two postnatal weeks (Zhou et al., 1999; Basille et al., 2000), a period of intense multiplication and migration of granule cells. Contestabile and colleagues showed that treatments of cerebellar granule precursors in vitro with PACAP38 inhibited proliferation by 25–35% (Contestabile et al., 2005).
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Recent publications suggest a possible involvement of PACAP signaling system in the etiology of anxiety and affective disorders (Reichenstein et al. 2008; Hammack et al. 2009; Kamnasaran 2003). Different research groups showed PACAP and its receptors’ expression profiles in distinct brain regions during development using various gene expression approaches (Waschek et al. 1998; Sheward et al., 1996; Zhou et al. 2000; Zhou et al. 1999). It has been also suggested that PACAP and its specific receptor PAC1 participate in the regulation of neurodevelopment (Waschek et al. 1998; Vaudry et al. 1999; Watanabe et al. 2006).
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Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to participate in the regulation of neuronal proliferation and differentiation. While these processes are considered to be mediated via PACAP's actions on the PACAP-specific receptor, PAC1R, the precise distribution of PAC1R during neurodevelopment has not yet to be elucidated in detail. The purpose of this study is to examine the distribution of PAC1R in the neurogenic region of the rostral migratory stream (RMS) from the apical subventricular zone (SVZa) to the olfactory bulb (OB) in infant mice using immunostaining. Co-immunostaining for PAC1R in a variety types of cell were carried out using different markers. These included the neural stem cell markers, nestin and glial fibrillary acidic protein (GFAP), a marker for migrating neuroblasts (doublecortin, DCX), a marker for immature neurons βIII-tubulin, (Tuj1), and a marker for mature neurons, neuronal nuclei (NeuN). PAC1R-like immunoreactivity (LI) was observed in the RMS. However, the intensity of PAC1R- LI was different depending on the regions which were investigated. PAC1R-LI was strong in nestin- and GFAP-positive cells in the SVZa and was also observed in NeuN-positive cells in the OB. However, the intensities of PAC1R-LI in DCX- and Tuj1-positive cells were weaker than the other markers. These results suggest that PACAP may participate in the neurodevelopment with the stage-specific expression of PAC1R and that PACAP plays important roles in neurons as well as in glial cells.
Role of PACAP in the physiology and pathology of the sympathoadrenal system
2008, Frontiers in Neuroendocrinology
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Soon after its discovery, it has been shown that PACAP exerts various trophic effects on cells of the nervous system (Table 1). PACAP and its type I receptor PAC1-R are expressed in the neural tube during early neurogenesis, suggesting that PACAP might be implicated in neural tube patterning and neurogenesis [23,165,175]. In particular, PACAP and PAC1-R are detected at early stages of sympathetic development, and PACAP exerts a potent mitogenic action and promotes differentiation and survival of sympathetic neuroblasts [23].
Sympathetic neurons and chromaffin cells derive from common sympathoadrenal precursors which arise from the neural crest. Cells from this lineage migrate to their final destination and differentiate by acquiring a catecholaminergic phenotype in response to different environmental factors. It has been shown that the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) and its PAC1 receptor are expressed at early stages of sympathetic development, and participate to the control of neuroblast proliferation and differentiation. PACAP also acts as a neurotransmitter to stimulate catecholamine and neuropeptide biosynthesis and release from sympathetic neurons and chromaffin cells, during development and in adulthood. In addition, PACAP and its receptors have been described in neuroblastoma and pheochromocytoma, and the neuropeptide regulates the differentiation and activity of sympathoadrenal-derived tumoral cell lines, suggestive of an important role in the pathophysiology of the sympathoadrenal lineage. Transcriptome studies uncovered genes and pathways of known and unknown roles that underlie the effects of PACAP in the sympathoadrenal system.
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Neural development is controlled by region-specific factors that regulate cell proliferation, migration and differentiation. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that exerts a wide range of effects on different cell types in the brain as early as the fetal stage. Here we review current knowledge concerning several aspects of PACAP expression in embryonic and neonatal neural tissue: (i) the distribution of PACAP and PACAP receptors mRNA in the developing brain; (ii) the characteristic generation of neurons, astrocytes and oligodendrocytes in brain areas where the PACAP receptor is expressed and (iii) the role of PACAP as a regulator of neural development, inducing differentiation and proliferation in association with other trophic factors or signal transduction molecules.

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Pituitary adenylate cyclase-activating polypeptide receptors during development: expression in the rat embryo at primitive streak stage

Abstract

Section snippets

Animals and tissue preparation

Authenticity of the experiments for the PACAP receptor

Expression of PACAP receptor in the early embryo

Conclusions

Acknowledgements

Trends Endocr. Metab.

Front. Neuroendocr.

Devl Brain Res.

Neuroscience

Regul. Pept.

Neurosci. Lett.

J. biol. Chem.

Biochem. biophys. Res. Commun.

J. biol. Chem.

Regul. Pept.

Neurosci. Lett.

Neuron

Biochim. biophys. Acta

Biochem. biophys. Res. Commun.

Biochem. biophys. Res. Commun.

J. chem. Neuroanat.

Brain Res.

Neurosci. Lett.

Brain Res.

Neurosci. Lett.

Peptides

Peptides

Biochim. biophys. Acta

Biochem. biophys. Res. Commun.

Biochem. biophys. Res. Commun.

Fedn Eur. biochem. Socs Lett.

Biochem. biophys. Res. Commun.

J. biol. Chem.

Regul. Pept.

J. biol. Chem.

Neurosci. Lett.

Neurosci. Lett.

Neurosci. Res.

Brain Res.

Regul. Pept.

Biochem. biophys. Res. Commun.

Neuroscience

Biochem. biophys. Res. Commun.

Biochem. biophys. Res. Commun.

Peptides

Biochem. biophys. Res. Commun.

Peptides

Brain Res.

Peptides

J. biol. Chem.

Postnatal development of the cerebellar cortex in the rat

J. comp. Neurol.

Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testes

Endocrinology

Pituitary adenylate cyclase-activating polypeptide (PACAP): discovery and current status of research

Regul. Pept.

PACAP functions as a neurotrophic factor

Ann. N. Y. Acad. Sci.