Altered cerebellar development in mice lacking pituitary adenylate cyclase-activating polypeptide

Previous studies have demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) exerts trophic effects during neurodevelopment. In particular, the occurrence of PACAP and its receptors in the cerebellum during pre- and postnatal periods suggests that it could play a crucial role in ontogenesis of this structure. To test this hypothesis, we compared the histogenesis of cerebellar cortex in wild-type and PACAP-knockout (PACAP-/-) mice at postnatal days (P)4 and 7. Morphometric analysis of PACAP-/- mice revealed a significant reduction in the thickness of the external granule cell layer at P4 and of the internal granule cell layer at P7. Expression of nestin, a neural precursor marker, and synaptophysin, a mature neuronal marker, was quantified by real-time PCR and Western blot. No modification of nestin expression was noticed between wild-type and PACAP-/- mice, but a substantial decrease in synaptophysin expression was observed in PACAP-/- mice at P4 and P7. Immunohistochemistry revealed a reduction in synaptophysin labelling in the molecular and internal granule cell layers of PACAP-/- mice at P7. Caspase-3 activation was significantly increased in PACAP-/- mice at P4 and P7. Autoradiographic studies revealed no difference in PACAP binding site distributions and PACAP was effective at stimulating cAMP production in both wild-type and PACAP-/- cultured granule cells. This study demonstrates that disruption of the PACAP gene induces alteration of the immature cerebellum. Neuronal differentiation of granule cells was delayed whereas cell death that naturally occurs during ontogeny was increased in PACAP-/- mice. These data provide the first evidence of a physiological role for PACAP during cerebellar development.     

Expression of the receptor for pituitary adenylate cyclase-activating polypeptide (PAC1-R) in reactive astrocytes

We generated transgenic mice that express an enhanced green fluorescent protein (EGFP) under the control of the mouse glial fibrillary acidic protein (GFAP) promoter. In one of the transgenic lines, the green fluorescence of EGFP was undetectable in almost all of the brain regions, including the neocortex, in untreated animals. However, when reactive astrogliosis was induced by cortical stab wounding, the strong fluorescence of EGFP was observed around the needle track but was not found in the corresponding area of the contralateral hemisphere. The EGFP-expressing cells had the morphological features of reactive astrocytes such as thick processes. The EGFP-expressing cells were found to overlap with the astroglial marker GFAP, but not with the microglial marker CD11b or the neuronal marker NeuN. Furthermore, there were some EGFP-expressing cells that expressed vimentin-like immunoreactivity, the specific marker for reactive astrocytes. These results strongly suggest that the EGFP-expressing cells are reactive astrocytes, but not resting astrocytes. Using these transgenic mice, immunostaining for the PAC1 receptor (PAC1-R) was performed. PAC1-R, which is a pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor, binds PACAP, which is known to have a wide variety of functions. An immunohistochemical study revealed the localization of PAC1-R in reactive astrocytes visualized with EGFP around the needle track at 5 days postsurgery.     

Distribution of pituitary adenylate cyclase-activating polypeptide and pituitary adenylate cyclase-activating polypeptide type I receptor mRNA in the chicken brain

To map in detail the brain areas in which pituitary adenylate cyclase-activating polypeptide (PACAP) may play a significant role in birds, the distribution of PACAP and PACAP type I receptor (PAC(1)-R) mRNA was examined throughout the entire chicken brain by using in situ hybridization histochemistry. Widespread distribution of both PACAP and its receptor mRNA was found. The telencephalic areas where the most intense signals for PACAP mRNA were found included the hyperstriatum accessorium, the hippocampus, and the archistriatum. In the diencephalon, a group of neurons that highly expressed PACAP mRNA was observed from the anterior medial hypothalamic nucleus to the inferior hypothalamic nucleus. Moderate expression was found in the paraventricular nucleus and the preoptic region. A second large group of neurons containing PACAP message was found within the nucleus dorsolateralis anterior thalami and extended caudally to the area around the nucleus ovoidalis and the nucleus paramedianus internus thalami. Furthermore, expression of PACAP message was observed within the bed nucleus of the pallial commissure, nucleus spiriformis medialis, optic tectum, cerebellar cortex, olfactory bulbs, and several nuclei within the brainstem (dorsal vagal and parabrachial complex, reticular formation). The highest expression of PAC(1)-R mRNA was found in the dorsal telencephalon, olfactory bulbs, lateral septum, optic tectum, cerebellum, and throughout the hypothalamus and thalamus. The presence of PACAP and PAC(1)-R mRNA in a variety of brain areas in birds suggests that PACAP mediates several physiologically important processes in addition to regulating the activity of the pituitary gland.     

Developmental regulation of pituitary adenylate cyclase-activating polypeptide and PAC(1) receptor mRNA expression in the rat central nervous system

As the brain develops, a homogeneous population of mitotically active progenitors generates the molecularly heterogeneous post-mitotic cells of the mature brain. The balance between cell division, growth arrest and differentiation of these progenitors undoubtedly requires the activation of a vast array of genes. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family. Within the nervous system, PACAP has been shown to stimulate neurite outgrowth, regulate neurotransmitter production and neuronal survival. These diverse biological actions are mediated through interaction with two types of receptors, a PACAP-selective receptor (PAC(1)-R) and receptors which interact almost equally with both VIP and PACAP. Since several lines of evidence suggest that PACAP acts as a neurotrophic factor, we sought to characterize PACAP and PAC(1)-R expression in the developing rat nervous system. The PAC(1)-R is expressed at very high levels in ventricular zones throughout the neuraxis. In addition to the embryonic enrichment in proliferative zones, PAC(1)-R expression is maintained in areas of neurogenesis in the adult central nervous system (CNS), namely, the subventricular zone of the olfactory bulb and hippocampal dentate gyrus. In contrast, PACAP is expressed primarily in the post-mitotic parenchyma. This temporal regulation and cellular distribution suggests that PACAP, through its interaction with the PAC(1)-R, may play a role in mammalian neurogenesis.     

Progesterone increases mRNA levels of pituitary adenylate cyclase-activating polypeptide (PACAP) and type I PACAP receptor (PAC(1)) in the rat hypothalamus

Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates pituitary hormone biosynthesis and secretion through its cognate receptors. PACAP also plays an important role in the regulation of ovarian steroid biosynthesis. If so, there might be a feedback regulation of hypothalamic PACAP synthesis by the pituitary and by ovarian steroids. In the present study, we used RNase protection assays to determine changes in mRNA levels of PACAP and type I PACAP receptor (PAC(1)) under the conditions of ovariectomy and replacement with ovarian steroids. Progesterone (P) alone or in combination with estradiol (E) induced significant increases in PACAP mRNA level in the medial basal hypothalamus (MBH) and PAC(1) mRNA levels in MBH and the preoptic area (POA). This finding suggests that feedback regulation takes place between the ovary and hypothalamic PACAP neurons. P is known to be a major regulatory feedback factor for hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons, but P receptor is not present in these neurons. Therefore, we examined a possible involvement of PACAP in the feedback regulatory pathway of P to LHRH neurons. After an antisense PAC(1) oligodeoxynucleotide (ODN) was i.c.v.-injected into the third ventricle of E and P-treated rats, LHRH mRNA levels were determined. The ODN markedly decreased the P-induced increase in the LHRH mRNA level. Taken together, the present data suggest that PACAP may play a role as a mediator in the regulation of LHRH synthetic machinery by stimulatory feedback of P.     

Morphine withdrawal is modified in pituitary adenylate cyclase-activating polypeptide type I-receptor-deficient mice

The pituitary adenylate cyclase-activating polypeptide type I-receptor (PAC1) is a G-protein-coupled receptor that is widely expressed in neurons of the central and peripheral nervous system. The strong expression of PAC1 in the second sensory neuron as well as in brainstem regions such as the locus coeruleus prompted us to elucidate the potential in vivo role of PAC1-mediated signalling in pain perception and opioid addiction using a PAC1-deficient mouse line. We observed a selective involvement of PAC1 in the mediation of visceral pain. While there was no impairment in acute somatic pain perception, PAC1-mutants exhibited a dramatically decreased response in the abdominal writhing test. These data in concert with data from the literature implicate PAC1 in the mediation of visceral and chronic pain. In addition, we observed that PAC1 did not influence the motivational aspects of opioid addictive properties, since morphine-induced rewarding effects and sensitization to locomotor responses were completely maintained in PAC1-deficient mice. However, there was a dramatic increase in physical withdrawal signs after naloxone-precipitated morphine withdrawal in PAC1 mutants. At the cellular level, electrophysiological examinations in locus coeruleus neurons from morphine-dependent wild-type and PAC1-deficient mice did not reveal any differences in firing rates. These data therefore suggested that most likely disruption of PAC1-mediated signalling in afferents towards the locus coeruleus but not within the intrinsic locus coeruleus system led to the enhancement of somatic withdrawal signs.     

Distribution of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide receptors (VPAC1, VPAC2, and PAC1 receptor) in the rat brain

To examine the distributions of VIP/PACAP receptors (VPAC1, VPAC2, and PAC1 receptors) in the brain and to identify the cell types that express these receptors, we performed immunohistochemistry and double immunofluorescence in the rat brain with specific antibodies. The immunohistochemistry revealed that the receptors had distinctive, complementary, and overlapping distribution patterns. High levels of the VPAC1 receptor were expressed in the cerebral cortex, hippocampal formation, deep cerebellar nuclei, thalamus, hypothalamus, and brainstem. The VPAC2 receptors were concentrated in the cerebral cortex, hippocampal formation, amygdalar regions, cerebellar cortex, deep cerebellar nuclei, hypothalamus, and brainstem. On the other hand, the PAC1 receptors had a more restricted distribution pattern in the brain, and high levels of the PAC1 receptors were confined to the cerebellar cortex, deep cerebellar nuclei, epithalamus, hypothalamus, brainstem, and white matter of many brain regions. Also, many fibers expressing the PAC1 receptors were observed in various areas, i.e., the thalamus, hypothalamus, and brainstem. The double immunofluorescence showed that the VIP/PACAP receptors were confined to the neuroglia as well as the neurons. All three types of the VIP/PACAP receptors were expressed in the astrocytes, and the PAC1 receptors were also expressed in the oligodendrocytes. These findings indicate that VIP and PACAP exert their functions through their receptors in specific locations in different combinations. We hope that this first demonstration of the distributions of the VIP/PACAP receptors provides data useful in the investigation of the mechanisms of the many functions of VIP and PACAP in the brain, which require further elucidation.     

Impaired long-term potentiation in vivo in the dentate gyrus of pituitary adenylate cyclase-activating polypeptide (PACAP) or PACAP type 1 receptor-mutant mice

The present study was conducted to clarify a role of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP type 1 receptor (PAC1R) in learning and memory function. We demonstrated long-term potentiation (LTP) in vivo in the dentate gyrus of PAC1R exon 2-deficient (PAC1R-/-) mice and heterozygous PACAP-deficient (PACAP+/-) mice using extracellular recording techniques. We used two paradigms of tetanic stimulation, suprathreshold and at threshold tetanus, which both induced LTP in vivo in PAC1R-/- and PACAP+/- mice. However, the population spike of 'at threshold' but not 'suprathreshold' LTP decreased significantly in PAC1R-/- and PACAP+/- mice. At threshold LTP of PACAP+/- mice was impaired greater than the one of PAC1R-/- mice. Thus, both PACAP and PAC1R could contribute to the establishment of LTP in a gene dosage-dependent manner, although PACAP rather than PAC1R might play a pivotal role in learning and memory function.     

Impaired somatodendritic responses to pituitary adenylate cyclase-activating polypeptide (PACAP) of supraoptic neurones in PACAP type I -receptor deficient mice

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor (PAC1 receptor) in regulating hypothalamic supraoptic neurones was investigated using PAC1 receptor-deficient male mice (PAC1-/-). The effects of PACAP on [Ca2+]i were investigated in freshly dissociated supraoptic neurones and on the somatodendritic release of vasopressin and oxytocin, examined on intact supraoptic nuclei. In supraoptic neurones from wild-type mice (PAC1+/+), 100 nm PACAP induced an increase in [Ca2+]i and release of vasopressin and oxytocin, whereas in heterozygous (PAC1+/-) and null-mutant mice (PAC1-/-), PACAP was much less effective. PACAP had no effect on these two parameters when applied to isolated neurohypophysial nerve terminals of PAC1+/+ and PAC1-/- mice, and rats. In conclusion, the PAC1 receptor is solely responsible for the PACAP-induced [Ca2+]i signalling and secretion of vasopressin and oxytocin in the somatodendritic region of supraoptic neurones.     

Neurotrophic and neuroprotective effects of pituitary adenylate cyclase-activating polypeptide (pACAP) on mesencephalic dopaminergic neurons

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is present in many regions of the adult and developing brain as are receptors for PACAP. PACAP stimulates different signalling cascades in neurons, involving cAMP, MAP kinase, and calcium. These characteristics suggest that PACAP may influence neuronal development. Here we have studied the effects of PACAP on mesencephalic dopaminergic neurons using primary cultures from embryonic rats. PACAP increased the number of tyrosine hydroxylase (TH)-immunoreactive neurons, elevated TH protein, and enhanced tritiated dopamine uptake in these cultures. Moreover, PACAP counteracted the effects of 6-hydroxydopamine treatments, which induce cell death of dopaminergic neurons. In situ hybridisation showed that both PACAP and PACAP receptor type 1 are present in developing and adult rat mesencephalon. These results show that PACAP has a neurotrophic action on dopaminergic neurons and partially protects them against 6-OHDA induced neurotoxicity. J. Neurosci. Res. 54:698–706, 1998. © 1998 Wiley-Liss, Inc.     

Signaling cascades involved in neuroprotection by subpicomolar pituitary adenylate cyclase-activating polypeptide 38

In neuronal/glial cocultures, pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) prevented neuronal death induced by gp120, lipopolysaccharide (LPS), or other toxic agents, but the dose response of the neuroprotective effect is bimodal, with a peak at a subpicomolar concentration and another peak at a subnanomolar to nanomolar concentration. Although the signaling cascade involved in neuroprotection by nanomolar concentration of the peptide has been shown to be mediated by activation of cAMP-dependent protein kinase and subsequent activation of mitogen-activated protein kinase (MAPK), the mechanism for neuroprotection by a subpicomolar level of PACAP38 remains elusive. In the present study, the signaling involved in neuroprotection by subpicomolar PACAP38 was studied in rat neuronal/glial cocultures. Addition of PACAP38 stimulated expression and activation of extracellular signal-related kinase-type MAPK with a peak response at 10⁻¹³ M; greater concentrations of the peptide induced lesser response. cAMP production also increased at subpicomolar levels of PACAP38, but the level remained unchanged at a level four to five times higher than the base level at concentration below 10⁻¹¹ M. cAMP then started increasing again dose-dependently in a range >10⁻¹¹ MPACAP38. Lipopolysaccharide (LPS)-induced neuronal death, indicated by increased release of neuronspecific enolase, was suppressed by PACAP38 in a bimodal fashion. Neuroprotection by 10⁻¹² M PACAP38 was completely abolished by a MAPK kinase-1 inhibitor, PD98059, and also partially suppressed by Rp-cAMP, a cAMP-dependent protein kinase inhibitor. Moreover, neuroprotection by a nanomolar level of PACAP38 was completely suppressed by Rp-cAMP but not affected by PD98059. We conclude that neuroprotection by subpicomolar PACAP38 is mainly mediated by the signaling pathway involving MAPK activation and partially regulated by cAMP-dependent protein kinase activation. Furthermore, PACAP38 stimulated expression of activity-dependent neuroprotective protein (ADNP), with a peak at 10⁻¹³ M. Greater doses of the peptide induced lesser response. However, 10⁻¹³ M PACAP38-stimulated expression of ADNP was not affected by PD98059. This suggests that neuroprotection by subpicomolar PACA38 might be mediated partially by expression of ADNP, but the major events for neuroprotection by subpicomolar PACAP38 remain to be identified.     

Signaling cascades involved in neuroprotection by subpicomolar pituitary adenylate cyclase-activating polypeptide 38

In neuronal/glial cocultures, pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) prevented neuronal death induced by gp120, lipopolysaccharide (LPS), or other toxic agents, but the dose response of the neuroprotective effect is bimodal, with a peak at a subpicomolar concentration and another peak at a subnanomolar to nanomolar concentration. Although the signaling cascade involved in neuroprotection by nanomolar concentration of the peptide has been shown to be mediated by activation of cAMP-dependent protein kinase and subsequent activation of mitogen-activated protein kinase (MAPK), the mechanism for neuroprotection by a subpicomolar level of PACAP38 remains elusive. In the present study, the signaling involved in neuroprotection by subpicomolar PACAP38 was studied in rat neuronal/glial cocultures. Addition of PACAP38 stimulated expression and activation of extracellular signal-related kinase-type MAPK with a peak response at 10-13 M; greater concentrations of the peptide induced lesser response. cAMP production also increased at subpicomolar levels of PACAP38, but the level remained unchanged at a level four to five times higher than the base level at concentrations below 10-11 M. cAMP then started increasing again dose-dependently in a range >10-11 M PACAP38. Lipopolysaccharide (LPS)-induced neuronal death, indicated by increased release of neuron-specific enolase, was suppressed by PACAP38 in a bimodal fashion. Neuroprotection by 10-12 M PACAP38 was completely abolished by a MAPK kinase-1 inhibitor, PD98059, and also partially suppressed by Rp-cAMP, a cAMP-dependent protein kinase inhibitor. Moreover, neuroprotection by a nanomolar level of PACAP38 was completely suppressed by Rp-cAMP but not affected by PD98059. We conclude that neuroprotection by subpicomolar PACAP38 is mainly mediated by the signaling pathway involving MAPK activation and partially regulated by cAMP-dependent protein kinase activation. Furthermore, PACAP38 stimulated expression of activity- dependent neuroprotective protein (ADNP), with a peak at 10-13 M. Greater doses of the peptide induced lesser response. However, 10-13 M PACAP38-stimulated expression of ADNP was not affected by PD98059. This suggests that neuroprotection by subpicomolar PACAP38 might be mediated partially by expression of ADNP, but the major events for neuroprotection by subpicomolar PACAP38 remain to be identified.     

Comparative distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites and PACAP receptor mRNAs in the rat brain during development

The distribution and density of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites as well as PACAP-specific receptor 1 (PAC1-R), vasoactive intestinal polypeptide/PACAP receptor 1 (VPAC1-R), and VPAC2-R mRNAs have been investigated in the rat brain from embryonic day 14 (E14) to postnatal day 8 (P8). Significant numbers of binding sites for the radioiodinated, 27-amino-acid form of PACAP were detected as early as E14 in the neuroepithelia of the metencephalon and the myelencephalon. From E14 to E21, the density of binding sites in the germinative areas increased by 3- to 5-fold. From birth to P12, the density of binding sites gradually declined in all neuroepithelia except in the external granule cell layer of the cerebellum, where the level of binding sites remained high during the first postnatal weeks. Only low to moderate densities of PACAP binding sites were found in regions other than the germinative areas, with the exception of the internal granule cell layer of the cerebellum, which contained a high density of sites. The localization of PACAP receptor mRNAs was investigated by in situ hybridization using [(35)S] uridine triphosphate-specific riboprobes. The evolution of the distribution of PAC1-R and VPAC1-R mRNAs was very similar to that of PACAP binding sites, the concentration of VPAC1-R mRNA being much lower than that of PAC1-R mRNA. In contrast, intense expression of VPAC2-R mRNA was observed in brain regions other than germinative areas, such as the suprachiasmatic, ventral thalamic, and dorsolateral geniculate nuclei. The discrete localization of PACAP binding sites as well as PAC1-R and VPAC1-R mRNAs in neuroepithelia during embryonic life and postnatal development strongly suggests that PACAP, acting through PAC1-R and/or VPAC1-R, may play a crucial role in the regulation of neurogenesis in the rat brain.     

Origin of pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive fibers innervating guinea pig parasympathetic cardiac ganglia

The present study investigated the origin of pituitary adenylate cyclase-activating polypeptide (PACAP) -immunoreactive (IR) fibers innervating guinea pig cardiac ganglia. Immunohistochemistry was performed on whole-mounts containing cardiac ganglia, and sections of stellate, nodose, and dorsal root ganglia (DRG, thoracic levels 1-4), and caudal medulla. In control preparations, only 4% of the cardiac neurons were PACAP-IR, although most cardiac ganglion cells were surrounded by a network of PACAP-IR fibers. After 3-7 days in explant culture, the number of PACAP-IR cardiac neurons increased approximately eightfold. However, virtually all PACAP-IR fibers surrounding the cardiac neurons had degenerated, demonstrating that the major source of the PACAP-IR fibers was extrinsic to the cardiac ganglia preparation. PACAP- and choline acetyltransferase (ChAT) immunoreactivity were colocalized in fibers within the stellate ganglia but not within neuropeptide Y (NPY) -IR cell bodies and fibers. PACAP-IR cells and fibers were present in the nodose ganglia. PACAP immunoreactivity also was present in fibers and primarily small neurons in thoracic DRGs. In situ hybridization demonstrated the presence of proPACAP mRNA within neurons in the region of the dorsal motor nucleus of the vagus and nucleus ambiguus. PACAP immunoreactivity was colocalized with ChAT immunoreactivity, but not with NPY immunoreactivity or SP immunoreactivity, in fibers surrounding neurons within cardiac ganglia. We conclude that PACAP-containing fibers innervating the postganglionic parasympathetic neurons in guinea pig cardiac ganglia are primarily preganglionic parasympathetic axons.     

Molecular characterization and gene expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) in the lizard brain

The pituitary adenylate cyclase-activating polypeptide (PACAP) is considered a pleiotropic neuropeptide in vertebrate physiology. The nucleotide sequence, the expression and the distribution of PACAP were determined in the brain of the lizard Podarcis sicula. RT-PCR showed that the brain of this reptile synthesizes an mRNA coding for PACAP. By performing in situ hybridization and immunohistochemistry techniques, a wide distribution of PACAP and its mRNA in neurons, nervous fibers and other cells was found. Phylogenetic sequence analysis indicates that lizard PACAP is highly conserved, resembling the vertebrate PACAP. Our data demonstrate that PACAP is not only highly preserved during vertebrate evolution but also suggest that PACAP could be implicated in a wide number of functions in the physiology of the reptile brain.     

Up-regulation of pituitary adenylate cyclase-activating polypeptide in urinary bladder pathways after chronic cystitis

These studies examined changes in the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) in micturition reflex pathways after chronic cystitis induced by cyclophosphamide (CYP). In control Wistar rats, PACAP immunoreactivity was expressed in fibers in the superficial dorsal horn at all segmental levels examined (L1, L2, and L4-S1). Bladder afferent cells (40-45%) in the dorsal root ganglia (DRG; L1, L2, L6, and S1) from control animals also exhibited PACAP immunoreactivity. After chronic, CYP-induced cystitis, PACAP immunoreactivity increased dramatically in spinal segments and DRG (L1, L2, L6, and S1) involved in micturition reflexes. The density of PACAP immunoreactivity was increased in the superficial laminae (I-II) of the L1, L2, L6, and S1 spinal segments. No changes in PACAP immunoreactivity were observed in the L4-L5 segments. Staining also increased dramatically in a fiber bundle extending ventrally from Lissauer's tract in lamina I along the lateral edge of the dorsal horn to the sacral parasympathetic nucleus in the L6-S1 spinal segments (lateral collateral pathway of Lissauer). After chronic cystitis, PACAP immunoreactivity in cells in the L1, L2, L6, and S1 DRG increased significantly (P 相似文献