Interactions of PACAP and ceramides in the control of granule cell apoptosis during cerebellar development.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal polypeptide superfamily. The PACAPergic system is actively expressed in the developing cerebellum of mammals. In particular, PACAP receptors are expressed by granule cell precursors suggesting a role of the peptide in neurogenesis of this cell type. Consistent with this hypothesis, several studies reported antiapoptotic effects of PACAP in the developing cerebellum. On the other hand, the sphingomyelin metabolites ceramides are recognized as important signaling molecules that play pivotal roles during neuronal development. Ceramides, which production can be induced by death factors such as FasL or TNFalpha, are involved in the control of cell survival during brain development through activation of caspase-dependent mechanisms. The present review focuses on the interactions between PACAP and ceramides in the control of granule cell survival and on the transduction mechanisms associated with the anti- and proapoptotic effects of PACAP and ceramides, respectively.

The factorial decomposition of the Mahalanobis distances in habitat selection studies.

The Mahalanobis distances have been introduced in habitat selection studies for the estimation of environmental suitability maps (ESMs). The pixels of raster maps of a given area correspond to points in the multidimensional space defined by the mapped environmental variables (ecological space). The Mahalanobis distances measure the distances in this space between these points and the mean of the ecological niche (i.e., the hypothesized optimum for the species) regarding the structure of the niche. The map of these distances over the area of interest is an estimated ESM. Several authors recently noted that the use of a single optimum for the niche of a species may lead to biased predictions of animal occurrence. They proposed to use instead a minimum set of basic habitat requirements, found by partitioning the Mahalanobis distances into a restricted set of biologically meaningful axes. However, the statistical approach they proposed does not take into account the environmental conditions on the area where the niche was sampled (i.e., the environmental availability), and we show that including this availability is necessary. We used their approach as a basis to develop a new exploratory tool, the Mahalanobis distance factor analysis (MADIFA), which performs an additive partitioning of the Mahalanobis distances taking into account this availability. The basic habitat requirements of a species can be derived from the axes of the MADIFA. This method can also be used to compute ESMs using only this small number of basic requirements, therefore including only the biologically relevant information. We also prove that the MADIFA is complementary to the commonly used ecological-niche factor analysis (ENFA). We used the MADIFA method to analyze the niche of the chamois Rupicapra rupicapra in a mountainous area. This method adds to the existing set of tools for the description of the niche.

The neuropeptide pituitary adenylate cyclase-activating polypeptide exerts anti-apoptotic and differentiating effects during neurogenesis: focus on cerebellar granule neurones and embryonic stem cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from ovine hypothalamus on the basis of its hypophysiotrophic activity. It has subsequently been shown that PACAP and its receptors are widely distributed in the central nervous system of adult mammals, indicating that PACAP may act as a neurotransmitter and/or neuromodulator. It has also been found that PACAP and its receptors are expressed in germinative neuroepithelia, suggesting that PACAP could be involved in neurogenesis. There is now compelling evidence that PACAP exerts neurotrophic activities in the developing cerebellum and in embryonic stem (ES) cells. In particular, the presence of PACAP receptors has been demonstrated in the granule layer of the immature cerebellar cortex, and PACAP has been shown to promote survival, inhibit migration and activate neurite outgrowth of granule cell precursors. In cerebellar neuroblasts, PACAP is a potent inhibitor of the mitochondrial apoptotic pathway through activation of the MAPkinase extracellular regulated kinase. ES cells and embryoid bodies (EB) also express PACAP receptors and PACAP facilitates neuronal orientation and induces the appearance of an electrophysiological activity. Taken together, the anti-apoptotic and pro-differentiating effects of PACAP characterised in cerebellar neuroblasts as well as ES and EB cells indicate that PACAP acts not only as a neurohormone and a neurotransmitter, but also as a growth factor.

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.

Pituitary adenylate cyclase-activating polypeptide promotes cell survival and neurite outgrowth in rat cerebellar neuroblasts.

High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors have been detected in the rat cerebellum during ontogenesis. In particular, PACAP receptors are actively expressed in immature granule cells, suggesting that PACAP may act as a neurotrophic factor in the developing rat cerebellum. In the present study, we have investigated the effect of PACAP on cell survival and neurite outgrowth in cultured immature cerebellar granule cells. In control conditions, cultured granule cells undergo programmed cell death. Exposure of cultured cells to PACAP for 24 and 48 h provoked a significant increase in the number of living cells. The effect of PACAP on cell survival was inhibited by the PACAP antagonist PACAP(6-38). Vasoactive intestinal polypeptide was approximately 1000 times less potent than PACAP in promoting cell survival. PACAP also induced a significant increase in the number of processes and in the cumulated length of neurites borne by cultured neuroblasts. The present results demonstrate that PACAP promotes cell survival and neurite outgrowth in cultured immature granule cells. Since PACAP and its receptors are expressed in situ in the rat cerebellar cortex, these data strongly suggest that PACAP plays a physiological role in the survival and differentiation of cerebellar granule cells.

Localization and characterization of PACAP receptors in the rat cerebellum during development: evidence for a stimulatory effect of PACAP on immature cerebellar granule cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are abundant in the brain and particularly in the cerebellum of adult rats. In contrast, the occurrence of PACAP binding sites has not been investigated during ontogenesis. The aim of the present study was to determine the distribution and biochemical characteristics of PACAP binding sites in the rat cerebellum during postnatal development, and to examine the effect of PACAP on immature cerebellar granule cells. Autoradiographic studies revealed that PACAP binding sites are transiently expressed in a germinative matrix of the cerebellar cortex, the external granule cell layer, and in the medulla, from postnatal days 8 to 25. A population of PACAP binding sites persisted in the internal granule cell layer in the mature cerebellum. Emulsion-coated cytoautoradiography, performed on cultured immature granule cells from eight-day-old rat cerebellum, demonstrated that transient PACAP binding sites are expressed by cerebellar immature granule cells. Biochemical characterization of binding revealed the occurrence of two classes of PACAP recognition sites exhibiting, respectively, high (Kd = 0.39 +/- 0.08 nM) and low (Kd = 21.2 +/- 9.4 nM) affinity for PACAP27. The two naturally occurring forms PACAP38 and PACAP27 were equipotent in competing for [125I]PACAP27 binding. In contrast, the [Des-His1]PACAP38 analog was eight times less efficient and vasoactive intestinal polypeptide only induced weak displacement of the binding. Exposure of cultured immature granule cells to PACAP27 resulted in a dose-dependent stimulation of the production of cAMP, indicating that PACAP binding sites represent authentic receptors positively coupled to adenylate cyclase. These results show that PACAP receptors are actively expressed in the cerebellum of rats during postnatal development. The presence of functional PACAP receptors in the external granule cell layer suggests that PACAP may play a role in the control of proliferation and/or differentiation of granule cells.

Pituitary adenylate cyclase-activating polypeptide stimulates both c-fos gene expression and cell survival in rat cerebellar granule neurons through activation of the protein kinase A pathway.

A high density of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors coupled to both adenylyl cyclase and phospholipase C is found in the external granule cell layer of the rat cerebellum during postnatal development. It has recently been reported that synthetic PACAP promotes cell survival and neurite outgrowth in immature granule cells. In the present study, we have investigated the transduction pathways that mediate the neurotrophic activity of PACAP in cultured granule cells from eight-day-old rat cerebellum. The effect of PACAP on cell survival was mimicked by dibutyryladenosine 3',5'-cyclic-monophosphate but not phorbol 12-myristate 13-acetate suggesting that only the adenylyl cyclase pathway is involved in the neurotrophic activity of PACAP. PACAP also induced a transient increase in c-fos messenger RNA level. The ability of PACAP to stimulate c-fos gene expression was mimicked by dibutyryladenosine 3',5'-cyclic-monophosphate but not phorbol 12-myristate 13-acetate. Similar effects of PACAP on granule cell survival were observed whether the cells were continuously incubated with PACAP for 48 h or only exposed to PACAP during 1 h. The protein kinase A inhibitor H89 significantly reduced the effect of PACAP on c-fos messenger RNA level whereas the specific protein kinase C inhibitor chelerythrine did not modify c-fos gene expression. These data indicate that the action of PACAP on cerebellar granule cell survival and c-fos gene expression are both mediated through the adenylyl cyclase/protein kinase A pathway. The observation that a short-term stimulation by PACAP can be converted into a long-lasting response indicates that the effect of the peptide on cell survival must involve immediate-early gene activation. The fact that a brief exposure to PACAP causes both c-fos gene expression and promotes cell survival strongly suggests that c-fos is involved in the trophic effect of PACAP on immature cerebellar granule cells.

Pharmacological, molecular and functional characterization of vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide receptors in the rat pineal gland.

Melatonin secretion from the mammalian pineal gland is strongly stimulated by noradrenaline and also by vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Three types of receptors for VIP and PACAP have been characterized so far: VIP1/PACAP receptors and VIP2/PACAP receptors, which possess similar high affinities for VIP and PACAP, and PACAP1 receptors which exhibit a 100-1000-fold higher affinity for PACAP. The aim of the present study was to characterize the receptor subtype(s) mediating the stimulatory effects of VIP and PACAP on melatonin synthesis in the rat pineal gland. Autoradiographic studies showed that PACAP and VIP were equally potent in displacing binding of radioiodinated PACAP27 from pineal sections. Amplification of pineal complementary DNAs by polymerase chain reaction using specific primers for the different receptor subtypes revealed that all three receptor messenger RNAs are expressed and that VIP1/PACAP receptor messenger RNA was predominant over VIP2/PACAP receptor messenger RNA. In vitro, VIP and PACAP stimulated melatonin synthesis with similar high potency and the effect of the two peptides were not additive. The selective VIP1/PACAP receptor agonists [R16]chicken secretin (1-25) and [K15, R16, L27]VIP(1-7)/growth hormone releasing factor(8-27) were significantly more potent than the selective VIP2/PACAP receptor agonist RO 25-1553 in stimulating melatonin secretion. The stimulatory effects of VIP and PACAP were similarly inhibited by the VIP1/PACAP antagonist [acetyl-His1, D-Phe2, K15, R16, L27]VIP(3-7)/growth hormone releasing factor(8-27). These data strongly suggest that VIP and PACAP exert a stimulatory effect on melatonin synthesis mainly through activation of a pineal VIP1/PACAP receptor subtype.

The neurotrophic activity of PACAP on rat cerebellar granule cells is associated with activation of the protein kinase A pathway and c-fos gene expression.

In vitro studies have shown that PACAP promotes cell survival and neurite outgrowth in immature cerebellar granule cells. In the present study, we have examined the transduction pathways involved in the neurotrophic activity of PACAP. Incubation of cultured granule cells with graded concentrations of PACAP produced a dose-dependent increase in c-fos mRNA level. The effects of PACAP on c-fos gene expression and granule cell survival were both mimicked by dbcAMP but not by PMA. The maximum effect of PACAP on c-fos gene expression was observed after 1 h of treatment. Similar effects of the peptide on granule cell survival were observed whether the cells were continuously incubated with PACAP for 48 h or only exposed to PACAP during 1 h. The PKA inhibitor H89 significantly reduced the effect of PACAP on c-fos mRNA level, whereas the specific PKC inhibitor chelerytrine had no effect. These data indicate that the action of PACAP on cerebellar granule cell survival and c-fos gene expression are both mediated through the adenylyl cyclase/PKA pathway.

PACAP acts as a neurotrophic factor during histogenesis of the rat cerebellar cortex.

During development of the rat cerebellum, PAC1 receptors are transiently expressed by neuroblasts of the external granule cell layer (EGL). We have previously shown that PACAP is a potent stimulator of granule cell survival in vitro. In the study reported in this paper, we have investigated the effect of PACAP on the development of the rat cerebellar cortex in vivo. PACAP induces a transient increase in the volume of the cerebellar cortex, with a maximum effect at postnatal day 12, which can be accounted for by an increase in the number of granule cells in the EGL, the molecular layer, and the internal granule cell layer (IGL). The effect of PACAP on the number of granule cells is blocked by the antagonist PACAP(6-38), which, by itself, produces a slight inhibition of the number of granule cells in the IGL. These data indicate that PACAP activates proliferation and/or inhibits programmed cell death of granule cells in the developing rat cerebellum. PACAP also stimulates neuronal migration from the EGL to the IGL. Thus, it appears that PACAP can act in vivo as a neurotrophic factor controlling histogenesis of the cerebellar cortex.

Ontogeny of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors in the rat cerebellum: a quantitative autoradiographic study.

Pituitary adenylate cyclase-activating polypeptide and PACAP receptors are both present in the rat cerebellar cortex, suggesting that PACAP may play an important role in the cerebellum. In the present study, the variation of the concentration of PACAP binding sites in the rat cerebellum was investigated during postnatal development by means of quantitative autoradiography, using [125I]PACAP27 as a radioligand. In the external granule cell layer and the medulla, the density of PACAP binding sites was high at birth, markedly decreased from postnatal day 8 (P8) to P25 and finally vanished at the end of the third postnatal week. In the internal granule cell layer and molecular layer, PACAP binding sites were first detected at P8. In the internal granule cell layer, the density of binding sites slightly decreased during development but remained elevated in adults. Conversely, in the molecular layer, PACAP binding sites rapidly decreased during the second and third postnatal weeks and virtually disappeared after P25. In all four layers of the cerebellar cortex, the autoradiographic labeling was displaced by PACAP27 (IC50 close to 10(-8) M), but was not affected by VIP. No significant changes in IC50 and Hill coefficient were noticed in the various layers throughout development. The present study shows that all four layers of the cerebellar cortex express PACAP binding sites during development. The evolution of the receptor concentration exhibited differential profiles in the various layers but the specificity characteristics of the recognition sites were identical in all four structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatostatin and pituitary adenylate cyclase-activating polypeptide (PACAP): two neuropeptides potentially involved in the development of the rat cerebellum.

Somatostatin and pituitary adenylate cyclase-activating polypeptide (PACAP) have been originally isolated from the ovine hypothalamus on the basis of their hypophysiotropic activities. There is now evidence that somatostatin and PACAP may play a role in the development of the central nervous system, particularly in the cerebellum. High concentrations of somatostatin and somatostatin receptors have been detected in the rat cerebellum during the first two postnatal weeks. Somatostatin binding sites are associated with a germinative matrix, the external granule cell layer, which generates the majority of the interneurons of the cerebellum. By using immature granule cells in primary culture, we could demonstrate that somatostatin binding sites are actually expressed by neuroblasts and correspond to authentic receptors negatively coupled to adenylate cyclase. Concurrently, studies on the distribution of PACAP receptors in the immature rat cerebellum showed the presence of a high concentration of binding sites in the external granule cell layer during the first two postnatal weeks. Pharmacological characterization of these binding sites showed that they correspond to type I PACAP receptors positively coupled to adenylate cyclase. The concomitant and transient expression of somatostatin and PACAP receptors by cerebellar neuroblasts in the external granule cell layer suggests that the two neuropeptides may be involved in the regulation of multiplication, migration and/or differentiation of neuroblasts. This hypothesis is also supported by the actions of somatostatin and PACAP on various transduction systems. In particular, the opposite effects of the two neuropeptides on adenylate cyclase activity suggest that somatostatin and PACAP may exert antagonistic actions.

[Pituitary adenylate cyclase-activating polypeptide]. / Pituitary adenylate cyclase-activating polypeptide.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been originally isolated from the sheep hypothalamus on the basis of its ability to stimulate cAMP formation in anterior pituitary cells. Post-translational processing of the PACAP precursor generates two biologically active molecular forms, PACAP38 and PACAP27, and a novel peptide called PACAP-related peptide whose activity remains unknown. The primary structure of PACAP has been remarkably conserved during evolution, from protochordates to mammals, suggesting that the peptide exerts important activities throughout the vertebrate phylum. The sequence of PACAP27 exhibits substantial similarities with those of vasoactive intestinal polypeptide (VIP), glucagon and secretin. The gene encoding the PACAP precursor is widely expressed in the brain and in various peripheral organs, notably in endocrine glands, the gastro-intestinal and uro-genital tracts and the respiratory system. In vivo and in vitro studies have shown that PACAP exerts multiple activities as a hormone, neurohormone, neurotransmitter or trophic factor. For instance, PACAP triggers the release of insulin and glucagon, activates steroidogenesis in the adrenal gland and gonads, and stimulates the secretion of most hypophysial cells. PACAP exerts a potent relaxant activity on smooth muscle fibers in blood vessels, lung and gut. In the brain, PACAP stimulates the electrical activity of various populations of neurons and increases tyrosine hydroxylase gene expression. Recent studies have shown that PACAP exerts a trophic activity during ontogenesis, notably in the adrenal medulla and in the central nervous system. The biological effects of PACAP are mediated through three distinct receptor subtypes which exhibit differential affinities for PACAP and VIP. The PAC1 receptor, which shows high selectivity for PACAP, is coupled to several transduction systems. In contrast, VPAC1 and VPAC2, which bind with the same affinity PACAP and VIP, are mainly coupled to the adenylyl cyclase pathway. The bronchodilatator and vasorelaxant effects of PACAP, as well as the antiproliferative and neuroprotective actions of the peptide, make it a valuable target for new drug development.

Pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide promote the genesis of calcium currents in differentiating mouse embryonic stem cells.

Identification of novel molecules that can induce neuronal differentiation of embryonic stem (ES) cells is essential for deciphering the molecular mechanisms of early development and for exploring cell therapy approaches. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are known to be implicated early during ontogenesis in cell proliferation and neuronal differentiation. The aim of the present study was to determine the effects of VIP and PACAP on functional differentiation of ES cells. Quantitative-reverse transcription-polymerase chain reaction analysis showed an inversion of the expression pattern of PAC1 and VPAC1 receptors with time. ES cells expressed genes encoding extracellular signal-regulated kinase 1 and 2 and c-jun amino terminal kinase1. ES cells also expressed T-type α1I and α1G, L-type α1C and α1D, and N-type α1B calcium channel subunit mRNAs. Both peptides modified the shape of undifferentiated ES cells into bipolar cells expressing the neuronal marker neuron-specific enolase (NSE). Immunostaining indicated that PACAP intensified T-type α1I subunit immunoreactivity, whereas VIP increased L-types α1C and α1D, as well as N-type α1B subunit. Electrophysiological recording showed that VIP and PACAP enhanced transient calcium current. Moreover, VIP generated sustained calcium current. These findings demonstrate that PACAP and VIP induce morphological and functional differentiation of ES cells into a neuronal phenotype. Both peptides promote functional maturation of calcium channel subunits, suggesting that they can facilitate the genesis of cellular excitability.

Distribution and functional characterization of pituitary adenylate cyclase-activating polypeptide receptors in the brain of non-human primates.

The distribution and density of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites have been investigated in the brain of the primates Jacchus callithrix (marmoset) and Macaca fascicularis (macaque) using [(125)I]-PACAP27 as a radioligand. PACAP binding sites were widely expressed in the brain of these two species with particularly high densities in the septum, hypothalamus and habenula. A moderate density of recognition sites was seen in all subdivisions of the cerebral cortex with a heterogenous distribution, the highest concentrations occurring in layers I and VI while the underlying white matter was almost devoid of binding sites. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed intense expression of the mRNAs encoding the short and hop-1 variants of pituitary adenylate cyclase-activating polypeptide-specific receptor (PAC1-R) in the cortex of both marmoset and macaque, whereas vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide mutual receptor, subtype 1 (VPAC1-R) and vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide mutual receptor, subtype 2 (VPAC2-R) mRNAs were expressed at a much lower level. In situ hybridization histochemistry showed intense expression of PAC1-R and weak expression of VPAC1-R mRNAs in layer IV of the cerebral cortex. Incubation of cortical tissue slices with PACAP induced a dose-dependent stimulation of cyclic AMP formation, indicating that PACAP binding sites correspond to functional receptors. Moreover, treatment of primate cortical slices with 100 nM PACAP significantly reduced the activity of caspase-3, a key enzyme of the apoptotic cascade. The present results indicate that PACAP should exert the same neuroprotective effect in the brain of primates as in rodents and suggest that PAC1-R agonists may have a therapeutic value to prevent neuronal cell death after stroke or in specific neurodegenerative diseases.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates adenylyl cyclase and phospholipase C activity in rat cerebellar neuroblasts.

The presence of receptors for the novel neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been recently demonstrated in the external granule cell layer of the cerebellum, a germinative matrix that generates the majority of cerebellar interneurons. In the present study, we have taken advantage of the possibility of obtaining a culture preparation that is greatly enriched in immature cerebellar granule cells to investigate the effect of PACAP on the adenylyl cyclase and phospholipase C transduction pathways. The two molecular forms of PACAP, i.e., 27-(PACAP27) and 38-(PACAP38) amino-acid forms of PACAP, induced a dose-dependent stimulation of cyclic AMP production in granule cells. The potencies of PACAP27 and PACAP38 were similar (ED50 = 0.12 +/- 0.01 and 0.23 +/- 0.07 nM, respectively), whereas vasoactive intestinal polypeptide (VIP) was approximately 100 times less potent. PACAP27 and PACAP38 also induced a dose-dependent stimulation of polyphosphoinositide breakdown (ED50 = 19.1 +/- 6.3 and 13.4 +/- 6.0 nM, respectively), whereas VIP had no effect on polyphosphoinositide metabolism. The effect of PACAP38 on inositol phosphate formation was significantly reduced by U-73122 and by pertussis toxin, indicating that activation of PACAP receptors causes stimulation of a phospholipase C through a pertussis toxin-sensitive G protein. In contrast, forskolin and dibutyryl cyclic AMP did not affect PACAP-induced stimulation of inositol phosphates. Taken together, the present results demonstrate that PACAP stimulates independently the adenylyl cyclase and the phospholipase C transduction pathways in immature cerebellar granule cells. These data favor the concept that PACAP may play important roles in the control of proliferation and/or differentiation of cerebellar neuroblasts.

Neurotrophic activity of pituitary adenylate cyclase-activating polypeptide on rat cerebellar cortex during development.

High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are present in the external granule cell layer of the rat cerebellum during postnatal development. In vitro studies have shown that PACAP promotes cell survival and neurite outgrowth on immature cerebellar granule cells in primary culture. In the present study, we have investigated the effect of PACAP on the development of the cerebellar cortex of 8-day-old rats. Incubation of cultured granule cells for 12 or 18 h with PACAP provoked a significant increase in the rate of incorporation of [(3)H]thymidine in cultured granule cells, suggesting that PACAP could stimulate the proliferation of granule cells. After 96 h of treatment, in vivo administration of PACAP provoked a transient increase in the number of granule cells in the molecular layer and in the internal granule cell layer. In contrast, PACAP did not affect the number of Purkinje cells. The augmentation of the number of granule cells evoked by PACAP was significantly inhibited by the PACAP receptor antagonist PACAP(6-38). Administration of PACAP also caused a significant increase in the volume of the cerebellar cortex. The present study provides evidence that PACAP can act in vivo as a trophic factor during rat brain development. Our data indicate that PACAP increases proliferation and/or inhibits programmed cell death of granule cells, as well as stimulating neuronal migration from the external granule cell layer toward the internal granule cell layer.

Long-term enhancement of REM sleep by the pituitary adenylyl cyclase-activating polypeptide (PACAP) in the pontine reticular formation of the rat.

In rats, rapid eye movement (REM) sleep can be elicited by microinjection of vasoactive intestinal polypeptide (VIP) into the oral pontine reticular nucleus (PnO). In the present study, we investigated whether this area could also be a REM-promoting target for a peptide closely related to VIP: the pituitary adenylyl cyclase-activating polypeptide (PACAP). When administered into the posterior part of the PnO, but not in nearby areas, of freely moving chronically implanted rats, PACAP-27 and PACAP-38 (0.3 and 3 pmol) induced a marked enhancement (60-85% over baseline) of REM sleep for 8 h that could be prevented by prior infusion of the antagonist PACAP-(6-27) (3 pmol) into the same site. Moreover, injections of PACAP into the centre of the posterior PnO resulted in REM sleep enhancement which could last for up to 11 consecutive days. Quantitative autoradiography using [125I]PACAP-27 revealed the presence in the PnO of specific binding sites with high affinity for PACAP-27 and PACAP-38 (IC50 = 2.4 and 3.2 nM, respectively), but very low affinity for VIP (IC50 > 1 microM). These data suggest that PACAP within the PnO may play a key role in REM sleep regulation, and provide evidence for long-term (several days) mechanisms involved in such a control. PAC1 receptors which have a much higher affinity for PACAP than for VIP might mediate this long-term action of PACAP on REM sleep.