The first two potential peptides of amnesiac have homology to mammalian pituitary adenylate cyclase activating peptide (PACAP) and the growth hormone releasing hormone (GHRH) (Feany, 1995).

Evolution of PACAP polypeptides

Growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) belong to the same superfamily of regulatory neuropeptides and have both been characterized on the basis of their hypophysiotropic activities. The molecular evolution of the GHRH/PACAP gene family from urochordates to mammals is described and the hypothesis is presented that the respective roles of GHRH and PACAP in the control of GH secretion are totally inverted in phylogenetically distant groups of vertebrates. In mammals, GHRH and PACAP originate from distinct precursors whereas, in all submammalian taxa investigated so far, including birds, amphibians and fish, a single precursor encompasses a GHRH-like peptide and PACAP. In mammals, GHRH-containing neurons are confined to the infundibular and dorsomedial nuclei of the hypothalamus while PACAP-producing neurons are widely distributed in hypothalamic and extrahypothalamic areas. In fish, both GHRH- and PACAP-immunoreactive neurons are restricted to the diencephalon and directly innervate the adenohypophysis. In mammals and birds, GHRH plays a predominant role in the control of GH secretion. In amphibians, both GHRH and PACAP are potent stimulators of GH release. In fish, PACAP strongly activates GH release whereas GHRH has little or no effect on GH secretion. The GHRH/PACAP family of peptides thus provides a unique model in which to investigate the structural and functional facets of evolution (Montero, 2000).

The levels of PACAP27 and PACAP38 were measured in the nervous, intestinal, excretory, and reproductive systems of the oligochaete (Annelida) Lumbricus polyphemus by radioimmunoassay. Although both PACAP27 and PACAP38 are significantly detectable in all of the examined tissues, the distribution of the peptides is very heterogeneous. Their highest concentrations are found in the cerebral ganglia and the ventral cord, followed by the alimentary tract and the nephridial system, respectively. Moreover, the reproductive system also contains a substantial amount of PACAP. The dominant form of the peptide discovered in the majority of tissues is PACAP27. Interestingly, about 10 times more PACAP27 than PACAP38 is found, with the latter representing only a fraction of PACAP-like immunoreactivity in the tissues of Lumbricus polyphemus (Somogyvari-Vigh, 2000).

Developmental expression of PACAP polypeptides

The marked similarity between the primary structures of human, other vertebrate, and the invertebrate tunicate PACAP suggests that PACAP is one of the most highly conserved peptides during the phylogeny of the metazoans. The distribution of PACAP-like immunoreactivity in the nervous system of three oligochaete (Annelida) worms was investigated using immunocytochemistry. The distribution pattern of immunoreactivity was similar in all three species (Lumbricus terrestris, Eisenia fetida, and Lumbricus polyphemus). The cerebral ganglion contains numerous immunoreactive cells and fibers. A few cells and fibers are found in the medial and lateral parts of the subesophageal and ventral cord ganglia. In the peripheral nervous system, immunoreactivity was found in the enteric nervous system, in epidermal sensory cells, and in the clitellum (Reglodi, 2000).

Expression of PACAP receptor

The distribution of PACAP-typeI-receptor (PACAP-I-R) mRNA and protein was studied in mouse using probes and a newly developed antiserum recognizing all known splice variants. RNase protection assays reveal the highest expression levels of PACAP-I-R mRNA in brain, in particular the hypothalamus and hippocampus. At the cellular level, in situ hybridization analysis demonstrates widespread distribution of PACAP-I-R mRNA in neurons throughout the brain, while glial cells do not express the gene. Highest expression levels of PACAP-I-R mRNA were observed in three regions: the limbic system, the hypothalamus, and the brainstem. In accordance with data obtained from in situ hybridization analysis, immunohistochemistry shows widespread distribution of PACAP-I-R like immunoreactivity in the neuropil. Rather strong immunoreactivity has been found in cerebellar and hippocampal mossy fibers where double immunolabelling reveals the presynaptic localization of the receptor protein. At the ultrastructural level, PACAP-I-R like immunoreactivity was observed around synaptic vesicles and close to the presynaptic grid in hippocampal mossy fiber terminals. This finding is in contradiction to the described postsynaptic localization of the PACAP-I-R in dendritic processes of hippocampal granule cells in rat. Due to their presynaptic induction, mossy fiber LTPs are distinctly different from LTPs in all other hippocampal regions. Therefore, the presynaptic localization of the PACAP-I-R in mossy fiber terminals may implicate this gene in influencing the synaptic strength of the mossy fiber pathway and hence memory consolidation (Otto, 1999).

Mutation of PACAP receptor

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor subtype 1 (PAC1) have been suggested to play a role in the modulation of learning and memory. However, behavioral evidence for altered mnemonic function due to altered PAC1 activity is missing. Therefore, the role of PAC1 in learning and memory was studied in mouse mutants lacking this receptor (PAC1 knock-out mice), tested in water maze two-choice spatial discrimination, one-trial contextual and cued fear conditioning, and multiple-session contextual discrimination. Water maze spatial discrimination was unaffected in PAC1 mutants, while a mild deficit was observed in multiple session contextual discrimination in PAC1 knock-out mice. Furthermore, PAC1 knock-out mice were able to learn the association between context and shock in one-trial contextual conditioning, but showed faster return to baseline than wild-type mice. Thus, the effects of PAC1 knock-out on modulating performance in these tasks were subtle and suggest that PAC1 only plays a limited role in learning and memory (Sauvage, 2000).

Solution structure and mutational analysis of PACAP binding to the extracellular domain of PAC1-RS

The pituitary adenylate cyclase-activating polypeptide (PACAP) receptor is a class II G protein-coupled receptor that contributes to many different cellular functions including neurotransmission, neuronal survival, and synaptic plasticity. The solution structure of the potent antagonist PACAP (residues 6'-38') complexed to the N-terminal extracellular (EC) domain of the human splice variant hPAC1-R-short (hPAC1-R(S)) was determined by NMR. The PACAP peptide adopts a helical conformation when bound to hPAC1-R(S) with a bend at residue A18' and makes extensive hydrophobic and electrostatic interactions along the exposed beta-sheet and interconnecting loops of the N-terminal EC domain. Mutagenesis data on both the peptide and the receptor delineate the critical interactions between the C terminus of the peptide and the C terminus of the EC domain that define the high affinity and specificity of hormone binding to hPAC1-R(S). These results present a structural basis for hPAC1-R(S) selectivity for PACAP versus the vasoactive intestinal peptide and also differentiate PACAP residues involved in binding to the N-terminal extracellular domain versus other parts of the full-length hPAC1-R(S) receptor. The structural, mutational, and binding data are consistent with a model for peptide binding in which the C terminus of the peptide hormone interacts almost exclusively with the N-terminal EC domain, whereas the central region makes contacts to both the N-terminal and other extracellular parts of the receptor, ultimately positioning the N terminus of the peptide to contact the transmembrane region and result in receptor activation (Sun, 2007).

Physiological response to PACAP polypeptides

PACAP is localized in nerve terminals that innervate arginine-vasopressin (AVP)-containing neurons in the rat hypothalamic supraoptic nucleus (SON). PACAP receptor (PACAPR) mRNA is expressed at high-levels in AVP-containing neurons in the SON, but at very low-levels in oxytocin-containing neurons. PACAPR-like immunoreactivity is found in SON and it is observed in the post-synaptic membranes as well as on the rough endoplasmic reticulum and cytoplasmic matrices in the magnocellular neurons. Doses of PACAP in the nanomolar range increase cytoplasmic Ca2+ concentrations ([Ca2+]i) in AVP-containing neurons; the increase in [Ca2+]i is inhibited by a protein kinase A blocker. These findings suggest that PACAP serves as a transmitter and/or modulator and the activation of PACAPR stimulates a cAMP-protein kinase A pathway which in turn evokes the Ca2+ signaling system. It is hypothesized that PACAP regulates the functions of AVP-containing neurons, which participate in the control of plasma osmolarity and blood pressure (Shioda, 1997).

PACAP is a member of the vasointestinal polypeptide gene family for which neurotrophic activity has been postulated. PACAP mRNA is expressed in the developing and adult hippocampus, which is the principal target region of septal cholinergic neurons. The effects of PACAP on septal cholinergic neurons was studied. In primary cultures from septum of embryonic and postnatal rats, PACAP increases the number of neurons immunohistochemically stained for the low-affinity nerve growth factor (NGF) receptor p75 and for the enzyme choline acetyltransferase (ChAT). PACAP also causes a corresponding increase in ChAT activity. In comparison, NGF has a greater effect than PACAP on the number of p75- and ChAT-positive neurons in these cultures. In vivo, following fimbria fornix transection, the number of immunohistochemically stained septal cholinergic neurons fell significantly to 18% in rats given continuous intracerebroventricular infusion of vehicle, whereas in rats given NGF the number of these neurons does not differ significantly from unoperated controls. In PACAP-treated rats the number was 48% of unoperated values, which represents a significant increase compared with vehicle-treated rats and a significant decrease compared with NGF-treated rats or unoperated controls. Double-staining experiments have shown that most ChAT-positive neurons in rat medial septum also express PACAP receptor 1. Together the results show that PACAP promotes the survival of septal cholinergic neurons in vitro, and after injury in vivo, suggesting that PACAP acts as a neurotrophic factor influencing the development and maintenance of these neurons (Takei, 2000).

In the brain, glutamatergic neurotransmission is terminated predominantly by the rapid uptake of synaptically released glutamate into astrocytes through the Na(+)-dependent glutamate transporters GLT-1 and GLAST and its subsequent conversion into glutamine by the enzyme glutamine synthetase (GS). To date, several factors have been identified that rapidly alter glial glutamate uptake by post-translational modification of glutamate transporters. The only condition known to affect the expression of glial glutamate transporters and GS is the coculturing of glia with neurons. Neurons regulate glial glutamate turnover via PACAP. In the cerebral cortex PACAP is synthesized by neurons and acts on the subpopulation of astroglia involved in glutamate turnover. Exposure of astroglia to PACAP increases the maximal velocity of [(3)H]glutamate uptake by promoting the expression of GLT-1, GLAST, and GS. Moreover, the stimulatory effects of neuron-conditioned medium on glial glutamate transporter expression are attenuated in the presence of PACAP-inactivating antibodies or the PACAP receptor antagonist PACAP 6-38. In contrast to PACAP, vasoactive intestinal peptide promotes glutamate transporter expression only at distinctly higher concentrations, suggesting that PACAP exerts its effects on glial glutamate turnover via PAC1 receptors. Although PAC1 receptor-dependent activation of protein kinase A (PKA) is sufficient to promote the expression of GLAST, the activation of both PKA and protein kinase C (PKC) is required to promote GLT-1 expression optimally. Given the existence of various PAC1 receptor isoforms that activate PKA and PKC to different levels, these findings point to a complex mechanism by which PACAP regulates glial glutamate transport and metabolism. Disturbances of these regulatory mechanisms could represent a major cause for glutamate-associated neurological and psychiatric disorders (Figiel, 2000).

Specific receptors for PACAP have been identified recently in different brain regions, including the hippocampus. The effects of PACAP-38 on the excitatory postsynaptic field potentials (fEPSPs) evoked at the Schaffer collateral-CA1 synapses has been examined. Brief bath application of PACAP-38 (0.05 nM) induces a long-lasting facilitation of the basal transmission. Enhancement of this response is occluded in part by previous high-frequency-induced long-term potentiation (LTP). PACAP-38 does not significantly alter the paired-pulse facilitation (PPF). PACAP-38 has been shown to have a presynaptic effect on the septohippocampal cholinergic terminals, which results in an increase in basal acetylcholine (ACh) release. To assess whether the PACAP-38 enhancement of CA1 synapses is related to the activation of the cholinergic system, the effect of this peptide in the presence of atropine, a muscarinic receptor antagonist, was examined. The enhancement of the fEPSPs by PACAP-38 is blocked by bath application of atropine. These results show that PACAP-38 induces facilitation of hippocampal synaptic transmission through activation of the cholinergic system via the muscarinic receptors (Roberto, 2000).

During the first postnatal week, glial cell production for the neocortex continues in the neocortical subventricular zone. During this time, the proenkephalin gene (PEnk) is expressed in numerous cells of the subventricular zone and of the adjacent neocortex. When neocortical astroglial cells are brought into dissociation culture, they also produce PEnk mRNA. The effect of PACAP38 on PEnk gene expression was examined in dissociation cultures as well as in slice cultures, which contained the subventricular zone and the adjacent neocortex. PACAP38 enhances the levels of PEnk mRNA in both culture systems. In dissociated astroglial cells, inhibition of protein kinase A, p44,42 mitogen-activated protein kinase as well as inhibition of the EGF-receptor tyrosine kinase by H89, PD98059 and AG1478, respectively, reduces the PACAP38-induced expression in a synergistic manner. In the neocortical part of the slice cultures, the effect of PACAP38 on PEnk gene expression is inhibited only by H89 and PD98059. Here, protein kinase A and p44,42 MAP kinases share a mechanism that increases the gene expression. Surprisingly, the expression of the PEnk gene in the glial progenitors of the subventricular zone as induced by PACAP38 is not affected by any of the three protein kinase inhibitors, but is blocked by the unspecific kinase inhibitor H7. It is concluded that PACAP38 induced the PEnk gene expression in both culture systems in a cell-type specific manner.

Caspase-3 knockout mice exhibit thickening of the internal granule cell layer of the cerebellum. Concurrently, it has been shown that intracerebral injection of pituitary adenylate cyclase-activating polypeptide (PACAP) induces a transient increase of the thickness of the cerebellar cortex. In the present study, the possible effect of PACAP on caspase activity has been investigated in cultured cerebellar granule cells from 8-day-old rat. Incubation of granule neurons with PACAP for 24 h promotes cell survival and prevents DNA fragmentation. Exposure of cerebellar granule cells to a specific caspase-3 inhibitor for 24 h markedly enhances cell survival and inhibits apoptotic cell death. Time-course studies have revealed that PACAP causes a prolonged inhibition of caspase-3 activity without affecting caspase-1. Administration of graded concentrations of PACAP for 3 h induces a dose-dependent inhibition of caspase-3 activity. Incubation of granule cells with both dibutyryl-cAMP (dbcAMP) and phorbol 12-myristate 13-acetate (PMA) mimics the inhibitory effect of PACAP on caspase-3. Cotreatment of cultured neurons with the protein kinase A inhibitor H89 and the protein kinase C inhibitor chelerythrine abrogate the effect of PACAP on caspase-3 activity. In contrast, the ERK kinase inhibitor U0126 does not affect the action of PACAP on caspase-3 activity. These data demonstrate that PACAP prevents cerebellar granule neurons from apoptotic cell death through a protein kinase A- and protein kinase C-dependent inhibition of caspase-3 activity (Vaudry, 2000).

Alcohol exposure during development can cause brain malformations and neurobehavioral abnormalities. In view of the teratogenicity of ethanol, identification of molecules that could counteract the neurotoxic effects of alcohol deserves high priority. Pituitary adenylate cyclase-activating polypeptide (PACAP) can prevent the deleterious effect of ethanol on neuronal precursors. Exposure of cultured cerebellar granule cells to ethanol inhibits neurite outgrowth and provokes apoptotic cell death. Incubation of granule cells with PACAP prevents ethanol-induced apoptosis, and this effect is not mimicked by vasoactive intestinal polypeptide, suggesting that PAC1 receptors are involved in the neurotrophic activity of PACAP. Ethanol exposure induces a strong increase of caspase-2, -3, -6, -8, and -9 activities, DNA fragmentation, and mitochondrial permeability. Cotreatment of granule cells with PACAP provokes a significant inhibition of all of the apoptotic markers investigated although the neurotrophic activity of PACAP could only be ascribed to inhibition of caspase-3 and -6 activities. These data demonstrate that PACAP is a potent protective agent against ethanol-induced neuronal cell death. The fact that PACAP prevents ethanol toxicity even when added 2 h after alcohol exposure, suggests that selective PACAP agonists could have potential therapeutic value for the treatment of fetal alcohol syndrome (Vaudry, 2002).

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potentiator of glucose-induced insulin secretion. PACAP binds to a PACAP-specific receptor (PAC1) and to VPAC receptors (VPAC1 and VPAC2), which share high affinity for vasoactive intestinal polypeptide (VIP). In the present study, the molecular expression of PACAP receptor isoforms and the signaling pathways involved in the insulin secretory effect of PACAP were investigated in isolated rat and mouse pancreatic islets. mRNA encoding PAC1-short, -hop, and -very short variants, as well as VPAC1 and VPAC2, were expressed in pancreatic islets. PACAP and VIP are equipotent in potentiating glucose-induced insulin release. Both peptides are also equipotent in increasing cAMP production, but PACAP is more efficient than VIP. Unlike carbachol, PACAP and VIP has no effect on inositol phosphate production. In the PAC1-deficient mouse, the insulinotropic effect of PACAP is reduced, and its differential effect on cAMP production is abolished, whereas the effects of VIP remain unchanged. These results clearly show that the insulinotropic effect of PACAP involves both VPAC and PAC1. The PAC1 variants expressed in rat and mouse pancreatic islets seem to be coupled to adenylate cyclase but not to PLC (Jamen, 2002).

PACAP, a neuropeptide that acts through G protein-coupled receptors, exerts neuroprotective effects upon many neuronal populations. However, the intracellular signaling mechanisms that account for PACAP's trophic effects are not well characterized. The possibility that PACAP uses neurotrophin signaling pathways has been tested. PACAP treatment results in an increase in TrkA tyrosine kinase activity in PC12 cells and TrkB activity in hippocampal neurons. The activation of TrkA receptors by PACAP requires at least 1 h of treatment and does not involve binding to nerve growth factor. Moreover, PACAP induces an increase in activated Akt through a Trk-dependent mechanism that results in increased cell survival after trophic factor withdrawal. The increases in Trk and Akt are blocked by K252a, an inhibitor of Trk receptor activity. In addition, transactivation of TrkA receptors by PACAP can be inhibited with PP1, an inhibitor of Src family kinases or BAPTA/AM, [1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester], an intracellular calcium chelator. Therefore, PACAP can exert trophic effects through a mechanism involving Trk receptors and utilization of tyrosine kinase signaling. This ability may explain several neuroprotective actions of PACAP upon neuronal populations after injury, nerve lesion, or neurotrophin deprivation (Lee, 2002).

Modulation of NMDA receptors by PACAP

A novel mechanism has been identified for modulation of the phosphorylation state and function of the N-methyl-d-aspartate (NMDA) receptor via the scaffolding protein RACK1. RACK1 binds both the NR2B subunit of the NMDA receptor and the nonreceptor protein-tyrosine kinase, Fyn. RACK1 inhibits Fyn phosphorylation of NR2B and decreases NMDA receptor-mediated currents in CA1 hippocampal slices. This study identified the signaling cascade by which RACK1 is released from the NMDA receptor complex and identified the consequences of the dissociation. Activation of the cAMP/protein kinase A pathway in hippocampal slices induces the release of RACK1 from NR2B and Fyn. This results in the induction of NR2B phosphorylation and the enhancement of NMDA receptor-mediated activity via Fyn. The neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP(1-38)) was identified as a ligand that induces phosphorylation of NR2B and enhances NMDA receptor potentials. Finally, it was found that activation of the cAMP/protein kinase A pathway induces the movement of RACK1 to the nuclear compartment in dissociated hippocampal neurons. Nuclear RACK1 in turn regulates the expression of brain-derived neurotrophic factor induced by PACAP(1-38). Taken together these results suggest that activation of adenylate cyclase by PACAP(1-38) results in the release of RACK1 from the NMDA receptor and Fyn. This in turn leads to NMDA receptor phosphorylation, enhanced activity mediated by Fyn, and to the induction of brain-derived neurotrophic factor expression by RACK1 (Yaka, 2003).

At CA1 synapses, activation of NMDA receptors (NMDARs) is required for the induction of both long-term potentiation and depression. The basal level of activity of these receptors is controlled by converging cell signals from G-protein-coupled receptors and receptor tyrosine kinases. Pituitary adenylate cyclase activating peptide (PACAP) is implicated in the regulation of synaptic plasticity because it enhances NMDAR responses by stimulating Gαs-coupled receptors and protein kinase A. However, the major hippocampal PACAP1 receptor (PAC1R) also signals via Gαq subunits and protein kinase C (PKC). In CA1 neurons, PACAP38 enhances synaptic NMDA, and evoked NMDAR, currents in isolated CA1 neurons via activation of the PAC1R, Gαq, and PKC. The signaling was blocked by intracellular applications of the Src inhibitory peptide Src(40-58). Immunoblots confirmed that PACAP38 biochemically activates Src. A Gαq pathway is responsible for this Src-dependent PACAP enhancement because it was attenuated in mice lacking expression of phospholipase C β1, it was blocked by preventing elevations in intracellular Ca2+, and it was eliminated by inhibiting either PKC or cell adhesion kinase β [CAKβ or Pyk2 (proline rich tyrosine kinase 2)]. Peptides that mimic the binding sites for either Fyn or Src on receptor for activated C kinase-1 (RACK1) also enhanced NMDAR in CA1 neurons, but their effects were blocked by Src(40-58), implying that Src is the ultimate regulator of NMDARs. RACK1 serves as a hub for PKC, Fyn, and Src and facilitates the regulation of basal NMDAR activity in CA1 hippocampal neurons (Macdonald, 2005).

PACAP polypeptides, behavior and learning

The action of PACAP 38 on one-way passive avoidance learning has been investigated. PACAP-38 was administered into the lateral brain ventricle and the latency of the passive avoidance response was measured 24 h later. In order to study the possible roles of various neurotransmitters in mediating the action of PACAP on the consolidation of passive avoidance learning, the animals were pre-treated with receptor blockers in doses that per se proved to be ineffective. PACAP facilitates the learning, the consolidation of learning and the retrieval of the passive avoidance response. The following receptor blockers attenuate the action of PACAP on this consolidation: haloperidol, phenoxybenzamine, propranolol and methysergide. An antagonist of PACAP 38, PACAP 6-38, and also nitro-L-arginine (the latter blocks the enzyme nitric oxide synthase) thereby inhibiting the formation of NO from L-arginine, completely block the action of PACAP 38 on consolidation. The following receptor blockers are ineffective: naloxone, bicuculline and atropine. The presented data suggest that PACAP 38 is able to improve the learning and memory processes in a passive avoidance paradigm. In this action, the PACAP 38 receptor and NO are important mediators. Dopaminergic, alpha- and beta-adrenergic mediation and serotonin receptors modify the action of PACAP 38, but they are probably not of great importance (Telegdy, 2000).

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been conserved remarkably during evolution and is widely expressed in the mammalian brain. In Drosophila, mutation of the PACAP homolog amnesiac results in behavioral defects, including impaired olfaction-associated learning and changes in ethanol sensitivity. The generation of mice lacking the PACAP gene (PACAP-/-) is reported. PACAP-/- mice are born in the expected Mendelian ratios but have a high early-mortality rate. The surviving adult PACAP-/- mice display remarkable behavioral changes; they exhibited hyperactive and explosive jumping behaviors in an open field, increased exploratory behavior, and less anxiety in the elevated plus maze, emergence, and novel-object tests. Analysis of PACAP-/- mice brains reveals that the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) is slightly decreased in the cortex and striatum compared with wild-type mice. The present study provides evidence that PACAP plays a previously uncharacterized role in the regulation of psychomotor behaviors (Hashimoto, 2001).

The results of behavioral experiments with PACAP-/- mice demonstrate that disruption of the PACAP gene in mice leads to perturbations in psychomotor behaviors, especially the exploratory component of locomotor behavior, implicating PACAP in psychotic brain functions. Furthermore, the 5-HIAA level was decreased slightly in the cortex and striatum of the PACAP-/- mouse brain (Hashimoto, 2001).

It is commonly believed that locomotor hyperactivity is associated with increased dopamine DA tone. In PACAP-/- mice, DA turnover is unchanged and the incidence of haloperidol-induced catalepsy is quite similar to that in wild-type mice. These findings suggest that the locomotor hyperactivity in PACAP-/- mice probably may not be a result of increased nigrostriatal dopaminergic activity. Alternatively, the importance of 5-HT in controlling the locomotor activity has been demonstrated in a study with 5-HT1B-receptor knockout mice. Moreover, a relative balance of the DA and 5-HT systems seems to be important for normal motor activity, and alterations in any of the parameters that control this delicate homeostatic situation might underlie hyperactive states. This may explain, at least in part, the locomotor hyperactivity of PACAP-/- mice (Hashimoto, 2001).

Several lines of evidence suggest that dysfunction of serotonergic pathways, especially those mediated by the 5-HT1A receptor (see Drosophila Serotonin receptor 1A) , is associated with anxiety-related traits. Although it has not been concluded that PACAP-/- mice are less anxious, if, indeed, they are less anxious, reduced 5-HT turnover (5-HIAA/5-HT ratio) cannot explain it. However, DA functions, particularly those mediated by the D4 receptor, are involved in novelty-related exploratory behavior, as reported in D4-receptor knockout mice. These mutant mice are less active in open-field tests and exhibit reduced exploration of novel stimuli in contrast to the phenotypes observed in PACAP-/- mice (Hashimoto, 2001).

One of the striking findings of the present study was that PACAP-/- mice showed abnormal jumping behavior in the open field arena. In NIH Swiss mice, an NMDA-receptor antagonist MK-801 is known to precipitate explosive episodic jumping behavior that can be attenuated by haloperidol. In addition, dysfunction of the NMDA receptor by MK-801 or the targeted disruption of its gene produces psychotic symptoms that closely resemble the positive and negative symptoms of schizophrenia. In view of the effects of MK-801, investigation of possible common mechanisms involved in the MK-801-induced abnormal behaviors and those of PACAP-/- mice would be warranted (Hashimoto, 2001).

Currently, it is accepted that multiple genes of small effect, rather than a single causative gene, act in concert with nongenetic factors to increase the risk of mental disorder. The present study shows that PACAP-/- mice display marked behavioral abnormalities without having marked changes in specific neuronal pathways in their brains. To date, no change has been at the level of gene expression of PACAP-receptor subtypes, tyrosine hydroxylase, and DA D2 receptor in the various brain regions of PACAP-/- mice. Of course, there are many other functional molecules for which expression levels should be determined. It is possible that PACAP-/- mice have several small but significant changes in the activity of neuronal networks, and that they collectively cause behavioral abnormalities (Hashimoto, 2001).

Because specific low-molecular-weight antagonists and agonists to the different PACAP-receptor subtypes are not available, the physiological role of PACAP as well as of each receptor subtype in brain function have not been addressed fully. PAC1 receptor-deficient mice did not show any apparent behavioral changes like those observed in PACAP-/- mice. PACAP interacts with three receptors: PACAP-preferring PAC1, VIP-shared VPAC1, and VPAC2 receptors. Lack of signal transmission of PACAP through the VPAC receptors may explain the behavioral changes in PACAP-/- mice (Hashimoto, 2001).

Several lines of evidence suggest that PACAP acts as a neurotrophic factor and plays a role in mammalian neurogenesis. For instance, the PAC1 receptor is expressed at very high levels in ventricular zones throughout the embryonic neuraxis, and PACAP likely regulates the development of the general features of the neuronal phenotype. Therefore, it is possible also that the lack of PACAP affects developmental processes, resulting in the observed behavioral abnormalities and in a high early mortality rate in PACAP-/- mice (Hashimoto, 2001).

Because it was demonstrated that the Drosophila mutant amnesiac, which displays behavioral defects, has a mutation in a neuropeptide gene, the in vivo role of the mammalian homolog PACAP has remained an open question. Disruption of the PACAP gene in mice leads to major alterations in psychomotor activity. Recent genetic linkage studies have suggested that a locus for schizophrenia as well as bipolar affective disorder is located on chromosome 18p11, where the human PACAP gene resides. It is now important to determine whether the mutation in the PACAP locus is implicated in disease in these select families (Hashimoto, 2001).

In conclusion, the present study proposes a role of PACAP-ergic neurons in regulating psychomotor behaviors acutely or developmentally. The PACAP-/- mouse should be a valuable tool to investigate both normal and pathological processes in which PACAP has been proposed to play a role (Hashimoto, 2001).

To ascertain whether very low dosages of pituitary adenylate cyclase-activating polypeptide (PACAP) influence learning in mammals, immediately after the acquisition trial of a passive avoidance response (PAR) paradigm, PACAP-38 was administered intracerebroventricularly at increasing dosages to different groups of rats. The mnemonic effects were measured by means of retention testing 48 and 96 h later. At intermediate PACAP-38 concentrations there was a significant mnemonic improvement of the PAR. The maximal effect was observed after the 0.2-ng PACAP-38 administration (longer step-through latencies). There was a lesser effect at the subsequent higher concentration, 2 ng. Higher dosages had no effects. It is concluded that PACAP-38 acts as an enhancer of mammalian mnemonic processes even at very low dosages. The positive effect follows an inverted U-shaped dose-response curve. The results may be of interest for the therapy of some neuropathological conditions (Sacchetti, 2001).

The olfactory bulb plays a critical role in odor discrimination and in processing olfactory cues controlling social behavior in mammals. Given that the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1) is highly expressed in the olfactory bulb, its role in regulating olfaction and social investigation was examined. Olfactory detection of nonsocial stimuli was similar in PAC1-deficient mice and wild-type (WT) littermates. In contrast, PAC1-deficient mice displayed markedly abnormal social behaviors. PAC1-deficient mice exhibit a faster decrease in social investigation after repeated exposure to social cues or ovariectomized female urine compared with WT mice. Moreover, PAC1-deficient females exhibit delayed affiliative behavior when housed with novel males, and PAC1-deficient males display excessive sexual mounting toward both females and males as well as reduced aggression and increased licking and grooming toward intruder males. In aggregate, these results uncover PAC1 signaling as an important factor in the development and/or functioning of neural pathways associated with pheromone processing and the regulation of social interactions in mice. In turn, these studies raise the potential clinical relevance of PACAP signaling dysfunctions in neuropsychiatric disorders characterized by social reciprocity impairments such as autism spectrum disorders (Nicot, 2004).

PACAP polypeptides and development

PACAP has been implicated in a broad variety of physiological processes. The PACAP precursor protein gives rise to three different peptides (the cryptic peptide, GHRH, and PACAP, respectively), and in this study their functional properties were dissected using Xenopus as a model system. PACAP and GHRH but not the cryptic peptide directly neuralize animal caps. In contrast to GHRH, the neuralizing effect mediated by PACAP is independent of the PKA pathway. Moreover, PACAP but not GHRH behaves like a BMP-4 antagonist. Blastocoel injection of PACAP-38 but not of the closely related peptides PACAP-27 and VIP leads to strong anteriorization of the injected embryos suggesting the possible involvement of a novel PACAP-preferring receptor (Otto, 2000).

The superior cervical ganglion (SCG) is a well-characterized model of neural development, in which several regulatory signals have been identified. Vasoactive intestinal peptide (VIP) has been found to regulate diverse ontogenetic processes in sympathetics, though functional requirements for high peptide concentrations suggest that other ligands are involved. Expression and functions of PACAP during SCG ontogeny are described, suggesting that the peptide plays critical roles in neurogenesis. PACAP and PACAP receptor [PAC(1)] mRNA's were detected at embryonic days 14.5 (E14.5) through E17.5 in vivo and virtually all precursors exhibited ligand and receptor, indicating that the system is expressed as neuroblasts proliferate. Exposure of cultured precursors to PACAP peptides, containing 27 or 38 residues, increases mitogenic activity 4-fold. Significantly, PACAP is 1000-fold more potent than VIP and a highly potent and selective antagonist entirely blocks effects of micromolar VIP, consistent with both peptides acting via PAC(1) receptors. Moreover, PACAP potently enhances precursor survival more than 2-fold, suggesting that previously defined VIP effects are mediated via PAC(1) receptors and that PACAP is the more significant developmental signal. In addition to neurogenesis, PACAP promotes neuronal differentiation, increasing neurite outgrowth 4-fold and enhancing expression of neurotrophin receptors trkC and trkA. Since PACAP potently activates cAMP and PI pathways and increases intracellular Ca(2+), the peptide may interact with other developmental signals. PACAP stimulation of precursor mitosis, survival, and trk receptor expression suggests that the signaling system plays a critical autocrine role during sympathetic neurogenesis (DiCicco-Bloom, 2000).

Accelerated evolution of the pituitary adenylate cyclase-activating polypeptide precursor gene during human origin

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a strong functional constraint during the course of evolution. However, through comparative sequence analysis, it was demonstrated that the PACAP precursor gene has undergone an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel neuropeptide might have originated during human evolution and functioned in the human brain. These data suggest that the PACAP precursor gene has undergone adaptive changes during human origin and may have contributed to the formation of human cognition (Wang, 2005).

amnesiac: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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