Molecular, cellular, and functional characterizations of pituitary adenylate cyclase-activating polypeptide and its receptors in the cerebellum of New and Old World monkeys.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) exerts trophic activities during cerebellar development, and a neuroprotective effect of PACAP has been demonstrated in pathological conditions such as stroke. However, all these data have been obtained in rodents, and neuroprotective effects of PACAP in primates remain unknown. Because of their evolutionary relationships with humans, monkeys represent powerful models for validating the therapeutic interest in PACAP. The objective of the present study was to characterize PACAP and its receptors in the cerebellum of two nonhuman primates. RT-PCR and in situ hybridization experiments revealed that PACAP is expressed in the cerebellum by Purkinje cells. Via immunohistochemistry, PACAP was detected in Purkinje cells and radial glial fibers. With regard to PACAP receptors, PAC1-R and VPAC1-R were detected by RT-PCR. In situ hybridization revealed a strong expression of PAC1-R and VPAC1-R in the granule cell layer (GCL), and VPAC1-R was also expressed in the Purkinje cell layer. A high density of PACAP binding sites was visualized in the GCL and the Purkinje cell layer. Competition studies indicated that, in the GCL, PACAP induced complete displacement of [(125)I]PACAP27 binding, whereas vasoactive intestinal polypeptide (VIP) was a weak competitor. In contrast, in the Purkinje cell layer, both PACAP and VIP displaced [(125)I]PACAP27 binding. Measurement of cAMP levels showed that PACAP is a powerful activator of adenylyl cyclase, whereas VIP is about 100-fold less potent. Altogether, these observations constitute the first demonstration of a functional PACAPergic system in monkey cerebellum. They strongly suggest that neuroprotective effects of PACAP can be transposed to primates, including human.

Neurotransmitter-mediated anxiogenic action of PACAP-38 in rats.

The action of PACAP-38 was studied by measuring the anxiogenic-anxiolytic behavior of rats in an elevated plus maze. PACAP-38 was administered into the lateral brain ventricle and the behavior of the animals was measured 3h later. The possible involvement of transmitters was measured by pretreating the animals with receptor blockers which alone did not influence the task, but in the doses used were effective with other neuropeptides. The receptor antagonist PACAP 6-38 (a PAC 1/VPAC2 receptor antagonist of PACAP-38 receptor), haloperidol (a non-selective dopamine receptor antagonist), phenoxybenzamine (an α1/α2ß-adrenergic receptor antagonist), propranolol(a ß-adrenergic receptor antagonist), bicuculline (a gamma-aminobutyric acid subunit A receptor antagonist), methysergide (a nonselective 5-HT2 serotonergic receptor antagonist), atropine (a nonselective muscarinic acetylcholine receptor antagonist), naloxone (a nonselective opioid receptor antagonist) and nitro-l-arginine which acts by blocking the enzyme nitric oxide synthase, thereby blocking the nitric oxide synthesis, were tested. The following parameters were measured: the time spent in open arms/the time spent in total entries. PACAP-38 decreased the ratio of time spent in open arms to the time spent in total entries, indicating anxiogenic action. The total number of entries was not altered significantly either by PACAP-38 or by the receptor blockers. The following receptor blockers diminished the action of PACAP-38: PACAP 6-38,haloperidol, methysergide, naloxone and nitro-l-arginine. Pretreatment with atropine, phenoxybenzamine, propranolol and bicuculline did not influence the action of PACAP-38 on the time spent in open arms. The results demonstrate that PACAP-38 administered into the lateral brain ventricle exerted anxiogenic action at 3 h following treatment. Pretreatment of the animals with various receptor blockers indicated that a nonselective dopaminergic receptor antagonist, 5HT2 serotonergic and opioid receptors, nitric oxide and PAC1 receptors are involved in the anxiogenic action induced by PACAP-38.

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.