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Pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to influence nervous system development. The aim of the present study was to investigate the effects of in ovo treatment with the PACAP antagonist PACAP6-38 during embryonic life (E8 and E16) on motor activity and social behavior in chicken. Our results showed that a single injection of PACAP6-38 during the first half of embryonic life caused subtle transient changes in general behavior and motor control when compared to saline-treated controls. Increased activity and reduced anxiety were observed also in a novel environment at 2 days after hatching. However, most of these behavioral differences disappeared by 2 weeks. PACAP6-38-treatment during the first half of embryonic life resulted in markedly reduced social behavior, which was still present at 2 weeks of age. Treatment during the second half of embryonic life resulted in no behavioral differences between control and PACAP6-38-treated chicken. PACAP content in different brain areas was not different between control and PACAP6-38-treated chicken at 5 days or 3 weeks of age, but it decreased significantly with age in both groups. In summary, our results show that PACAP6-38 treatment at E8 caused transient changes in motor behavior, and long-lasting reduction in social behavior.

Vasoactive intestinal polypeptide (VIP) occurs in high concentrations throughout the gut and the nervous system. The presence of VIP has been shown in a number of species, mainly by immunohistochemistry. The aim of the present study was to develop a new, highly specific VIP radioimmunoassay to investigate the distribution of VIP in the central nervous system of various vertebrate and invertebrate species. Different areas of the brain and spinal cord were removed from rats, chickens, turtles, frogs and fishes. The cerebral ganglia and the ventral ganglionic chain were investigated in the earthworm. The tissue samples were processed for VIP radioimmunoassay. Our results show that the antiserum used in the radioimmunoassay turned to be C-terminal specific, without significant affinity to other members of the VIP peptide family. Detection limit of the assay was 0.1 fmol/ml. Highest concentrations were found in the turtle diencephalon, followed by other brain areas in the turtle and rat. All other brain areas in the examined species contained significant levels of VIP. Immunoreactivity was also shown in the cerebral and ventral ganglia of the earthworm. In summary, our results show comparative quantitative distribution in representative species of the phylogenetic line, using the same experimental conditions.

One of the recently found functions of pituitary adenylate cyclase activating polypeptide (PACAP) is the modulation of circadian rhythms. Widespread distribution of PACAP-containing neurons and receptors has been shown in the chicken. Recently, we have demonstrated that PACAP levels oscillate in a circadian manner in the chicken brain. Daily variation in PACAP levels might be influenced by several regulatory mechanisms. Among the structures that may regulate PACAP levels, one candidate is the pineal gland. Therefore, in the present study, we investigated the effect of pinealectomy on the levels of PACAP in the chicken brain. Animals were kept under 12:12-h light–dark schedule. Pinealectomy was performed at 3 weeks of age; sham-operated animals were used as controls. The animals were sacrificed at 15 and 24 h 1 week after pinealectomy. The brainstem and diencephalon were removed, and tissue samples were processed for PACAP and cAMP radioimmunoassay (RIA). PACAP and cAMP levels showed nighttime elevations in both the sham-operated and pinealectomized animals, except for the PACAP content in the diencephalon of pinealectomized chicken. PACAP levels of pinealectomized animals were significantly higher in the diencephalon and brainstem as compared to the control animals at both time-points. Levels of cAMP correlated well with levels of PACAP. The present results provide evidence that the pineal gland has an inhibitory impact on PACAP-neurons in the chicken brainstem and diencephalon.

Levels of PACAP38 were measured in different areas of the chicken brain under various lighting conditions by radioimmunoassay (RIA). Selected groups of animals were maintained under light for 14 h alternating with 10 h of darkness (LD), reversed lighting conditions (DL) and constant light (LL) or constant dark (DD). Daily variations of PACAP levels were observed in the brainstem, diencephalon, telencephalon and retina. In the brainstem and diencephalon, levels of PACAP increased during subjective nighttime, except in the DL group where levels were elevated between 15–21 h. In the telencephalon, the lowest level of PACAP was measured between 12–21 h except in the DL group where two peaks occurred at 18 and 03 h. In the retina, all 4 groups showed a similar level and pattern, with lowest levels during midday hours. No daily variation was observed in the pineal gland. According to the present observations, it is suggested that PACAP levels differ in several areas of the chicken brain under various lighting conditions and photic stimuli do not appear to be the main regulators of the circadian variations of PACAP.

The present article investigated the levels of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) in the brains of rats and chickens 12, 36, and 84 h after starvation. PACAP levels increased in both species, 12 h after food deprivation in rats, and with a 24-h delay in chickens. VIP levels showed a more complex pattern: a gradual increase in the hypothalamus and telencephalon, and a significant decrease in the brain stem of rats. In chickens, a decrease was observed in every brain area after 36 h of starvation. These data show that PACAP and VIP are differentially regulated and are involved in the regulatory processes under a food-restricted regimen, and are differentially altered in nocturnal and diurnal species.

The aim of the present article was to investigate the occurrence and temporary changes of pituitary adenylate cyclase-activating polypeptide (PACAP)-38 and vasoactive intestinal peptide (VIP) in various brain areas of chicken embryos by means of radioimmunoassay. The highest concentrations of PACAP-38 were measured in the brain stem followed by the hypothalamus, cerebellum, and telencephalon. PACAP-38 levels were significantly higher than those of VIP in all examined brain areas. The levels of both PACAP-38 and VIP showed a tendency to decrease until hatching during embryonic development of the chicken.

PACAP plays an important role during development of the nervous system and is also involved in memory processing. The aim of the present study was to investigate the function of PACAP in chicken embryonic olfactory memory formation by blocking PACAP at a sensitive period in ovo. Chicken were exposed daily to strawberry scent in ovo from embryonic day 15. Control eggs were treated only with saline, while other eggs received a single injection of the PACAP antagonist PACAP6-38 at day 15. The consumption of scented and unscented water was measured daily after hatching. Animals exposed to strawberry scent in ovo showed no preference. However, chickens exposed to PACAP6-38, showed a clear preference for plain water, similarly to unexposed chicken. Our present study points to PACAP's possible importance in embryonic olfactory memory formation.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a ubiquitous member of the VIP/secretin/glucagon bioactive peptide family. The distribution, concentration of PACAP, and its role in the control of rhythmic melatonin (MT) secretion from chicken pineal gland were studied. In the chicken pineal gland (ChPG), 40ng PACAP/g tissue was measured with radioimmunoassay. No midday-midnight differences in the PACAP content of the ChPG could be detected. Immunohistochemical studies of ChPG showed PACAP immunoreactive nerve fibers in the perivascular space and around the pinealocytes. Neither PACAP-labeled perikarya nor PACAP immunopositivity in the pinealocytes could be detected. In five day perifusion experiments, carried out under darkness, both MT and cAMP showed circadian rhythmic release pattern from explanted ChPG. One hour exposure of ChPG to PACAP induced transient (3-4h) elevation of MT and cAMP release. The responses were dose-dependent in the range from 1 to 100nM PACAP concentrations. The magnitude of the response was independent on the phase of the daily cycle in which PACAP was applied. cAMP levels during normal daily cycle and also PACAP-induced cAMP responses always preceded similar changes of MT by about an hour suggesting that cAMP is an intracellular intermediate in controlling MT release from the ChPG. At the same time PACAP, similarly to VIP, did not shift the phase of the in vitro circadian MT rhythm of the pineals. Our data reveal, that PACAP is present in nerve fibers in the chicken pineals and pineal cells contain functioning PACAP-sensitive receptors. PACAP apparently exerts a modulatory effect of the rhythmic MT release from the chicken pineal but does not modify the intrinsic biological clock in the avian pineal gland. Thus, cAMP-mediated intracellular mechanisms in ChPG are not components of the pineal circadian clock, but intermediaries between the clock-mechanism and MT release and may also be components of clock-independent MT release modifiers.

Pituitary adenylate cyclase activating polypeptide (PACAP) occurs in two molecular forms: PACAP-38 and PACAP-27. Soon after the isolation and chemical characterization of PACAP, the first radioimmunoassay (RIA) methods have been developed, but it is a still rarely used laboratory technique in the field of PACAP research. The aim of the present study was to develop a novel, highly specific PACAP-38 assay to investigate the quantitative distribution of PACAP-38 in the central nervous system of various vertebrate species under the same technical and experimental conditions. Different areas of the brain and the spinal cord were removed from rats, chickens and fishes and the tissue samples were processed for PACAP-38 RIA. Our results indicate that the antiserum used in the RIA is C-terminal specific, without affinity for other members of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon peptide family. The average ID50 value was 48.6±3.4 fmol/ml determined in 10 consecutive assays. Detection limit for PACAP-38 proved to be 2 fmol/ml. PACAP-38 immunoreactivity was present in the examined brain areas of each species studied, with highest concentration in the rat diencephalons. High levels of PACAP-38 were also detected in the rat telencephalon, followed by spinal cord and brainstem. The central nervous system of the fish also contained considerable concentrations of PACAP-38, whereas lowest concentrations were measured in the central nervous system of the chicken.