PAC1 receptor localization in a model nervous system: light and electron microscopic immunocytochemistry on the earthworm ventral nerve cord ganglia.

The presence and pattern of pituitary adenylate cyclase activating polypeptide (PACAP) type I (PAC1) receptors were identified by means of pre- and post-embedding immunocytochemical methods in the ventral nerve cord ganglia (VNC) of the earthworm Eisenia fetida. Light and electron microscopic observations revealed the exact anatomical positions of labeled structures suggesting that PACAP mediates the activity of some interneurons, a few small motoneurons and certain sensory fibers that are located in ventrolateral, ventromedial and intermediomedial sensory longitudinal axon bundles of the VNC ganglia. No labeling was located on large interneuronal systems such as dorsal medial and lateral giant axon systems and ventral giant axons. At the ultrastructural level labeling was mainly restricted to endo- and plasma membranes showing characteristic unequal distribution in various neuron parts. An increasing abundance of PAC1 receptors located on both rough endoplasmic reticulum and plasma membranes was seen from perikarya to neural processes, indicating that intracellular membrane traffic might play a crucial role in the transportation of PAC1 receptors. High number of PAC1 receptors was found in both pre- and postsynaptic membranes in addition to extrasynaptic sites suggesting that PACAP acts as neurotransmitter and neuromodulator in the earthworm nervous system.

PKA-Bad-14-3-3 and Akt-Bad-14-3-3 signaling pathways are involved in the protective effects of PACAP against ischemia/reperfusion-induced cardiomyocyte apoptosis.

The neuropeptide PACAP (pituitary adenylate cyclase activating polypeptide) and its receptors are widely expressed in the nervous system and various other tissues. PACAP has well-known anti-apoptotic effects in neuronal cell lines. Recent data suggest that PACAP exerts anti-apoptotic effects also in non-neuronal cells. The peptide is present in the cardiovascular system, and has various distinct effects. The aim of the present study was to investigate whether PACAP is protective against in vitro ischemia/reperfusion-induced apoptosis in cardiomyocytes. Cultured cardiomyocytes were exposed to 60 min ischemia followed by 120 min reperfusion. The addition of PACAP1-38 significantly increased cell viability and decreased the ratio of apoptotic cells as measured by MTT test and flow cytometry. PACAP induced the phosphorylation of Akt and protein kinase A. In the present study we also examined the possible involvement of Akt- and protein kinase A-induced phosphorylation and thus inactivation of Bad, a pro-apoptotic member of the Bcl-2 family. It was found that ischemia significantly decreased the levels of phosphorylated Bad, which was counteracted by PACAP. Furthermore, PACAP increased the levels of Bcl-xL and 14-3-3 protein, both of which promote cell survival, and decreased the apoptosis executor caspase-3 cleavage. All effects of PACAP1-38 were inhibited by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP has protective effects against ischemia/reperfusion-induced cardiomyocyte apoptosis and provides new insights into the signaling mechanisms involved in the PACAP-mediated anti-apoptotic effects.

Changes in open-field activity and novelty-seeking behavior in periadolescent rats neonatally treated with monosodium glutamate.

Monosodium glutamate (MSG) treatment of neonatal rodents leads to degeneration of the neurons in the arcuate nucleus, inner retinal layers and various other brain areas. It also causes various changes in the motor activity, sensory performance and learning abilities. We have previously shown that MSG treatment delays the appearance of some reflexes during neurobehavioral development and leads to temporary changes in reflex performance and motor coordination. Investigation of novelty-seeking behavior is of growing importance for its relationship with sensitivity to psychomotor stimulants. Perinatal administration of numerous toxic agents has been shown to influence novelty-seeking behavior in rats, but little is known about the influence of neonatal MSG treatment on the novelty-seeking behavior. The aim of the present study was to compare changes in locomotor, spontaneous exploratory and novelty-seeking behavior in periadolescent rats neonatally treated with MSG. Newborn rats were treated with 4 mg/g MSG subcutaneously on postnatal days 1, 3, 5, 7 and 9. Open-field behavior was tested at 2, 3, 4, 6 and 8 weeks of age. We found that MSG administration led to only temporary increases in locomotor behavior, which was more pronounced during the first few postnatal weeks, followed by a subtle hypoactivity at 2 months of age. Novelty-seeking was tested in four 5-min trials at 3 weeks of age. Trial 1 was in an empty open-field, two identical objects were placed in the arena during trial 2 and 3, and one of them was replaced to a novel object during trial 4. We found that the behavioral pattern of MSG-treated rats was the opposite in all tested signs in the novelty exploration test compared to control pups. In summary, our present study shows that neonatal MSG treatment leads to early temporary changes in the locomotor activity followed by hypoactivity at 2 months of age. Furthermore, MSG-treated rats show a markedly disturbed novelty-seeking behavior represented by altered activity when subjected to a novel object.

Protective effects of PACAP in excitotoxic striatal lesion.

The present article investigated the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) treatment in a quinolinic acid (QA)-induced unilateral lesion of the striatum, a model of Huntington;s disease (HD). PACAP was given locally, preceding the lesion. Behavioral analysis was performed after 1, 10, and 30 days, when motor activity and asymmetrical signs were evaluated. Three weeks after the treatment, a catalepsy test was performed by haloperidol administration, and finally histological assessment of the striatum was done. Our results show that PACAP treatment attenuated the behavioral deficits and reduced the number of lesioned neurons in the striatum.

Comparative study of the effects of PACAP in young, aging, and castrated males in a rat model of Parkinson's disease.

We have previously shown that PACAP ameliorates the neurological symptoms and reduces the dopaminergic cell loss in young male rats, in a 6-hydroxydopamine (6-OHDA)-induced lesion of the substantia nigra, a model of Parkinson's disease. In the present study, we compared the effects of PACAP in young, aging, and castrated males. Our results show that PACAP significantly reduced the dopaminergic cell loss in young and aging males. In castrated males, 6-OHDA did not induce such a severe cell loss, and it was not altered by PACAP. However, PACAP effectively ameliorated behavioral symptoms in all groups, with a degree of recovery depending on age and endocrine status.

Effects of systemic PACAP treatment in monosodium glutamate-induced behavioral changes and retinal degeneration.

The present article investigated effects of systemic pituitary adenylate cyclase-activating polypeptide (PACAP) treatment in monosodium glutamate (MSG)-induced retinal degeneration and neurobehavioral alterations in neonatal rats. It was found that the dose of PACAP that effectively enhances neurobehavioral development in normal rats was able to counteract the retarding effect of MSG on righting, forelimb placing, and grasp reflexes and caused a significant amelioration of the righting and gait reflex performance and motor coordination at 2 weeks of age. In the retina, significant amelioration of neuronal loss in the inner retinal layers was achieved, but it was much less than that observed by local administration.

Pituitary adenylate cyclase activating polypeptide protects cardiomyocytes against oxidative stress-induced apoptosis.

Pituitary adenylate cyclase activating polypeptide (PACAP) has well-known neuroprotective effects, and one of the main factors leading to neuroprotection seems to be its anti-apoptotic effects. The peptide and its receptors are present also in the heart, but whether PACAP can be protective in cardiomyocytes, is not known. Therefore, the aim of the present study was to investigate the effects of PACAP on oxidative stress-induced apoptosis in cardiomyocytes. Our results show that PACAP increased cell viability by attenuating H2O2-induced apoptosis in a cardiac myocyte culture. PACAP also decreased caspase-3 activity and increased the expression of the anti-apoptotic markers Bcl-2 and phospho-Bad. These effects of PACAP were counteracted by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP is able to attenuate oxidative stress-induced cardiomyocyte apoptosis.

Effect of PACAP in 6-OHDA-induced injury of the substantia nigra in intact young and ovariectomized female rats.

Pituitary adenylate cyclase activating polypeptide (PACAP) has neuroprotective effects in various neuronal cultures and in models of brain pathologies in vivo. Among others, it protects dopaminergic neurons in vitro, against 6-OHDA- and rotenone-induced injury. Recently, we have shown that PACAP reduces dopaminergic cell loss and ameliorates behavioral outcome following unilateral 6-OHDA-induced injury of the substantia nigra in male rats. However, after castration, PACAP led only to a slight amelioration of the behavioral symptoms. The aim of the present study was to investigate the degree of neuroprotection exerted by PACAP in female rats, using the same model. It was found that PACAP had no effect on the dopaminergic cell loss in intact female rats, only caused amelioration of certain acute behavioral signs. In contrast, PACAP effectively increased dopaminergic cell survival and decreased behavioral deficits in ovariectomized females. These results indicate that the neuroprotective effect of PACAP in a rat model of Parkinson's disease is gender-specific.

Development of neurological reflexes and motor coordination in rats neonatally treated with monosodium glutamate.

Monosodium glutamate (MSG) treatment of neonatal rats causes neuronal degeneration in various brain areas and leads to several neurochemical, endocrinological and behavioral alterations. However, relatively little is known about the development of neurological reflexes and motor coordination of these animals. Therefore, the aim of the present study was to examine the neurobehavioral development of newborn rats treated with MSG. Rats received MSG at postnatal days 1, 3, 5, 7, and 9. Appearance of neural reflexes and reflex performance as well as motor coordination were examined for 5 weeks after birth. The efficacy of MSG treatment was confirmed by histological examination of the arcuate nucleus. We found that MSG treatment delayed the appearance of forelimb placing, forelimb grasp and righting reflexes, besides the retarded somatic development. The treated pups performed surface righting in significantly longer times. Also, worse performance was observed in the foot-fault and rota-rod tests. However, MSG-treated rats reached control levels by the end of the fifth postnatal week. These results show that MSG treatment does not cause permanent alterations in the neurobehavioral development, only delays the appearance of some reflexes and leads to temporary changes in reflex performance and motor coordination signs.

Gastrin- and cholecystokinin-like immunoreactivities in the nervous system of the earthworm.

The distribution of cholecystokinin and gastrin-like immunoreactive cell bodies and fibers in the nervous system of 2 annelid worms, Lumbricus terrestris and Eisenia fetida, has been studied by means of immunohistochemistry. The cerebral ganglion contains 170-250, the subesophageal ganglion contains 120-150, and the ventral ganglia contain 50-75 cholecystokinin immunoreactive cells, that represent 8-12%, 8-10% and 4-5% of the total cell number, respectively. The anti-gastrin serum stained 330-360 nerve cells in the cerebral, 32-46 in the subesophageal and 7-25 in the ventral cord ganglia, representing 15-16%, 2-3% and 0.5-2% of the total cell number. Immunopositivity was found with both antisera in the enteric nervous system, where the stomatogastric ganglia and the enteric plexus contain immunoreactive cells and fibers. Immunopositive cells were found in the epithelial and subepithelial cells, as well as in nerve cells innervating the muscular layer of the gastrointestinal tube. Various epidermal sensory cells also displayed strong immunoreactivity. According to our findings and the results of several functional studies, it is suggested that in annelids cholecystokinin- and gastrin-like peptides may be involved in digestive regulation, sensory processes and central integrating processes.

Distribution of PACAP-like immunoreactivity in the nervous system of oligochaeta.

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. We investigated the distribution of PACAP-like immunoreactivity in the nervous system of three oligochaete (Annelida) worms with 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 were 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.

Immunohistological detection of degenerating CRF-immunoreactive nerve fibers in the median eminence after lesion of paraventricular nucleus of the rat. A light and electron microscopic study.

Corticotropin releasing factor (CRF)-immunoreactive neurons were detected in the paraventricular nuclei (PVN) of the rat brain, using both the traditional and the recently developed silver-gold intensified PAP methods at light and electron microscopic levels. The latter technique was more sensitive, compared to the classical PAP method, and proved to be highly specific at the ultrastructural level. The immunolabeled perikarya showed smooth or rough contoured fusiform or multipolar shape. Bilateral surgical destruction of PVN caused a gradual decrease in the number of CRF-immunopositive fibers of the median eminence. Following the second post-operative week, CRF-immunoreactivity practically disappeared from this area. In the case of unilateral lesion of PVN, the diminution of immunoreactivity was restricted to the ipsilateral side of the median eminence-pituitary stalk region. Applying the silver-gold intensified PAP method to electron microscopy, the detection of immuno-labeled degenerating fibers became possible, among morphologically similar, densely degenerating, but unlabeled, profiles. This study reports that CRF fibers to the capillary system of the median eminence of the rat originate principally from PVN.

Pituitary adenylate cyclase activating polypeptide is highly abundant in the nervous system of anoxia-tolerant turtle, Pseudemys scripta elegans.

The levels of the pituitary adenylate cyclase activating polypeptide (PACAP) were measured in the central nervous system and in peripheral organs of the anoxia-tolerant freshwater turtle, Pseudemys scripta elegans by radioimmunoassay. The concentration of PACAP38 was strikingly high in the central nervous system and lower but considerable immunoreactivity was detected in the peripheral organs. Levels of PACAP38 in the turtle brain exceed those measured in rat and human brain areas by 10-100-fold. Based on these exceptionally high levels of PACAP and the known neuroprotective role of the peptide, it can be suggested that PACAP38 plays a role in the extraordinary resistance of the turtle brain from anoxia-induced neuronal damage.

Tissue distribution of PACAP27 and -38 in oligochaeta: PACAP27 is the predominant form in the nervous system of Lumbricus polyphemus.

The levels of pituitary adenylate cyclase-activating polypeptide (PACAP)27 and -38 were measured in the nervous, intestinal, excretory, and reproductive systems of Lumbricus polyphemus by radioimmunoassay. Although both PACAP27 and -38 were significantly detectable in all of the examined tissues, the distribution of the peptides was very heterogeneous. Their highest concentrations were found in the cerebral ganglia and the ventral cord, followed by the alimentary tract and the nephridial system, respectively. Moreover, the reproductive system also contained a substantial amount of PACAP. The dominant form of the peptide discovered in the majority of tissues was PACAP27. Interestingly, about 10 times more PACAP27 than PACAP38 was found, with the latter representing only a fraction of PACAP-like immunoreactivity in the tissues of Lumbricus polyphemus.

Effects of pretreatment with PACAP on the infarct size and functional outcome in rat permanent focal cerebral ischemia.

PACAP exerts neuroprotective effects under various neurotoxic conditions in vitro. In vivo, it reduces brain damage after global and transient focal ischemia. The present study investigated whether PACAP has neuroprotective effects when applied before the onset of permanent ischemia. Rats were given bolus injections of PACAP38 intracerebroventricularly, and then underwent permanent middle cerebral artery occlusion. The results show that 2 microg of PACAP significantly reduced the infarct size measured 12 and 24h after the onset of ischemia. No further reduction was obtained by a 7-day pretreatment. PACAP also ameliorated certain sensorimotor deficits. Our present study provides further evidence for the neuroprotective effects of PACAP, and implies that it might be a promising preventive therapeutic agent in ameliorating ischemic brain damage.

Changes in diurnal fluctuations of plasma thyroid stimulating hormone and corticosterone following anterior hypothalamic deafferentation in the rat.

Systemic thyroid stimulating hormone (TSH) and corticosterone (B) levels were measured in serial plasma samples withdrawn from 8 adult female rats before and for several weeks after anterior hypothalamic cuts (FC), made with a modified Halász knife (1.5 mm radius). With lights on from 5:00 to 19:00 h the rats were bled by rapid venipuncture at 9:00 and 20:00 h 14 days before and 5 days after FC and by chronic intra-atrial cannulas at 9:00, 13:00, 17:00 and 20:00 h on days 25, 30 and 40 or 50 post-FC. The frontal cuts, localized later by histology to sites just rostral to the suprachiasmatic nucleus in 3 rats and just caudal to the nucleus in the other 5 brains, altered the charactersitic diurnal drop in plasma TSH and rise in B but did not eliminate marked fluctuations in hormone levels. The new hormonal platterns could not be related specifically to FC loci and appeared to be a dynamic process for each rat. At 5 days after FC the diurnal patterns were either obscured or shifted in phase. The most consistent finding at subsequent sampling intervals was that TSH and B plasma levels in each animal showed parallel fluctuations (during 9:00--20:00 h) as though synchronized in phase. The data suggest that interruption of anterior hypothalamic connections in the female rat is compatible with fluctuations in plasma TSH and B which are unique to a particular animal and may be obscured by group analysis.

Examination of sensorimotor performance following middle cerebral artery occlusion in rats.

Middle cerebral artery occlusion (MCAO) in rats is the most commonly used stroke model. Besides the infarct size, assessment of sensorimotor performance has become increasingly important in neuroprotective drug research. However, contradictions exist about procedures for testing functional outcome following MCAO. The aim of the present study was to evaluate a relatively simple set of neurological tests based on the most commonly used scoring systems, and to describe the functional recovery and correlation with the infarct size in rats sacrificed 2 or 14 days after permanent or transient MCAO. The smaller infarct size of rats with transient occlusion was reflected in the neurological scores only during the first 6h. By day 14, no recovery occurred in postural signs, lateral resistance and spontaneous activity, other signs showed different degrees of recovery. Correlation with the infarct size was found only on certain days in gait disturbance, placing reactions, daily body weight and spontaneous activity. According to our observations, the most commonly used sensorimotor tests provide a useful initial screening of functional deficits, but these tests most probably measure deficits caused by infarction of the core area. It is suggested that these tests should be completed by more refined tests when testing a neuroprotective drug which reduces the infarct size in penumbral areas.

Co-localization of serotonin and FMRFamide-like immunoreactivities in the central nervous system of the earthworm, Eisenia fetida.

In the present study co-localization of serotonin and FMRFamide-like immunoreactivities was studied in the central nervous system of the earthworm, Eisenia fetida. Alternate serial sections were stained with antisera raised against serotonin and FMRFamide, using the peroxidase-antiperoxidase method. The immunostained consecutive sections were compared by light-microscopy and on reconstructed images using NIH Image and Neurolucida programs. Although there was a large number of serotonin and FMRFamide-like immunoreactive neurons in the central nervous system of Eisenia, co-localization of immunostaining was relatively rare. In the cerebral ganglion no cells were found that stained for both antisera. In the subesophageal and ventral cord ganglia the neurons reacting for both antisera represented approximately 17-23% and 10-14% of the serotonin- and 7-11% and 9-17% of the FMRF-amide-like immunoreactive cells, respectively. It is suggested, that FMRFamide-related peptides act as neuromodulators and may also influence the activity of serotonergic neurons.

Immunohistochemical and ultrastructural study of the enteric nervous system of earthworm, Lumbricus terrestris L.

Light and electronmicroscopic data reveal the presence of a well developed nerve plexus in the gut of the earthworm. The plexus contains subepithelial solitary nerve cells and fibers and an extensive neuropil among the muscle cells. There are two types of nerve cells in the enteric plexus. The first type contains mainly dense-core vesicles, and exhibits glyoxylic-acid induced fluorescence. Since none of these cells showed serotonin immunoreactivity, they are probably noradrenergic or dopaminergic. The second type contains large dense granules, suggesting that these cells are peptidergic (neurosecretory). A part of these cells are substance P immunoreactive, however no NPY, CGRP, or proctolin immunopositive cells were found. Ultrastructurally seven types of nerve fibers can be distinguished in the neuropil. Their distribution shows great variability within parts of the enteric canal. The observation that only two types of nerve cells are located within the gut makes it probable that some of the axons are extrinsic. According to immunohistological studies they may come from the stomatogastric system or from the segmental nerves. This is further supported by the fact that there is a well-developed subepithelial serotoninergic plexus in the fore-gut. Two types of neuromuscular junctions can be visualized in the muscular layer. The first type, representing a phylogenetically earlier form, exhibits wide junctional gap and pre- or postjunctional membrane thickening. The second type is the close contact. There are significantly more junctions observed in the fore-gut than in other parts of the gut.

Immunohistochemical study of the nervous system in earthworm (Lumbricus terrestris L.).

In the present work it we describe serotonin, noradrenaline, proctolin, neuropeptide Y, substance P, and calcitonin gene related peptide immunoreactive structures in earthworm. A few large serotonin immunoreactive perikarya are located in brain and in the stomatogasric ganglia and many of them in the segmental ganglia. A serotonin immunoreactive fiber plexus can be seen beneath the epithelium of the body wall, both the sensory papillae and chaetae contain serotonin immunoreactive elements. Some of the sensory cells are serotonin immunoreactive, too. A serotonin immunoreactive network can ben found in the enteric network of the fore- and mid-gut. Only a few noradrenaline immunoreactive cells are observed in the caudal part of the brain, while their number in the segmental ganglia is high. Proctolin-, and substance P immunoreactive cells are small and numerous in the brain without any preference in their location. Such nerve cells are widely distributed in the ganglia of the stomatogastric system, in the subesophageal and in segmental ganglia. Many surface epithelial (probably sensory) cells are proctolin immunoreactive. Substance P immunoreactive nerve cells can also be located in the entire length of the enteric plexus together with substance P immunoreactive fibers. No neuropeptide Y- or calcitonin gene related peptide immunoreactive structures can be seen in the brain. A relative small number of neuropeptide Y- and calcitonin gene related peptide immunoreactive perikarya, and a rich network of neuropeptide Y- and calcitonin gene related peptide immunoreactive fibers can be detected in the subesophageal ganglion. According to preliminary studies, neuropeptide Y is probably co-localized with serotonin.