Distribution of hypothalamic, hippocampal and other limbic peptidergic neuronal cell bodies giving rise to retinopetal fibers: anterograde and retrograde tracing and neuropeptide immunohistochemical studies.

In our present work utilizing the retrograde or anterograde transport of tracers (biotinylated dextran amine and Fluorogold, respectively) we have provided direct evidence for the cells of origin of the limboretinal pathway in rats and their termination in the retina using light microscopic approach. Administration of biotinylated dextran amine into the vitreous body resulted in nerve cell body labeling in several structures: the supraoptic and paraventricular nuclei, the hippocampus (CA1, CA3), the dentate gyrus, the indusium griseum, the olfactory tubercle, and the medial habenula, all of them belong to the limbic system. We estimated that the total number of retrogradely labeled cells is 1495+/-516. We have seen fiber labeling in the retinorecipient suprachiasmatic nucleus and in the primary visual center, the lateral geniculate body, but labeled nerve cell bodies in these structures were never seen. Iontophoretic application of Fluorogold into the hippocampal formation, where the major part of the biotinylated dextran amine-labeled cell bodies was observed, resulted in labeled fibers in the optic nerve and in the retina indicating that the retrogradely labeled cells in the hippocampus and the dentate gyrus among others are the cells of origin of the centrifugal visual fibers. Sections showing biotinylated dextran amine labeling were stained for vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactivity using immunohistochemistry. Some biotinylated dextran amine-labeled cells also showed vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactivity. We conclude that the limboretinal pathway exists and that the cells of origin are partially vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactive.

Identification of autonomic neuronal chains innervating gingiva and lip.

The major goals of this present study were 1) to further clarify which parasympathetic ganglion sends postganglionic fibers to the lower gingiva and lip that may be involved in the inflammatory processes besides the local factors; 2) to separately examine the central pathways regulating sympathetic and parasympathetic innervation; and 3) to examine the distribution of central premotor neurons on both sides. A retrogradely transported green fluorescent protein conjugated pseudorabies virus was injected into the lower gingiva and lip of intact and sympathectomized adult female rats. Some animals received virus in the adrenal medulla which receive only preganglionic sympathetic fibers to separately clarify the sympathetic nature of premotor neurons. After 72-120h of survival and perfusion, the corresponding thoracic part of the spinal cord, brainstem, hypothalamus, cervical, otic, submandibular and trigeminal ganglia were harvested. Frozen sections were investigated under a confocal microscope. Green fluorescence indicated the presence of the virus. The postganglionic sympathetic neurons related to both organs are located in the three cervical ganglia, the preganglionic neurons in the lateral horn of the spinal cord on ipsilateral side; premotor neurons were found in the ventrolateral medulla, locus ceruleus, gigantocellular and paraventricular nucleus and perifornical region in nearly the same number on both sides. The parasympathetic postganglionic neurons related to the gingiva are present in the otic and related to the lip are present in the otic and submandibular ganglia and the preganglionic neurons are in the salivatory nuclei. Third order neurons were found in the gigantocellular reticular and hypothalamic paraventricular nuclei and perifornical area.

Atomoxetine reverses locomotor hyperactivity, impaired novel object recognition, and prepulse inhibition impairment in mice lacking pituitary adenylate cyclase-activating polypeptide.

Attention-deficit/hyperactivity disorder (ADHD) is a complex neurobehavioral disorder that is characterized by attention difficulties, impulsivity, and hyperactivity. A non-stimulant drug, atomoxetine (ATX), which is a selective noradrenaline reuptake inhibitor, is widely used for ADHD because it exhibits fewer adverse effects compared to conventional psychostimulants. However, little is known about the therapeutic mechanisms of ATX. ATX treatment significantly alleviated hyperactivity of pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient (PACAP(-/-)) mice with C57BL/6J and 129S6/SvEvTac hybrid background. ATX also improved impaired novel object recognition memory and prepulse inhibition in PACAP(-/-) mice with CD1 background. The ATX-induced increases in extracellular noradrenaline and dopamine levels were significantly higher in the prefrontal cortex of PACAP(-/-) mice compared to wild-type mice with C57BL/6J and 129S6/SvEvTac hybrid background. These results suggest that ATX treatment-induced increases in central monoamine metabolism may be involved in the rescue of ADHD-related abnormalities in PACAP(-/-) mice. Our current study suggests that PACAP(-/-) mice are an ideal rodent model with predictive validity for the study of ADHD etiology and drug development. Additionally, the potential effects of differences in genetic background of PACAP(-/-) mice on behaviors are discussed.

Immunohistochemical demonstration of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, in the ovine hypothalamus.

We recently reported isolation, characterization and synthesis of a novel ovine hypothalamic peptide with 38 residues which stimulates accumulation of cAMP in rat anterior pituitary cell cultures. The peptide was named PACAP38 (pituitary adenylate cyclase-activating polypeptide with 38 residues). The presence of another peptide corresponding to the N-terminal 1-27 residues (PACAP27) was also demonstrated. Both PACAP38 and PACAP27 have an amidated C-terminus. Antisera against synthetic PACAP27 were generated in rabbits. These antisera were tested for titer and specificity in enzyme-linked immunosorbent assay. One of the antisera (no. 88121-3) exhibited a high titer of antibody, which was specific to PACAP27 and PACAP38 with exception of slight cross-reactivity with ovine CRF (oCRF). Therefore, the antibodies against oCRF were removed from the antiserum using a solid phase method. Removal of oCRF antibodies was confirmed by enzyme-linked immunosorbent assay. A dense immunoreactive fiber network was found in both external and internal zones of the median eminence and pituitary stalk. The fibers were demonstrated to be in close contact with the hypophysial portal capillaries. The preabsorption of antiserum with vasoactive intestinal polypeptide or with the mixture containing TRH, LHRH, oCRF, ovine GH-releasing factor, somatostatin, and bovine thyroglobulin did not affect the immunostaining. On the other hand, the preabsorption of antiserum with an excess of PACAP27 or PACAP38 abolished the immunostaining. Therefore, the staining is considered specific for PACAP27 and PACAP38. Stained fibers were also present in the posterior pituitary. A dense fiber network was observed and the lateral hypothalamus the fibers appeared to cling to unstained neuronal cell bodies and their dendrites. In the lateral septum the fibers surrounded some blood vessels. Immunolabeled cell bodies were found in the paraventricular and supraoptic nuclei. These findings support the view that PACAP may play a multifunctional role, including that of a hypophysiotropic hormone, neurotransmitter, neuromodulator, and vasoregulator.

Presence of immunoreactive vasoactive intestinal polypeptide in anterior pituitary of normal male and long term estrogen-treated female rats: a light microscopic immunohistochemical study.

The presence and synthesis of vasoactive intestinal polypeptide (VIP) has been demonstrated in the rat anterior pituitary. It was recently confirmed that immunoreactive VIP is present in the anterior pituitary, and in a thyroid-deficient state, VIP could be detected by light microscopic immunohistochemistry. It has also been suggested that VIP plays a stimulatory role in PRL secretion. To gain more detailed information on the localization of VIP and the conditions that alter the synthesis of VIP, we examined VIP immunoreactivity using immunohistochemistry in pituitaries of normal male and cycling female rats and in those states in which PRL secretion was enhanced (pregnancy, lactation, long term estrogen treatment, and pituitary implanted under kidney capsule of normal or estrogen-treated female rats). In situ, implanted and cultured pituitary cells were stained for VIP immunoreactivity using the peroxidase-antiperoxidase method. VIP immunoreactivity was observed in about 45% of the male rats, in all estrogen-treated female rats, in the implanted pituitaries under the kidney capsule (three of five from estrogen-treated and one of five from intact females, respectively), and in the pituitary cell cultures derived from estrogen-treated rats. Using a double labeling procedure we have also observed PRL immunoreactivity in a small population of the VIP-positive cells. These results suggest a positive regulatory role of estrogen in expression of the VIP gene. The physiological and pathophysiological significance of VIP in PRL secretion, however, remains to be clarified.

Pituitary adenylate cyclase activating peptide: a novel vasoactive intestinal peptide-like neuropeptide in the gut.

Pituitary adenylate cyclase activating peptide (PACAP) is a vasoactive intestinal peptide (VIP)-like hypothalamic peptide occurring in two forms, PACAP-27 and the C-terminally extended PACAP-38. The predicted rat and human PACAP sequence is identical to the isolated ovine one. In the present study, the occurrence and distribution of PACAP-like peptides were examined in the gut of several species by immunocytochemistry and immunochemistry using an antibody raised against PACAP-27. PACAP-like immunoreactivity was observed in nerve fibers in the gut wall of all species examined (chicken, mouse, rat, hamster, guinea-pig, ferret, cat, pig, sheep and man). In the chicken and human gut, immunoreactive fibers were numerous in all layers. In the other species examined the fibers were predominantly found in the myenteric ganglia and smooth muscle. Delicate PACAP-immunoreactive fibers were seen in the gastric mucosa of mouse, rat, hamster and man but not in the other species examined. The chicken proventriculus harbored numerous PACAP-immunoreactive endocrine cells which were identical with the serotonin-containing cells storing gastrin-releasing peptide. PACAP-immunoreactive nerve cell bodies were numerous in the submucous ganglia and moderate in number in the myenteric ganglia of the human gut. They were few in the intramural ganglia of the other species examined. Extrinsic denervation (performed on segments of rat and guinea-pig small intestine) did not visibly affect the PACAP innervation, indicating an intramural origin of most PACAP-immunoreactive fibers. Double immunostaining for VIP and PACAP revealed co-existence of the two peptides in nerve cell bodies and nerve fibers of the human and chicken gut and in fibers in the gastric mucosa of mouse and rat. In all other species examined and in all other locations in the gut PACAP-immunoreactive nerve cell bodies and nerve fibers were distinct from those storing VIP; many of them contained gastrin-releasing peptide instead. Immunochemistry revealed PACAP-like peptides in gut extracts of all species studied; upon high performance liquid chromatography the immunoreactive material co-eluted with synthetic PACAP-27. The distribution of PACAP-immunoreactive nerve cell bodies and nerve fibers in the gut wall suggests their involvement in the regulation of both motor and secretory activities.

Solid-phase adsorption method for removing undesired antibodies from polyclonal antiserum.

We have developed a novel and simple method, requiring only a small amount of antigen, for removal of undesired antibodies from antiserum. The method was established using a well-characterized antiserum against rat luteinizing hormone (anti-rLH). Wells of polystyrene tissue culture plates were coated with rat LH (rLH). Anti-rLH diluted 1:3000 was added to rLH-coated wells and shaken to remove LH antibodies. Control anti-rLH was treated in a similar manner in non-rLH-coated wells. Both antisera were tested by immunocytochemistry on rat pituitaries. Antiserum from rLH-coated wells stained no cells, whereas the control serum stained cells that were morphologically typical of LH cells. The effectiveness of this antibody removal was also confirmed in a modified ELISA. In another experiment, anti-rLH and anti-hTSH beta sera were mixed. The final dilution of both antisera was 1:10,000. Anti-rLH was removed by the purification method described. Completeness of antibody removal was confirmed by a double-immunohistochemical staining of rat pituitary in which sections were first stained by the PAP method and then stained with an immunofluorescence procedure after elution of the first antigen-antibody complex. The mixed antiserum incubated in rLH-coated wells did not stain LH cells. There was no co-localization between the LH immunopositivity demonstrated by an anti-rLH serum using immunofluorescence and cells immunostained with the purified antiserum using the PAP method. As indicated in ELISA, the titer of the TSH beta antiserum was not decreased compared to that of the untreated, mixed control antiserum, and the LH antibodies were eliminated by the treatment. This new purification method has four distinct advantages: (a) antiserum is not treated chemically; (b) it requires only a small amount of antigen compared with the amount required for affinity chromatography; (c) neither the undesired antigen-antibody complex(es) nor an excess amount of antigen is present in the purified antiserum; and (d) removal of undesired antibodies can be monitored by ELISA.

New aspects of the neuroendocrine role of PACAP.

The presence of PACAP was revealed in the anterior pituitary with RIA, HPLC, and with the demonstration of its mRNA. The level of PACAP mRNA in the anterior pituitary is the highest during the proestrous LH surge. In our immunohistochemical studies we were able to demonstrate PACAP immunoreactive cells in the anterior pituitary. The shape and the distribution of PACAP immunoreactive cells were very similar to that of the gonadotropes; however, the number of PACAP cells was less than that of LH cells. Additionally, another PACAP-positive cell population with small diameter appeared in the proestrous stage, during pregnancy and lactation. Double labeling revealed that the major part of large PACAP cells exhibited LH immunoreactivity and those with a small diameter contained PRL. It is not clear whether the pituitary- or the hypothalamic-born PACAP, or both, influence pituitary LH and PRL secretion. I.c.v. administration of PACAP just prior to the critical period in the proestrous stage inhibited the expected ovulation and blocked the proestrus LH and PRL surge, although i.v. administration of PACAP had no effect. PACAP antiserum did not interfere with ovulation when i.c.v. or i.v. injection was used. Our results support the view that PACAP has a role in the control of LH and PRL secretion during the estrous cycle, pregnancy, and lactation. The inhibitory effect of PACAP on ovulation is mediated through the hypothalamus.

PACAP38 and PACAP27 administered intracerebroventricularly have an opposite effect on LH secretion.

The effect of PACAP38 on the LH surge and ovulation was compared with that of PACAP27 and VIP in the same model. The peptides were administered intracerebroventricularly before the critical period of the proestrous stage. PACAP38 was able to inhibit ovulation and to prevent the preovulatory LH surge; however, PACAP27 did not inhibit the ovulation and VIP inhibited the ovulation in 2/11 animals. In those animals of the last two groups in which ovulation occurred, the preovulatory LH surge was higher than in control rats. It is speculated that the opposite effect of PACAP38 and PACAP27 on the preovulatory LH surge and ovulation is possibly mediated through different receptors.

Immunohistochemical localization of PACAP in the ovine digestive system.

The localization of immunoreactive PACAP (PACAP-IR) in the entire length of the sheep gastrointestinal tract and the pancreas was studied by an immunohistochemical method. PACAP-IR-containing nerve fibers innervated the longitudinal smooth muscle layer of the mucosa in the esophagus, stomach, and small and large intestines, the muscular layer of the stomach and intestine, Brunner's gland of the duodenum, and the walls of small arteries. PACAP-IR fibers also innervated the exocrine acini, islets of Langerhans, and the small arteries in the connective tissue septa of the pancreas. These findings suggest a regulatory role of PACAP in the digestive system.

VIP fibers in rat optic chiasm and optic nerve arising from the hypothalamus.

This is the first report showing VIP fibers in the optic chiasm and the optic nerves of intact rats. These fibers form a fan-shaped dorso-medial bundle in the optic nerves. After colchicine injection into the vitreous body VIP fibers could be followed farther in the optic nerve toward the eye when compared to intact rats. After removal of eyes (enucleation) the VIP fiber-bundle became more prominent and VIP immunoreactive perikarya appeared in the supraoptic and para ventricular nuclei. When five-nine months after the enucleation Phaseolus vulgaris leucoagglutinin was administered to the paraventricular or supraoptic area, the anterogradely transported tracer was demonstrated in the optic nerve. These observations suggest the existence of a hypothalamic projection to the eye, which is, at least in part, VIP immunoreactive.

PACAP colocalizes with luteinizing and follicle-stimulating hormone immunoreactivities in the anterior lobe of the pituitary gland.

Pituitary adenylate cyclase activating polypeptide (PACAP) and its close relative vasoactive intestinal polypeptide (VIP) were demonstrated in the anterior pituitary gland. The cells which exhibited PACAP immunoreactivity were oval or round shaped. Their distribution was similar to that of gonadotropes but the number of PACAP immunoreactive cells was less. Double labeling revealed that PACAP immunoreactivity partially colocalized with luteinizing and follicle-stimulating hormone; however, colocalization with other pituitary hormone immunoreactivities was not demonstrated. Our results suggest an autocrine or paracrine role of PACAP in the regulation of pituitary functions.

Immunohistochemical localization of the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), in human and primate hypothalamus.

A 38 residue neuropeptide was recently isolated from ovine hypothalamus in our laboratory, and named pituitary adenylate cyclase activating polypeptide (PACAP38) based on its biological activity. Rabbit antisera against synthetic PACAP27 were characterized by ELISA for immunohistochemical use. PACAP-immunoreactive neuronal elements having similar distributions were demonstrated in both human and spider monkey hypothalami. Many PACAP-immunoreactive cell bodies were present in the supraoptic and paraventricular nuclei. Immunopositive nerve fiber networks were stained throughout the hypothalamus, including in both external and internal zones of the tuber cinereum, close to the transition of the pituitary stalk (median eminence). These results suggest that PACAP plays multifunctional roles as a hypophysiotropic hormone, neurohypophysial hormone, neurotransmitter or neuromodulator in higher vertebrate species including man.

Pituitary adenylate cyclase activating polypeptide (PACAP) is present in human and cat gastric glands.

In the present work we have studied the occurrence of pituitary adenylate cyclase activating polypeptide (PACAP) in human and cat stomach mucosa using immunohistochemistry. As seen under a light microscope, there were many large rounded and ovoid cells that were PACAP immunopositive, mainly in the neck of the gastric glands of both species. The immunopositive material was predominant in the perinuclear area. The PACAP immunolabeling was specific because the preincubation of the antiserum with PACAP abolished the immunostaining. In human samples under electron microscope, the PACAP immunoreactive cells have shown the characteristics of parietal cells. In faintly stained cells, the localization of DAB reaction product was associated with the surface of the intracellular canaliculi. Cell labeling could not be observed besides parietal cells.

Comparative study on the appearance of various bioactive peptides in foregut derivates during the ontogenesis.

Bioactive peptides have an important multifunctional role in the gastrointestinal tract. In the present study we have investigated the dynamism of the appearance of PACAP (pituitary adenylate cyclase activating polypeptide), VIP (vasoactive intestinal polypeptide), gastrin, and secretin immunoreactivities in human foregut derivates during the ontogenesis using an immunohistochemical approach. None of these peptides were observed in the foregut derivates of an 8-week-old embryo. VIP immunoreactive nerve fibers appeared by the 11th week in the smooth muscle layers of the stomach. No other peptide immunoreactivities were observed of this stage. In 18- and 20-week old fetuses PACAP, secretin, and gastrin immunoreactive cells appeared in the developing glands of the stomach. In the duodenum gastrin immunoreactivity was present in the Lieberkühn's glands and secretin immunoreactive cells were seen between the surface epithelial cells. In the pancreas secretin immunoreactivity was found in the Langerhans islets; however, PACAP immunreactivity was observed in the exocrine portion. The distribution of VIP fibers did not change during the fetal life and it was similar to the adult pattern. According to our results the appearance of PACAP, secretin, and gastrin in the developing glands suggests their role in the proliferation and differentiation of the epithelial derivates.

Effect of PACAP on LH release studied by cell immunoblot assay depends on the gender, on the time of day and in female rats on the day of the estrous cycle.

We have previously demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) can be released from cultured rat anterior pituitary cells and when added to the medium in physiological concentration it releases LH from individual gonadotropes. In the present work, we studied whether the release of PACAP and the responsiveness of LH cells to PACAP depend on the gender, on the time of day when the animals were sacrificed, and in females on the stage of the estrous cycle. Anterior pituitary cells were cultured on nitrocellulose membrane. We found that the number of PACAP releasing cells was higher in proestrous than in diestrous female or in male rats and their number was always higher in the evening than at the other times. The effect of PACAP on LH cells was stimulatory in the morning of proestrus and diestrus. In proestrous rats, PACAP did not influence LH release in the afternoon or the evening, but in diestrous rats it decreased it in the afternoon and the evening. In males, there was a decrease of LH due to PACAP treatment at 10 and 20 h; however, PACAP did not influence LH at 16 h. It was concluded that in vivo PACAP might be involved in the circadian and episodic release of LH at pituitary level.

What may be the anatomical basis that secretin can improve the mental functions in autism?

Autism was first described and characterized as a behavioral disorder more than 50 years ago. The major abnormality in the central nervous system is a cerebellar atrophy. The characteristic histological sign is a striking loss or abnormal development in the Purkinje cell count. Abnormalities were also found in the limbic system, in the parietal and frontal cortex, and in the brain stem. The relation between secretin and autism was observed 3 years ago. Clinical observations by Horváth et al. [J. Assoc. Acad. Minor. Physicians 9 (1998) 9] supposed a defect in the role of secretin and its receptors in autism. The aim of the present work was to study the precise localization of secretin immunoreactivity in the nervous system using an immunohistochemical approach. No secretin immunoreactivity was observed in the forebrain structures. In the brain stem, secretin immunoreactivity was observed in the mesencephalic nucleus of the trigeminal nerve, in the superior olivary nucleus, and in scattered cells of the reticular formation. The most intensive secretin immunoreactivity was observed in the Purkinje cells of the whole cerebellum and in some of the neurons of the central cerebellar nuclei. Secretin immunoreactivity was also observed in a subpopulation of neurons in the primary sensory ganglia. This work is the first immunohistochemical demonstration of secretin-immunoreactive elements in the brain stem and in primary sensory ganglia.

Neonatal PACAP administration in rats delays puberty through the influence of the LHRH neuronal system.

The onset of puberty is a concerted action of many factors which leads to cyclic LHRH release in rats. It has been demonstrated that; in common with vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase activating polypeptide (PACAP) is also involved in the differentiation of the central nervous system. In our previous work, it was shown that a single PACAP injection into neonatal female rats delayed puberty. In the present work, neonatal administration of PACAP delayed the vaginal opening and decreased the weight of anterior pituitaries, the number of expelled ova at the first ovulation and the intensity of LHRH immunostaining in the septo-preoptico-infundibular system. PACAP antiserum had a reverse effect on LHRH immunoreactivity. The other studied parameters in the latter group remained unchanged compared to control rats. It was concluded that neonatal PACAP administration delayed the onset of puberty through the influence of the LHRH neuronal system.

Cell immunoblot assay study demonstrating the release of PACAP from individual anterior pituitary cells of rats and the effect of PACAP on LH release.

The presence of pituitary adenylate cyclase activating polypeptide (PACAP) was previously demonstrated in the anterior pituitary by radioimmunoassay, immunohistochemistry, and reverse transcript-polymerase chain reaction (RT-PCR). With the use of cell immunoblot assay (CIBA), when the pituitary cells were cultured on nitrocellulose membrane, the release of PACAP by individual anterior pituitary cells was observed. The released peptide, trapped by the nitrocellulose membrane forming a blot around the cells, was demonstrated by immunocytochemistry. Double labeling revealed that a part of PACAP-immunoreactive cells can release LH as well. With the use of sandwich enzyme immunoassay (S-EIA), it was found that the concentration of PACAP in the anterior pituitaries is 10(-10) M. In cell culture in a similar concentration, PACAP stimulated the LH release from female gonadotropes, but did not influence it from male ones. The stimulated release of LH was indicated by the enhancement in the diameter of LH blots compared to the untreated control cultures. We concluded that PACAP may be released from the anterior pituitary cells in a concentration which would be able to influence LH release not only in vitro but under in vivo conditions as well. The effect of PACAP on LH release was different in female and male pituitary cultures.

Is the knife-cut in the hypothalamus a permanent barrier to regrowth of nerve fibers? -an affirmative answer.

The regenerative capacity of nerve fibers was studied in adult female rats. Horseradish peroxidase was injected into the anterior hypothalamus lateral to the suprachiasmatic nucleus 4 days, 6 weeks and 4 months, respectively, following an archiform retrochiasmatic knife-cut. The trajectory of the stained fibers was examined on horizontal sections of the hypothalamus. No nerve fibers could be seen sprouting across the scar-tissue of the knife-cut regardless of the survival time. In one rat (6-week survival time) a bundle of fine nerve fibers turned in a medial direction at the caudal end of the knife-cut, suggesting that sprouting fibers were destined to reinnervate parts of the deafferented medial-basal hypothalamus.