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.

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.

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.

Effect of the lack of light impulses on the hypothalamic PACAP and C-fos immunoreactivities in rats.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the secretin family. It is widely distributed in the central and peripheral nervous systems. The highest concentration of PACAP was found in the hypothalamus. In the present work it has been studied whether PACAP is involved in the mediation of photic stimuli to the anterior pituitary gland. We have examined the effect of the lack of light impulses on the hypothalamic PACAP and C-fos immunoreactivities. In adult rats 10 days after the removal of the eyes (surgical enucleation) and in those received monosodium glutamate treatment neonatally (chemical enucleation). The PACAP immunostaining enhanced in the hypothalamic magnocellular nuclei and in the extemal zone of the median eminence. C-fos immunoreactivity also enhanced in a few hypothalamic nuclei 2 hours after the surgical enucleation indicating that the lack of light impulses activated hypothalamic neurons which, in turn, might stimulate the release of PACAP into the portal circulation. It has been concluded that PACAP may be involved in photoendocrine regulations.

PACAP participates in the regulation of the hormonal events preceeding the ovulation.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the secretin family. It was isolated and characterized in 1989. Its neuroendocrine role was demonstrated in vivo and in vitro systems. It seems that in vivo the effect of PACAP on the gonadotrop hormone secretion depends on the route of administration. It was reported that intravenous (i.v.) injection of PACAP elevated, while intra-cerebro-ventricular (i.c.v.) administration depressed plasma LH levels. In the present study it was demonstrated that PACAP, administered i.c.v. before the critical period of the proestrous stage, blocked the ovulation and prevented the proestrous LH surge in rats. The blocking effect of PACAP is not directly mediated by endogenous opioids because the antagonizing effect of Naloxone, an opioid receptor antagonist, was questionable. Under our experimental conditions we could not confirm the stimulating effect of i.v. administered PACAP.

Present status of knowledge about the distribution and colocalization of PACAP in the forebrain.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a recently discovered member of the secretion family. 1. PACAP is a well conserved peptide during the phylogenesis. It has two bioactive amidated forms: PACAP38 and PACAP27 with 38 and 27 residues, respectively. 2. PACAP and its receptors are widely distributed in the central and peripheral nervous systems and in non-neural tissues. 3. In the central nervous system PACAP immunoreactive neuronal elements have been observed in the hypothalamus (magno- and parvocellular cell groups), both layers of the median eminence, the septum, the thalamus, the amygdaloid complex, the hippocampus, and various regions of the cortex. 4. In the periphery, PACAP was found in small sensory and parasympathetic neurons. 5. In the hypothalamus PACAP partially colocalizes with oxytocin- and tyrosine hydroxylase-immunoreactivities. In the septum there is no colocalization between the two immunoreactivities, but PACAP- and tyrosine hydroxylase-immunoreactive fibers were often found to establish synaptic contacts with the same, unlabeled dendrite. It was reported that in the periphery, in sensory neurons PACAP colocalized with substance-P and in parasympathetic neurons with acetylcholin. 6. PACAP functions as a neurotransmitter, hypothalamic releasing factor, posterior pituitary hormone, and trophic factor of the nervous tissue. PACAP also participates in neuro-immunoendocrine mechanisms.

Comparative distribution of immunoreactive pituitary adenylate cyclase activating polypeptide and vasoactive intestinal polypeptide in rat forebrain.

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are structurally similar, share the same high affinity site in same peripheral tissues and increase the intracellular content of adenylate cyclase. To establish which neural circuits are signaling with each of these two peptides, we systematically compared the immunohistochemical distribution of PACAP and VIP in selected rat forebrain regions using previously characterized antiserum. The PACAP antiserum recognized both PACAP27 and PACAP38, and PACAP immunoreactivity was unaffected by preincubation with various other peptides. PACAP-immunoreactive perikarya and fibers were observed in both hypothalamic and extrahypothalamic regions. In the hypothalamus PACAP perikarya were located in the supraoptic, paraventricular, anterior commissural, periventricular, and perifornical nuclei. In intact rats PACAP immunolabeled fibers were present in the internal zone of the median eminence and posterior pituitary. One week after hypophysectomy the intensity of staining in the internal zone was enhanced and immunoreactive fibers appeared in the external zone of the median eminence. Two or 3 weeks later a dense fiber network was observed around the portal capillaries in the external zone, and immunoreactive material further accumulated in the fibers of the internal zone. PACAP-immunoreactive perikarya and fibers were also observed in several extrahypothalamic regions including central thalamic nuclei, amygdaloid complex, bed nucleus of stria terminalis, septum, hippocampus and cingulate, and entorhinal cortices. In the lateral septum and entorhinal cortex PACAP fibers surrounded unstained neuronal cell bodies and small blood vessels. In intact rats, VIP-immunoreactive perikarya were present in all regions of the cerebral cortex, hippocampus, amygdaloid complexus and in the suprachiasmatic nucleus, but not in the paraventricular and supraoptic nuclei. In colchicine-treated rats the VIP perikarya appeared in the preoptic area and paraventricular nucleus. The fibers were organized in two main pathways: the stria terminalis and an ascending pathway from the suprachiasmatic nucleus to the paraventricular area. Hypophysectomy induced the appearance of VIP-immunoreactive fibers in the internal zone of the median eminence and perikarya in the supraoptic and paraventricular nuclei in addition to the suprachiasmatic nucleus. The dissimilar distributions of PACAP and VIP suggest that PACAP neural circuits are independent of that of VIP in the rat forebrain. These findings support possible multifunctional roles for PACAP as a posterior pituitary hormone, a hypophysiotrophic factor, and a neurotransmitter/neuromodulator.

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.

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.

Pituitary gonadotropin-releasing hormone binding sites in persistent estrous rats.

Pituitary gonadotropin-releasing hormone (GnRH) binding sites were studied in persistent estrous rats. For comparison females at different stages of the estrous cycle, ovariectomized animals, and intact males were examined. Persistent estrus was induced by a hypothalamic knife cut, administering testosterone to neonatal rats, or by constant illumination. Rats showing spontaneously such alteration were also used in the experiment. Besides pituitary GnRH-binding sites, the GnRH content of the median eminence and plasma luteinizing hormone, follicle-stimulating hormone, estradiol, and progesterone concentrations were determined. Pituitary GnRH receptors changed during the estrous cycle with the highest concentrations observed on proestrus. In rats in persistent estrus, pituitary GnRH-binding sites varied between proestrous and estrous values of cyclic females. Ovariectomy resulted in a significant increase in receptors. Depending on how the persistent estrous syndrome was induced, very different amounts of GnRH were found in the median eminence of the various groups. Our data indicate that the GnRH-binding sites on pituitary cells of persistent estrous rats are in essence not changed.

Hypothalamus and puberty.

The effect of a transitory increase in plasma FSH and LH levels during the prepubertal period on puberty has been investigated. Twenty-day-old rats had been bilaterally ovariectomized and 24 hr later they received two ovaries of infantile rats beneath the kidney capsule. These rats exhibited precocious puberty. Animals into which two additional ovaries had been transplanted first and the next day their own ovaries removed showed puberty at the same time as controls. Additional investigations provided evidence that the ovariectomized and ovary implanted rats plasma FSH and LH levels were in the control range four days after implantation. The findings support the assumption that the hypothalamic lesion-induced precocious puberty is due rather to a transitory enhanced release of gonadotropin releasing hormone by the lesion than to the destruction of sex-steroid sensitive structures inhibiting the release of gonadotropic hormones in prepubertal rats.

Neural control of ovulation.

Pituitary gonadotrophin function is controlled to a great extent by the central nervous system and by the feedback action (positive and negative) of sex steroids. Neural structures involved in this mechanism may be divided into two levels. The first level is represented by the nervous structures releasing gonadotrophin-releasing hormone (GnRH) in a pulsatile manner into the portal circulation. The gene encoding the precursor protein for GnRH has been described recently. The precursor protein appears to be composed of 92 amino acids, in which the GnRH decapeptide is preceded by a signal peptide and followed by a peptide termed GAP for GnRH-associated peptide. The GnRH-synthesizing neurones and the nervous structures synchronizing the GnRH discharge (pulse generator) in the monkey, and probably also in the human, reside in the medial basal hypothalamus, which appears to have a highly integrated structure. The GnRH pulse generator is influenced by nervous structures outside the medial basal hypothalamus (second level of control) as well as by ovarian and other hormones. These influences probably impinge directly or indirectly on the hypothalamic oscillator. Concerning the chemical nature of the substances mediating the action, a large number of neurotransmitters and neuropeptides have been reported to influence GnRH secretion.

Effect of various hypothalamic deafferentations injuring different parts of the GnRH pathway on ovulation, GnRH content of the median eminence, and plasma LH and FSH levels.

Various supra- and retrochiasmatic cuts injuring different parts of the septo-preoptico-infundibular GnRH pathway were made in adult female rats, and their effects on ovulation, median eminence (ME) GnRH content and on plasma LH and FSH levels were studied. Extended retrochiasmatic frontal cut just behind the optic chiasm, or a frontal cut in front of the suprachiasmatic nucleus presumably interrupting the whole GnRH pathway blocked ovulation, led to persistent estrus with polyfollicular ovaries, and reduced the ME GnRH content to 10 and 32%, respectively, expressed in percentage of unoperated control value. Severance of the GnRH pathway on one side or partial interruption of the pathway on the two sides in the retrochiasmatic area did not interfere with ovulation, and the ME GnRH content was 50% or more of the control value. Disconnection of the GnRH fibers coming from the septum resulted in a more than 30% decrease in the GnRH content of the ME, but did not block ovulation. Two separate symmetrical lateral cuts in the suprachiasmatic area leaving intact the presently known GnRH pathway reduced the ME GnRH content to 40% of the intact value, but did not interfere with ovulation. Plasma LH and FSH levels of the animals with different knife cuts were considerably dissociated and there were no apparent correlations between brain interventions and the concentrations of these two hormones in the blood. Our previous findings together with the present data suggest that: half of the GnRH pathway, medial or lateral bundles of the pathway on the two sides are sufficient for ovulation and cyclic gonadotrophic function; persistent estrus develops if the ME GnRH content is below 40% of the control value, and about 60% of the ME GnRH originates outside the preoptic, supra- and retrochiasmatic region. Half of this 60% may come from the septum and the vertical part of the diagonal band of Broca, the other half from the region in front of the preoptic area. The remaining 40% presumably arises from the preoptic (7-10%), supra- (15-20%) and retrochiasmatic region (8-10%).

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.

Direct neural connection from the medial preoptic area to the hypothalamic arcuate nucleus of the rat.

Direct neural connections from the medial preoptic area (MPOA) to the arcuate nucleus were studied on light and electron microscope level by a multistep experimental procedure. The hypothalamic deafferentation technique of Halász and Pupp (1965) was used in itself or was combined with electrolytic lesions. In order to eliminate all fibers of more rostral source traversing the medial preoptic area an extended rostral preoptic deafferentation was made, and a survival time of at least 3 weeks was chosen to allow for the complete disappearance of the degenerated fragments from the arcuate nucleus. In the main experimental group the medial preoptic area was destroyed by an electrolytic lesion 3 or more weeks following such rostral preoptic deafferentations. In these animals degenerated fibers and terminals certainly of preoptic origin were found distributed bilaterally, with a predominance on the side of the lesion. - These data suggest that axons originating from medial preoptic neurons terminate in the arcuate nucleus, thus constituting a preoptico-tuberal pathway.

Data on the absence of axon terminals of medial preoptic area neurons in the surface zone of the median eminence.

In order to investigate whether neurons of the medial preoptic area (MPOA) project to the surface zone (zona palisadica) of the median eminence (ME) and proximal part of the pituitary stalk, an electrolytic lesion or a frontal cut was placed in the pre- and suprachiasmatic region of the rat hypothalamus and the mentioned zone examined under the electron microscope. Degenerated nerve profiles were not observed int the zona palisadica following a lesion restricted to the MPOA or after a frontal cut at the posterior border of the MPOA. Altered elements were seen only in those cases in which the posterior part of the suprachiasmatic region was destroyed. The present data indicate that neurons of the MPOA do not terminate in the zona palisadica of the ME but presumably end on the nerve cells of the medial basal hypothalamus.

Data on protein synthesis of deafferented hypothalamic arcuate neurons.

Protein synthesizing activity of the rat hypothalamic arcuate nucleus following partial or total deafferentation of the medial basal hypothalamus was studied by light and electron microscopic autoradiography when administering tritiated leucine into the lateral ventricle. There were significantly more grains over the arcuate nucleus 21 days after disconnection of this hypothalamic region than over the intact nucleus. Isolation of a temporal cortical region induced similar changes in the isolated area, although this effect was not so pronounced as in the arcuate region. Data suggest that the protein synthesizing activity of arcuate neurons increases significantly after interruption of neural connections of the medial basal hypothalamus. It is assumed that the effect is primarily due to transneuronal alteration and/or interruption of inhibitory afferents.