Morphological characterization of relationship between gap junctions and gonadotropin releasing hormone nerve terminals in the rat median eminence.

The present study aimed to reveal possible morphological relationships between gonadotropin-releasing hormone (GnRH) nerve terminals and gap junctions in the median eminence. Coronal brain sections from castrated male rats were dual immunostained with GnRH and either connexin 26, 32, or 43, and examined by confocal laser microscopy. Connexin 43-immunoreactive puncta were distributed between GnRH-immunoreactive fibers, and some of them were colocalized with GnRH-immunoreactivities. Dual immunostaining with connexin 43 and glial fibrillary acidic protein revealed that most of the puncta were located in astrocytes. At the immunoelectron microscopic level, connexin 43-immunoreactivities were mainly located on the plasma membranes of glial-like processes. Few connexin 26- or connexin 32-immunoreactivities were found in the median eminence. The present results indicate the possibility that gap junctions play a role in the GnRH release at the median eminence.

Excitatory amino acids act on the median eminence nerve terminals to induce gonadotropin-releasing hormone release in female rats.

The present study is designed to examine the terminal regulation of gonadotropin-releasing hormone (GnRH) release by excitatory amino acids in the median eminence of ovariectomized (OVX) rats. In in vitro experiments, median eminence tissues were superfused in the medium containing glutamate or excitatory amino acid agonists, such as N-methyl-d,l-aspartate or kainate. These drugs induced a Ca2+-dependent GnRH release from median eminence fragments. The agonists also stimulated GnRH release from superfused synaptosome prepared from the median eminence tissues in a Ca2+-dependent manner. In the immunocytochemical study, immunoreactivity for glutamate or its ionotropic receptor subtypes, such as NR1, GluR1, GluR2/3, GluR6/7, and KA2, was examined in the median eminence of OVX rats under electron microscopy. Immunoreactivities for glutamate or its receptor subtypes were observed on the nerve terminals, most of which were located in close proximity to the other nerve terminals without forming synaptic contacts. In addition, quite a few synaptic contacts which were immunopositive for GluR1, GluR2/3, KA2, or glutamate were found in this area. The present results indicate that excitatory amino acids stimulate GnRH release by acting at the nerve terminals of the median eminence in a Ca2+-dependent manner in the absence of gonadal steroid. The effect of excitatory amino acids in this area might be mediated by glutamate receptors mainly in nonsynaptic fashion, such as by volume transmission.

Confocal laser scanning and electron-microscopic analyses of the relationship between VIP-like and GnRH-like-immunoreactive neurons in the lateral septal-preoptic area of the pigeon.

The lateral septum and the preoptic area of birds comprise neurons immunoreactive (ir) for vasoactive intestinal polypeptide (VIP) and gonadotropin-releasing hormone (GnRH). By use of immunohistochemical single- and double-labeling techniques, we have investigated the distribution and the connections of these two types of peptidergic neurons in the lateral septal-preoptic area of the pigeon at both the light- and electron-microscopic levels. An accumulation of VIP-like-ir neurons, some of which are cerebrospinal fluid-contacting neurons, is found in the area adjacent to the ventromedial walls of the lateral ventricles in the lateral septum corresponding to the medial part of the lateral septal organ. VIP-like-ir terminals are scattered throughout the lateral septal-preoptic area, which also contains GnRH-like-ir cell bodies. The number of GnRH-like-ir cell bodies in the lateral septum is smaller than that of the VIP-like-ir neurons. GnRH-like-ir cells have a simple bipolar or multipolar shape and a beaded axon that emerges from the soma or one of the proximal dendrites. Confocal laser scanning microscopy has shown VIP-like-ir terminals in close apposition to GnRH-like-ir cell bodies in the lateral septal-preoptic area. Furthermore, the electron-microscopic double-immunolabeling has revealed synaptic contacts between VIP-like-ir axon terminals and GnRH-like-ir cell bodies or dendrites. These contacts, however, do not show synaptic specializations. The present results suggest that functional interactions take place between VIP and GnRH neurons in the lateral septal-preoptic area of the pigeon and that these interactions are involved in mediating photoperiodic responses.

Evidence for terminal regulation of GnRH release by excitatory amino acids in the median eminence in female rats: a dual immunoelectron microscopic study.

The present study was designed to determine whether excitatory amino acids directly act on the gonadotropin-releasing hormone (GnRH) nerve terminals to release the peptide. The median eminence taken from ovariectomized rats was dual immunostained with GnRH and ionotropic glutamate receptor subtypes (NR1, GIuR1, GluR2/3, GluR6/7 and KA2), and their colocalization was examined under electron microscopy. The connection of fibers immunopositive for GnRH and glutamate was also examined. Of the glutamate receptor subtypes, NR1- and KA2-immunoreactivities were colocalized with GnRH-immunoreactivity in nerve terminals of the median eminence. In addition, some glutamate-immunopositive nerve terminals were shown to abut the many GnRH-immunopositive nerve terminals. No synaptic contacts were observed on these immunopositive nerve terminals. These results suggest that GnRH release is regulated at the GnRH nerve terminals by excitatory amino acids in a non-synaptic manner in the median eminence.

Immunocytochemical identification of pinopsin in pineal glands of chicken and pigeon.

Pinopsin is a blue-sensitive photoreceptive molecule possibly involved in photic entrainment of the circadian pacemaker in the chicken pineal gland. To characterize pinopsin as a circadian photoreceptor, antibodies were raised against the C-terminal portion of pinopsin. As expected from the divergence of the amino acid sequence of this region, the resultant antibody cross-reacted with neither chicken rhodopsin nor red-sensitive cone pigment (chicken red). In Western blot analysis, the antibody stained a single band of 42-kDa protein in a detergent-extract of chicken pineal membranes, suggesting that pinopsin (calculated molecular weight, 38187) might be glycosylated and/or palmitoylated. Immunocytochemical examination of pineal sections of the chicken and the pigeon with this antibody revealed strong positive images for most of the membrane structures in the lumen of the follicles. This antibody also stained string- and bulb-shaped structures of the chicken parafollicular cells, the morphology of which resembles those of retinal photoreceptor cells. In contrast to the predominant distribution of pinopsin, a monoclonal antibody specific for chicken red stained a smaller number of membrane structures in the lumen of chicken pineal follicles. These results strongly suggest that the chicken pineal gland contains at least two types of photoreceptive molecules, pinopsin (major) and chicken red (minor). We show that the former molecule is localized in parafollicular pinealocytes and in the outer segments of pinealocytes that make contact with the follicular lumen.

Immunoelectron-microscopic investigation of the subcellular localization of pinopsin in the pineal organ of the chicken.

Pinopsin is a photoreceptive molecule cloned from the chicken pineal organ. An antibody highly specific for pinopsin was applied in light- and electron-microscopic immunocytochemical studies of the pineal organ of 1 to 2-month-old chickens. Intense immunoreactivity was found in the follicular lumen at the light-microscopic level. In addition, small immunoreactive spherical or fibrous structures were diffusely distributed at the parafollicular aspect of the pineal organ. To identify immunoreactive elements precisely, we used pre-embedding immunoelectron microscopy. These studies revealed immunoreactive outer segments of pinealocytes arranged closely side by side in the follicular lumina. The thin initial portion of the outer segment arose from a basal body located in the inner segment. Immunoreactive pear-shaped outer segments occupied small lumina. Follicular lumina displayed immunonegative arrays of whorl-like lamellar membranes. Occasionally, these immunonegative structures were surrounded by immunoreactive concentric lamellar complexes. In the parafollicular pineal parenchyma, long slender cilium-like structures or enlarged cilia and concentric lamellar arrays showed intense immunoreactivity. All immunoreactive structures observed in this study were considered to represent outer segments of pinealocytes of the chicken pineal organ.

In vivo microdialysis studies of pineal and ocular melatonin rhythms in birds.

Pineal and retinal melatonin has an important role in the control of avian circadian rhythms. In order to study the mechanisms of circadian rhythms of melatonin synthesis in the pineal and in the eye, in vivo microdialysis was applied to these organs. In both pigeons and Japanese quails, pineal and ocular melatonin levels were high during the dark and low during the day under light-dark (LD) cycles. These rhythms persisted under constant dim light (LLdim) conditions indicating the circadian nature of pineal and ocular melatonin release. Light has two effects on melatonin synthesis. One is acute inhibition of melatonin synthesis and the other is entrainment of circadian melatonin rhythms. We have examined photoreceptors mediating these effects in the pigeon. The results have indicated that the eyes are not involved in light-induced suppression and photic entrainment of pineal melatonin release, and pineal photoreceptors themselves are likely to mediate these effects. Concerning ocular melatonin, retinal photoreceptors seem to mediate light-induced suppression and photic entrainment and no evidence supporting mediation of extraretinal photoreceptors was obtained. Because dopamine is implicated in retinal melatonin synthesis, we measured dopamine and melatonin release simultaneously from the eye of pigeon. In contrast to melatonin rhythms, dopamine increased during the day and decreased during the dark. This antiphase relationship between melatonin and dopamine persisted in LLdim, suggesting an interaction between these two rhythms. The results of an intraocular injection of dopamine or melatonin in the phase of melatonin and dopamine rhythms indicated that the interaction is required for maintaining the antiphase relationship between the two rhythms.

Effect of fasting and immobilization stress on estrogen receptor immunoreactivity in the brain in ovariectomized female rats.

The present study examined the effect of 48-h fasting and 1-h immobilization on estrogen receptor immunoreactivity in selected hypothalamic areas and the nucleus of the solitary tract (NTS) in ovariectomized rats. Fasting induced an increase in ER-immunoreactive cells in the paraventricular nucleus (PVN), periventricular nucleus (PeVN) and NTS compared with the unfasted control group. Similarly, immobilization caused an increase in ER-positive cells in the same areas, PVN, PeVN and NTS, versus the non-immobilized group. There was no significant increase in the number of ER-immunoreactive cells in the preoptic area (POA), arcuate nucleus (ARC) or ventromedial hypothalamic nucleus (VMH) following fasting and immobilization. Our previous work in ovariectomized rats with estrogen microimplants in the brain revealed that the PVN and A2 region of the NTS are the feedback sites of estrogen in activating the neural pathway to suppress pulsatile LH secretion during 48-h fasting. The result in the food-deprived rats suggests that estrogen modulation of the suppression of LH secretion during fasting is partly due to the increase in estrogen receptors in the PVN and A2 region. The physiological significance of the increase in neural ER following immobilization remains to be elucidated.

Vagus nerve mediates the increase in estrogen receptors in the hypothalamic paraventricular nucleus and nucleus of the solitary tract during fasting in ovariectomized rats.

The effect of total subdiaphragmatic vagotomy on estrogen-receptor immunoreactivity (ERIR) in the paraventricular nucleus (PVN) and nucleus of the solitary tract (NTS) was examined in fasted ovariectomized rats to clarify the peripheral inputs mediating fasting-induced increase in ERIR in these two nuclei. Vagotomy abolished the effect of 48-h fasting on the expression of ER in these two areas. The result indicates that the neural signal(s) that increase the expression of ER in the PVN and A2 region of the NTS following 48-h fasting is transmitted through the vagus. The involvement of the vagus in the fasting-induced increase in ER in the PVN and A2 region may also be the same neural pathway involved in the suppression of pulsatile luteinizing hormone secretion in fasted female rats.

Immunocytochemical identification of serotonergic supraependymal nerve fibers in the third ventricle of the house musk shrew, Suncus murinus.

Supraependymal fibers of the house musk shrew (Suncus murinus) were examined by conventional scanning electron microscopy (SEM), backscattered electron (BSE) imaging of enzyme immunohistochemistry and by immunotransmission electron microscopy in the dorsal part of the third ventricular wall. In this region, ependymal cells were not so heavily ciliated and conventional SEM studies showed two main categories of supraependymal fibers. The first type consisted of long fibers fasciculated which were distributed over the ventricular surface between the anterior commissure and the subfornical organ. The second category was a thin fiber which was observed on the ependymal luminal surface. Some of these fibers had varicoses or terminal-like swellings. This type of supraependymal fiber seemed to originate in the first type of fiber bundles. To confirm the nature and the distribution of serotonin-immunoreactive supraependymal fibers, BSE imaging using immunohistochemical reactions was used. Serotonin-immunoreactive structures were shown as highlighted structures by means of a backscattered electron mode. These investigations revealed that the majority of both types of supraependymal fibers observed by conventional SEM contained serotonin. A moderate number of serotonergic supraependymal fibers was observed on the ventricular surface of the subfornical organ. Immunohistochemical studies using Vibratome sections of identical ventricular regions revealed the presence of serotonin-immunoreactive processes, with the use of light- and electron-microscopy. They were distributed in in the third ventricle just adjacent to the ependymal luminal surfaces. These fibers contained immunoreactive large cored vesicles and immunonegative small clear vesicles.

Ultrastructure of cerebrospinal fluid-contacting neurons immunoreactive to vasoactive intestinal peptide and properties of the blood-brain barrier in the lateral septal organ of the duck.

Immuno-electron-microscopic investigations of cerebrospinal fluid (CSF)-contacting neurons immunoreactive to vasoactive intestinal peptide in the duck lateral septum have revealed that this cell type gives rise to an adventricular dendrite terminating with a bulbous swelling in the lateral ventricle. The swelling bears a cilium and contains mitochondria and immunolabeled dense-core vesicles. Two types of processes emerge from the basal part of the perikaryon. The first has a large diameter, contains diffusely distributed immunoreaction, and receives synaptic input, indicating that this process is a basal dendrite. The other type is of a beaded appearance, displays immunolabeled dense-core vesicles, and represents the axon of the CSF-contacting neuron. VIP-immunoreactive terminal formations are located within the neuropil of the lateral septum and the nucleus accumbens. Some of them form synaptic contacts with immunonegative profiles. No VIP-immunoreactive terminal formations are seen in the perivascular spaces of the lateral septum. Tracer experiments with horseradish peroxidase have revealed that the blood-brain barrier is lacking in the lateral septal organ and nucleus accumbens of the duck. Capillaries, arterioles, and venoles of this region are coated by nonfenestrated endothelial cells connected by "leaky" junctions, allowing the tracer to penetrate from the lumen into the perivascular space and further into the intercellular clefts of the neuropil. Our immuno-electron-microscopic investigations show that VIP-immunoreactive CSF-contacting neurons of the lateral septum closely resemble CSF-contacting neurons occurring in other brain regions, e.g., the hypothalamus. The arrangement of VIP-immunoreactive terminal formations suggests that, in the lateral septum, the VIP-like neuropeptide serves as a neurotransmitter (-modulator). The lack of a blood-brain barrier in the lateral septal organ and the nucleus accumbens raises the possibility that this region is a window in the avian brain allowing exchange of information between the central nervous system and the bloodstream; it thus resembles a circumventricular organ.

Electron-microscopic investigations of vasoactive intestinal peptide (VIP)-like immunoreactive terminal formations in the lateral septum of the pigeon.

Vasoactive intestinal peptide (VIP)-like immunoreactive terminal fields were examined in the lateral septum of the pigeon by means of immunocytochemistry. According to light-microscopic observations, these projections originated from VIP-like immunoreactive cerebrospinal fluid (CSF)-contacting neurons, which are located in the ependymal layer of the lateral septum and form a part of the lateral septal organ. The processes of these cells gave rise to dense terminal-like structures in the lateral septum. Pre-embedding immuno-electron microscopy revealed that VIP-like immunoreactive axon terminals had synaptoid contacts with perikarya of small VIP-immunonegative neurons of the lateral septum, which were characterized by an invaginated nucleus, numerous mitochondria, a well-developed Golgi apparatus, endoplasmic reticulum and a small number of dense-core vesicles (about 100 nm in diameter). VIP-like immunoreactive axons were also seen in contact with immunonegative dendrites in the lateral septum. In both axosomatic and axodendritic connections, VIP-like immunoreactive presynaptic terminals contained large dense-core vesicles, clusters of small vesicles and mitochondria. These findings suggest that VIP-immunoreactive neurons of the lateral septal organ project to small, presumably peptidergic nerve cells of the lateral septum and that the VIP-like neuropeptide serves as a neuromodulator (-transmitter) in this area.

Vasoactive intestinal peptide-immunoreactive cerebrospinal fluid-contacting neurons in the reptilian lateral septum/nucleus accumbens.

By means of immunocytochemical demonstration of vasoactive intestinal peptide (VIP) an accumulation of cerebrospinal fluid (CSF)-contacting neurons was found in a circumscribed region of the nucleus accumbens/lateral septum of eleven reptilian (chelonian, lacertilian, ophidian, crocodilian) species. Basal processes of these cells contribute to a subependymal plexus whose density displays considerable interspecific variation. VIP-immunoreactive nerve fibers occur also in the lateral septum and the nucleus accumbens where they encompass immunonegative cells in a basket-like pattern. The CSF-contacting neurons are surrounded by columnar ependymocytes frequently arranged in a pseudostratified manner. These specialized arrays of ependymal cells, however, occupy a more extended area than the VIP-immunoreactive CSF-contacting neurons and can be traced from the rostro-ventral pole of the lateral ventricle to the interventricular foramen. These observations suggest the existence of a telencephalic site of CSF-contacting neurons which may be more widespread than hitherto thought and which may participate in a circumventricular system of the lateral ventricle. Previous studies mainly performed with birds indicate that the VIP-immunoreactive CSF-contacting neurons of the nucleus accumbens might form a part of the "encephalic" (extraretinal and extrapineal) photoreceptor. However, further experiments are required to test this supposition since the VIP-immunoreactive neurons of the nucleus accumbens remained unlabeled by antibodies against bovine rodopsin and chicken cone-opsin in all eleven species analysed in this investigation.

Glycoconjugate histochemistry of the subcommissural organ in the domestic chicken: lectin histochemistry using the avidin-biotin-peroxidase complex (ABC) method.

The properties and distribution of the secretory substance in the ependymal cells of the subcommissural organ in the domestic chicken were examined with seven different lectins, using the avidin-biotin-peroxidase complex method. On the ventricular surface of this organ, LFA, PNA, RCA-I and WGA reacted positively. In the apical cytoplasm, Con A, LFA, RCA-I and WGA gave positive reactions. On the other hand, only Con A and WGA gave positive reactions in the basal cytoplasm. In the hypendymal vascular wall, LFA, RCA-I and WGA reacted positively. Neuraminidase digestion completely inhibited reactions with LFA and reduced the WGA reaction within the apical cytoplasm. At the same time, this digestion disclosed a moderately positive reaction with PNA in the apical cytoplasm. The significant findings of this study may be summarized as follows: 1) the ependymal cells of the subcommissural organ in the domestic chicken synthesize a complex oligosaccharide for a secretory product which is discharged into the ventricular cavity; 2) in the basal cytoplasm and basal processes, only oligosaccharides containing large amount of mannose are present.

Kinky coat, a new autosomal recessive mutation in the musk shrew, Suncus murinus.

A kinky-coat mutant was discovered at the fifth generation of the BAN strain originating from wild musk shrews (Suncus murinus) in Bangladesh. Mating experiments indicated that the kinky-coat character is controlled by a single autosomal recessive gene designated kc (kinky coat), which is not allelic to the gene ch (curly hair) previously reported in the Tr strain derived from wild musk shrews on Taramajima Island, Japan. Because the kc/kc homozygotes were fully fertile and viable, the kc gene should be useful as a genetic marker in linkage studies. In external appearance, homozygotes were characterized by curly vibrissae, somewhat unkept coat hair, and wavy long hair on the tail. Both the length and width of coat hair did not differ significantly between homozygous and normal shrews. Light microscopic observations showed that shafts of the kc coat hair are wavy and often have small swellings with disorganization of the medullary structure. Scanning electron microscopic examinations further revealed that the shafts of the vibrissae, coat hair, and tail hair have abnormalities such as longitudinal fissures, twists, and hollows. It is clear that these modifications caused waviness or curling of the shafts of the three kinds of hairs observed.

Immunocytochemical demonstration of serotonin-immunoreactive cerebrospinal fluid-contacting neurons in the paraventricular organ of pigeons and domestic chickens.

The paraventricular organs (PVO) of the pigeon and domestic chicken contain at least three types of serotonin-immunoreactive (serotonin-ir) CSF-contacting neurons. Type 1 neurons were predominant. They had two bipolar extending processes. The somata were mostly found in the pars hypendymalis. Type 2 neurons were characterized by thin and long apical processes. Their perikarya were found in the pars distalis of the PVO or the more lateral area of this organ. Type 3 neurons were considerably smaller and had round somata. They were mostly bipolar with thin and short dendritic processes and thin basal processes. A small number of this type was conspicuous along the cranial peripheral region of the PVO. In addition to the PVO area, aggregations of small, bipolar serotonin-ir CSF-contacting neurons were shown in the most caudal wall of the third ventricle of both species, distributed medially or paramedially. Immunoelectron microscopy revealed many dense granules in apical ventricular processes and perikarya. Synaptic connections were frequently observed on basal processes.

Scanning electron microscopy of the ovary in the house musk shrew, Suncus murinus.

The structure of the house musk shrew (Suncus murinus) ovary, prepared by the Osmium-DMSO-Osmium method, was examined with a scanning electron microscope. Many ovarian follicles in various stages of development can be seen in the musk shrew ovary, but fully matured follicles (Graafian follicles) are never observed. In the inner layer of zona granulosa of the preantral follicles, the follicular cells containing a large amount of glycogen deposition in the cytoplasm are observed. Folded and collapsed zona pellucida surrounding the remnant of the degenerated oocyte, which is thought to be a residual form of atretic follicle, is observed in a small, crypt-like depression on the surface of the ovary where it has been split by cracking.

Ultrastructural and cytochemical studies of the subcommissural organ in the domestic chicken with special reference to the apical secretion.

Ultrastructural and cytochemical studies were undertaken with special reference to the apical secretion in the ependymal cells of the subcommissural organ in the domestic chicken. Glucose-6-phosphatase (G6Pase) and acid phosphatase (AcPae) were used as cytochemical markers for the endoplasmic reticulum (ER) and lysosomes, respectively. Periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) cytochemistry was employed to detect glycoconjugates. Most dense bodies were negative with AcPase staining. Dilated cisternal of ER were reacted with G6Pase cytochemistry. Golgi saccules and several kinds of spherical bodies were stained with PA-TCH-SP cytochemistry. Our results suggest that: 1) most dense bodies in the apical cytoplasm belong to the secretory granules discharged into the ventricular cavity, 2) dilated cisternae of the ER serve as the storage site of the substance which eventually becomes the secretory product, however, contents of the ER are not directly released into the ventricular cavity. Findings of this studies lead us to speculate that there are two intracellular transportation via different compartments: one is a transport via small vesicles from the perinuclear ER to the Golgi apparatus and the other is a transport bypassing the Golgi apparatus via cisternae of ER from the perinuclear ER to the supra-Golgi region. The substances in these two structures seem to be intermingled with each other and matured into secretory granules. This granules may be discharged into the ventricular cavity by exocytosis.

Supraependymal cells and fibers in the third ventricle of the domestic chicken. A scanning electron microscopic study.

Supraependymal cells, fibers and what are presumed to be neuronal bulb-like projections were found in the third ventricle of the domestic chicken with a scanning electron microscope. At least two types of supraependymal cells were found: neuron-like cells and phagocyte-like cells. The former were predominantly seen in the area of the paraventricular organ and infundibular recess. The latter were abundant on the ventricular surface of the median eminence and subfornical organ. Bulb or club-like projections thought to be the dendritic terminals of CSF-contacting neurons were observed in the area of the paraventricular organ and infundibular recess. Similar structures were observed at the preoptic recess as well. The supraependymal neuronal components found in the domestic chicken differed from those of mammals in several respects: 1. the wall of the third ventricle was devoid of supraependymal fibrous plexus except for that of the paraventricular organ; 2. bulb-like projections were abundant in the area of the paraventricular organ; 3. supraependymal neuron-like cells were unipolar or bipolar in appearance. These data underline the dissimilarity of the CSF-contacting neuronal system of birds and mammals.

Scanning electron microscopic studies on the embryonic development of the surface structures of the paraventricular organ in the Brown Leghorn chicken.

The development of the surface specialization of the paraventricular organ (PVO) was studied in the domestic chicken from the 10th embryonic day to the day of hatching by scanning electron microscopy. On the 10th embryonic day, the ventricular surface of the PVO was found to be covered with many oval-shaped processes. On the 12th embryonic day, an additional kind of elongated processes appeared in the dorsal area of the ventricular surface of the organ. From the 16th to 18th embryonic day, such elongated processes were present on the entire ventricular surface of the PVO. At the same stage, the elongated processes in the dorsal portion of the PVO began to form small, meshed networks over the surface of the ependyma. Both the oval-shaped processes and the elongated processes are thought to be dendritic terminals of the PVO neurons.