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We performed a morphological study in type 2 diabetes mellitus (DM) model rats by focusing on the connective tissue papillae (CTPe) and capillary loops (CLs) of filiform papillae in the lingual dorsum in the type 2 DM model rats (GK rats). The normal group comprised nine 8-week-old male Wistar rats. The DM group comprised nine 8-week-old male GK rats. Image analysis of light microscopic specimens in CTPe was performed (height and cross-sectional area). Image analysis of scanning electron microscopic specimens (acrylic plastic injection method) in CLs was performed (number, thickness, height, interval between the tops of the two CLs, and crossing ratio between ascending and descending crura (CRC)). We compared these values between both groups (Student's t test). The former analysis revealed that the height and cross-sectional area of CTPe were smaller in the DM group than in the normal group. The latter analysis revealed that the thickness, height, and CRC of the CLs were smaller in the DM group than in the normal group. However, no significant differences were detected in the number and interval of CLs between both groups. Therefore, we concluded that DM caused regressive change in CTPe and CLs.

Age-related inhibition of salivary secretion has been demonstrated in rats, and the nitric oxide (NO) present in the supraoptic nucleus (SON) and the medial septal area has been reported to play an inhibitory role in the regulation of salivary secretion. In the present study, we investigated the age-related changes occurring in the NO synthase (NOS)-expressing neurons in the SON, which is related to the production of NO, and discussed the interrelation between the age-related changes in the NOS-expressing neurons and the age-related inhibition of salivary secretion. Nissl staining and reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry were performed for young adult and aged rats. Quantitative analysis was also performed using the Nissl-stained and NADPH-d-positive neurons. Although the numbers of the Nissl-stained neurons did not change, significant age-related increases were detected in cell number, cell size and reactive density of the NADPH-d-positive neurons. Therefore, the production of NO in the SON neurons increased with age. We concluded that the age-related increase in the NO in the SON might be a factor that contributes to the age-related inhibition of salivary secretion.

It has been reported that the spinal trigeminal nucleus caudalis (Sp5C), which receives nociceptive information from the oro-facial regions, has four laminae. To clarify the role of glial cells in the transmission of the nociceptive information, the present study was conducted to examine the detailed distribution of astrocytes in each lamina and also to investigate a morphological interrelationship between the astrocytes and nerve endings in the rat Sp5C. After the preparation of the serial cryostat sections, immunohistochemistry for glial fibrillary acidic protein (GFAP) was employed to identify the astrocytes, and immunohistochemistry for substance P (SP), calcitonin gene-related peptide (CGRP), was used for the nerve endings. We also employed double-labeling immunofluorescence and electron microscopic immunohistochemistry for the GFAP/SP or GFAP/CGRP. GFAP-positive reactions were observed in all laminae of the Sp5C, and SP- or CGRP-positive nerve endings were observed in the lamina I and II. Additionally, we clarified the presence of GFAP/SP- or GFAP/CGRP-positive reactions by the double-labeling immunofluorescence and demonstrated the morphological interrelationship between the astrocytes and nerve endings by the double-labeling electron microscopic immunohistochemistry. These findings suggest that astrocytes might play some roles in the transmission of nociceptive information from the oro-facial region.

Age-related changes in astrocytes, tanycytes and the microvasculature were investigated in the median eminence (ME) of the anterior hypothalamo-hypophyseal system of 2- and 24-month old rats by means of immunocytochemistry and scanning electron microscopy. A computer-assisted image processing system showed a significant increase of the glial fibrillary acidic protein (GFAP) immunoreactivity in the internal layer (IL) and the perivascular region of the external layer (EL) of the ME with age. However, no remarkable changes in either layer could be detected employing vimentin immunoreactivity. An immunoelectron microscopic study revealed that the number of the GFAP-immunoreactive processes markedly increased with age in regions surrounding the capillaries of the EL. Therefore, it may be considered that, in both layers, the astrocytic cell bodies and associated glial processes exhibit a significant increase with age. Investigation of the microvasculature under a scanning electron microscope demonstrated the development and the sinusoidal progress of the external capillary plexus with age, but these changes were not observed in the internal capillary network of the ME. In conclusion, the results of the present study showed a close correlation between the increase of the GFAP-immunoreactive astrocytes and the development of the external capillary plexus. In addition, there was a close correlation between the increase in the GFAP-immunoreactive astrocytes and the sinusoidal progress.

Transection of the rat facial nerve (FN) leads to a series of retrograde changes within the facial nuclei, which has been called the axon reaction. This reaction includes the rapid activation of both astrocytes and microglia and a change in the gene expression in axotomized motoneurons. In the present study, we investigated the expression of the transforming growth factor-β (TGF-β) superfamily member [bone morphogenetic protein (BMP)-2, 4, 6 and TGF-β1] in the axotomized facial nuclei using in situ hybridization. The aim of the present study was to clarify whether the BMP-2, 4, 6 and TGF-β1 are related to the regenerative and degenerative processes in the axotomized facial nuclei. The results showed that BMP-2 and TGF-β1 mRNA increased gradually after axotomy, and reached the maximal values on day 7 post injury. BMP-6 was expressed abundantly in the facial motoneurons, and no obvious differences were shown in the gene expression after transection of FN. However, no significant signals for BMP-4 in motoneuron were detected at any time point. These results revealed that some members of the TGF-β superfamily such as BMP-2 and TGF-β1, might be involved in glial activation and motoneuron survival, and might be potential neurotrophic factors of FN.

The subject of the present study was a tongue of the ring-tailed lemur (Lemur catta), which was provided by the Cooperation Research Program of the Primate Research Institute, Kyoto University. Using a scanning electron microscope, we observed the fine structures of the lingual papillae, which are distributed on or just under the surface of the body of the tongue of this animal. We compared our results with other primates and other mammalian species, and discussed the evolution of the papillae from the viewpoint of comparative anatomy.The filiform and fungiform papillae were observed on the dorsum of the tongue body in the ring-tailed lemur. The filiform papillae are arranged in a circular pattern chracteristic of the primates. Each circle consists of 13-18 cone-like papillae. There are some differences in size and shape of the papillae between the anterior and posterior parts of the tongue. The general morphology is similar both in the epithelial and connective tissue papillae. In contrast, the fungiform papillae show a hemispheric shape on the epithelium, while they are of a cactus-like shape in the connective tissue. This feature of the fungiform papillae is quite similar to those observed in several species of insectivores and lagomorphs.According to the present study, together with the results by previous authors, it appears that in lemurs, which are classified in prosimians, the filiform papillae show a morphology chracteristic of the order primates, but the fungiform papillae still retain a primitive feature relative to the Anthropoidea.

The present study examined a novel function of PRL-releasing peptide (PrRP) on the neuroendocrine. PrRP-immunoreactive nerve fibers and nerve terminals were located in the vicinity of the somatostatin (SOM)-neurons in the hypothalamic periventricular nucleus (PerVN). Immuno-electron microscopy revealed that PrRP-immunoreactive nerve terminals made synaptic contacts with nonimmunoreactive neuronal elements in the PerVN. Intracerebroventricular (icv) administration of PrRP induced immediate early gene, NGFI-A, in SOM-neurons in the PerVN. Double-labeling in situ hybridization showed that some parts of SOM-neurons in the PerVN expressed PrRP receptor messenger RNA. Therefore, some parts of SOM-neurons in the PerVN are considered to be directly innervated by PrRP via PrRP receptor. In addition to the above morphological characteristics, icv administration of PrRP decreased plasma GH levels. Such inhibitory effects of PrRP on the secretion of GH from the anterior pituitary were diminished by depletion or neutr...

It has been shown that apoptosis is involved in normal embryonic development. The aim of the present study was to elucidate the role and alteration of apoptosis in the pathogenesis of cleft palate induced by retinoic acid (RA) and the ectomesenchymal stem (EMS) cells influenced by RA. RA was administered by gavage to pregnant C57BL/6N strain mice in the experimental group, and the control group received oil alone. Pregnant mice were killed at set periods of time thereafter and histologically analyzed. EMS cells explanted from the palatal shelves of embryonic mice were cultured and characterized by immunohistochemistry, growth curves and population-doubling time. The alterations of apoptosis of EMS cells and developing palatal shelves influenced by RA were evaluated by the terminal deoxynucleotidyl transferase-mediated UTP-biotin nick end-labeling (TUNEL) method. RA-treated mice showed formation of cleft palates resulted from the small size of the palatal shelves and their failure to lift. TUNEL staining showed that the number of apoptotic mesenchymal cells in palatal shelves in the RA-treated mice was increased significantly when compared with the control group. The primary culture of EMS cells proceeded successfully. The population-doubling time of RA-treated cells was much longer compared with non-treated EMS cells. RA also dramatically increased the number of apoptotic cells in EMS cells in vitro. We concluded that EMS cells are the crucial cells in palate development. RA could inhibit the proliferation and induced the apoptosis of EMS cells. The inhibition of growth and excess apoptosis of EMS cells may contribute to the formation of cleft palate and other orofacial congenital malformations.

The relationship between neuronal nitric oxide synthase (n-NOS), and heme oxygenase-2 (HO-2), the enzyme synthesizing CO, in the myenteric plexuses of the canine esophagus were examined by means of double staining technique of NADPH-diaphorase (NADPH-d) histochemistry and HO-2 immunohistochemistry. The coexistent ratio was examined regarding three portions of the esophagus; the cervical, intrathoracic and subdiaphragmatic portions. In the myenteric neurons of all portions, n-NOS frequently coexisted with HO-2 but HO-2 only partially coexisted with n-NOS. Thus, NO cooperates with CO but CO has some functions independent of NO. Many NADPH-d reactive nerve fibers were detected but there were very few HO-2 immunoreactive nerve fibers around the blood vessels and exocrine glands in the esophagus. As a neurotransmitter, NO may be more important than CO because many NADPH-d reactive nerve fibers could be detected in the esophagus. Another possibility is that CO may act as a neuromodulator, rather than as a neurotransmitter.

To investigate the possibility of the neurosecretion of Bax-like protein in the adult rat, we examined its distribution in the hypothalamo-posterior pituitary system using light and electron microscopic immunohistochemistry. We also measured the serum Bax-like protein using an enzyme immunoassay. Neurons in the paraventricular nucleus (PVN) and supraoptic nucleus of the hypothalamus exhibited intense Bax-like immunoreactivity (Bax-IR). Bax-like immunoreactive axons projected to the posterior pituitary through the internal layer of the median eminence. By immunoelectron microscopy, Bax-IR was observed in the granulated vesicles and in the membrane of the roughsurfaced endoplasmic reticulum in the neuronal perikarya in the medial magnocellular division of PVN. It was also detected in the secretory vesicles in the nerve terminals of the posterior lobe of the pituitary gland. Double-labeling histochemistry revealed that almost all Bax-like immunoreactive substances were present in the oxytocin neurons, and many Bax-like immunoreactive neurons were nitro-oxidergic neurons. The enzyme immunoassay revealed that the average Bax-like protein concentration in the serum was 39.0±9.6 pmol/ml in the adult male rat. Colchicine treatment significantly increased the densities of Bax-IR in the PVN and decreased the serum Bax-like protein levels. From these results, Bax-like protein is considered to be produced in the hypothalamic oxytocin neurons, transported through axoplasmic flow and secreted into systemic circulation at the posterior pituitary as a "neurohormone"

The development of astroglial cells and the effect of the retinohypothalamic tract on it were studied by vimentin and glial fibrillary acidic protein (GFAP) immunocytochemistry in the suprachiasmatic nucleus (SCN) of the rat. At the embryonic stage, vimentin-immunoreactive (VIM-IR) radial glia, precursors of astrocytes, were dominant. However, their filaments vanished in the first few postnatal days. Instead of VIM-IR glial filaments, GFAP-immunoreactive (GFAP-IR) astrocytes appeared at E20 and grew rapidly from the P3 stage. GFAP immunoreactivity in the ventrolateral portion of the SCN (VLSCN) was measured using a computer-assisted image analyzing system. In normal rats, GFAP immunoreactivity showed a stepwise pattern with two slopes at P3-P4 and P20-P25. Bilaterally eye-enucleated rats operated on the day of birth showed lower GFAP immunoreactivity than normal rats and the GFAP immunoreactivity did not increase between P20 and P25 when GFAP-IR glial processes rapidly expand. Electron microscopic investigation at P50 (adult stage) revealed that neurons in the VLSCN had often direct apposition without astroglial processes and the frequency of this finding was significantly higher in eye-enucleated rats than in the control rats. These findings strongly suggest that the postnatal development of astroglial elements, particularly the expansion of GFAP-IR processes in the SCN, is regulated by retinohypothalamic projection.

Astroglial elements in the suprachiasmatic nucleus (SCN) in golden hamsters and rats were investigated by GFAP immunocytochemistry and a computer−assisted analyzing system. Serial frontal sections, including the SCN region, were prepared and GFAP immunoreactivity in the following areas were measured as optical density (O.D.) per area (O.D./μm2); (1) the whole SCN region, (2) the ventrolateral portion (VLSCN), where vasoactive intestinal peptide (VIP) neurons were mainly distributed, (3) the dorsomedial portion, where arginine vasopressin (AVP) neurons were mainly distributed, and (4) as determined by unilateral intraocular injection of cholera toxin B subunit, the area where retinohypothalamic tract (RHT) fibers were distributed. In the rat, GFAP immunoreactivity was observed throughout the SCN, particularly in the VLSCN, while in the golden hamster, astroglial elements enclosed the whole region of the SCN. Inaddition, quantitative analysis revealed that the O. D. of the GFAP immunoreactivity in the VLSCN, where VIP neurons and the RHT fibers were distributed, were significantly higher than those in other regions of the SCN. These results strongly suggested that the astroglial elements, VIP neurons and RHT fibers have close morphological interactions, although delicate differences were demonstrated in both species.

We studied NGFI-A gene expression in response to photic stimulation in the rat suprachiasmatic nucleus (SCN) using in situ hybridization histochemistry. This gene expression spread within the SCN and extended dorsally into the anterior hypothalamus after 30 min-1 h of light exposure at circadian time (CT) CT18. It appeared first in the ventrolateral SCN where the retinohypothalamic tract (RHT) innervates, then it expanded dorsomedially in the SCN and beyond the SCN to the anterior hypothalamus. However, stimulation for 2 h light exposure decreased its expression in the SCN. NGFI-A expression in the somatostatin neurons in the periventricular nucleus increased from 8.7% to 41% with increasing exposure time from 5 to 30 min. NGFI-A mRNA expression in the SCN was suppressed by pretreatment with baclofen, the GABAB receptor agonist. The spread of photic information from the retina to the SCN was visualized at immediate early gene level not only in the SCN but also in the area beyond the SCN. Somatostatin neurons in the periventricular nucleus which project to the external layer of the median eminence and are involved in regulation of growth hormone release showed NGFI-A gene expression corresponding to the duration of photic stimulation. Photic-induced NGFI-A gene expression in the SCN was also shown to be regulated by GABAergic transmission via GABAB receptors. These NGFI-A gene-expressing cells in the SCN may be involved in the circadian entrainment by light and some of those outside the SCN may participate in the regulation of neuroendocrine function.

This study was performed to examine the differences in expression of heme oxygenase protein with age using immunocytochemistry. We compared the contents of HO-1 and HO-2 between young and aged rats using immunocytochemical methods. Stronger HO-1 expression was detected in the internal layer of the median eminence (ME) of aged than of young rats. Moreover, the cells expressing HO-1 were larger in the aged than the young animals. Electron microscopy indicated these cells with HO-1-like immunoreactivity (HO-1-LI) to be astrocytes. These findings suggested that the expression of HO-1 increased in the ME with age. The significance of this increased expression of HO-1 with age will be discussed briefly.

Arginine vasopressin peptide and messenger RNA expression were examined at the cellular level in the magnocellular and parvocellular neurons in the rat paraventricular nucleus after dehydration and rehydration, employing immunocytochemistry andin situ hybridization histochemistry on the same tissue sections. Most magnocellular vasopressinergic neurons of control animals expressed both vasopressin-like immunoreactivity and messenger RNA. However, neurons negative for vasopressin-like immunoreactivity but expressing messenger RNA were also detected, and their number increased during dehydration. In contrast, almost all of the parvocellular vasopressinergic neurons of dehydrated animals expressed vasopressin messenger RNA alone, with continued increase in their number after rehydration, despite return of the number of magnocellular vasopressinergic neurons to the control level. Vasopressin messenger RNA and corticotropin releasing factor-like immunoreactivity were co-localized in the same parvocellular neurons, and vasopressin-immunoreactive nerve terminals were detected in the external zone of the median eminence. These findings suggest that magno- and parvocellular vasopressinergic neurons are differentially activated during dehydration/rehydration. Osmotic stimuli activate all magnocellular vasopressinergic neurons, but the effect is not simultaneous in all of these neurons. Parvocellular vasopressinergic neurons are also activated by the stress of dehydration which effect appears to last longer than in the magnocellular system.

The morphological interactions between astroglial and neuronal elements were elucidated in the rat suprachiasmatic nucleus (SCN) by light and electron microscopic immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP), vasoactive intestinal peptide (VIP) and arginine-vasopressin (AVP). Throughout the SCN, particularly in its ventral portion, GFAP-like-immunoreactive (GFAP-LI) astroglial elements were found. These astrocytes displaying GFAP-like immunoreactivity occasionally contained fairly well-developed organelles. Some of these astrocytes were found as satellite cells in close contact with non-immunoreactive neuronal perikarya and processes. Around the neurons, GFAP-LI astroglial processes were also observed to cover some portions of presynaptic and postsynaptic elements. In addition, these astroglial elements were seen between two neuronal somata and pericytes of blood capillaries as glial endfeet. By double labeling immunoelectron microscopy using antibodies against GFAP/VIP and GFAP/AVP, some portions of VIP-like-immunoreactive or AVP-like-immunoreactive neuronal somata and processes were found to be engulfed by GFAP-LI astroglial processes. The possible functional roles of the morphological interactions between astroglial and neuronal elements are discussed.

The suprachiasmatic nucleus (SCN) is considered to be a circadian oscillator that regulates a set of phys iological aspects of behavior, including sleep-wakefulness and hormone release in mammalian species. In this review, we describe recent research that has begun to reveal the functional organization of the SCN. The SCN, which consists of a bilateral pair of tiny nuclei located just above the optic chiasm, contains several kinds of peptidergic neurons, but vasoactive intestinal peptide (VIP), arginine vasopressin (AVP), and somatostatin (SOM) neurons are the main components. VIP neurons and AVP neurons show distinctly different locations in the SCN; the former are found in the ventrolateral portion, whereas the latter are localized in the dorsomedial portion. VIP neurons receive all neuronal inputs from other regions of the CNS, such as those evoked by photic stimulation via the retinal ganglion cells and those relayed by 5HT inner vation from the raphe nuclei. VIP neurons relay their information to other kinds of neurons in the SCN, such as AVP and SOM neurons. VIP neurons, thus, may play a significant role in entrainment of circadian rhythm. VIP, AVP, SOM, and their mRNAs show rhythmic fluctuations that are predicted by this model; VIP and its mRNA show diurnal variation under the influence of photic stimulation, whereas AVP, SOM, and their mRNAs show endogenous rhythms. Immediate early genes (lEGs), such as c-fos mRNA, are also expressed in VIP neurons in the SCN, and IEG expression in the cells appears to be modified by photic stimuli. Together with transplantation studies showing that exogenous SCN tissue tends to restore circadian rhythm in arrhythmic animals, these results are beginning to clarify the function of the SCN in setting, maintaining, and resetting the biological clock. NEUROSCIENTIST 3:215-225, 1997

Photic induction of NGFI-A gene expression was investigated in the rat suprachiasmatic nucleus (SCN) using in situ hybridization histochemistry. Following light exposure for 30 min, NGFI-A mRNA appeared in the ventral portion of the rostral SCN, in the ventrolateral and in part of the dorsomedial portion at the middle level, and in the lateral portion of the caudal SCN. The distribution of NGFI-A mRNA was wider than that of c-fos mRNA which was confined to the ventrolateral portion at the middle level of the SCN. By double labeling in situ hybridization, approximately half of NGFI-A mRNA-positive cells in the SCN were shown to coexpress vasoactive intestinal peptide (VIP) mRNA, while 16% of cells positive for c-fos mRNA coexpressed VIP mRNA. These findings indicate that the broadness of NGFI-A mRNA and c-fos mRNA expression after photic stimulation are different. NGFI-A gene induced in these cells of the SCN including VIP neurons may be involved in circadian entrainment by light.

In the present study, interactions between lutenizing hormone releasing hormone (LHRH) -like immunoreactive (LHRH-LI) nerve endings and processes of tanycytes and/or astrocytes in the median eminence (ME) particularly its external layer were investigated by double labeling immunoelectron microscopy employing antibodies against glial fibrillary acidic protein (GFAP) and LHRH. GFAP-like immunoreactivity was observed in some tanycytes throughout the ME and/or astrocytes in the subependymal layer of the ME. In the palisade layer of the ME, GFAP-like immunoreactive (GFAP-LI) elements were frequently seen to make intimate contact with LHRH-LI nerve endings. LHRH-LI nerve endings and processes of the tanycytes and/or astrocytes were also found to direct contact with the basement membrane of the pericapillary space. And they were partially engulfed each other in the vicinity of the perivascular region. The above findings strongly suggest that the processes of tanycytes and/or astrocytes with intimate communication of cerebrospinal fluid in the third ventricle may play a role in regulation of secretion of LHRH into the portal vessels.

Neuronal activity of the suprachiasmatic nucleus (SCN) is known to be regulated by two major extrinsic factors conveyed by three anatomically distinct pathways to the SCN: photic stimulus by the direct retinohypothalamic tract (RHT) and the indirect geniculohypothalamic tract (GHT), and information from the brainstem by ascending forebrain serotonergic (5-hydroxytryptamine: 5-HT) tract. It has been shown that VIP mRNA level in neurons of the SCN is altered by external light, but remains stable in constant darkness. In the present study, by using the in situ hybridization technique combined with computer-assisted image analysis, we examined VIP mRNA expression in the SCN of rats in which the two major factors were eliminated, i.e. photic stimulus by exposing animals in total darkness and 5-HT transmission by three-day successive administration of p-chlorophenyl-alanine methylester (an inhibitor of tryptophan hydroxylase, 200 mg/kg, daily). In saline-treated controls, VIP mRNA levels remained almost constant throughout the day. In contrast, in PCPA-treated rats, a significant rhythm of VIP mRNA was observed with a peak at CT 4 and a trough at CT 20. These observations suggest that the removal of photic and 5-HT influence induces VIP mRNA rhythm in the SCN, indicating that VIP mRNA is controlled not only by photic information but also by the circadian clock.

The influence of serotonergic inputs on the levels of vasoactive intestinal peptide (VIP) mRNA in the rat suprachiasmatic nucleus (SCN) was examined by in situ hybridization combined with morphometrical analysis. Depletion of serotonin by p-chlorophenylalanine methyl ester (300 mg/kg i.p. daily for 3 days) caused a marked decrease in VIP mRNA signal levels in the ventrolateral part of the SCN. This finding suggests that serotonergic input controls VIP production at the mRNA level.

Localization of nitric oxide synthase (NOS) in the nodose ganglia of the dog, rat and guinea pig was investigated. A double-staining technique of NOS immunohistochemistry and NADPH-diaphorase (NADPHd) histochemistry was used; then the ratio of NADPHd-positive and NOS-positive cells to the total cells was calculated. The distribution of positive cells within the canine nodose ganglion was also investigated. NADPHd-positive neurons were detected in all the ganglia. Three intensities of reactivity to NADPHd histochemistry (strong, weak or negative) were detected in the neurons of all three species. There were more cells that stained strongly for NADPHd in the rat, but fewer in the dog and guinea pig, indicating that a species difference may exist. NADPHd-positive neurons were less abundant in the rostral third of the canine nodose ganglion than in the middle or caudal thirds. NADPHd reactivity was completely co-localized to the cells that demonstrated neuronal NOS immunoreactivity in the canine nodose ganglion. Thus, NADPHd histochemical reactivity may be a reliable marker of NOS in the nodose ganglion.

We investigated the effects of bone morphogenetic protein-2 (BMP-2) and some other BMPs on regeneration of peripheral motor nerves in vivo. The facial nerves of 24 New Zealand rabbits were crushed to examine a series of retrograde changes in the facial nuclei and axons, in what has been called the “axon reaction”. The facial nerves of the experimental group were treated with epineurial coaptation and BMP-2 after the injury. Nerves not treated with BMP-2 were regarded as controls. The expression of BMP-2 was investigated by in situ hybridisation in the neurons of facial nuclei. The electrophysiology, image analysis and transmission electron microscopy were used to evaluate the level of the recovery of facial nerves. The results showed that the axons in the experimental group were thicker and denser than those in the control group four weeks later. The expression of BMP-2 in the neurons of facial nuclei increased after injury. The electron microscopic observations showed that the axons’ degeneration in the experimental group was less than that in the control group. Despite the morphological difference between the two groups, there was no apparent difference between them in nerve conduction velocity. These findings suggest that BMP-2 might be involved in the regeneration of facial nerves, and might function as a potential neurotrophic factor.

We present a very rare case of right partial and double internal jugular veins, found in an 86-year-old Japanese female cadaver during a student dissection practice session in 2002 at Osaka Dental University. In this case, the right internal jugular vein separated into medial and lateral branches at a level with the middle of the fifth cervical vertebra. Both branches had the same thickness as an internal jugular vein and poured into the right subclavian vein. A slender venous space slit was formed by these two branches and the right subclavian vein. The inferior belly of the right omohyoid muscle and the inferior root of the right ansa cervicalis passed through the superior region in this venous space. To our knowledge, this case has never been reported previously. Therefore, we attempted to investigate the incidence based on existing references for similar cases and speculated on the development based on our findings. We considered the medial branch was the right internal jugular vein and the lateral branch was the communicating branch between the external and internal jugular veins.

Theexpression of vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) in the suprachiasmatic nucleus (SCN) changes depending on light. VIP mRNA increases and GRP mRNA decreases in the light phase, while they do not show change without light. In the present study we investigated the involvement of serotonin (5-HT) in the expression of VIP and GRP messenger RNA in the SCN of the rat. The decrease in VIP mRNA and the increase in GRP mRNA in the light phase were amplified by 5-HT depletion using 5,6-dihydroxytryptamine injected into the lateral ventricle. These enhancements due to 5-HT depletion were reversed to control levels by applying 5-HT 1B agonists TFMPP and CGS12066A, but not a 5-HT 1A /5-HT 7 agonist, 8-OH-DPAT. The 5-HT 1B receptor is known to exist on the terminals of the retinohypothalamic tract (RHT). Therefore, next we investigated the morphological relationship of RHT and 5-HT terminals by double-labeling immunocytochemistry and demonstrated that 5-HT-immunoreactive fibers and cholera toxin B subunit-labeled RHT terminals were intermingled in the ventrolateral SCN, and 5-HT axon processes had close contact with RHT terminals. Collectively, these pharmacological and morphological results suggest that 5-HT afferents from raphe nuclei modulate VIP and GRP expression in neurons of the ventrolateral SCN by activating the 5-HT 1B receptor in the RHT.

In mammals, the biological clock (circadian oscillator) is situated in the suprachiasmatic nucleus (SCN), a small bilaterally paired structure just above the optic chiasm. Circadian rhythms of sleep-wakefulness and hormone release disappear when the SCN is destroyed, and transplantation of fetal or neonatal SCN into an arrhythmic host restores rhythmicity. There are several kinds of peptide-synthesizing neurons in the SCN, with vasoactive intestinal peptide, arginine vasopressin, and somatostatine neurons being most prominent. Those peptides and their mRNA show diurnal rhythmicity and may or may not be affected by light stimuli. Major neuronal inputs from retinal ganglion cells as well as other inputs such as those from the lateral geniculate nucleus and raphe nucleus are very important for entrainment and shift of circadian rhythms. In this review, we describe morphological and functional interactions between neurons and glial elements and their development. We also consider the expression of immediate-early genes in the SCN after light stimulation during subjective night and their role in the mechanism of signal transduction. The reciprocal interaction between the SCN and melatonin, which is synthesized in the pineal body under the influence of polysynaptic inputs from the SCN, is also considered. Finally, morphological and functional characteristics of clock genes, particularly mPers, which are considered to promote circadian rhythm, are reviewed.

The retinal projections to gastrin-releasing peptide (GRP)-expressing neurons in the rat suprachiasmatic nucleus (SCN) were investigated by double immunofluorescence and immunoelectron microscopy. Optic nerve terminals labeled by cholera toxin B subunit (CTb) which was transported from the retinal ganglion cells were intermingled with GRP-immunoreactive cell bodies and processes in the ventrolateral portion of the SCN. Ultrastructural analysis revealed that CTb-immunoreactive retinal terminals made synaptic contacts with GRP-immunoreactive dendritic processes. These results demonstrated that photic information is directly input from the optic nerve to GRP neurons in the SCN and these GRP neurons may be involved in circadian entrainment by light.

Age-related decreases in circadian oscillating activity are speculated to be one of the causes of psychiatric symptoms. To explore the effects of aging on vasoactive intestinal peptide (VIP) synthesis in the suprachiasmatic nucleus (SCN), we investigated the changes in VIP mRNA levels in aged rats compared with young-adult rats under a light/dark cycle using in situ hybridization combined with microcomputer-based imaging analysis. In the young-adult rats, total signals of VIP mRNA in the light-phase showed a significant decrease compared with those on the dark-phase. The VIP signal level in the aged rats was markedly lower than that in young-adults in both light and dark phases. Moreover, in the aged rats, there were no significant differences in VIP mRNA level between the light and dark phases. These results suggest that gene expression of VIP neurons, a main component of the circadian oscillating system, becomes disturbed in the aged rat brain.

Photic stimulus and serotonin (5-hydroxytryptamine; 5-HT) are two factors known to regulate vasoactive intestinal peptide (VIP) synthesis in the suprachiasmatic nucleus (SCN). To explore the role of 5-HT in the photic stimulus-induced change in VIP synthesis, we investigated the changes in level of VIP mRNA under a 12 h light/12 h dark cycle following depletion of 5-HT by intraperitoneal administration of p -chlorophenylalanine (PCPA) methyl ester (200 mg/kg concentration) for 3 successive days. To estimate VIP mRNA expression, we performed in situ hybridization using imaging plates combined with microcomputer-based imaging analysis. In light-phase, total signals of VIP mRNA from the PCPA-treated rats showed a signifanct decrease compared with those from the saline-treated control rats. However, in dark-phase, there were no significant decreases between the PCPA-treated rats and the saline-control rats. The present results strongly suggest that 5-HT neuronal inputs to the SCN interfere with the effect of photic stimulus on VIP synthesis at the mRNA level.

Pituitary adenylate cyclase-activating polypeptide-like immunoreactive (PACAP-LI) neuronal elements in the rat hypothalamus including the median eminence (ME) were investigated by light and electron microscopic immunocytochemistry. PACAP-LI neuronal perikarya with well-developed cell organelles and dense granules were distributed mainly in the magnocellualr portion of the paraventricular nucleus and throughout the entire supraoptic nucleus. In the ME, numerous PACAP-LI neuronal processes were found in the internal layer (IL), and immunoreactive terminals containing dense granules, vesicles and mitochondria were detected around portal capillaries which penetrated into the IL from the external layer. Thereafter, PACAP is released into the portal capillaries in the IL, transported to the anterior pituitary and plays a role in the stimulation of adenylate cyclase of anterior pituitary cells.

The midline of the rostral ventral medulla (RVM) is the portion in which many serotonin (5-HT) neurons of the nucleus raphe magnus and the rostral nucleus raphe pallidus are located and where dense catecholaminergic (CA) fibers are distributed. In this study, we investigated the connection between spinally projecting 5-HT neurons and CA fibers in the rat RVM by light and electron microscopic immunocytochemistry. First, light microscopic immunocytochemistry using a triple labeling method revealed that the 5-HT-immunoreactive (IR) neuron containing retrograde tracer from the cervical cord was intimately surrounded by tyrosine hydroxylase (TH)-IR fibers. Second, silver-gold intensified TH-IR axon terminals were found to make synaptic contacts with 5-HT-IR neuronal perikarya and dendrites by double labeling immunoelectron microscopy. These morphological findings suggest that spinally projecting 5-HT neurons, presumed to be involved in pain modulation or sympathetic autonomic control, are directly regulated by CA neurons at the level of the RVM.