Morphological evidence for direct interaction between kisspeptin and gonadotropin-releasing hormone neurons at the median eminence of the male goat: an immunoelectron microscopic study.

Kisspeptin has been thought to play pivotal roles in the control of both pulse and surge modes of gonadotropin-releasing hormone (GnRH) secretion. To clarify loci of kisspeptin action on GnRH neurons, the present study examined the morphology of the kisspeptin system and the associations between kisspeptin and GnRH systems in gonadally intact and castrated male goats. Kisspeptin-immunoreactive (ir) and Kiss1-positive neurons were found in the medial preoptic area of intact but not castrated goats. Kisspeptin-ir cell bodies and fibers in the arcuate nucleus (ARC) and median eminence (ME) were fewer in intact male goats compared with castrated animals. Apposition of kisspeptin-ir fibers on GnRH-ir cell bodies was very rare in both intact and castrated goats, whereas the intimate association of kisspeptin-ir fibers with GnRH-ir nerve terminals was observed in the ME of castrated animals. Neurokinin B immunoreactivity colocalized not only in kisspeptin-ir cell bodies in the ARC but also in kisspeptin-ir fibers in the ME, suggesting that a majority of kisspeptin-ir fibers projecting to the ME originates from the ARC. A dual immunoelectron microscopic examination revealed that nerve terminals containing kisspeptin-ir vesicles made direct contact with GnRH-ir nerve terminals at the ME of castrated goats. There was no evidence for the existence of the typical synaptic structure between kisspeptin- and GnRH-ir fibers. The present results suggest that the ARC kisspeptin neurons act on GnRH neurons at the ME to control (possibly the pulse mode of) GnRH secretion in males.

Microstructure characteristics of the cornea in birds and mammals.

In this study, the microstructure of the cornea was compared among chickens (Gallus gallus), jungle crows (Corvus macrorhynchos), rats (Rattus norvegicus) and rabbits (Oryctolagus cuniculus). The density of keratocytes in the mammals was over 3 times that in the birds. The size of the keratocytes in the birds and rat were significantly lower than those in the rabbit. Using scanning and transmission electron microscopy, the bundles of collagen fibers in the birds were found to be well arranged, while those in the mammals were arranged randomly. The collagen lamellae of the birds were significantly thicker than those of the mammals, and the numbers of collagen lamellae in the birds were significantly smaller than in the mammals. The center-to-center distances between the collagen fibrils of the chicken and rabbit were significantly larger than those of the crow and rat. The densities of collagen fibrils in the chicken and rabbit were significantly less than those of the crow and rat.

Seasonal increase in olfactory receptor neurons of the Japanese toad, Bufo japonicus, is paralleled by an increase in olfactory sensitivity to isoamyl acetate.

Japanese toads (Bufo japonicus) migrate to and from breeding sites in the early spring, possibly guided by olfactory cues. We previously showed that the electrical activity of olfactory receptor neurons (ORNs) in the toads was enhanced in the breeding period. We undertook morphological and physiological studies of the olfactory epithelium to determine whether any cellular substrate of the epithelium underlies the enhanced electrical activity of ORNs. The ORNs of the toads were labeled by antiserum to olfactory marker protein (OMP), and the morphology of the labeled cells and their distribution in the epithelium were examined throughout the year. The OMP-positive cells, distributed mainly in the basal and intermediate layers of the epithelium, were most numerous in the early breeding period. Cell proliferation in the epithelium detected by 5-bromo-2'-deoxyuridine labeling was most elevated in this period. The electrical activity of ORNs was examined by recording the electroolfactogram (EOG) in the toads throughout the year. Statistical analysis showed a positive correlation between the density of OMP-positive cells in the epithelium and the amplitude of the EOG responses. A greater number of ORNs in the breeding period possibly aids the toads in migrating to their breeding sites.

The morphological change of supporting cells in the olfactory epithelium after bulbectomy.

Transmission electron microscopy was used to study the responses of the supporting cells of the olfactory epithelium at 1-5 days after surgical ablation of the olfactory bulb (bulbectomy). In intact olfactory epithelium, lamellar smooth endoplasmic reticulum and rod-shaped mitochondria were distinctly observed in the supporting cells. On the first day after bulbectomy, bending of the microvilli and an increase in the smooth endoplasmic reticulum were observed. Cristae of the mitochondria became obscure, and the density of the mitochondrial matrix decreased. On the second day after bulbectomy, the number of microvilli decreased, broad cytoplasmic projections that contained cytoplasmic organelles protruded into the luminal side, and the mitochondria were swollen. On the fifth day after bulbectomy, microvilli seemed to be normal and some cells had large cytoplasmic projections that protruded toward the lumen of the nasal cavity. Within the cytoplasmic projections of the supporting cells, a large lamellar and reticular-shaped smooth endoplasmic reticulum was evident. Mitochondria exhibited almost normal morphology. The current findings demonstrate that morphological changes occur in the supporting cells after bulbectomy. This new evidence hypothesizes that these changes represent events that contribute to the regeneration of the olfactory epithelium after bulbectomy.

Histological properties of the nasal cavity and olfactory bulb of the Japanese jungle crow Corvus macrorhynchos.

The nasal cavity and olfactory bulb (OB) of the Japanese jungle crow (Corvus macrorhynchos) were studied using computed tomography (CT) and histochemical staining. The nasal septum divided the nasal cavity in half. The anterior and maxillary conchae were present on both sides of the nasal cavity, but the posterior concha was indistinct. A small OB was present on the ventral surface of the periphery of the cerebrum. The OB-brain ratio--the ratio of the size of the OB to that of the cerebral hemisphere--was 6.13. The olfactory nerve bundles projected independently to the OB, which appeared fused on gross examination. Histochemical analysis confirmed the fusion of all OB layers. Using a neural tracer, we found that the olfactory nerve bundles independently projected to the olfactory nerve layer (ONL) and glomerular layer (GL) of the left and right halves of the fused OB. Only 4 of 21 lectins bound to the ONL and GL. Thus, compared with mammals and other birds, the jungle crow may have a poorly developed olfactory system and an inferior sense of olfaction. However, it has been contended recently that the olfactory abilities of birds cannot be judged from anatomical findings alone. Our results indicate that the olfactory system of the jungle crow is an interesting research model to evaluate the development and functions of vertebrate olfactory systems.

Vomeronasal neurons promote synaptic formation on dendritic spines but not dendritic shafts in primary culture of accessory olfactory bulb neurons.

To investigate the morphological changes of accessory olfactory bulb (AOB) neurons arising from pheromonal signals, a coculture system of AOB neurons and vomeronasal (VN) neurons had been established. Our previous study indicates that under coculture condition, the density of dendritic spines of an AOB neuron is less and the individual spine-head volume is larger than those under monoculture condition. In this study, to determine whether these differences in the dendrites of AOB neurons reflect the differences in synapse formation and synaptic properties, we observed these cultured cells by electron microscopy. Various synapses were observed under each culture condition. Synapses were classified on the basis of their postsynaptic structure and the size of postsynaptic density (PSD) was measured. Under the coculture condition with VN neurons, synapses on dendritic spines, which formed between AOB neurons, were observed frequently. In contrast, many synapses were formed on dendritic shafts under monoculture condition. The PSD of asymmetrical synapses on the spines under coculture condition was larger than that under monoculture condition. Moreover, some dendrodendritic reciprocal synapses were found only in coculture. We confirmed synapse formation between VN axons and AOB dendrites by immunohistochemical electron microscopy; thus, the characteristics of synapses between AOB neurons are considered to be modified by the synaptic contacts with VN axons.

Morphological and histochemical study of the nasal cavity and fused olfactory bulb of the brown-eared bulbul, Hysipetes amaurotis.

The brown-eared bulbul (Hysipetes amaurotis) is commonly found in Japan where it is regarded as a harmful bird that causes damage to agricultural products. Few studies have investigated the sensory apparatus of this bird, and consequently little is known of the sensory modalities it uses. Here we analyzed the anatomical and histological properties of the nasal cavity and olfactory bulb (OB) of the bulbul in order to investigate the functional level of olfaction in this species. Although both anterior and maxillary conchae were observed in the bulbul nasal cavity, there was no structure equivalent to the posterior concha. The OB located on the ventral side of the anterior extremity of the cerebrum and the ratio of olfactory bulb size to that of the cerebral hemisphere were very small. Interestingly, the left and right OBs were completely fused at the midline of the cerebrum. Furthermore, certain types of lectins that bind to the olfactory nerve of vertebrates with a well-developed sense of smell also bound positively to the olfactory nerve and glomerular layers of the bulbul OB. These findings suggest that the brown-eared bulbul has an anatomically and functionally less well developed sense of smell compared to other avian species. Although the molecular and developmental mechanisms underlying the fusion of the OB remain unknown, we suggest that the fused OB may offer a unique model for studying the evolution and development of the central olfactory nervous system in vertebrates.

A postpartum separation induces c-Fos expression in the supramammillary nucleus of lactating rats.

AIM: Elucidation of the neural mechanism of maternal behaviors is a medically and biologically important research task. The rat is the laboratory animal most extensively analyzed for maternal behaviors. However, the neural mechanism that maintains the motivation of postpartum rats for maternal behaviors has not yet been elucidated. In this study, we aimed to identify brain regions involved in the maintenance of motivation for maternal behaviors by detecting brain regions that exhibit changes in nerve activity when the mother rat is separated from her pups. METHODS: Lactating mother rats were separated from their pups on postpartum day 3 and kept away from the pups for a certain period of time, and brain regions that exhibited changes in nerve activity when the rats were separated from their pups and those that exhibited changes in nerve activity when the pups are returned were detected by immunohistochemistry using anti-c-Fos antibody, a marker for increased nerve activity. RESULTS: Rats that were separated from their pups and with the pups returned later showed increases in the number of c-Fos immunoreactive (c-Fos-IR) cells in the medial preoptic area (MPA), the bed nucleus of the stria terminalis (BST), the caudal portion of posterior hypothalamic area (PH) and the supramamillary nucleus (SUM). In mother rats permanently separated from their pups, only the PH and SUM exhibited an increase in the number of c-Fos-IR cells. CONCLUSION: In rats, the SUM is involved in aversive memory and changes in the postpartum anxiety level. The observed increase in the number of c-Fos-IR cells in the SUM of mother rats separated from their pups suggests that the nerve activity change in the SUM, which is involved in aversive memory and anxiety, is involved in the maintenance of maternal behaviors.

Xenopus V1R vomeronasal receptor family is expressed in the main olfactory system.

To date, over 100 vomeronasal receptor type 1 (V1R) genes have been identified in rodents. V1R is specifically expressed in the rodent vomeronasal organ (VNO) and is thought to be responsible for pheromone reception. Recently, 21 putatively functional V1R genes were identified in the genome database of the amphibian Xenopus tropicalis. Amphibians are the first vertebrates to possess a VNO. In order to determine at which point during evolution the vertebrate V1R genes began to function in the vomeronasal system, we analyzed the expression of all putatively functional V1R genes in Xenopus olfactory organs. We found that V1R expression was not detected in the VNO but was specifically detected in the main olfactory epithelium (MOE). We also observed that V1R-expressing cells in the MOE coexpressed Gi2, thus suggesting that the V1R-Gi2-mediated signal transduction pathway, which is considered to play an important role in pheromone reception in the rodent VNO, exists in the amphibian MOE. These results suggest that V1R-mediated signal transduction pathway functions in Xenopus main olfactory system.

Localization of G protein alpha subunits and morphology of receptor neurons in olfactory and vomeronasal epithelia in Reeve's turtle, Geoclemys reevesii.

Most vertebrates have two nasal epithelia: the olfactory epithelium (OE) and the vomeronasal epithelium (VNE). The apical surfaces of OE and VNE are covered with cilia and microvilli, respectively. In rodents, signal transduction pathways involve G alpha olf and G alpha i2/G alpha o in OE and VNE, respectively. Reeve's turtles (Geoclemys reevesii) live in a semiaquatic environment. The aim of this study was to investigate the localization of G proteins and the morphological characteristics of OE and VNE in Reeve's turtle. In-situ hybridization analysis revealed that both G alpha olf and G alpha o are expressed in olfactory receptor neurons (ORNs) and vomeronasal receptor neurons (VRNs). Immunocytochemistry of G alpha olf/s and G alpha o revealed that these two G proteins were located at the apical surface, cell bodies, and axon bundles in ORNs and VRNs. Electron microscopic analysis revealed that ORNs had both cilia and microvilli on the apical surface of the same neuron, whereas VRNs had only microvilli. Moreover G alpha olf/s was located on only the cilia of OE, whereas G alpha o was not located on cilia but on microvilli. Both G alpha olf/s and G alpha o were located on microvilli of VNE. These results imply that, in Reeve's turtle, both G alpha olf/s and G alpha o function as signal transduction molecules for chemoreception in ORNs and VRNs.

p-Chloroamphetamine-induced rat ejaculation is not associated with the preoptic nucleus or medial nucleus amygdala.

Aim: In the rat, intraperitoneal injection of p-chloroamphetamine (PCA), which releases central 5-hydroxytryptamine (5-HT) from serotonergic nerve terminals, induces ejaculation, even in the absence of an estrus female or female-related smell information. It is well known that the medial preoptic nucleus (MPN) and the medial nucleus amygdala (MEA) play a major role in the control of male sexual behavior in mammals. We examined whether or not neuronal activity of the MPN and/or the MEA was associated with PCA-induced ejaculation. Methods: Using c-Fos immunohistochemistry, we demonstrated a difference in the neural activities of the MPN and the MEA for ejaculation during copulation with an estrus female and ejaculation by PCA injection. Results: Increased numbers of c-Fos-immunoreactive (c-Fos-IR) cells were found in the MPN and the MEA in the brains of the mating animals, whereas in the brains of the animals undergoing PCA-induced ejaculation there was no increase in the number of c-Fos-IR cells in the MPN and a small increase in the MEA. Conclusion: Based on these results, ejaculation induced by PCA is not associated with the MPN. Moreover, the MEA is not the main act for this ejaculation. (Reprod Med Biol 2008; 7: 37-43).

Expression of a vomeronasal receptor gene (V1r) and G protein alpha subunits in goat, Capra hircus, olfactory receptor neurons.

Most mammals have two distinct olfactory epithelia, the olfactory epithelium (OE) and vomeronasal epithelium (VNE), containing, respectively, olfactory receptor neurons (ORNs) and vomeronasal receptor neurons (VRNs). Olfactory receptors (ORs), which couple to G alpha olf, are generally expressed by ORNs, whereas two vomeronasal receptor families (V1rs and V2rs) coupled respectively to G alpha i2 and G alpha o, are expressed by VRNs. Previously, we reported that one goat V1rs (gV1ra1) is expressed by ORNs and VRNs. To investigate the characteristics of vomeronasal-receptor-expressing ORNs in mammals we performed double-label in situ hybridization for gV1ra1, G alpha i2, G alpha olf, olfactory marker protein (OMP), and growth association protein 43 (GAP43). Goat V1r-expressing ORNs are categorized into two types situated in different areas of the epithelium. The first type of V1r-expressing ORN coexpressed G alpha i2, but not OMP or GAP43. The second type of V1r-expressing ORN expresses G alpha olf and OMP, but not G alpha i2 or GAP43. These findings suggest that the two types of V1r-expressing ORN in goat OE function using different G protein alpha subunits for chemoreception.

Distribution of Notch1-expressing cells and proliferating cells in mouse vomeronasal organ.

Vomeronasal receptor neurons (VRNs) proliferate and differentiate continuously in the vomeronasal organ (VNO) throughout life. In adult mice, new VRNs are generated mainly in the marginal region, located in the boundary region between sensory and nonsensory epithelia. The Notch signaling pathway is involved in differentiation in the developing nervous system. To understand the Notch signaling pathway involved in generating VRNs, we focused on the relationship between the expression pattern of Notch1 and the localization of proliferating cells in both developing and regenerating mice VNO, and examined the Notch signaling pathway involved in the development of VNO by in situ hybridization of Notch1 and immunocytochemistry of 5-bromo-2'-deoxyuridine. During embryonic and neonatal development, proliferating cells and Notch1-expressing (+) cells were observed evenly throughout VNO. A large number of proliferating cells and Notch1 (+) cells were observed in embryonic VNO, but gradually decreased during development. The localization of proliferating cells was similar to that of Notch1 (+) cells at each developmental stage. In adult VNO, there are a few proliferating cells and Notch1 (+) cells, which were only in the marginal region of VNO. Seven days after removal of the accessory olfactory bulb (AOB), VRNs proliferated throughout VNO. Although the number of Notch1 (+) cells also increased in VNO, the majority of these were concentrated in the dorsal region of VNO, suggesting that it has two types of differentiating cell. These results suggest that Notch1 plays a role in the differentiation of VRNs during development and regeneration of VRNs after removal of AOB.

The olfactory bulb and the number of its glomeruli in the common marmoset (Callithrix jacchus).

The olfactory system has been well studied in mammals such as mice and rats. However, few studies have focused on characterizing this system in diurnal primates that rely on their sense of smell to a lesser extent due to their ecological environment. In the present study, we determined the histological organization of the olfactory bulb in the common marmoset (Callithrix jacchus). We then constructed 3-dimensional models of the glomeruli of the olfactory bulb, and estimated the number of glomeruli. Olfactory glomeruli are the functional units of olfactory processing, and have been investigated in detail using mice. There are approximately 1800 glomeruli in a mouse hemibulb, and olfactory sensory neurons expressing one selected olfactory receptor converge onto one or two glomeruli. Because mice have about 1000 olfactory receptor genes, it is proposed that the number of glomeruli in mammals is nearly double that of olfactory receptor genes. The common marmoset carries only about 400 intact olfactory receptor genes. The present study revealed that the number of glomeruli in a marmoset hemibulb was approximately 1500-1800. This result suggests that the number of glomeruli is not positively correlated with the number of intact olfactory receptor genes in mammals.

Rheb activation disrupts spine synapse formation through accumulation of syntenin in tuberous sclerosis complex.

Rheb is a small GTP-binding protein and its GTPase activity is activated by the complex of Tsc1 and Tsc2 whose mutations cause tuberous sclerosis complex (TSC). We previously reported that cultured TSC neurons showed impaired spine synapse morphogenesis in an mTORC1-independent manner. Here we show that the PDZ protein syntenin preferentially binds to the GDP-bound form of Rheb. The levels of syntenin are significantly higher in TSC neurons than in wild-type neurons because the Rheb-GDP-syntenin complex is prone to proteasomal degradation. Accumulated syntenin in TSC neurons disrupts spine synapse formation through inhibition of the association between syndecan-2 and calcium/calmodulin-dependent serine protein kinase. Instead, syntenin enhances excitatory shaft synapse formation on dendrites by interacting with ephrinB3. Downregulation of syntenin in TSC neurons restores both spine and shaft synapse densities. These findings suggest that Rheb-syntenin signalling may be a novel therapeutic target for abnormalities in spine and shaft synapses in TSC neurons.

Expression of vomeronasal receptor genes in Xenopus laevis.

In the course of evolution, the vomeronasal organ (VNO) first appeared in amphibians. To understand the relationship between the VNO and the vomeronasal receptors, we isolated and analyzed the expression of the vomeronasal receptor genes of Xenopus laevis. We identified genes of the Xenopus V2R receptor family, which are predominantly expressed throughout the sensory epithelium of the VNO. The G-protein Go, which is coexpressed with V2Rs in the rodent VNO, was also extensively expressed throughout the vomeronasal sensory epithelium. These results strongly suggest that the V2Rs and Go are coexpressed in the vomeronasal receptor cells. The predominant expression of the Xenopus V2R families and the coexpression of the V2Rs and Go imply that V2Rs play important roles in the sensory transduction of Xenopus VNO. We found that these receptors were expressed not only in the VNO, but also in the posterolateral epithelial area of the principal cavity (PLPC). Electron microscopic study revealed that the epithelium of the PLPC is more like that of the VNO than that of the principal and the middle cavity. These results suggest that in adult Xenopus the V2Rs analyzed so far are predominantly expressed in the vomeronasal and vomeronasal-like epithelium. The analysis of V2R expression in Xenopus larvae demonstrates that V2Rs are predominantly expressed in the VNO even before metamorphosis.

Expression and regulation of the immediate-early gene product Arc in the accessory olfactory bulb after mating in male rat.

Recent studies of the accessory olfactory bulb have shown that the expression of immediate-early genes, e.g., c-fos, c-jun and egr-1, can be used as a marker of neuronal activity in response to pheromonal cues. In this study, we analyzed the expression pattern, in response to mating, of the novel immediate-early gene product Arc (an activity-regulated cytoskeleton-associated protein). Arc is hypothesized to play a role in activity-dependent neuronal plasticity in the hippocampus. In a control group of male rats, only a small number of Arc-immunoreactive cells were observed in the accessory olfactory bulb. In a mating group, however, a marked increase in the number of Arc-immunoreactive cells was observed only in the granule cell layer of the accessory olfactory bulb. The increase in the number of Arc-immunoreactive cells after mating was similar to that observed for other immediate-early genes. However, for the mating group, the increase in Arc-positive cells was limited to the granule cell layer. Granule cells have been shown to exhibit a strong synaptic plasticity in response to pheromonal stimulation. From these findings we suggest that Arc plays an important role in neuronal plasticity in the accessory olfactory bulb.

Long-term exposure to low levels of formaldehyde increases the number of tyrosine hydroxylase-immunopositive periglomerular cells in mouse main olfactory bulb.

Multiple chemical sensitivity (MCS) in response to a long-term low-level chemical exposure is as yet an unclarified disorder. To determine the role of olfactory function in the induction of MCS, immunocytochemical analysis of the main olfactory bulb (MOB) was performed after exposure of mice to low levels of formaldehyde. A long-term exposure resulted in an increase in the number of tyrosine hydroxylase-immunopositive periglomerular cells and may affect the neuronal function of the MOB.

Rapid induction of Arc is observed in the granule cell dendrites in the accessory olfactory bulb after mating.

The activity-regulated cytoskeleton-associated protein (Arc), encoded by the immediate early gene arc, is enriched in the brain and is hypothesized to play a role in the activity-dependent neuronal plasticity in the hippocampus. In the present study, the time course of Arc expression during the post-mating period was determined immunocytochemically, and the localization of Arc in the neurons in the accessory olfactory bulb (AOB) of female mice after mating was analyzed using immunocytochemical electron microscopy. Transient increases in the number of Arc-immunoreactive cells were observed in the glomerular, mitral/tufted cell and granule cell layers of the AOB after mating. In particular, the increase in the granule cell layer was remarkable, and larger than the increases in the other layers. In addition, electron microscopic observation revealed that Arc immunoreactivity was in the dendrites of the granule cells 1.5 h after mating. These results indicate that expression of Arc protein is induced rapidly and transiently in granule cell dendrites after mating. It is postulated that Arc protein has a role in the neuronal plasticity of the AOB after mating.

A comparative immunocytochemical study of development and regeneration of chemosensory neurons in the rat vomeronasal system.

Vomeronasal neurons undergo continuous neurogenesis during development and after neuronal injury. We used immunocytochemical methods to compare different stages of the vomeronasal organ development to those of regeneration following vomeronasal nerve transection. At E15 and at 6 to 10 days after injury, nestin-positive cells were observed throughout the sensory epithelium. We did not find nestin immunoreactivity to be localized to the boundary region of the epithelium. The early appearance and wide distribution of nestin-positive cells suggests that they represent chemosensory precursor cells that develop and migrate vertically in the epithelium. Vomeronasal receptor cells degenerated 6 to 8 days after nerve transection, but axon terminals in the accessory olfactory bulb (AOB) continued to show the presence of the chemosensory specific marker (OMP) for up to ten days, a significant finding observed in this study. It is likely that the distance from the site of nerve transection may contribute to differences in the time course of anterograde and retrograde axon degradation. OMP-positive neurons were observed in the normal adult epithelium and to a much lesser extent 10-60 days after recovery from nerve transection. Axons from regenerated receptor cells did not reach the AOB during this time period. This failure to reestablish connections with target cells in the AOB could explain why OMP-positive cells were rarely observed among the regenerated cells in the vomeronasal epithelium.