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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.

A sex difference has been reported in the responsiveness of the vomeronasal (VN) system to pheromones. In the present study, to clarify a direct and acute influence of 17β-estradiol (E2) on the accessory olfactory bulb (AOB) neurons, we investigated the effect of E2 on dendritic spines in cultured AOB cells derived from male and female neonatal rats. After 17-18 days in vitro (DIV), cultured AOB cells were transfected with GFP expression vectors. At 21-23 DIV, cells were treated with E2, and time-lapse images of transfected AOB neurons identified as granule cells were taken under a confocal laser scanning microscope for 3h. The dendritic spine head area of granule cells was quantitatively evaluated, and spine heads were classified into larger (≥ 1 μm²) and smaller (<1 μm²) ones before E2-treatment (0 h). In cultured cells derived from both sexes, the larger spines were not significantly changed at 1, 2 and 3 h after E2-treatment. In contrast, E2-treatment significantly enlarged the head area of the smaller spines of granule cells derived from the female, whereas E2 did not cause any significant effects on those from the male. Our results provide evidence for the sexually-dimorphic effect of E2 on spine development in AOB granule cells., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2011 S. Karger AG, Basel.)

One of the major challenges in brain research is to unravel a network of molecules, neurons, circuits and systems that are responsible for dynamic and hierarchical brain functions. To understand molecular events that occur in synapses could be an important key to exploring the mechanism of information processing. A spatiotemporal recording method is required to observe neuronal activities in a particular local circuit and to resolve single synaptic potential with high resolution. As alternative methods, real-time imaging using fluorescent probes and optical recording methods are also a powerful approach for investigating the molecular dynamics of biological events in neurons in vitro and in vivo. Recently, optical imaging techniques have become of great importance to visualize the molecular dynamics in a micron-sized compartment of a single neuron such as neuronal synapse. In general, the presynaptic axon forms synapses at the postsynaptic site on the dendritic spines in the mammalian central nervous system. Subsets of the synapses undergo a series of enduring changes in spine shape and density as well as alterations in electrophysiological functions. Here we describe recent optical imaging studies conducted by elaborate methods and techniques that provide evidence for the link between neural activity and molecular dynamics., (Copyright 2008 S. Karger AG, Basel.)

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.

Bunazosin hydrochloride is a potent and selective alpha1-adrenoceptor antagonist that has been clinically used both as a systemic antihypertensive as well as an ocular hypotensive drug. In a number of studies, we have examined some effects of bunazosin hydrochloride that might indicate its potential as an anti-glaucoma drug. In normal rabbit eyes, topically instilled bunazosin hydrochloride reached the posterior retina by local penetration at concentrations sufficient to attenuate the phenylephrine- or endothelin-1 (ET-1)-induced constriction of retinal arteries. Furthermore, bunazosin hydrochloride improved the impairment of optic nerve head (ONH) blood flow, the prolongation of visual-evoked potentials (VEP) implicit time, the enlargement of the optic disk cup, and the decrease in the number of retinal ganglion cell layer cells induced by repeated injections of ET-1 in rabbits. Topically instilled bunazosin hydrochloride improved the reductions in ONH capillary blood flow and VEP amplitude induced in rabbit eyes by nitric oxide synthase inhibition. In rat primary retinal cultures, bunazosin hydrochloride reduced glutamate-induced neuronal cell death, presumably through a Na+ channel blocking effect. In healthy humans, topically instilled bunazosin hydrochloride reportedly increases blood velocity in the ONH, retina and choroid, without significantly altering either blood pressure or heart rate. These results indicate that bunazosin hydrochloride exerts both an improvement effect within the ocular circulation and a direct neuroprotective effect. Hence, bunazosin hydrochloride may be useful as a therapeutic drug against ischemic retinal diseases (such as glaucoma and retinal vascular occlusive diseases) that are associated with disturbances of the ocular circulation.

We previously established a primary culture system of the accessory olfactory bulb (AOB) to investigate the functional roles of individual types of neuron in pheromonal signal processing. However, the detailed characteristics of cultured AOB neurons were not yet apparent. In the present study, we address the cytological aspects of cultured AOB neurons using immunocytochemical staining methods. Cultured AOB neurons were compared with cultured main olfactory bulb (MOB) neurons in neuronal composition, maturational time course, and cell size. The number of total neurons, measured by microtubule-associated protein 2 (MAP2) immunostaining, progressively decreased, and glutamic acid decarboxylase positive (GAD+) interneurons were scarcely changed in their number in both AOB and MOB cultures over the culture periods. In contrast, the number of tyrosine hydroxylase positive (TH+) neurons in AOB cultures showed a slight, but significant, increase over time in culture, while those in MOB cultures remarkably decreased. The numbers of total neurons and GAD+ neurons were significantly greater in AOB cultures than in MOB cultures at all investigated time points. However, the numbers of TH+ neurons were lower at 7 days in vitro (DIV) and greater at 21 DIV in AOB cultures than in MOB cultures. The somatic sizes of all types of neurons at 14 DIV were significantly larger in AOB cultures than in MOB cultures. Furthermore, the frequency distributions of somatic sizes of total, GAD+, and TH+ neurons were significantly different between AOB and MOB cultures. These subtle differences in vitro may reflect in vivo differences between the AOB and MOB.

The characteristics of functional changes of GABAergic synapses between cultured rat cortical neurons were observed by monitoring intracellular calcium level ([Ca2+]in) during development in vitro. After 5 days in vitro (DIV), cultured cortical neurons spontaneously exhibited synchronous oscillatory changes in [Ca2+]in, which were derived from synaptic activity. Exposure to bicuculline, antagonist of gamma-aminobutyric acid (GABA)(A) receptors, caused a marked decrease in the frequency of [Ca2+]in oscillations at 7-20 DIV. Although the frequency of spontaneous oscillations increased during this culture period, the ratio of the decrease in the frequency following bicuculline treatment did not significantly change. Thereafter, to investigate the detailed morphological changes of GABAergic synapses during development in vitro, the cultured neurons were immunostained with antibodies to glutamic acid decarboxylase (GAD), synaptophysin and GABA(A) receptor and were observed under a confocal laser microscope. Most of the GAD-positive puncta colocalized with synaptophysin-positive puncta and were opposed to GABA(A) receptor-positive structures. The images of GAD-positive puncta were reconstructed from the confocal three-dimensional data to analyze their number, volume, and surface area. The number of these puncta increased with culture time at 7-20 DIV. Although the volume of individual GAD-positive puncta did not significantly change, the surface area decreased in a time-dependent manner over the culture period. This system that we developed enabled us to investigate in detail the morphological and functional changes of GABAergic synapses during neuronal development.

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.

Aggregation of disease proteins is believed to be a central event in the pathology of polyglutamine diseases, whereas the relationship between aggregation and neuronal death remains controversial. We investigated this question by expressing mutant huntingtin (htt) with a defective adenovirus in different types of neurons prepared from rat cerebral cortex, striatum or cerebellum. The distribution pattern of inclusions is not identical among different types of primary neurons. On day 2 after infection, cytoplasmic inclusions are dominant in cortical and striatal neurons, whereas at day 4 the ratio of nuclear inclusions overtakes that of cytoplasmic inclusions. Meanwhile, nuclear inclusions are always predominantly present in cerebellar neurons. The percentage of inclusion-positive cells is highest in cerebellar neurons, whereas mutant htt induces cell death most remarkably in cortical neurons. As our system uses htt exon 1 protein and thus aggregation occurs independently from cleavage of the full-length htt, our observations indicate that the aggregation process is distinct among different neurons. Most of the neurons containing intracellular (either nuclear or cytoplasmic) aggregates are viable. Our findings suggest that the process of mutant htt aggregation rather than the resulting inclusion body is critical for neuronal cell death.

Our previous study morphologically revealed that the adult goat vomeronasal (VN) system was different from the rodent and opossum one, and at least two types of VN systems exist in mammals. However, it remains unknown whether the developments in both types of VN systems are ontogenetically distinct and when the goat VN system is established. In this study, we morphologically observed the fetal development of the goat accessory olfactory bulb (AOB) and VN neuron. In the fetus, Gi2-expressing VN terminals terminated at glomeruli throughout the AOB, and no immunoreactivities for Go were detected in the nerve terminals reaching into AOB. The layer structure of AOB rapidly developed in the latter half of gestation. In the VN organ (VNO), at the middle stage of gestation, the dendritic processes of VN neuron were exposed in the VN lumen, and scattered and thin microvilli existed on the protrusion of the VN neuron. In the apical part of dendritic processes, no clear vesicle existed. However, the immunohistochemistry of an olfactory marker protein (OMP) revealed that a few VN neurons with OMP exist in VN sensory epithelium (VSE) before birth, although marked immunoreactivities were detected in adult VSE. Fetal VN neurons appeared to be underdeveloped. These results suggest that the goat VN system is ontogenetically distinct from the rodent and opossum VN systems, and is underdeveloped before birth. The goat VN system will develop and mature during the early postnatal period similar to the rodent and opossum VN systems.

To investigate the roles of the GABAergic inhibitory system of accessory olfactory bulb (AOB) in pheromonal memory formation, we have developed a primary culture system of AOB neurons, which had numerous excitatory and inhibitory synapses. Using this culture system of AOB neurons, we examined the correlation in rats between neuronal excitation and synaptic morphology by bicuculline-induced disinhibition of cultured AOB neurons. The exposure to bicuculline induced long-lasting oscillatory changes in the intracellular calcium level ([Ca2+]in) of cultured non-GABAergic multipolar neurons, which were identified as mitral/tufted cells (MT cells). These MT cells exhibited the appearance of dendritic filopodia structures after a 10-min treatment with bicuculline. By labelling presynaptic terminals with FM4-64, the appearance of new presynaptic terminals was clearly observed on newly formed filopodia after 120 min treatment with bicuculline. These results suggest that bicuculline-induced [Ca2+]in oscillation of MT cells induces the growth of filopodia and subsequently the formation of new presynaptic terminals. Furthermore, tetrodotoxin or the deprivation of extracellular calcium blocked bicuculline-induced synapse formation. The present results indicate that the long-lasting [Ca2+]in oscillation caused by bicuculline-induced disinhibition of cultured MT cells is significantly implicated in the mechanism underlying synapse formation on cultured AOB neurons. Our established culture system of AOB neurons will aid in clarifying the mechanism of synapse formation between AOB neurons and the molecular mechanism of pheromonal memory formation.

Purpose: To investigate whether conditioned medium from embryonic rat retinas can induce differentiation of adult rat hippocampus-derived neural stem cells (AHSCs) into neurons and glia in vitro., Methods: AHSCs were cultured in 3 types of media: standard culture medium, conditioned medium from embryonic rat retina, and standard culture medium with retinoic acid. Neuronal and glial differentiation of the cultured cells was assessed by cell growth analysis, flow cytometric analysis, immunofluorescent staining, and RT-PCR analysis., Results: Cells cultured in the standard medium showed very little neuronal and glial differentiation. The cells cultured in the conditioned medium and the medium with retinoic acid showed neuronal morphology and growth inhibition. They also expressed mature neuronal markers and glial markers. In addition, the cells cultured in the conditioned medium expressed Thy-1, HPC-1, and calbindin, which were not found in the previous studies with postnatal retinas in vivo. Those cultured in the medium with retinoic acid expressed HPC-1 and calbindin, but not Thy-1., Conclusions: Conditioned medium from embryonic rat retina contains factors that induce neuronal and glial cell differentiation of AHSCs, and promote up-regulation of some types of retinal cell markers., (Copyright 2003 S. Karger AG, Basel)

Previously, we established a culture system of the accessory olfactory bulb in order to investigate the functional role of each accessory olfactory bulb neurons in pheromonal signal processing. In the present study, we developed a co-culture system of cultured accessory olfactory bulb neurons with partially dissociated cells of the vomeronasal organ. The dissociated cells of the vomeronasal organ form spherical structures surrounding a central cavity in culture, referred to as the vomeronasal pockets. The projection and activity of olfactory receptor neurons affect the differentiation and maturation of main olfactory bulb neurons. It was also reported induction of tyrosine hydroxylase expression in main olfactory bulb neurons when they were co-cultured with explants of the olfactory epithelium. Thus, we investigated the effects of co-culture with vomeronasal pockets on the differentiation and/or maturation of cultured accessory olfactory bulb neurons in relation to tyrosine hydroxylase expression. The number of tyrosine hydroxylase-containing neurons developmentally increased over time in the accessory olfactory bulb culture. This increase was significantly enhanced by coculture with vomeronasal pockets. Interestingly, a significant change in tyrosine hydroxylase expression was not observed when main olfactory bulb neurons were co-cultured with vomeronasal pockets. Moreover, significant changes in tyrosine hydroxylase expression were not observed when accessory olfactory bulb neurons were co-cultured with olfactory epithelium explants, as was previously observed in co-culture of main olfactory bulb neurons and olfactory epithelium explants. These results suggest that the differentiation and/or maturation of accessory olfactory bulb neurons is modified by vomeronasal organ neurons via specific interactions between the sensory organ and its target.

PTB-like protein (PTBLP) is a new homologue of pyrimidine tract binding protein (PTB), and has been cloned as a possible autoantigen in cancer-associated retinopathy. PTBLP has two functional domains, the nuclear localization signal and the RNA recognition motifs (RRMs). Full-length PTBLP (PTBLP-L) has four RRMs, and its alternative splicing product (PTBLP-S) lacks the third and fourth RRMs. Although PTBLPs are expressed in neuronal tissues, the function of PTBLPs has not been determined. We have studed whether PTBLP plays a role in neuronal differentiation using PC12 cells. During the process of nerve growth factor-induced neuronal differentiation of PC12 cells, PTBLP-L was down-regulated whereas PTBLP-S was up-regulated. Transfection of PTBLP-L into PC12 cells led to the suppression of neuronal differentiation. In PTBLP-S transfected cells, however, this suppression was not evident. When both PTBLP-L and PTBLP-S were co-transfected, the suppressive effect of PTBLP-L decreased. In differentiated cells, PTBLP-S localized in the nucleus and PTBLP-L was found dispersed throughout the cytoplasm and neuronal growth cone. These findings suggest that PTBLP-L acts as a negative regulator of neuronal differentiation and PTBLP-S acts as a competitor of PTBLP-L.

Neural stem cells are capable of differentiating along multiple central nervous system cell-type lineages, and their use as graft material has provided new strategies for the treatment of neuronal damage. We transplanted adult rat hippocampus-derived neural stem cells into eyes of adult rats that underwent ischemia-reperfusion injury. As control, the cells were also injected into normal rats eyes without ischemic insult. The rats were sacrificed at 1, 2, 4, and 8 weeks, and the eyes were examined histochemically. In eyes with the insult, the transplanted cells were well integrated into the host retinas and expressed Map2ab. In the control, none of the cells migrated into the retina. These results suggest that neural stem cells may be used as donor cells for transplantation to repair ischemic-injured retina.

We have quantified the optical signals of synaptically induced neural activities in an in vitro brain slice preparation in terms of electrophysiological signals. The qualification was done using electrophysiologically well known neural activities in the CA1 area of rat hippocampal slices stained with externally applied fluorescent voltage-sensitive dye (VSD; Di-4-ANEPPS). Together with a newly designed CCD-based digital high-speed camera system and epi-fluorescent optics, our improvements were made on a protocol for staining using a newly designed chamber system. These improvements enabled us to make stable and reliable recordings of optical signals and electrophysiological measurements without affecting the physiological status and to make a quantitative comparison between them. The time course and amplitude of the optical signal showed fair agreement with intracellular and extracellular recordings, and was stable over 2 h. The optical signal followed synaptically induced long-term potentiation (LTP) as monitored by the electrophysiological signals. A regional difference in the amount of LTP was found in optical signals and was confirmed in the electrophysiological signals. These results demonstrate the capabilities of our improved method as an alternative but more potent tool to measure the neuronal activities of brain slice in addition to electrophysiological method.

Goats have a well-developed vomeronasal (VN) system and exhibit pheromone-induced reproductive facilitation, but there are no reports on the projection pattern of VN neurons in this species. Rodent, guinea pig and opossum accessory olfactory bulbs (AOBs) have been shown to have a segregated pattern of projection of the VN neurons, which express the two alpha-subtypes of the G-protein, namely Gi2 and Go, to the rostral and caudal regions of the AOB, respectively. In this study we investigated the projection pattern of VN nerve terminals by immunocytochemical staining of the goat vomeronasal organ (VNO) and the AOB with antibodies to Gi2 and Go. Gi2-immunoreactivity was found on the luminal surface of the sensory epithelium of the VNO, and in the VN nerve and glomerular layer throughout the AOB. On the other hand, Go-immunoreactivity was not identified in either the VNO or the VN nerve layer of the AOB. These results indicate that the projection pattern of VN neurons from the VNO to the AOB in the goat is considerably different from that in rodents which show a distinct segregated pattern.

The heterogeneous structure of the accessory olfactory bulb (AOB) has been demonstrated immunocytochemically. In this study, we analyzed the expression of an immediate-early gene protein, c-Fos, as a marker of neuronal activity in response to chemosensory cues was analyzed. The number of c-Fos-immunoreactive (Fos-ir) cells was measured in the rostral and caudal zones of the AOB in male ICR mice after exposure to the soiled bedding of female mice. The results revealed no significant difference in the number of Fos-ir cells in the caudal zone of the AOB between exposure to the soiled bedding of female ICR mice (ICR group) and exposure to that of female Balb mice (Balb group). In the rostral zone, however, the number of Fos-ir cells in the glomerular layer and granule cell layer was larger in the ICR group than in the Balb group. The difference in the expression of c-Fos in response to different pheromonal stimuli between the rostral and caudal zones in the mouse AOB has been shown for the first time in this study. These results strongly suggest that the heterogeneous structure of the AOB has an important role in the perception and processing of pheromones.

We have designed and constructed a high-speed CCD imaging system for optically detecting neural activity from preparations stained externally with a voltage-sensitive dye, and have used this system to image evoked and epileptiform neural activity in the rat somatosensory cortex. The imaging system uses a commercially available 1/3-in. CCD chip, and it can continuously capture images for more than 8 s, at 1000 frames/s, with a spatial resolution of 128 x 62 pixels. The spatial/temporal resolution of the CCD sensor is variable by changing the geometry of on-chip binning pixels, which can be controlled by a PC/AT computer. Dye bleaching correction was not necessary for long-term imaging of epileptiform neural events, since the sensitivity of the CCD sensor was increased by combining the signal from adjacent pixels.

Localization of neuronal nitric oxide synthase-immunoreactivity (nNOS-IR) in the median eminence of female rats (n=4) was examined by electron microscopy to explore the possibility that nitric oxide is involved in the terminal regulation of neurosecretory peptides such as GnRH. Under light microscopy, a dense distribution of nNOS-IR was observed in this region. Electronmicroscopically, nNOS-IR was found in glial elements and nerve terminals containing dense-core vesicles. We also found a few nNOS-immunopositive synapses, in which intense immunoreactivity was found on the postsynaptic density and mitochondrial membrane. The localization of nNOS-IR in nerve terminals and glial elements in the median eminence might indicate that nNOS plays a role in regulating the release of neurosecretory peptide., (Copyright 1998 Elsevier Science B.V.)

The expression of Brn-2, a central nervous systems (CNS)-specific POU domain transcription factor, in the developing mouse neocortex was examined with an anti-Brn-2 antibody. Brn-2 protein was first detected in CNS on embryonic day (E) 11.5, and remained strong until E15.5. From E11.5 to postnatal day (P) 0, a high level of Brn-2 expression was observed in the subventricular zone, the intermediate zone, and the outer layer of the neocortex, but not in the ventricular zone. In the double-staining experiments, most of the Brn-2 positive cells were also positive for NCAM-H, an adhesion molecule specific to post-mitotic neurons. Furthermore, BrdU-labeling experiments demonstrated the presence of Brn-2 protein exclusively in postmitotic cells. These results indicated that, in the developing neocortex, Brn-2 expression is up-regulated after the final cell division. Therefore, this transcription factor may be involved in the migration and/or maturation process of the immature neuronal cells.

It has been proposed that synchronous oscillations of groups of neurons corresponding to sensory information and changes in temporal pattern of oscillations are important for processing of the information in the cortex. However, it has not been determined yet how the temporal or spatial pattern of such oscillations are regulated. We observed spontaneous synchronous oscillations of Ca2+ transients, which were caused by bursts of action potentials of neurons, even in cultured cortical neurons. The frequency of synchronous Ca2+ oscillations increased with development of synapses in cultured neurons and was highly correlated to the number of synapses formed in the same culture.

Cerebral cortical cells from fetal rats (18 days) were cultured for 3, 7, 14, 21, 28, 35 days in vitro (DIV) and the development of synapses was examined morphologically by electron microscopy. At 3 DIV, no synapses could be recognized. An immature type of synapse was found at 7 DIV which thereafter developed morphologically. The length of the synaptic contact zone (SCZ) increased with DIV from 271 +/- 11.4 nm (mean +/- SEM) at 7 DIV to 410 +/- 11.4 nm at 35 DIV. The number of synaptic vesicles per terminal also increased with DIV: 10.0 +/- 1.2 at 7 DIV, 35.7 +/- 3.4 at 21 DIV, and 53.3 +/- 4.5 at 35 DIV. The time course of numerical density of synapses was examined quantitatively by electron microscopy. Synaptic density was very low at 7 DIV. It was significantly increased at 14 DIV and thereafter showed variable changes. Four culture series showed decreases after 14 DIV, but one series showed a further increase at 21 DIV followed by a decrease at 28 DIV. The mean density of synapses at each DIV was as follows: 1780 +/- 86/10(6) microns3 at 7 DIV, 4244 +/- 595/10(6) microns3 at 14 DIV, 2285 +/- 674/10(6) microns3 at 21 DIV, 2552 +/- 646/10(6) microns3 at 28 DIV, and 2080 +/- 532/10(6) microns3 at 35 DIV. Neuronal cell density was counted in each culture to calculate the relative number of synapses per neuron. The cell density decreased with age from 301 +/- 51/10(6) microns3 at 7 DIV to 39 +/- 9/10(6) microns3 at 35 DIV.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of differential rearing on synaptic morphology and neuronal size were examined in the medial amygdaloid nucleus (MAN) of adult rats. Forty-day-old male rats were housed in one of three ways: individually (isolated condition, IC); with four males per cage (unisexual condition, UC); or with two males and two females per cage (social condition, SC). After 2 months, the animals were prepared for electron and light microscopy. Although there was no statistically significant difference in number of synapses per unit volume in MAN, the number of perforated (P) synapses, which are characterized by discontinuities in the postsynaptic density, were significantly greater in the UC and the SC than in the IC. The length of synaptic contact zone of P synapses was also longer in both the UC and the SC compared with the IC, whereas the length of nonperforated synapses was longer only in the SC. Soma area was also larger in the SC compared with the IC. These results demonstrate that exposure to different rearing conditions, in which the pheromonal environment can be substantially different, can induce striking morphological changes in both synapses and neurons in the MAN of adult rats.

Among all the neural cells in fetal rat brain culture developing neurons showed the highest rate of infection by Japanese encephalitis virus (JEV). JEV specifically bound to these cells as measured by immuno-staining. These results indicate that developing neurons are the major target of JEV, and that the initial specific binding of virus to these cells may be one of the reasons for the neurotropism of JEV.

Recently we have been studying gonadotropin-releasing hormone (GnRH) cells of the terminal nerve (TN) in the dwarf gourami, which may serve as a good model system for the study of neuromodulator functions. Here we report on the ultrastructural characterization of TN-GnRH cells using postembedding immunoelectron microscopy. The GnRH immunoreactivities could be demonstrated on dense-cored vesicles (DCVs) in cell bodies, fibers and varicosities. However, we could find no evidence of GnRH-immunoreactive synapses which are characterized by active zones. This may suggest that GnRH is released non-synaptically from DCV-containing fiber varicosities and that it exerts its modulatory action on GnRH receptors located on nearby as well as distant target neurons.

Cerebral cortical cells from fetal rats (14 days) were cultured in vitro (7-104 days). Localization of synaptophysin was examined immunocytochemically using a monoclonal antibody (SY-38) against synaptophysin. Synaptophysin-immunoreactive (SP-IR) spots were distributed on the surface of the cells. Electron-microscopic observation showed that the SP-IR staining was localized at presynaptic terminals, indicating that the SP-IR spots observed will, fluorescence microscopy represent presynaptic terminals. The time-course of the distribution of SP-IR spots was examined. SP-IR spots could not be observed in cells at 7 days in vitro (DIV). At 14 DIV, SP-IR spots could be first observed on the surface of cell bodies and neurites. Thereafter, the density of SP-IR spots on the cell bodies and neurites increased with DIV. At 42 and 104 DIV, SP-IR spots were almost continued, surrounding the neurites and cell bodies of the cultured cells. These immunocytochemical studies suggest that the dissociated 14-day fetal cerebral cortical neurons make synapses by at least 14 DIV and that the density of synapses increases with time.

In a study on Fischer rats, all animals infected with Japanese encephalitis virus (JEV) before the age of 13 days died, but animals infected after the age of 14 days did not die, confirming the age-dependent resistance to JEV infection in the rat brain. A study of the kinetics of JEV infection in the developing rat brain disclosed that JEV antigen disappeared in a particular pattern, i.e., from the deeper layers to the upper layers of the motor cortex, which paralleled neuronal maturation in the cortex. Fifteen-day-old rats, which were resistant to JEV infection, received intracerebral transplants of neurons taken from 19-day embryos. When these animals were infected with JEV after transplantation, viral antigen was detected only in the embryonal neurons soon after transplantation. Thus, it can be concluded that the susceptibility to JEV infection in the rat brain is closely associated with neuronal immaturity.