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A ROCK inhibitor Fasudil is widely administered to relieve vasospasm in patients after subarachnoid hemorrhage in Japan. We investigated the difference of Fasudil and Y-27632, a common ROCK inhibitor, on neurite regeneration in culture and axonal regeneration after injuring the optic nerve (OpN) in cats. The optimal dose of Y-27632, determined by counting the number and length of neurites in retinal explants, was found to be 100 microM: the only effect of Fasudil was to promote extension of glial processes. We next examined the effects of Fasudil (10 microM-100 microM) and Y-27632 (10 microM-300 microM) on axonal regeneration in the crushed OpN model in vivo. Immediately after crushing the left OpN, Fasudil or Y-27632 was injected into the vitreous and the crushed site. Injection of 10 microM and 100 microM Y-27632 induced extension of the optic axons beyond the crush site, with the latter dosage giving stronger regeneration. Very few axons passed beyond the crush site in the optic nerve with phosphate-buffered saline injection, and no axons elongated in the OpN with Fasudil injection.

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.

To study neuroprotective effects of bunazosin hydrochloride which is an alpha(1)-adrenoceptor antagonist used as an ocular hypotensive drug compared to other alpha(1)-adrenoceptor antagonists, and its mechanism of action. We evaluated the neuroprotective effects of bunazosin hydrochloride or seven other alpha(1)-adrenoceptor antagonists against glutamate-induced cell death in rat primary retinal cultures. We also evaluated the binding inhibition of bunazosin hydrochloride for 24 different receptors/channels and its effects on the Na(+) influx into cells induced by veratridine or glutamate. Bunazosin hydrochloride significantly inhibited glutamate-induced cell death at concentrations of 1 and 10 microM. Cells were also protected when treated with some alpha(1)-adrenoceptor antagonists, but not by the others. Bunazosin hydrochloride showed a high inhibition for Na(+) channels and inhibited the Na(+) influx induced by veratridine or glutamate. These findings indicate that in retinal cultures bunazosin hydrochloride has a neuroprotective effect against glutamate-induced cell death and that the inhibition of Na(+) channels by bunazosin hydrochloride may be partly responsible for this effect.

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.

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.

Postnatal cytogenesis in rat somatosensory cortex was examined by the technique combining light microscopic autoradiography with electron microscopy. Tritiated thymidine was injected intraperitoneally to rats on different days (0-21 days). All animals were sacrificed on the twenty-fifth day after birth. Coronal slices including somatosensory cortex were embedded in epoxy resin. Semithin sections for autoradiography and ultrathin ones for electron microscopy were made alternately. The labeled cortical cells were found mainly in the cases injected with [3H]thymidine during the first and the second weeks. Examination of laminar distribution of the labeled cells revealed that the cells in deeper laminae were labeled on earlier postnatal days than those in more superficial laminae. The labeled cells were examined with electron microscope to identify their nature. By this, it was revealed that ultrastructural morphology of the labeled cells were that of glial cells (either astrocyte or oligodendrocyte). Time and space pattern of this neocortical postnatal gliogenesis shows the tendency of the inside-out sequence, though the pattern is not as distinctive as that of prenatal neocortical neurogenesis. The relationships between the pattern of gliogenesis and the maturation of cortical neurons is suggested.

The rearrangement of terminations from the bed nucleus of stria terminalis (BST) was examined in the medial amygdaloid nucleus (MAN) at 2 months following the lesion of the accessory olfactory bulb (AOB) using an electron microscopy and degeneration study. At 2 days following a BST lesion, the number of degenerating synapses was 0.7 +/- 0.1 (mean +/- S.E.M.) per unit area (2500 microns2) in the molecular layer, and 3.0 +/- 0.3 in the cellular part. At 2 months after an AOB lesion, the degenerating synapses from the AOB had completely disappeared from the MAN. The BST was then lesioned at 2 months after the AOB lesion and, at 2 days following this BST lesion, the degenerating synapses were counted in MAN. The numbers observed were 3.3 +/- 0.6 per unit area in the molecular layer and 4.5 +/- 0.4 in the cellular part. Therefore, the number of these degenerating synapses increased significantly within the molecular layer, though, in the cellular part the number of synapses was not significantly elevated over control. No differences in postsynaptic profiles (ratio of synapses on dendritic spine to dendritic shaft) were observed after the AOB lesion. These results indicate that the BST fibers formed new synapses in the molecular layer following the denervation of AOB fibers. The possibility of new synapse formation by other afferent fibers in addition to the AOB fibers is discussed as is the relationship between lesion induced synaptic reorganization and functional recovery after injury.

The time course of the appearance of degenerating synapses and the loss and reappearance of intact synapses was investigated in the medial amygdaloid nucleus (MAN) following lesions of the accessory olfactory bulb (AOB). Degenerating synapses were concentrated in the molecular layer of MAN. A quantitative electron microscopic analysis of the molecular layer was performed at 2, 4, 8, 16, 32 and 64 days after the placement of kainic acid injections within AOB. At each survival time, counts of synapses were made from photomicrographic montages. The degenerating synapses were observed at 2 days after lesion. Their density reached a peak at 2 or 4 days after the lesion and decreased thereafter. No degenerating synapses were found at 64 days following the lesion. Shrinkage of the thickness of molecular layer was observed following the AOB lesion. This was most pronounced at 4 or 8 days after the lesion at which time the thickness was 70-75% of the contralateral molecular layer. The shrinkage receded moderately (85% of contralateral layer at 64 days after lesion). The compensated synaptic density of intact synapses was reduced to less than half of the control density at 4 or 8 days after the lesion. Thereafter synaptic density increased gradually and reached more than 80% of control density at 64 days after lesion. The recovery in number of intact synapses following the lesion suggests the possibility of synaptic reorganization by remaining afferent fibers.

It has been shown that male rats demonstrate a preference for the odor of the female rat. This preference has been suggested to be a function of the accessory olfactory pathway. Thus, changes in preference of the male rat for the odor of the female were examined following accessory olfactory bulb (AOB) removal. Preference decreased to about 30% of that seen in the intact rat during the 10 days following AOB removal. Thereafter, it increased gradually over one month survival time up to approximately 70% of the level demonstrated by the intact rat. The rats whose main olfactory bulb and AOB were removed did not show any recovery at any survival time. The time course of the behavioral recovery after AOB removal was compared with the time course of the recovery of synaptic density which was observed in the medial amygdaloid nucleus after AOB removal. The relationship between functional recovery and reorganization of synaptic connections is discussed.

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC4.6.1.2.) was histochemically demonstrated in rat brain with light and electron microscopes by using a specific monoclonal antibody to soluble guanylate cyclase from rat brain. Under a light microscope, intense reactions were seen in caudate-putamen complex, neocortex and cerebellar cortex. Immunoreactive cells were mainly some types of neurons such as small neurons in caudate-putamen complex, Purkinje cells in cerebellar cortex and pyramidal cells in neocortex. Some astroglial cells were also stained. Not all neurons or glial cells exhibited the positive guanylate cyclase reactivity. Electron microscopic examination revealed that guanylate cyclase was localized within postsynaptic components (perikaryon and dendrites) in neurons and in the cytoplasm and plasma membrane of astroglia. Presynaptic terminals were free of reaction. The observation supports a possibility that cyclic GMP is involved in the postsynaptic events of neuronal transmission and the regulation of intracellular processes.

Ultrastructural studies have aided in identifying the principal cell types and the layers of the olfactory bulb in goldfish. The concentrically arranged four layers are: the primary olfactory nerve layer (most peripheral), the glomerular layer, the mitral cell layer, and the granule cell layer (deepest). Three types of cell bodies were differentiated by their size and cytoplasmic organelles: mitral cells, granule cells and short axon cells. The axons of the primary olfactory nerve formed synaptic contacts with mitral cell dendrites in the glomeruli. Reciprocal synapses were observed between the granule cell dendrites and the mitral cell dendrites in the mitral cell layer. Terminals of the centrifugal fibers forming synaptic contacts with the granule cell dendrites were identified in the granule cell layer. The relationships between the cellular and synaptic organizations and the neural mechanism of olfaction are discussed.

The rearrangement of the terminations of intra-amygdaloid fibers was examined in the medial amygdaloid nucleus (MAN) following denervation of the fibers from the accessory olfactory bulb (AOB) of adult rat using immunohistochemistry of anterogradely transported lectin (Phaseolus vulgaris leucoagglutinin, PHA-L). The AOB was removed unilaterally by suction at two months before the PHA-L injection. The PHA-L was injected bilaterally into the posteromedial region of the amygdala (posteromedial amygdaloid nucleus and amygdala-hippocampal transitional area). After the tissue was processed by the routine immunohistochemical method, the immunologically labeled axons and axon terminals in the MAN were observed with the light microscope. On the control side, labeled axons were observed in the cellular part of the MAN, but they were not found in the molecular layer in which the fibers from the accessory olfactory bulb terminate. On the side of the AOB removal, the labeled axons were found not only in the cellular part but also in the molecular layer. Electron microscopic observations showed that the labeled axon terminals made synaptic contacts in the molecular layer. These results indicate that terminals of intra-amygdaloid fibers expand from the cellular part to the molecular layer in the MAN and make synaptic contacts in this layer following the denervation of the AOB fibers.

Changes of terminal connections of projection fibers from area 2 of the sensory cortex to the motor cortex following chronic lesion in the thalamus were examined using the electron microscope. The lesioned areas included nucleus ventralis anterior, n. ventralis lateralis and rostral part of n. ventralis posterolateralis. The synaptic sites were identified using the Golgi impregnation method to identify postsynaptic neurons in the motor cortex and the axonal degeneration method to identify presynaptic terminals of fibers originating from area 2. The following results were obtained. (1) The number of degenerating terminals per unit area in the motor cortex was increased to nearly twice that in normal animals. (2) The number of degenerating terminals synapsing with stellate cells was not increased but stayed more or less the same as in normal animals. (3) The number of degenerating terminals contacting pyramidal cells increased substantially, to more than twice that in normal animals. (4) These newly formed synapses were found on proximal dendritic shafts of the pyramidal cells in both layers III and V, suggesting that these synapses occupied the spaces where the thalamocortical terminals were located. (5) The functional significance of these newly formed synapses was discussed in relation to the recovery of motor function following thalamic lesion.

Rats were injected with [3H]thymidine on different postnatal days (PD 0 approximately PD 16) and sacrificed after 6 h. Parietal cortices were embedded in epoxy resin and then semithin sections for autoradiography and ultrathin ones for electron microscopy were made alternately. On the day of birth (PD 0), about 70% of total labeled cells within the cortical gray, which were proved to be glial cells, were observed in the inner half of the cortical width. On PD 4, however, the intracortical distribution of labeled cells was reversed, i.e. about 70% of labeled cells were found in the outer half. Thereafter, on PD 6 and PD 8, no significant difference of the number of the labeled cells could be noted between the outer and the inner halves of the cortical gray. The pattern of glial proliferation in the parietal cortex of early postnatal rat thus showed an inside-out tendency, although not so distinctive as that of prenatal neurogenesis in the neocortex. Electron microscopy of the labeled cells revealed that on earliest days, they showed the condensation of nuclear chromatin and abundant free ribosomes, both suggesting the immaturity of cells, while, on later days, the nuclear chromatin became dispersed and free ribosomes decreased, indicating the progress of cellular differentiation as glial cells.