Actin bundles in perineurial cells of rat spinal nerves

The overall distribution of the actin cytoskeleton in perineurial cells of rat spinal nerves was examined by confocal laser and thin-section electron microscopy. Confocal laser microscopy of whole-mount nerves stained with fluorescent-labelled phalloidin revealed two types of actin bundles in perineurial cells; stress fiber-type actin bundles and circumferential actin bundles. The degree of development of the actin cytoskeleton varied in different segments of different nerves. Stress fiber-type actin bundles were also immunostained for myosin and vinculin and were well-developed in the perineurial cells of large-sized nerves and dorsal root ganglia, whereas they were poor in spinal nerve root sheaths within the subarachnoid space. In peripheral nerves, stress fiber-type actin bundles tended to be arranged transverse to the nerve axis. Circumferential actin bundles were localized along intercellular junctions, which were immunostained with several junctional proteins such as –catenin, occludin and ZO-1. Thin-section electron microscopy confirmed the distribution pattern of actin bundles observed by confocal laser microscopy. These findings suggest that actin bundles may play some roles in structurally stabilizing the perineurium by providing mechanical support for the cell layers as well as cell junctions to maintain perineurial integrity and form diffusion barriers in peripheral nerves.     

Distribution of Ca(2+)/calmodulin-dependent protein kinase I beta 2 in the central nervous system of the rat

We have examined the distribution of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-6 (BMP-6) in the brain of rats subjected to a mild and reversible ischemic damage produced by a 20-min occlusion of both carotid arteries without occlusion of the vertebral arteries. We have selected this model to study how the expression of trophic factor of the TGF-beta superfamily changes in neurons that recover from a transient insult. Immunocytochemical analysis showed a loss of TGF-beta1 in neurons of all hippocampal subfields immediately after the ischemic period, followed by a recovery of immunoreactivity in CA1 and CA3 neurons after reperfusion. BMP-6 immunoreactivity was also lost in most hippocampal neurons, but immunostaining became particularly intense in the interstitial space after both ischemia and reperfusion. An interstitial localization of BMP-6 was also observed in the cerebral cortex, particularly after reperfusion. Mild ischemia also induced substantial changes in the expression of TGF-beta1 and BMP-6 within the cerebellar cortex. In control animals, these factors appeared to be localized in granule cells (TGF-beta1) and Purkinje cells (both), whereas the molecular layer was not immunopositive. Both TGF-beta1 and BMP-6 were highly expressed in the interstitial spaces of the cerebellar cortex either 20 min after ischemia or 20 min after reperfusion. Taken collectively, these results suggest that a mild and reversible ischemia stimulates the release of BMP-6 from neurons into the interstitial space. We speculate that BMP-6, besides functioning during brain development, may also regulate neuronal resistance to insults of the adult brain.     

Expression of Ca2+/calmodulin-dependent protein kinase (CaMK) Ibeta2 in developing rat CNS

We observed the onset time and distribution pattern of beta2 isoform of Ca2+/calmodulin-dependent protein kinase I (CaMKIbeta2) in the CNS of the rat during the embryonic period until birth using an immunohistochemical method. The expression of CaMKIbeta2 started at embryological day 10 when the three primary brain vesicles and neural tube are generated from the neural plate. During the embryonic period, highly immunoreactive products were ubiquitously detected in neurons in the CNS, although neurons in the caudate-putamen and globus pallidus were faintly immunostained or immunonegative. High expression of CaMKIbeta2 persisted in the olfactory bulb, lymbic system, neocortex, septal nuclei, amygdala complex, some hypothalamic nuclei, pontine nuclei, Purkinje cells and granule cells in the cerebellar cortex through the developing period. At the subcellular level, CaMKIbeta2 was strongly expressed in nuclei of neurons but faintly in their cytoplasm, suggesting that this protein has an important role in the nuclear signaling pathway.This study demonstrates that expression of CaMKIbeta2 begins at the earliest developmental stage of the rat CNS and persists through the developing period.