Shin-ichi Murase

Migration of Perilesional Microglia after Focal Brain Injury and Modulation by CC Chemokine Receptor 5: An In Situ Time-Lapse Confocal Imaging Study

Microglia rapidly become reactive in response to diverse stimuli and are thought to be prominent participants in the pathophysiology of both acute injury and chronic neurological diseases. However, mature microglial reactions to a focal lesion have not been characterized dynamically in adult vertebrate tissue. Here, we present a detailed analysis of long-distance perilesional microglial migration using time-lapse confocal microscopy in acutely isolated living slices from adult brain-injured mice. Extensive migration of perilesional microglia was apparent by 24 h after injury and peaked at 3 d. Average instantaneous migration speeds of ∼5 μm/min and peak speeds >10 μm/min were observed. Collective, directed migration toward the lesion edge was not observed as might be expected in the presence of chemoattractive gradients. Rather, migration was autonomous and could be modeled as a random walk. Pharmacological blockade of the cysteine-cysteine chemokine receptor 5 reduced migration velocity and the number of perilesional migratory microglia without affecting directional persistence, suggesting a novel role for chemokines in modulation of discrete migratory parameters. Finally, activated microglia in the denervated hippocampal stratum oriens did not migrate extensively, whereas human immunodeficiency virus-1 tat-activated microglia migrated nearly twice as fast as those at the stab lesion, indicating a nonuniform microglial response to different stimuli. Understanding the characteristics and specific molecular mechanisms underlying microglial migration after neural injury could reveal novel targets for therapeutic strategies for modulating neuroinflammation in human diseases.

Apoptosis regulates the number of Schwann cells at the premyelinating stage

Abstract: At the premyelinating stage, the Schwann cells of peripheral nerves are able to recognize the axon, to arrange themselves along it in a nonoverlapping manner, and finally to establish a one‐to‐one cell‐axon relationship. The mechanism that regulates these processes is not known in detail. We found the existence of a significant Schwann cell apoptosis in vivo of rat postnatal sciatic nerve, peaking around postnatal day 3. More than 50% of the neonatal Schwann cells cultured in axon‐free medium undergo a rapid apoptosis. The apoptosis can be suppressed by addition of survival factors such as Neu differentiation factors or by increasing the adhesion of Schwann cells to substratum. We suggest that in neonatal nerves in vivo, Schwann cells are highly susceptible to apoptosis, but they are saved from death by contact with axons. The dramatic increase in number of Schwann cells between postnatal day 0 and 3 overcomes the number of axons available for them. Consequently the Schwann cells that fail to contact an axon undergo apoptosis. In conclusion, the number of Schwann cells in the developing nerves is regulated by the apoptosis and clearly depends on the survival signals from axons.

Expression pattern of integrin ?1 subunit in purkinje cells of rat and cerebellar mutant mice

We found that integrin β1 subunit (INTβ1)‐immunoreactive Purkinje cells first appeared caudally at postnatal day (PD) 6 of rat and most Purkinje cells gradually became positive by PD 12. The expression of INTβ1 was then suppressed in some of these cells, so that the positive Purkinje cells in the adult were organized into parasagittal bands interposed by negative cells throughout the vermis and hemispheres. When Purkinje cells were deprived of their climbing fiber innervation by inferior cerebellar pedunculotomy or by transplantation of cerebellar anlagen into the anterior eye chamber, the subsequent patterning of INTβ1‐positive Purkinje cells was not changed. In both reeler and weaver mice, the INTβ1‐positive parasagittal bands were observed, however, the Purkinje cells in the staggerer mice did not express INTβ1 at any stage. These data suggest that the expression of INTβ1 in Purkinje cells is genetically programmed in the developing cerebellum, and that the afferent synaptic inputs by climbing and parallel fibers are not prerequisites for INTβ1 expression in Purkinje cells. Therefore, the unique distribution patterns of INTβ1‐positive Purkinje cells provides a new marker for postnatal development of rodent cerebella.

Integrin ?1 localization in murine central and peripheral nervous system

The distribution of neurons expressing integrin α1 subunit protein (INTα1) was examined in adult mouse tissues of not only the central nervous system, but also the sympathetic ganglia, and the adrenal gland by immunohistochemistry and immunoelectron microscopy. INTα1‐positive neurons were observed in most tissues examined, and most of them were found to coexpress tyrosine hydroxylase (TH) except for Purkinje cells and hippocampal neurons. Expression of INTα1 was also observed in the malpositioned cortical neurons in reeler mutants, and appeared not to be affected by the aberrant cell migration of the reeler cortical neurons. In situ hybridization showed that the expression of INTα1 mRNA was correlated with synthesis of the INTα1 protein in each case, and this finding indicated that expression of the protein was controlled by transcriptional regulation of the INTα1 gene. J. Comp. Neurol. 395:161–176, 1998.

Concomitant expression of genes encoding integrin αvβ5 heterodimer and vitronectin in growing parallel fibers of postnatal rat cerebellum: A possible role as mediators of parallel fiber elongation

External granule cells in the premigratory zone and the upper molecular layer of neonatal rat cerebellum elongate their neurites (parallel fibers) bidirectionally before and during migration into the internal granular layer. In the present study, it is shown that integrin αvβ5 heterodimer (INTαvβ5) is expressed in parallel fibers in these layers at postnatal days 3–20, but not in migrating granule cells or mature parallel fibers. Vitronectin (VN), the dominant ligand for INTαvβ5, was concomitantly detected in the premigratory zone and the upper molecular layer during this period. Several other subunits including α1–6 and β1–4 were not detected. When granule cells were prepared from postnatal cerebella and cultured for a few days, the parallel fibers elongated well in response to VN, but the granule cells did not migrate on VN. This fiber elongation was specifically inhibited by both anti‐INTαvβ5 antibody and peptides containing Arg‐Gly‐Asp (RGD), a sequence responsible for cell adhesion mediated by VN. Neither control integrin antibody against integrin αvβ3 heterodimer nor control peptides containing Arg‐Gly‐Glu (RGE) showed an inhibitory effect on fiber elongation. These observations strongly suggest that the INTαvβ5 VN receptor plays a role in the elongation of parallel fibers from granule cells during cerebellar histogenesis, but its expression is not required for their maintenance or granule cell migration. INTαvβ5 could be considered as a new marker of parallel fibers during cerebellar development. J. Comp. Neurol. 397:199–212, 1998.

Neuronal Expression of Macrophage Colony Stimulating Factor in Purkinje Cells and Olfactory Mitral Cells of Wild-type and Cerebellar-mutant Mice

Macrophage colony stimulating factor (M-CSF) is known to be the most effective growth factor for macrophage and microglial proliferation. In the brain tissue system, M-CSF is mainly produced in astrocytes and microglia, but is not known to occur in neurons. In the present paper, we examined the distribution of neurons expressing M-CSF in the mouse brain by immuno-histochemistry and in situ hybridization. We observed M-CSF immunoreactivity in both the cerebellum and the olfactory bulb. These positive cells were found to be Purkinje cells in the cerebellum, and mitral cells in the olfactory bulb. M-CSF mRNA expression was also confirmed to occur in these cells. Purkinje cells of reeler and weaver mutants showed M-CSF expression as seen in wild-type mice; however, those in the staggerer mutant did not. This expression in wild-type mice first appeared at postnatal day 7 and continued stably thereafter. When Purkinje cells were deprived of their climbing fibre innervation by inferior cerebellar pedunculotomy or by transplantation of cerebellar anlagen into the anterior eye chamber, the expression of M-CSF remained unchanged. These data indicate that expression of M-CSF in Purkinje cells is controlled by an intrinsic mechanism and could, therefore, be a new marker of postnatal development in rodent cerebella. The absence of M-CSF expression in the staggerer mutant is possibly due to developmental arrest in the early postnatal period.

Transplantation of embryonic olive in the climbing-fiber-deprived adult rat cerebellum: synaptogenesis on host Purkinje dendritic spines by donor climbing fibers.

Synaptic formation by donor climbing fibers on the host Purkinje spines was observed in our experiments. In the adult rat cerebellum where the inferior olive and climbing fibers had been destroyed by intraperitoneal injection of 3-acetylpyridine, the medullary embryonic 14-16-day (E14-E16) tissue containing the olive was grafted. After 3 weeks, climbing-fiber-type preterminals bearing closely-packed round vesicles were found that established synaptic contacts on dendritic spines of the host Purkinje cells. Quantitative analysis indicates the increment of newly-formed climbing presynaptic terminals, the number of which was statistically significant (P less than 0.01).