Haruo Okado

Extension of glial processes by activation of Ca2+-permeable Ampa receptor channels

AMPA type-glutamate receptor channels (AMPARs) assembled without the GluR2 (GluR-B) subunit are characterized by high Ca2+ permeability, and are expressed abundantly in cerebellar Bergmann glial cells. Here we show that the morphology of cultured Bergmann glia-like fusiform cells derived from the rat cerebellum was changed by manipulating expression of Ca2+-permeable AMPARs using adenoviral vector-mediated gene transfer. Converting endogenous Ca2+-permeable AMPARs into Ca2+-impermeable channels by viral-mediated transfer of GluR2 gene induced retraction of glial processes. In contrast, overexpression of Ca2+-permeable AMPARs markedly elongated glial processes. The process extension was blocked by 2,3-Dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX), a specific antagonist of AMPAR. These results indicate that glutamate regulates the morphology of glial processes by activating Ca2+-permeable AMPARs.

Bcl-2 expression by retrograde transport of adenoviral vectors with Cre-loxP recombination system in motor neurons of mutant SOD1 transgenic mice.

We investigated genes expression by retrograde axonal transport of replication-defective adenoviruses carrying genes for LacZ (AdLacZ) and Bcl-2 in motor neurons of transgenic mice expressing mutant human Cu/Zn superoxide dismutase (SOD1) gene containing a substitution of alanine for glycine at position 93. We found that intramuscular injection of AdLacZ into the tongue of mutant SOD1 transgenic mice and their wild-type littermates at various ages results in high expression of the transgene and similar time course of expression in hypoglossal cranial nerve nuclei, suggesting no difference in the behavior of the transgene expression between the two groups. Subsequently, we employed a molecular switching cassette for Bcl-2 designed to express Bcl-2 by Cre-loxP recombination using adenoviral vectors, and examined the COS7 and primary neuronal cells with the mutant SOD1 gene. The overexpression of Bcl-2 in both cells and the neuronal protection against staurosporine-induced apoptosis were observed, after dual infection of adenoviral vectors with cassette for Bcl-2 (AxCALNLBcl-2) and Cre recombinase (AxCANCre). After inoculation of AxCALNLBcl-2 followed by AxCANCre into the tongue of both mutant SOD1 transgenic mice and wild-type littermates, Bcl-2 was detected in both the injection site and the hypoglossal nuclei of brainstems, suggesting that this was the result of retrograde transport of AxCALNLBcl-2 and AxCANCre and expression of Bcl-2 by Cre recombinase in the hypoglossal nuclei. This strategy for delivery of exogenous genes such as Bcl-2 will be useful for studying neuronal death/survival and introducing foreign genes into postmitotic motor neurons, and in gene therapy for motor neuron diseases such as ALS.

Callosal commissural neurons of Dab1 deficient mutant mouse, yotari

The yotari mouse is an autosomal recessive mutant mouse, caused by mutation of disabled homolog 1 (Dab1) gene. The mutant mouse is recognized by unstable gait and tremor and by early deaths around at the time of weaning. The cytoarchitectures of cerebeller and cerebral cortices and hippocampal formation of the yotari mouse are abnormal. These malformations strikingly resemble those of reeler mouse. In the present study we examined the callosal commissural (CC) neurons of yotari, reeler and normal mice with the injection of recombinant adenovirus into the frontal area 1 (Fr1) to find some possible phenotypes specific for the yotari mouse. The distribution pattern of CC neurons of the yotari was similar to that of the reeler: retrogradely labeled CC neurons were seen throughout all depths of the contralateral Fr1. However, the present statistical analysis revealed that the difference of the mean intracortical position of the CC neurons between the yotari and the reeler is significantly different (Students t-test), suggesting that the phenotype of the yotari is clearly different from that of the reeler.

Expression of AMPA receptors in rat superior colliculus and effect of orbital enucleation

We analyzed the distribution and the morphological characteristics of neurons expressing AMPA-type glutamate receptor subunits (GluR1 and GluR2) in the superficial partition (stratum zonale (SZ), stratum griseum superficiale (SGS) and stratum opticum (SO)) of the rat superior colliculus. GluR1-expressing neurons had round or ovoid somata in SGS and round or fusiform somata and primary dendrites extending tangential or horizontal side in SO. On the other hand, GluR2-expressing neurons mainly corresponded to vertical fusiform cells with vertically oriented dendrites in SGS and medium-sized stellate or ovoid cells with many primary dendrites in SO. The results suggest that the expressions of GluR1 and GluR2 are differentially regulated in individual neurons of the superficial partition. To analyze the effect of retinal deafferentation on the expression of the GluRs, we performed unilateral orbital enucleations in rats within a week after birth. Thirty days after retinal lesioning, lower expression of GluR2 mRNA was observed in the neurons of the contralateral side as compared with that of the ipsilateral side in SO, but not in SGS. These results indicate that GluR2 expression in the SO neurons is regulated by the correct afferentation from the retina.

Expression of recombinant NMDA receptors in hippocampal neurons by adenoviral-mediated gene transfer

N-methyl-d-aspartate (NMDA) receptors have attracted a great deal of attention because they are intimately involved in brain development, synaptic plasticity and a variety of neurological disorders. The ability to artificially alter the properties of NMDA receptors in central nervous system (CNS) neurons would be useful for elucidating the physiological roles of these receptors. It would also raise the possibility of gene therapy of neurological diseases caused by malfunction of NMDA receptors. In this study, we constructed three recombinant adenoviruses encoding rat NMDA receptor subunit cDNAs, NMDAR1 (NR1), NMDAR2B (NR2B) and mutant NR1(N598R) in which the asparagine (N) site of the wild-type NR1 was replaced with arginine (R) by site-directed mutagenesis. PC12 cells co-infected with recombinant adenoviruses bearing NR1 and NR2B cDNAs expressed conventional NMDA receptors that were permeable to Ca2+ and sensitive to Mg2+, whereas those with viruses bearing NR1(N598R) and NR2B cDNAs expressed Ca2+-impermeable and Mg2+-insensitive receptors. When rat hippocampal neurons in culture were infected with NR1(N598R) and NR2B viruses, both Ca2+ permeability and Mg2+ sensitivity of NMDA receptors were markedly reduced in the infected neurons. Excitatory postsynaptic currents (EPSCs) mediated by NMDA receptors also became much less sensitive to Mg2+. Thus, the NR1(N598R)/NR2B receptors were more dominant than the native NMDA receptors in the infected neurons, and the former receptors introduced by the adenoviral vectors functioned as postsynaptic receptors. These results indicate that the functional properties of postsynaptic NMDA receptors can be manipulated by gene transfer technology using adenoviral vectors.