The function of striatal adenosine A2Areceptors (A2ARs) is well recognized because of their high expression levels and the documented antagonistic interaction between A2ARs and dopamine D2receptors in the striatum. However, the role of extrastriatal A2ARs in modulating psychomotor activity is largely unexplored because of the low level of expression and lack of tools to distinguish A2ARs in intrinsic striatal versus nonstriatal neurons. Here, we provided direct evidence for the critical role of A2ARs in extrastriatal neurons in modulating psychomotor behavior using newly developed striatum-specific A2AR knock-out (st-A2AR KO) mice in comparison with forebrain-specific A2AR KO (fb-A2AR KO) mice. In contrast to fb-A2AR KO (deleting A2ARs in the neurons of striatum as well as cerebral cortex and hippocampus), st-A2AR KO mice exhibited Cre-mediated selective deletion of the A2AR gene, mRNA, and proteins in the neurons (but not astrocytes and microglial cells) of the striatum only. Strikingly, cocaine- and phencyclidine-induced psychomotor activities were enhanced in st-A2AR KO but attenuated in fb-A2AR KO mice. Furthermore, selective inactivation of the A2ARs in extrastriatal cells by administering the A2AR antagonist KW6002 into st-A2AR KO mice attenuated cocaine effects, whereas KW6002 administration into wild-type mice enhanced cocaine effects. These results identify a critical role of A2ARs in extrastriatal neurons in providing a prominent excitatory effect on psychomotor activity. These results indicate that A2ARs in striatal and extrastriatal neurons exert an opposing modulation of psychostimulant effects and provide the first direct demonstration of a predominant facilitatory role of extrastriatal A2ARs.

We previously demonstrated by site-directed mutagenesis analysis that the amino acid residues at positions 62 and 214 to 216 in the N-terminal region of mouse hepatitis virus (MHV) spike (S) protein are important for receptor-binding activity (H. Suzuki and F. Taguchi, J. Virol. 70:2632-2636, 1996). To further identify the residues responsible for the activity, we isolated the mutant viruses that were not neutralized with the soluble form of MHV receptor proteins, since such mutants were expected to have mutations in amino acids responsible for receptor-binding activity. Five soluble-receptor-resistant (srr) mutants isolated had mutations in a single amino acid at three different positions: one was at position 65 (Leu to His) (srr11) in the S1 subunit and three were at position 1114 (Leu to Phe) (srr3, srr4, and srr7) and one was at position 1163 (Cys to Phe) (srr18) in the S2 subunit. The receptor-binding activity examined by a virus overlay protein blot assay and by a coimmunoprecipitation assay showed that srr11 S protein had extremely reduced binding activity, while the srr7 and srr18 proteins had binding activity similar to that of wild-type cl-2 protein. However, when cell surface receptors were used for the binding assay, all srr mutants showed activity similar to that of the wild type or only slightly reduced activity. These results, together with our previous observations, suggest that amino acids located at positions 62 to 65 of S1, a region conserved among the MHV strains examined, are important for receptor-binding activity. We also discuss the mechanism by which srr mutants with a mutation in S2 showed high resistance to neutralization by a soluble receptor, despite their sufficient level of binding to soluble receptors.

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This fact indicates the possibility that the difference in MHV susceptibility between BALB/c and SJL mice is determined by the virus-binding activity of the receptor. To test this possibility, we have examined MHV susceptibility in mice with the homozygous MHVR1 gene (R1/R1 genotype), mice with the MHVR1 and MHVR2 genes (R1/R2 genotype), and mice with the homozygous MHVR2 gene (R2/R2 genotype) produced by cross and backcross mating between BALB/c and SJL mice. All 63 F 2 and backcrossed mice with the MHVR1 gene (R1/R1 and R1/R2) were susceptible to MHV infection, and all 57 with the homozygous MHVR2 gene (R2/R2) were resistant. We have also examined the MHV receptor genotypes of several mouse strains that were reported to be susceptible to MHV infection. All of those mice had the MHVR1 gene. These results suggest the possibility that the viral receptor determines the susceptibility of the whole animal to MHV infection.

To study the role of NMDA receptors in dopamine signaling of the striatum, the brain area that receives glutamatergic inputs from various cortical areas and most dopaminergic inputs, we generated striatum-specific NMDA receptor-deficient mice. The mutant pups showed reduced food intake and retarded growth starting at the second postnatal week and died on approximately postnatal day 20 (P20). The time course of postnatal lethality is similar to that of compound mutant, double knockout of dopamine D1/D2 receptors, or genetically engineered dopamine-deficient mouse. In vivo electrophysiological recordings in the mutant pups showed that frequencies in the range of gamma oscillation were reduced in the striatal circuits. Moreover, the number of functional dopamine receptors in the striatum as measured by D1-and D2-binding experiments was greatly diminished in the mutants as compared with control animals. A consequence of diminished dopamine binding in the striatum manifested in an increase of locomotor activity. The administration of D1/D2 agonists paradoxically reduced the hyperactivity of the mutant mice as compared with an increase in locomotor activity in control mice. These results demonstrate that the NMDA receptor plays an essential role in the integration of dopamine signaling in the striatum and that is required in behavioral function.T he striatum receives glutamatergic projections from virtually all areas of the cerebral cortex and dense dopaminergic projections from the substantia nigra and ventral tegmental area (1, 2). The principal neurons of the striatum, medium spiny neurons (MSNs), are GABAergic projection neurons. These neurons are classified into two groups: the MSNs that express D1 dopamine receptors responsible for a direct pathway and the MSNs that express D2 dopamine receptors responsible for an indirect pathway (3). These MSNs constitute 90-95% of the striatal neuronal population (4) and contain high levels of NMDA receptors (5, 6).During development, dopamine receptor expressions (both D1 and D2) are already abundant by the late embryonic stage, however functional dopamine receptor binding is low at birth and progressively increases to reach adult levels between postnatal day (P)14 and P21 (7). This delayed appearance of functional dopamine receptors in the MSNs of the striatum suggests that maturation of functional dopamine receptors is regulated by an unknown mechanism, other than the mere availability of the dopamine receptor mRNAs.During postnatal weeks, it has been shown that the NMDA receptor-mediated currents develop later in the MSNs compared with other brain regions such as the hippocampus. There is a Ϸ2-fold increase from the first to the third postnatal week (8). Recently, molecular interactions between NMDA receptors and dopamine receptors have been reported. For example, NMDA receptors trap diffusible D1 receptors by direct physical interaction (9-11). The interaction between NR2B, a subunit of the NMDA receptor, and the D2 receptor can also disrupt the association of Ca 2ϩ /cal...

Although most inbred mouse strains are highly susceptible to mouse hepatitis virus (MHV) infection, the inbred SJL line of mice is highly resistant to its infection. The principal receptor for MHV is murine CEACAM1 (mCEACAM1). Susceptible strains of mice are homozygous for the 1a allele of mCeacam1, while SJL mice are homozygous for the 1b allele. mCEACAM1a (1a) has a 10-to 100-fold-higher receptor activity than does mCEACAM1b (1b). To explore the hypothesis that MHV susceptibility is due to the different MHV receptor activities of 1a and 1b, we established a chimeric C57BL/6 mouse (cB61ba) in which a part of the N-terminal immunoglobulin (Ig)-like domain of the mCeacam1a (1a) gene, which is responsible for MHV receptor function, is replaced by the corresponding region of mCeacam1b (1b). We compared the MHV susceptibility of these chimeric mice to that of SJL and B6 mice. B6 mice that are homozygous for 1a are highly susceptible to MHV-A59 infection, with a 50% lethal dose (LD 50 ) of 10 2.5 PFU, while chimeric cB61ba mice and SJL mice homozygous for 1ba and 1b, respectively, survived following inoculation with 10 5 PFU. Unexpectedly, cB61ba mice were more resistant to MHV-A59 infection than SJL mice as measured by virus replication in target organs, including liver and brain. No infectious virus or viral RNA was detected in the organs of cB61ba mice, while viral RNA and infectious virus were detected in target organs of SJL mice. Furthermore, SJL mice produced antiviral antibodies after MHV-A59 inoculation with 10 5 PFU, but cB61ba mice did not. Thus, cB61ba mice are apparently completely resistant to MHV-A59 infection, while SJL mice permit low levels of MHV-A59 virus replication during self-limited, asymptomatic infection. When expressed on cultured BHK cells, the mCEACAM1b and mCEACAM1ba proteins had similar levels of MHV-A59 receptor activity. These results strongly support the hypothesis that although alleles of mCEACAM1 are the principal determinants of mouse susceptibility to MHV-A59, other as-yet-unidentified murine genes may also play a role in susceptibility to MHV.

Postmortem studies have revealed a downregulation of the transcription factor Pax5 in GABAergic neurons in bipolar disorder, a neurodevelopmental disorder, raising the question whether Pax5 in GABAergic neurons has a role in normal brain development. In a genetic approach to study functions of Pax5 in GABAergic neurons, Pax5 was specifically deleted in GABAergic neurons from Pax5 floxed mice using a novel Gad1-Cre transgenic mouse line expressing Cre recombinase in Gad1-positive, i.e. GABAergic neurons. Surprisingly, these mice developed a marked enlargement of the lateral ventricles at approximately seven weeks of age, which was lethal within 1-2 weeks of its appearance. This hydrocephalus phenotype was observed in mice homozygous or heterozygous for the Pax5 conditional knockout, with a gene dosagedependent penetrance. By QTL (quantitative trait loci) mapping, a 3.5 Mb segment on mouse chromosome 4 flanked by markers D4Mit237 and D4Mit214 containing approximately 92 genes including Pax5 has previously been linked to differences in lateral ventricular size. Our findings are consistent with Pax5 being a relevant gene underlying this QTL phenotype and demonstrate that Pax5 in GABAergic neurons is essential for normal ventricular development.

IntroductionThe efficacy of natalizumab was evaluated in Japanese patients with relapsing-remitting multiple sclerosis (RRMS) in a 24-week, phase 2 bridging study. An open-label, 2-year extension study from this trial was conducted to assess the safety and efficacy of natalizumab treatment in Japanese patients.MethodsA total of 97 patients (43 previously on placebo; 54 previously on natalizumab) who had completed the bridging study were treated with 300 mg natalizumab every 4 weeks. Multiple sclerosis relapses, changes in Expanded Disability Status Scale (EDSS) scores, and adverse events were assessed at regular intervals. Anti-natalizumab and anti-JC virus (JCV) antibodies were measured.ResultsAfter 2 years of natalizumab treatment, the mean adjusted annualized relapse rate was 0.30 (95% confidence interval [CI]: 0.18–0.52) among previously-on-placebo patients and 0.13 (95% CI: 0.05–0.29) among previously-on-natalizumab patients. The mean change in EDSS score from baseline to week 120 was −0.03 among previously-on-placebo patients and −0.18 among previously-on-natalizumab patients. In both groups, >90% of patients experienced ≥1 adverse event. Two previously-on-placebo patients developed persistently positive anti-natalizumab antibodies. Approximately 65% of all patients tested positive for anti-JCV antibodies at open-label treatment initiation. No deaths or progressive multifocal leukoencephalopathy cases were reported.ConclusionsThe efficacy and safety findings from this 2-year open-label extension study are comparable to and confirm the results of other clinical trials of natalizumab conducted in non-Asian patient populations, and provide longer-term evidence of efficacy and safety in Japanese patients.Trial registrationClinicalTrials.gov identifier NCT01416155.FundingBiogen.