Biomedical subjects
Publications and source records attributed to Kenji Sakimura.
Carbonic anhydrase inhibitors AZA, EZA, and 4-acetamidobenzsulfonamide were found to inhibit human AQP4-M23 mediated water transport by 80%, 68%, and 23%, respectively, at 20 microM in an in vitro functional assay. AZA was found to have an IC50 against AQP4 of 0.9 microM. Phloretin was inactive under the same conditions.
Brain-derived neurotrophic factor (BDNF) is involved in synaptic development and plasticity, and alterations in BDNF expression or signaling are implicated in drug addiction and psychiatric diseases, such as depression and schizophrenia. In this study, we administered phencyclidine to postnatal and adult rats with different time schedules, and determined the correlations between BDNF expression and the behavioral effects. Both single and repeated phencyclidine injections into adult rats induced BDNF up-regulation in the corticolimbic system and a decrease in prepulse inhibition, both of which were transient. In contrast, subchronic postnatal administration increased BDNF protein and mRNA levels in the hippocampus and entorhinal cortex, which were sustained until 8 weeks of age. In parallel, the postnatal rats treated with phencyclidine developed a persistent decrease in prepulse inhibition at the adult stage. The chronic BDNF increase appeared to contribute to the prepulse inhibition abnormality, as subchronic BDNF infusion into the hippocampus of normal rats mimicked the prepulse inhibition deficits. This study suggests that phencyclidine exposure during brain development induces sustained BDNF up-regulation in the limbic system with a biological link to sensorimotor gating deficits.
Although the properties of ketamine appear to be well characterized, there is a lot of ambiguity in the literature regarding its analgesic effects. After careful selection of proper experimental conditions and drug doses, we systematically characterized the effects of systemic ketamine on acute somatic nociception in mice and examined the role of the NMDA receptor epsilon1 subunit in mediating its analgesia. Intraperitoneal administration of ketamine was not analgesic in any of the phasic pain assays (thermal, mechanical, electrical) applied to C57BL/6 (wild-type) and NMDA receptor epsilon1 subunit knockout (mutant) mice. Surprisingly, rather than being analgesic for thermal nociception, ketamine showed pronociceptive properties in case of low-intensity heat stimulation in wild-type mice. In the formalin test (tonic pain), ketamine significantly reduced phase 2 nociceptive behavior in both wild-type and mutant mice. These data indicate that in wild-type mice ketamine has no analgesic effect on phasic pain in normal somatic tissues, but alleviates tonic pain after inflammation. Such analgesic spectrum of ketamine can be fully explained by its NMDA receptor antagonist properties. The results for the mutant mice suggest that the epsilon1 subunit of the NMDA receptor does not mediate the analgesic effects of ketamine in tonic pain.
Transmembrane alpha-amino-3-hydroxyl-5-isoxazolepropionate (AMPA) receptor regulatory proteins (TARPs) play pivotal roles in AMPA receptor trafficking and gating. Here we examined cellular and subcellular distribution of TARP gamma-8 in the mouse brain. Immunoblot and immunofluorescence revealed the highest concentration of gamma-8 in the hippocampus. Immunogold electron microscopy demonstrated dense distribution of gamma-8 on the synaptic and extrasynaptic surface of hippocampal neurons with very low intracellular labeling. Of the neuronal surface, gamma-8 was distributed at the highest level on asymmetrical synapses of pyramidal cells and interneurons, whereas their symmetrical synapses selectively lacked immunogold labeling. Then, the role of gamma-8 in AMPA receptor expression was pursued in the hippocampus using mutant mice defective in the gamma-8 gene. In the mutant cornu ammonis (CA)1 region, synaptic and extrasynaptic AMPA receptors on dendrites and spines were severely reduced to 35-37% of control levels, whereas reduction was mild for extrasynaptic receptors on somata (74%) and no significant decrease was seen for intracellular receptors within spines. In the mutant CA3 region, synaptic AMPA receptors were reduced mildly at asymmetrical synapses in the stratum radiatum (67% of control level), and showed no significant decrease at mossy fiber-CA3 synapses. Therefore, gamma-8 is abundantly distributed on hippocampal excitatory synapses and extrasynaptic membranes, and plays an important role in increasing the number of synaptic and extrasynaptic AMPA receptors on dendrites and spines, particularly, in the CA1 region. Variable degrees of reduction further suggest that other TARPs may also mediate this function at different potencies depending on hippocampal subregions, input sources and neuronal compartments.
Input-dependent left-right asymmetry of NMDA receptor epsilon2 (NR2B) subunit allocation was discovered in hippocampal Schaffer collateral (Sch) and commissural fiber pyramidal cell synapses (Kawakami et al., 2003). To investigate whether this asymmetrical epsilon2 allocation is also related to the types of the postsynaptic cells, we compared postembedding immunogold labeling for epsilon2 in left and right Sch synapses on pyramidal cells and interneurons. To facilitate the detection of epsilon2 density difference, we used epsilon1 (NR2A) knock-out (KO) mice, which have a simplified NMDA receptor subunit composition. The labeling density for epsilon2 but not zeta1 (NR1) and subtype 2/3 glutamate receptor (GluR2/3) in Sch-CA1 pyramidal cell synapses was significantly different between the left and right hippocampus with opposite directions in strata oriens and radiatum; the left to right ratio of epsilon2 labeling density was 1:1.50 in stratum oriens and 1.44:1 in stratum radiatum. No significant difference, however, was detected in CA1 stratum radiatum between the left and right Sch-GluR4-positive (mostly parvalbumin-positive) and Sch-GluR4-negative interneuron synapses. Consistent with the anatomical asymmetry, the amplitude ratio of NMDA EPSCs to non-NMDA EPSCs in pyramidal cells was approximately two times larger in right than left stratum radiatum and vice versa in stratum oriens of epsilon1 KO mice. Moreover, the amplitude of long-term potentiation in the Sch-CA1 synapses of left stratum radiatum was significantly larger than that in the right corresponding synapses. These results indicate that the asymmetry of epsilon2 distribution is target cell specific, resulting in the left-right difference in NMDA receptor content and plasticity in Sch-CA1 pyramidal cell synapses in epsilon1 KO mice.
The gamma subunit of voltage-dependent Ca2+ channels (VDCCs) is characterized by molecular diversity and regulation of AMPA-type glutamate receptors as well as VDCCs. In the present study, we examined expressions for the VDCCgamma1-8 subunit mRNAs in developing and adult mouse brains by in situ hybridization. In adult brains, the gamma2 and gamma7 subunit mRNAs were widely expressed in various grey matter regions with the highest level in cerebellar Purkinje cells and granule cells. The gamma3 and gamma8 subunit mRNAs predominated in the telencephalon, with the latter being at striking levels in the hippocampus. The gamma4 subunit mRNA was enriched in the olfactory bulb, striatum, thalamus and hypothalamus. The gamma5 subunit mRNA was abundant in the olfactory bulb, hippocampal CA2, thalamus, inferior colliculus and Bergmann glia. Transcripts of these subunits were detected in embryonic brains: some showed well-preserved spatial patterns (gamma2, gamma5, gamma7 and gamma8), while others underwent developmental up- (gamma3) or down-regulation (gamma4). In contrast, the gamma1 and gamma6 subunit mRNAs were negative or very low throughout brain development. Therefore, the present study has revealed spatial diversity in gene expression for individual VDCCgamma subunits, presumably reflecting functional diversity of this protein family and their differential involvement in neural function.
Precise topological matching of presynaptic and postsynaptic specializations is essential for efficient synaptic transmission. Furthermore, synaptic connections are subjected to rearrangements throughout life. Here we examined the role of glutamate receptor (GluR) delta2 in the adult brain by inducible and cerebellar Purkinje cell (PC)-specific gene targeting under the pure C57BL/6 genetic background. Concomitant with the decrease of postsynaptic GluRdelta2 proteins, presynaptic active zones shrank progressively and postsynaptic density (PSD) expanded, resulting in mismatching between presynaptic and postsynaptic specializations at parallel fiber-PC synapses. Furthermore, GluRdelta2 and PSD-93 proteins were concentrated at the contacted portion of mismatched synapses, whereas AMPA receptors were distributed in both the contacted and dissociated portions. When GluRdelta2 proteins were diminished, PC spines lost their synaptic contacts. We thus identified postsynaptic GluRdelta2 as a key regulator of the presynaptic active zone and PSD organization at parallel fiber-PC synapses in the adult brain.
Endogenous polyamines like spermine are known to have four distinct effects on recombinant N-methyl-d-aspartate (NMDA) receptor channels: voltage-dependent inhibition, glycine-dependent stimulation, glycine-independent stimulation and decreased affinity to the agonist (l-glutamate). These effects are highly dependent on the constituting epsilon subunits (epsilon1-epsilon4) of the recombinant NMDA receptor channels. Agmatine reportedly inhibits native NMDA receptor channels in cultured hippocampal neurons. In the present investigation, the effects of agmatine on the epsilon/zeta heteromeric NMDA receptor channels expressed in Xenopus laevis oocytes were examined using the two-electrode voltage clamp method. Agmatine inhibited the four epsilon/zeta (epsilon1/zeta1, epsilon2/zeta1, epsilon3/zeta1 and epsilon4/zeta1) channels with similar sensitivity (an IC50 value of about 300microM at -70mV). This effect was dependent on the membrane potential and was more pronounced at hyperpolarized membrane potentials (voltage-dependent inhibition). Agmatine did not exhibit other stimulatory (glycine-dependent and -independent effects) or inhibitory (decreased affinity to l-glutamate) effects. These properties are similar to the pharmacological profile of well-characterized NMDA receptor channel blockers like phencyclidine and ketamine. Thus, regarding the effects on the NMDA receptor channels, agmatine is not like other endogenous polyamines rather it acts as a channel blocker.
The N-methyl-D-aspartate (NMDA) receptor NR3B subunit co-assembles with NR1 and NR2 subunits to form a receptor complex with distinct channel properties. In the present study, we investigated the effects of co-expression of the NR3B subunit on the anesthetic sensitivities of NMDA receptors for NR1/NR2 channels expressed in Xenopus oocytes. Although the NR3B subunit prominently reduced the current amplitude of NR1/NR2A-B channels, the sensitivities of NR1/NR2A-B channels to Mg2+, ketamine, isoflurane, nitrous oxide, and ethanol were not altered by coexpression of the NR3B subunit. These results suggest that the anesthetic sensitivities of NMDA receptors do not depend on the presence or absence of the NR3 subunit. Mutations of two amino acid residues in the NR3B subunit at positions homologous to the N and N + 1 sites in the NR1 and NR2 subunits, which constitute the blocking sites for Mg2+ and ketamine, did not affect the sensitivities of NR1/NR2B/NR3B channels to Mg2+, ketamine and isoflurane. Thus, the amino acid residues at the N and N + 1 sites in NR3 subunits are unlikely to be involved in the formation of channel blocking sites in NR1/NR2/NR3 channels.
N-methyl-D-aspartate (NMDA) receptors have been suggested to be critical for the development of central sensitization, which may amplify postoperative pain. NMDA receptors are formed by GluRzeta (NR1) with any one of four GluRvarepsilon1-4 (NR2A-D) subunits. To clarify the involvement of NMDA receptors in postoperative pain, we examined the effect of the GluRepsilon2-selective antagonist (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenyl piperidino)-1-propanol (CP-101,606) on postoperative pain caused by plantar incision. We also applied the postoperative pain model to GluRepsilon1 and GluRepsilon4 knockout mice. CP-101,606 administered intrathecally 30 min prior to incision significantly increased mechanical withdrawal thresholds 2 h and 1-3 days after surgery and reduced postoperative pain dose-dependently. Neither GluRvarepsilon1 nor GluRepsilon4 knockout mice showed a difference in withdrawal thresholds as compared with wild-type mice. Pretreatment with CP-101,606 did not produce an additive analgesic effect in the mice. These results demonstrate that GluRepsilon2-containing NMDA receptors are involved in postoperative pain and that CP-101,606 may be effective in reducing it.
In NMDA receptors, GluRepsilon/NR2 subunits strictly require the GluRzeta1/NR1 subunit to exit from endoplasmic reticulum (ER) to the cell surface in vitro and to the postsynapse in vivo, whereas C terminus-dependent self-surface delivery has been demonstrated for the GluRzeta1 subunit in vitro. To test whether this leads to C terminus-dependent self-postsynaptic expression in neurons in vivo, we investigated the GluRzeta1 subunit in cerebellar granule cells lacking two major GluRepsilon subunits, GluRepsilon1/NR2A and GluRepsilon3/NR2C. In the mutant cerebellum, synaptic labeling for the GluRzeta1 subunit containing the C2 (GluRzeta1-C2) or C2' (GluRzeta1-C2') cassette was reduced at mossy fiber-granule cell synapses to the extrasynaptic level. The loss was not accompanied by decreased transcription and translation levels, increased extrasynaptic labeling, or ER accumulation. Quantitative immunoblot revealed substantial reductions in the mutant cerebellum of GluRzeta1-C2 and GluRzeta1-C2'. The most severe deficit was observed in the postsynaptic density (PSD) fraction: mutant levels relative to the wild-type level were 12.3 +/- 3.3% for GluRzeta1-C2 and 17.0 +/- 4.6% for GluRzeta1-C2'. The GluRzeta1 subunit carrying the C1 cassette (GluRzeta1-C1) was, although low in cerebellar content, also reduced to 12.7 +/- 3.5% in the mutant PSD fraction. Considering a trace amount of other GluRepsilon subunits in the mutant cerebellum, the severe reductions thus represent that the GluRzeta1 subunit, by itself, is virtually unable to accumulate at postsynaptic sites, regardless of C-terminal forms. By protein turnover analysis, the degradation of the GluRzeta1 subunit was accelerated in the mutant cerebellum, being particularly rapid for that carrying the C2 cassette. Therefore, accompanying expression of GluRepsilon subunits is essential for postsynaptic localization and protein stability of the GluRzeta1 subunit.
Stargazin (gamma-2) is disrupted in the ataxic and epileptic mutant mouse, stargazer (stg). The striking defect in the stg cerebellum is the lack of functional AMPA receptors on granule cells. Recently, it has been reported that gamma-2 and its related molecules are crucial for the surface expression, synaptic targeting and recycling of AMPA receptors, being termed collectively as the transmembrane AMPA receptor regulatory proteins (TARPs). However, it is still unclear whether TARPs directly modulate AMPA receptor activity. Here we report that coexpression of GluRalpha1 (GluR1) with gamma-2 using HEK293 cells and Xenopus oocytes markedly enhanced glutamate-induced currents. This effect was far beyond the increase of AMPA receptor surface expression and accompanied by increased glutamate affinity and subunit cooperativity. Other member of TARPs (gamma-3, gamma-4, and gamma-8) also enhanced the current response through the AMPA receptors. The enhancing effect by gamma-2 coexpression was further observed for homomeric GluRalpha2 (GluR2) channels, which, when expressed alone, are known to produce only a small or negligible current response. These results suggest that gamma-2 not only promotes AMPA receptor surface expression but also directly modulates AMPA receptor activity.
Rapid administration of large doses of ammonia leads to death of animals, which is largely prevented by pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists. The present study focuses on a subunit(s) of NMDA receptor involved in ammonia-induced death by use of NMDA receptor GluRepsilon subunit-deficient (GluRepsilon(-/-)) mice and the selective GluRepsilon2 antagonist CP-101,606. Acute ammonia intoxication was induced in mice (eight per group) by a single intraperitoneal (i.p.) injection of ammonium chloride. Appearance of neurological deteriorations depended on the doses of ammonium chloride injected. While wild-type, GluRepsilon1(-/-), GluRepsilon4(-/-), and GluRepsilon1(-/-)/epsilon4(-/-) mice all died by ammonium chloride at 12 mmol/kg during the first tonic convulsions, two of eight GluRepsilon3(-/-) mice survived. Pretreatment of wild-type mice with CP-101,606 prevented two mice from ammonia-induced death. Pretreatment of GluRepsilon3(-/-) mice with CP-101,606 prevented the death of three mice and prolonged the time of death of non-survivors. Similarly, the neuronal form of nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI) as well as the nonselective NOS inhibitor L-NMMA, but not the inducible NOS inhibitor 1400W, partially prevented the death of mice and prolonged the period of death. Furthermore, ammonium chloride prolonged the increase in intracellular free Ca2+ concentration ([Ca2+]i) and subsequent NO production induced by NMDA in the cerebellum. These results suggest that activation of NMDA receptor containing GluRepsilon2 and GluRepsilon3 subunits and following activation of neuronal NOS are involved in acute ammonia intoxication which leads to death of animals.
Ketamine is an IV anesthetic with N-methyl-d-aspartate receptor (NMDAR)-blocking properties. However, it is still unclear whether ketamine's general anesthetic actions are mediated primarily via blockade of NMDAR. Functional NMDARs are composed by the assembly of a GluRzeta1 (NR1) subunit with GluRepsilon (GluRepsilon1-4; NR2A-D) subunits, which confer unique properties on native NMDARs. We hypothesized that animals deficient in GluRepsilon1, an abundant and ubiquitously postnatally expressed NMDAR subunit, might be resistant to the effects of ketamine. Here, we evaluated a righting reflex to determine the general anesthetic/hypnotic potency of ketamine administered intraperitoneally to GluRepsilon1 knockout mice and compared these results with those for wild-type mice. Mutant mice were more resistant to ketamine than control mice. Unexpectedly, mutant mice were also more resistant to pentobarbital, which is thought not to interact with NMDAR at clinically relevant concentrations. Although these data in no way eliminate the possibility of the involvement of the NMDAR GluRepsilon1 subunit in mediation of ketamine anesthesia/hypnosis, they suggest the difficulties with interpretation of altered anesthetic sensitivity in knockout animal models.
Noxious afferent input following tissue damage and inflammation triggers a state of neuronal hyperexcitability-a phenomenon of central sensitization-which manifests behaviorally as allodynia and hyperalgesia. At the molecular level, maintenance of central sensitization is largely dependent on the N-methyl-D-aspartate receptor (NMDAR) activation. NMDARs are composed of GluRzeta1 (NR1) and one of four GluRepsilon (NR2) subunits, which determine the functional properties of native NMDARs. Although there is accumulating evidence to implicate GluRepsilon 2-containing NMDARs in pain mechanisms, the functional significance of GluRepsilon 1-containing NMDARs in this setting has not been examined in detail. Here, we used hind paw injection of formalin, complete Freund's adjuvant and a nerve injury model to investigate the effects of GluRepsilon 1 subunit gene deletion on pain-related behavior in mice. In all of the models tested, GluRepsilon 1-deficient mice exhibited responses similar to wild-type controls. These results suggest that GluRepsilon 1 disruption does not result in altered nociceptive behavior in mice. Although the contribution of other nociceptive pathways cannot be ruled out, we speculate that the preserved function of GluRepsilon 2-containing NMDARs could explain unaltered nociceptive behavior in mutant mice.
The common gamma chain, which was originally identified as a component of interleukin-2 receptors (IL-2R), plays a key role in differentiation of T lymphocytes and natural killer (NK) cells. In the present study, cDNA of the porcine common gamma chain gene and its genomic DNA were molecularly cloned and characterized. The porcine common gamma chain gene was found to consist of 8 exons, spanning approximately 3.7 kb, and to encode a 368-amino acid polypeptide. The amino acid sequence showed 82.4%, 71.1%, 86.1%, and 84.8% similarities with that of human, murine, bovine, and canine chains, respectively. The common gamma chain gene was assigned to swine chromosome Xq13 by FISH analysis and was consistent with the result of radiation hybrid (RH) mapping. When various porcine tissues were examined for the expression of this gene, the expression was observed in lymphocytes and lymphocyte-related tissues. Since GATA, T cell factor-1 (TCF-1), Ets-1, activated protein2 (AP-2), and Ikaros2 binding motifs were demonstrated in the 5' upstream region of this gene, promoter activity was investigated using luciferase gene as a reporter. The results indicate that the Ets-1 binding motif in the segment from -95 to -59 (major transcription initiation site: +1) was an essential cis-acting regulatory element for the common gamma chain gene in lymphoid cells.
Organized synapse formation on to Purkinje cell (PC) dendrites by parallel fibers (PFs) and climbing fibers (CFs) is crucial for cerebellar function. In PCs lacking glutamate receptor delta2 (GluRdelta2), PF synapses are reduced in number, numerous free spines emerge, and multiple CF innervation persists to adulthood. In the present study, we conducted anterograde and immunohistochemical labelings to investigate how CFs innervate PC dendrites under weakened synaptogenesis by PFs. In the GluRdelta2 knock-out mouse, CFs were distributed in the molecular layer more closely to the pial surface compared with the wild-type mouse. Serial electron microscopy demonstrated that CFs in the knock-out mouse innervated all spines protruding from proximal dendrites of PCs, as did those in the wild-type mouse. In the knock-out mouse, however, CF innervation extended distally to spiny branchlets, where nearly half of the spines were free of innervation in contrast to complete synapse formation by PFs in the wild-type mouse. Furthermore, from the end point of innervation, CFs aberrantly jumped to form ectopic synapses on adjacent spiny branchlets, whose proximal portions were often innervated by different CFs. Without GluRdelta2, CFs are thus able to expand their territory along and beyond dendritic trees of the target PC, resulting in persistent surplus CFs by innervating the distal dendritic segment. We conclude that GluRdelta2 is essential to restrict CF innervation to the proximal dendritic segment, by which territorized innervation by PFs and CFs is properly structured and the formation of excess CF wiring to adjacent PCs is suppressed.