Biomedical subjects

K Sakimura

Publications and source records attributed to K Sakimura.

At least 37 records · Page 2Linked to original sources

PubMed1999

Direct inhibition of the N-methyl-D-aspartate receptor channel by high concentrations of opioids.

BACKGROUND: Electrophysiologic and receptor binding studies showed that some opioids have noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist properties. METHODS: The effects and mechanisms of action of various opioid compounds were examined on four kinds of heteromeric NMDA receptor channels, namely the epsilon1/zeta1, epsilon2/zeta1, epsilon3/zeta1, and epsilon4/zeta1 channels, expressed in Xenopus oocytes. Furthermore, the action sites of opioids on NMDA receptor channels were investigated by site-directed mutagenesis. RESULTS: Meperidine inhibited four kinds of channels to a similar extent with inhibitor concentrations for half-control response (IC50s) of 210-270 microM. Morphine, fentanyl, codeine, and naloxone also inhibited NMDA receptor channels with affinities comparable to meperidine. Opioid inhibition exhibited voltage dependence and was quite effective at negative potentials. Opioids also shifted the inhibition curve of Mg2+ to the right. Furthermore, replacement of the conserved asparagine residue with glutamine in the channel-lining segment M2 of the zeta1 subunit, which constitutes the block sites of Mg2+ and ketamine, reduced the sensitivity to opioids, whereas that of the epsilon2 subunit barely affected the opioid sensitivity. CONCLUSIONS: These results, together with previous findings, suggest that the low-affinity NMDA receptor antagonist activity is a common characteristic of various opioid compounds, and that the inhibition is a result of channel-block mechanisms at the site, which partially overlaps with those of Mg2+ and ketamine. This antagonist property of opioids may be clinically significant in the spinal cord following epidural or intrathecal administration, after which the cerebrospinal fluid concentrations of some opioids reach the high micromolar level.

Amino Acid Sequence↗

PubMed1998

Increased thresholds for long-term potentiation and contextual learning in mice lacking the NMDA-type glutamate receptor epsilon1 subunit.

The NMDA-type glutamate receptor (GluR) channel, composed of the GluRepsilon and GluRzeta subunits, plays a key role in synaptic plasticity in the CNS. The mutant mice lacking the GluRepsilon1 subunit exhibited a reduction in hippocampal long-term potentiation (LTP), but a stronger tetanic stimulation restored the impairment and the saturation level of LTP was unaltered. These results suggest an increase of threshold for LTP induction in the GluRepsilon1 mutant mice. After a series of backcrosses we established a GluRepsilon1 mutant mouse line with a 99.99% pure C57BL/6 genetic background. The performance of the mutant mice in tone- and context-dependent fear conditioning tests was comparable with that of the wild-type mice. However, a significant difference in the extent of contextual learning became apparent when the chamber exposure time before footshock was shortened. Furthermore, there was a significant difference in freezing responses immediately after footshock on the conditioning day between the wild-type and mutant mice, and the difference was not restored by longer chamber exposure in contrast to the contextual learning on the next day of the conditioning. These results suggest that the GluRepsilon1 subunit of the NMDA receptor channel is a determinant of thresholds for both hippocampal LTP and contextual learning and plays differential roles in two forms of contextual fear memories.

Animals↗

PubMed1998

Sensitivity of the N-methyl-D-aspartate receptor channel to butyrophenones is dependent on the epsilon2 subunit.

The effects of three kinds of butyrophenones, haloperidol, droperidol and spiperone, on the N-methyl-D-aspartate (NMDA) receptor channel were examined on the epsilon1/zeta1, epsilon2/zeta1, epsilon3/zeta1 and epsilon4/zeta1 heteromeric NMDA receptor channels, expressed in Xenopus oocytes. Micromolar concentrations of haloperidol selectively inhibited the epsilon2/zeta1 channel, whereas the epsilon1/zeta1, epsilon3/zeta1 and epsilon4/zeta1 channels were enhanced or minimally affected by higher concentrations of haloperidol. Similarly, droperidol and spiperone inhibited the epsilon2/zeta1 channel more strongly than the other epsilon/zeta channels, although sensitivities of the epsilon2/zeta1 channel to droperidol and spiperone were lower than those to haloperidol. These results suggest that the sensitivities of the NMDA receptor channels to butyrophenones are dependent on the epsilon2 subunit. Furthermore, the replacement with glutamine of the conserved asparagine residue in segment M2, which constitutes the Mg2+ block sites, of the epsilon2 and zeta1 subunits (the mutations epsilon2-N589Q and zeta1-N598Q, respectively) reduced the sensitivities to haloperidol. The mutation zeta1-N598Q reduced the sensitivities to haloperidol more effectively than the mutation epsilon2-N589Q. These results, together with previous findings, suggest that the haloperidol block sites of the NMDA receptor channel partially overlap the Mg2+ block sites.

Amino Acid Substitution↗

PubMed1998

Distribution and development of NMDA receptor activities at hippocampal synapses examined using mice lacking the epsilon1 subunit gene.

The effects of targeted disruption of the gene encoding N-methyl-D-aspartate (NMDA) receptor epsilon1 subunit were examined in hippocampal CA1 pyramidal cell synapses and compared with the effects in the CA3 region. The mutation resulted in the significant reduction of NMDA receptor activities at the synapses in the CA1 stratum oriens, as had been observed in the CA1 stratum radiatum which we reported before. This result was in sharp contrast to our previous observation that in the CA3 region, the epsilon1 mutation suppressed NMDA receptors at the synapses in the stratum radiatum but not in the stratum oriens. It is suggested that the subunit composition of NMDA receptors may not be determined simply by the location within a pyramidal cell, but by other factors such as properties of synaptic inputs. We also examined the postnatal development of long-term potentiation (LTP) in the CA3 region. The development of LTP at the CA3 stratum radiatum synapses closely followed the development of the epsilon1 subunit, and the epsilon1 mutation strongly suppressed this LTP, suggesting that the targeted disruption of the epsilon1 subunit may not be compensated by other epsilon subunits. The LTP at the CA3 stratum oriens synapses was not significantly affected by the mutation at any age.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1998

Role of the carboxy-terminal region of the GluR epsilon2 subunit in synaptic localization of the NMDA receptor channel.

The synaptic localization of the N-methyl-D-aspartate (NMDA) type glutamate receptor (GluR) channel is a prerequisite for synaptic plasticity in the brain. We generated mutant mice carrying the carboxy-terminal truncated GluR epsilon2 subunit of the NMDA receptor channel. The mutant mice died neonatally and failed to form barrelette structures in the brainstem. The mutation greatly decreased the NMDA receptor-mediated component of hippocampal excitatory postsynaptic potentials and punctate immunofluorescent labelings of GluR epsilon2 protein in the neuropil regions, while GluR epsilon2 protein expression was comparable. Immunostaining of cultured cerebral neurons showed the reduced punctate staining of the truncated GluR epsilon2 protein at synapses. These results suggest that the carboxy-terminal region of the GluRepsilon2 subunit is important for efficient clustering and synaptic localization of the NMDA receptor channel.

Afferent Pathways↗

PubMed1998

Selective scarcity of NMDA receptor channel subunits in the stratum lucidum (mossy fibre-recipient layer) of the mouse hippocampal CA3 subfield.

Hippocampal synapses express two distinct forms of the long-term potentiation (LTP), i.e. NMDA receptor-dependent and -independent LTPs. To understand its molecular-anatomical basis, we produced affinity-purified antibodies against the GluRepsilon1 (NR2A), GluRepsilon2 (NR2B), and GluRzeta1 (NR1) subunits of the N-methyl-D-aspartate (NMDA) receptor channel, and determined their distributions in the mouse hippocampus. Using NMDA receptor subunit-deficient mice as the specificity controls, section pretreatment with proteases (pepsin and proteinase K) was found to be very effective to detect authentic NMDA receptor subunits. As the result of modified immunohistochemistry, all three subunits were detected at the highest level in the strata oriens and radiatum of the CA1 subfield, and high levels were also seen in most other neuropil layers of the CA1 and CA3 subfields and of the dentate gyrus. However, the stratum lucidum, a mossy fibre-recipient layer of the CA3 subfield, contained low levels of the GluRepsilon1 and GluRzeta1 subunits and almost excluded the GluRepsilon2 subunit. Double immunofluorescence with the AMPA receptor GluRalpha1 (GluR1 or GluR-A) subunit further demonstrated that the GluRepsilon1 subunit was colocalized in a subset, not all, of GluRalpha1-immunopositive structures in the stratum lucidum. Therefore, the selective scarcity of these NMDA receptor subunits in the stratum lucidum suggests that a different synaptic targeting mechanism exerts within a single CA3 pyramidal neurone in vivo, which would explain contrasting significance of the NMDA receptor channel in LTP induction mechanisms between the mossy fibre-CA3 synapse and other hippocampal synapses.

Animals↗

PubMed1997

Impaired parallel fiber-->Purkinje cell synapse stabilization during cerebellar development of mutant mice lacking the glutamate receptor delta2 subunit.

The glutamate receptor delta2 subunit (GluRdelta2) is specifically expressed in cerebellar Purkinje cells (PCs) from early developmental stages and is selectively localized at dendritic spines forming synapses with parallel fibers (PFs). Targeted disruption of the GluRdelta2 gene leads to a significant reduction of PF-->PC synapses. To address its role in the synaptogenesis, the morphology and electrophysiology of PF-->PC synapses were comparatively examined in developing GluRdelta2 mutant and wild-type cerebella. PCs in GluRdelta2 mutant mice were normally produced, migrated, and formed spines, as did those in wild-type mice. At the end of the first postnatal week, 74-78% of PC spines in both mice formed immature synapses, which were characterized by small synaptic contact, few synaptic vesicles, and incomplete surrounding by astroglial processes, eliciting little electrophysiological response. During the second and third postnatal weeks when spines and terminals are actively generated, the percentage of PC spines forming synapses attained 98-99% in wild type but remained as low as 55-60% in mutants, and the rest were unattached to any nerve terminals. As a result, the number of PF synapses per single-mutant PCs was reduced to nearly a half-level of wild-type PCs. Parallelly, PF stimulation less effectively elicited EPSCs in mutant PCs than in wild-type PCs during and after the second postnatal week. These results suggest that the GluRdelta2 is involved in the stabilization and strengthening of synaptic connectivity between PFs and PCs, leading to the association of all PC spines with PF terminals to form functionally mature synapses.

Animals↗

PubMed1997

Synapse-selective impairment of NMDA receptor functions in mice lacking NMDA receptor epsilon 1 or epsilon 2 subunit.

1. We have explored the effects of targeted disruption of the N-methyl-D-aspartate (NMDA) receptor epsilon 1 or epsilon 2 subunit gene on NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) and long-term potentiations (LTPs) at the two types of synapse in mouse hippocampal CA3 pyramidal neurons: those formed by the commissural/associational (C/A) and fimbrial (Fim) inputs. 2. Electrophysiological experiments were performed in hippocampal slices prepared from both wild-type and epsilon 1- or epsilon 2-disrupted mice using extracellular and whole-cell patch recording techniques. To assess the epsilon 1, epsilon 2 and zeta 1 subunit expression at cellular levels, we performed non-isotopic in situ hybridization with digoxigenin-labelled cRNA probes. 3. We could record EPSCs in response to the stimulations to either of the C/A and Fim afferents from a single CA3 pyramidal neuron. The epsilon 1, epsilon 2 and zeta 1 subunits were expressed together in individual CA3 neurons. 4. The epsilon 1 subunit disruption selectively reduced NMDA EPSCs and LTP in the C/A-CA3 synapse without significantly affecting those in the Fim-CA3 synapse, whereas the epsilon 2 subunit mutation diminished NMDA EPSCs and LTP in the Fim-CA3 synapse with no appreciable functional modifications in the C/A-CA3 synapse. 5. These results suggest that NMDA receptors with different subunit compositions function within a single CA3 pyramidal cell in a synapse-selective manner.

Animals↗

PubMed1997

Absence of prostaglandin E2-induced hyperalgesia in NMDA receptor epsilon subunit knockout mice.

1. We have previously found that intrathecal administration of prostaglandins E2 (PGE2) and D2 (PGD2) into conscious mice induced hyperalgesia by the hot plate test. The present study investigated the involvement of N-methyl-D-aspartate (NMDA) receptor in the prostaglandin-induced hyperalgesia by use of mice tacking NMDA receptor epsilon 1, epsilon 4, or epsilon 1/epsilon 4 subunits. 2. PGE2 induced hyperalgesia over a wide range of doses from 50 pg to 500 ng kg-1 in wild-type mice. But PGE2 could not induce hyperalgesia in epsilon 1, epsilon 4, or epsilon 1/epsilon 4 subunit knockout mice. 3. The NMDA receptor antagonist D-(-)-2-amino-5-phosphonovaleric acid (D-AP5), the non-NMDA receptor antagonist 7-D-glutamylaminomethyl sulphonic acid (GAMS), and the nitric oxide synthase inhibitor N epsilon-nitro-L-arginine methyl ester (L-NAME) inhibited the PGE2-induced hyperalgesia in wild-type mice. 4. PGD2 induced hyperalgesia at doses of 25 ng to 250 ng kg-1 in both wild-type and epsilon 1/epsilon 4 subunit knockout mice. The substance P receptor antagonist OP 96.345 blocked the PGD2-induced hyperalgesia in wild-type and epsilon 1/epsilon 4 subunit knockout mice. 5. These results demonstrate that the pathways leading to hyperalgesia are different between PGD2 and PGE2, and that both epsilon 1 and epsilon 4 subunits of the NMDA receptor are involved in the PGE2-induced hyperalgesia.

Animals↗

PubMed1996

Functional correlation of NMDA receptor epsilon subunits expression with the properties of single-channel and synaptic currents in the developing cerebellum.

NMDA receptor (NMDAR) subunits epsilon 1-epsilon 4 are expressed differentially with respect to brain region and ontogenic period, but their functional roles still are unclear. We have compared an epsilon 1 subunit-ablated mutant mouse with the wild-type to characterize the effect of epsilon subunit expression on NMDAR-mediated single-channel currents and synaptic currents of granule cells in cerebellar slices. Single-channel and Western blot analyses indicated that the epsilon 2 subunit disappeared gradually during the first postnatal month in both wild-type and mutant mice. Concomitantly, the voltage-dependent Mg2+ block of NMDAR-mediated EPSCs (NMDA-EPSCs) was decreased. Throughout the developmental period studied, postnatal day 7-24 (P7-P24), the decay time course of NMDA-EPSCs in epsilon 1 mutant (-/-) mice was slower than in wild-type mice. We suggest that the expression of the epsilon 3 subunit late in development is responsible for a reduction in the sensitivity of NMDA-EPSCs to block by extracellular Mg2+ and that receptors containing the epsilon 1 subunit determine the fast kinetics of the NMDA-EPSCs.

Age Distribution↗

PubMed1996

Instability of mutant Cu/Zn superoxide dismutase (Ala4Thr) associated with familial amyotrophic lateral sclerosis.

In about 20-25% of cases of familial amyotrophic lateral sclerosis (FALS) patients have mutations in the Cu/Zn superoxide dismutase (SOD1) gene. The mechanism through which the mutations in the SOD1 gene cause ALS still remain unknown. We performed pulse-chase experiments using a system for the transient expression of human SOD1 in COS7 cells to examine whether the Ala4Thr mutation, which we previously reported, decreases the stability of SOD1. The expression vector (pEF-BOS) carrying the wild-type or mutant (Ala4Thr) human SOD1 cDNA was transfected into COS7 cells, and transiently expressed human SOD1 was then metabolically radiolabeled. Half-lives of the wild-type and the Ala4Thr mutant SOD1 were determined to be 78 h and 18 h, respectively. These results suggest that the Ala4Thr mutation in SOD1 decreases the stability of SOD1 and that this instability may play an important role in the pathogenesis of the degeneration of motor neurons in FALS.

Amyotrophic Lateral Sclerosis↗

PubMed1996

Age-dependent reduction of hippocampal LTP in mice lacking N-methyl-D-aspartate receptor epsilon 1 subunit.

The effects of targeted disruption of the N-methyl-D-aspartate (NMDA) receptor epsilon 1 subunit gene were studied during the postnatal development of epsilon 1-disrupted mutant mice. Using the mice at the ages of 2-3, 5-6 and 9-10 weeks, we examined NMDA receptor channel-mediated synaptic currents and long-term potentiation (LTP) in CA1 pyramidal neurons of hippocampal slices. NMDA receptor channel currents, expressed as the ratios to non-NMDA receptor channel currents, decreased with the age in both wild-type and mutant mice, but the values in the mutant mice was approximately half of those of the wild-type mice at all ages examined. The LTP in the mutant mice was also reduced, but in contrast to the NMDA receptor channel currents, the extent of the reduction in the LTP was age-dependent. The reduction was marginal at the age of 2-3 weeks, and became progressively prominent to adulthood, with the potentiation being 26% of that of the wild-type mice at 9-10 weeks.

Age Factors↗

PubMed1996

Gene structure and chromosomal localization of the mouse NMDA receptor channel subunits.

Multiple espilon subunits are major determinants of the diversity of the N-methyl-D-aspartate (NMDA) receptor channel. The four epsilon subunit mRNAs exhibit distinct expression patterns in the brain. In an attempt to elucidate the molecular basis of selective and characteristic expression of the NMDA receptor channel subunits, we have isolated the gene encoding the mouse NMDA receptor epsilon 3 subunit and have determined its structural organization. The epsilon 3 subunit gene spans 17.5 kb and consists of 14 exons. The major transcription start site is 439 bp upstream of the ATG initiation codon as determined by primer extension and S1 nucleas protection analyses. Two polyadenylation sites are 397 (or 398) and 402 bp downstream of the termination codon. The 5'-flanking region of the epsilon 3 subunit gene contains GC-rich segments including consensus sequences for binding of the transcription factors Spl and EGR-1. The murine chromosomal locations of the five NMDA receptor channel subunits, the epsilon 1 (Grin2a), epsilon 2 (Grin2b), epsilon 3 (Grin2c), epsilon 4 (Grin2d) and zeta 1 (Grinl) subunits, were determined using an interspecific backcross mapping panel derived from crosses of [(C57BL/6JxM. spretus) F1xC57BL/6J] mice. Each of these genes mapped to a single chromosome location. The mapping results assigned the five loci to five different mouse autosomes, indicating that they have become well dispersed among mouse chromosomes.

Amino Acid Sequence↗

PubMed1996

Impairment of suckling response, trigeminal neuronal pattern formation, and hippocampal LTD in NMDA receptor epsilon 2 subunit mutant mice.

Multiple epsilon subunits are major determinants of the NMDA receptor channel diversity. Based on their functional properties in vitro and distributions, we have proposed that the epsilon 1 and epsilon 2 subunits play a role in synaptic plasticity. To investigate the physiological significance of the NMDA receptor channel diversity, we generated mutant mice defective in the epsilon 2 subunit. These mice showed no suckling response and died shortly after birth but could survive by hand feeding. The mutation hindered the formation of the whisker-related neuronal barrelette structure and the clustering of primary sensory afferent terminals in the brainstem trigeminal nucleus. In the hippocampus of the mutant mice, synaptic NMDA responses and longterm depression were abolished. These results suggest that the epsilon 2 subunit plays an essential role in both neuronal pattern formation and synaptic plasticity.

Animals↗

PubMed1995

The sensitivity of AMPA-selective glutamate receptor channels to pentobarbital is determined by a single amino acid residue of the alpha 2 subunit.

Clinical concentrations of pentobarbital inhibit the alpha-amino-3-hydroxy-5- methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor (GluR) channels. Recently, the AMPA-selective GluR channels that contained the alpha 2 subunit were shown to be more sensitive to pentobarbital block than those without the alpha 2 subunit. Here we demonstrated that replacement by glutamine of the arginine residue in putative transmembrane segment M2 of the alpha 2 subunit (mutation alpha 2-R586Q) drastically reduced the pentobarbital sensitivity of the alpha 2 heteromeric channel to the level comparable to those of the alpha 1 and alpha 2-R586Q homomeric channels. These results suggest that the arginine residue in segment M2 of the alpha 2 subunit is the critical determinant of the sensitivities of the AMPA-selective GluR channels to pentobarbital.

Amino Acid Sequence↗

PubMed1995

Identification of four different forms of syntaxin 3.

cDNAs for four different forms of syntaxin 3 were cloned from a mouse brain cDNA library, and the proteins encoded by these clones were named syntaxin 3A (previous syntaxin 3), 3B, 3C and 3D. Syntaxin 3B contained a different sequence in the carboxyl terminal region from that of syntaxin 3A. The amino terminal region of syntaxin 3C contained an 18 amino acid sequence instead of a 34 amino acid sequence present in syntaxins 3A and 3B. Syntaxin 3D consisted of only 86 amino acids and lacked any putative transmembrane segments. These forms of syntaxin 3 are probably generated by alternative splicing of the primary transcript of syntaxin 3 gene. Cytoplasmic portions of syntaxins 3A and 3B but not of syntaxin 3C or 3D bound to Munc-18/n-sec1.

Alternative Splicing↗

PubMed1995

Effects of propofol on various AMPA-, kainate- and NMDA-selective glutamate receptor channels expressed in Xenopus oocytes.

Effects of a general intravenous anesthetic 2,6-diisopropylphenol (propofol) on various glutamate receptor (GluR) channels were examined on the alpha 1 and alpha 1/alpha 2 GluR channels selective for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), the beta 2/gamma 2 GluR channels selective for kainate, and the epsilon 2/zeta 1 and epsilon 3/zeta 1 N-methyl-D-aspartate (NMDA) receptor channels expressed in Xenopus oocytes. Propofol suppressed the current responses of the alpha 1/alpha 2, beta 2/gamma 2, epsilon 2/zeta 1 and epsilon 3/zeta 1 channels in a dose-dependent manner, whereas it enhanced the current responses of the alpha 1 channel. The extents of inhibition were in the order epsilon 2/zeta 1 > epsilon 3/zeta 1 > beta 2/gamma 2 > alpha 1/alpha 2 channels. During perfusion of 500 microM propofol, the alpha 1/alpha 2, beta 2/gamma 2, epsilon 2/zeta 1 and epsilon 3/zeta 1 channels were progressively suppressed. Furthermore, 10 min perfusion of 20 microM propofol inhibited the epsilon 2/zeta 1 channel by 24%. These results suggest that clinical concentrations (approximately 35 microM) of propofol suppress the NMDA receptor channels slightly.

Animals↗

PubMed1995

Reduced hippocampal LTP and spatial learning in mice lacking NMDA receptor epsilon 1 subunit.

The NMDA (N-methyl-D-aspartate) receptor channel is important for synaptic plasticity, which is thought to underlie learning, memory and development. The NMDA receptor channel is formed by at least two members of the glutamate receptor (GluR) channel subunit families, the GluR epsilon (NR2) and GluR zeta (NR1) subunit families. The four epsilon subunits are distinct in distribution, properties and regulation. On the basis of the Mg2+ sensitivity and expression patterns, we have proposed that the epsilon 1 (NR2A) and epsilon 2 (NR2B) subunits play a role in synaptic plasticity. Here we show that targeted disruption of the mouse epsilon 1 subunit gene resulted in significant reduction of the NMDA receptor channel current and long-term potentiation at the hippocampal CA1 synapses. The mutant mice also showed a moderate deficiency in spatial learning. These results support the notion that the NMDA receptor channel-dependent synaptic plasticity is the cellular basis of certain forms of learning.

Animals↗