Rescue of abnormal phenotypes in delta2 glutamate receptor-deficient mice by the extracellular N-terminal and intracellular C-terminal domains of the delta2 glutamate receptor.

The delta2 glutamate receptor (GluRdelta2) is expressed predominantly in cerebellar Purkinje cells. GluRdelta2 knock-out mice show impaired synaptogenesis and loss of long-term depression (LTD) at parallel fiber/Purkinje cell synapses, and persistent multiple climbing fiber (CF) innervation of Purkinje cells, resulting in severe ataxia. To identify domains critical for GluRdelta2 function, we produced various GluRdelta2 deletion constructs. Using lentiviral vectors, those constructs were expressed in Purkinje cells of GluRdelta2-deficient mice at postnatal day (P) 6 or 7, and rescue of abnormal phenotypes was examined beyond P30. Most constructs failed to rescue the defects of GluRdelta2-deficient mice, mainly because they were not efficiently transferred to the postsynaptic sites. However, a construct carrying only the extracellular N-terminal domain (NTD) and the intracellular C-terminal domain (CTD) linked with the fourth transmembrane domain of GluRdelta2 (NTD-TM4-CTD) caused incomplete, but significant rescue of ataxia, consistent with relatively better transport of the construct to the synapses. Notably, the expression of NTD-TM4-CTD in GluRdelta2-deficient Purkinje cells restored abrogated LTD, and aberrant CF territory in the molecular layer. Although the expression of NTD-TM4-CTD failed to rescue persistent multiple CF innervation of GluRdelta2-deficient Purkinje cells, a similar construct in which only TM4 was replaced with a transmembrane domain of CD4 successfully rescued the multiple CF innervation, probably due to more efficient transport of the protein to postsynaptic sites. These results suggest that NTD and CTD are critical domains of GluRdelta2, which functions substantially without conventional ligand binding and ion channel structures.

Inhibitory effects of the antiepileptic drug ethosuximide on G protein-activated inwardly rectifying K+ channels.

Antiepileptic drugs protect against seizures by modulating neuronal excitability. Ethosuximide is selectively used for the treatment of absence epilepsy, and has also been shown to have the potential for treating several other neuropsychiatric disorders in addition to several antiepileptic drugs. Although ethosuximide inhibits T-type Ca(2+), noninactivating Na(+), and Ca(2+)-activated K(+) channels, the molecular mechanisms underlying the effects of ethosuximide have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+) channels (GIRK, or Kir3) play an important role in regulating neuronal excitability, heart rate and platelet aggregation. In the present study, the effects of various antiepileptic drugs on GIRK channels were examined first by using the Xenopus oocyte expression assay. Ethosuximide at clinically relevant concentrations inhibited GIRK channels expressed in Xenopus oocytes. The inhibition was concentration-dependent, but voltage-independent, and time-independent during each voltage pulse. However, the other antiepileptic drugs tested: phenytoin, valproic acid, carbamazepine, phenobarbital, gabapentin, topiramate and zonisamide, had no significant effects on GIRK channels even at toxic concentrations. In contrast, Kir1.1 and Kir2.1 channels were insensitive to all of the drugs tested. Ethosuximide also attenuated ethanol-induced GIRK currents. These inhibitory effects of ethosuximide were not observed when ethosuximide was applied intracellularly. In granule cells of cerebellar slices, ethosuximide inhibited GTPgammaS-activated GIRK currents. Moreover, ADP- and epinephrine-induced platelet aggregation was inhibited by ethosuximide, but not by charybdotoxin, a platelet Ca(2+)-activated K(+) channel blocker. These results suggest that the inhibitory effects of ethosuximide on GIRK channels may affect some of brain, heart and platelet functions.

Glial glutamate transporters maintain one-to-one relationship at the climbing fiber-Purkinje cell synapse by preventing glutamate spillover.

A glial glutamate transporter, GLAST, is expressed abundantly in Bergmann glia and plays a major role in glutamate uptake at the excitatory synapses in cerebellar Purkinje cells (PCs). It has been reported that a higher percentage of PCs in GLAST-deficient mice are multiply innervated by climbing fibers (CFs) than in the wild-type (WT) mice, and that CF-mediated EPSCs with small amplitude and slow rise time, designated as atypical slow CF-EPSCs, are observed in these mice. To clarify the mechanism(s) underlying the generation of these atypical CF-EPSCs, we used (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA), an inhibitor of glial glutamate transporters. After the application of PMB-TBOA, slow-rising CF-EPSCs were newly detected in WT mice, and their rise and decay kinetics were different from those of conventional fast-rising CF-EPSCs but similar to those of atypical CF-EPSCs in GLAST-deficient mice. Furthermore, both slow-rising CF-EPSCs in the presence of PMB-TBOA in WT mice and atypical CF-EPSCs in GLAST-deficient mice showed much greater paired-pulse depression compared with fast-rising CF-EPSCs. In addition, both of them were more markedly inhibited by gamma-d-glutamyl-glycine, a low-affinity competitive antagonist of AMPA receptors. These results indicated that both of these types of EPSCs were mediated by a low concentration of glutamate released from neighboring CFs. Based on all of these findings, we suggest that glial transporters prevent glutamate released from a single CF from spilling over to neighboring PCs other than the synaptically connected PC, and play an essential role in the maintenance of the functional one-to-one relationship between CFs and PCs.

Facilitated activation of metabotropic glutamate receptors in cerebellar Purkinje cells in glutamate transporter EAAT4-deficient mice.

Around excitatory synapses in cerebellar Purkinje cells (PCs), GLAST and EAAT4 are expressed as predominant glial and neuronal glutamate transporters, respectively. EAAC1, another subtype of neuronal glutamate transporter, is also expressed in PCs. EAAT4 is co-localized with metabotropic glutamate receptors (mGluRs) at perisynaptic sites in excitatory synapses in PCs, and this neuronal transporter was reported to be involved in the regulation of mGluR activation induced by the stimulation of parallel fibers (PFs). However, it remains to be elucidated whether only EAAT4 is specifically involved in mGluR activation among the glutamate transporters expressed near excitatory synapses in PCs. Here we examined mGluR-mediated excitatory postsynaptic currents (mGluR-EPSCs) evoked by PF stimulation in cerebellar slices of mice deficient in EAAT4, EAAC1, or GLAST. PF-evoked mGluR-EPSCs showed larger amplitude and faster rising kinetics in EAAT4-deficient mice than in the wild-type mice. In contrast, there was no significant difference in either the amplitude or the rising kinetics of mGluR-EPSCs in GLAST- or EAAC1-deficient mice compared to wild-type mice. We conclude that EAAT4 is most closely involved in mGluR activation in PCs among the glutamate transporters.

Differential roles of glial and neuronal glutamate transporters in Purkinje cell synapses.

Glutamate transporters are essential for terminating excitatory neurotransmission. Two distinct glutamate transporters, glutamate-aspartate transporter (GLAST) and excitatory amino acid transporter 4 (EAAT4), are expressed most abundantly in the molecular layer of the cerebellar cortex. GLAST is expressed in Bergmann glial processes surrounding excitatory synapses on Purkinje cell dendritic spines, whereas EAAT4 is concentrated on the extrasynaptic regions of Purkinje cell spine membranes. To clarify the functional significance of the coexistence of these transporters, we analyzed the kinetics of EPSCs in Purkinje cells of mice lacking either GLAST or EAAT4. There was no difference in the amplitude or the kinetics of the rising and initial decay phase of EPSCs evoked by stimulations of climbing fibers and parallel fibers between wild-type and EAAT4-deficient mice. However, long-lasting tail currents of the EPSCs appeared age dependently in most of Purkinje cells in EAAT4-deficient mice. These tail currents were never seen in mice lacking GLAST. In the GLAST-deficient mice, however, the application of cyclothiazide that reduces desensitization of AMPA receptors increased the peak amplitude of the EPSC and prolonged its decay more markedly than in both wild-type and EAAT4-deficient mice. The results indicate that these transporters play differential roles in the removal of synaptically released glutamate. GLAST contributes mainly to uptake of glutamate that floods out of the synaptic cleft at early times after transmitter release. In contrast, the main role of EAAT4 is to remove low concentrations of glutamate that escape from the uptake by glial transporters at late times and thus prevents the transmitter from spilling over to neighboring synapses.

Roles of glial glutamate transporters in shaping EPSCs at the climbing fiber-Purkinje cell synapses.

Glial glutamate transporters, GLAST and GLT-1, are co-localized in processes of Bergmann glia (BG) wrapping excitatory synapses on Purkinje cells (PCs). Although GLAST is expressed six-fold more abundantly than GLT-1, no change is detected in the kinetics of climbing fiber (CF)-mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(-/-) mice compared to the wild-type mice (WT). Here we aimed to clarify the mechanism(s) underlying this unexpected finding using a selective GLT-1 blocker, dihydrokainate (DHK), and a novel antagonist of glial glutamate transporter, (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA). In the presence of cyclothiazide (CTZ), which attenuates the desensitization of AMPA receptors, DHK prolonged the decay time constant (tau(w)) of CF-EPSCs in WT, indicating that GLT-1 plays a partial role in the removal of glutamate. The application of 100 nM PMB-TBOA, which inhibited CF-mediated transporter currents in BG by approximately 80%, caused no change in tau(w) in WT in the absence of CTZ, whereas it prolonged tau(w) in the presence of CTZ. This prolonged value of tau(w) was similar to that in GLAST(-/-) mice in the presence of CTZ. These results indicate that glial glutamate transporters can apparently retain the fast decay kinetics of CF-EPSCs if a small proportion ( approximately 20%) of functional transporters is preserved.

Muscarine-induced increase in frequency of spontaneous EPSCs in Purkinje cells in the vestibulo-cerebellum of the rat.

Cholinergic projections are relatively sparse in the cerebellum compared with other parts of the brain. However, some mossy fibers in the vestibulo-cerebellum are known to be cholinergic. To clarify the functional roles of cholinergic mossy fibers in the vestibulo-cerebellum, we investigated the effects of acetylcholine (ACh) on the membrane electrical properties of both granule cells and Purkinje cells in slices of the cerebellar vermis of the rat using whole-cell patch-clamp techniques. The bath application of ACh induced a marked increase in the frequency of spontaneous EPSCs (sEPSCs) in Purkinje cells specifically in the vestibulo-cerebellum. This effect of ACh was mimicked by muscarine but not by nicotine. It was abolished by application of either tetrodotoxin or the antagonist of AMPA receptors, indicating that the ACh-induced enhancement of sEPSCs occurred indirectly via the activation of neurons sending glutamatergic projections to Purkinje cells. In approximately 15% of granule cells tested in the vestibulo-cerebellum, muscarine elicited membrane depolarization accompanied by a decrease in membrane conductance and increased the neuronal excitability. The muscarine-induced depolarization of granule cells in the vestibulo-cerebellum was attributable to the inhibition of standing-outward K+ currents (IKSO) most likely via the activation of muscarinic M3 receptors. Taken together, these results indicate that ACh increases the firing frequency of granule cells by inhibiting IKSO, which in turn increases the frequency of sEPSCs in Purkinje cells in the rat vestibulo-cerebellum.

Postsynaptic expression of a new calcium pathway in hippocampal CA3 neurons and its influence on mossy fiber long-term potentiation.

Long-term potentiation (LTP) in the CA1 region of the hippocampus is induced by postsynaptic Ca(2+) influx via NMDA receptors (NMDARs). However, this synaptic plasticity occurs independently of NMDARs when Ca(2+)-permeable AMPA receptors (AMPARs) are expressed at postsynaptic sites using various genetic techniques, indicating that an increase in Ca(2+) level at critical postsynaptic sites, regardless of its entry pathway, triggers the induction of LTP at CA1 synapses. In contrast, NMDARs are sparsely distributed on mossy fiber (MF) synapses in CA3 hippocampal neurons, and most evidence favors the presynaptic mechanism for LTP induction, although some reports suggested a postsynaptic mechanism. In this study, we examined whether Ca(2+) influx through the newly produced postsynaptic receptors during high-frequency stimulation affects the induction of MF LTP. For this purpose, we expressed Ca(2+)-permeable AMPARs in CA3 pyramidal neurons by Sindbis viral-mediated gene transfer of the unedited form of the glutamate receptor 2 (GluR2Q) subunit, as a new pathway for postsynaptic Ca(2+) entry, in rat hippocampal organotypic cultures. Virally expressed myc-tagged GluR2Q was detected at the complex spines known as the thorny excrescences, which serve as postsynaptic targets for MF synaptic input, on the proximal apical dendrites of CA3 pyramidal cells. Furthermore, endogenous Ca(2+)-impermeable AMPARs at MF synapses were converted into Ca(2+)-permeable receptors by GluR2Q expression. However, the postsynaptic expression of Ca(2+)-permeable AMPARs had no significant influence on the two types of MF LTP induced by different stimulus protocols. These results supported the notion that MF LTP is independent of postsynaptic Ca(2+).

Effects of a novel glutamate transporter blocker, (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), on activities of hippocampal neurons.

Glutamate transporters rapidly take up synaptically released glutamate and maintain the glutamate concentration in the synaptic cleft at a low level. (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA) is a novel glutamate transporter blocker that potently suppresses the activity of glial transporters. TFB-TBOA inhibited synaptically activated transporter currents (STCs) in astrocytes in the stratum radiatum in rat hippocampal slices in a dose-dependent manner with an IC50 of 13 nM, and reduced them to approximately 10% of the control at 100 nM. We investigated the effects of TFB-TBOA on glutamatergic synaptic transmission and cell excitability in CA1 pyramidal cells. TFB-TBOA (100 nM) prolonged the decay of N-methyl-D-aspartic acid receptor (NMDAR)-mediated excitatory postsynaptic currents (EPSCs), whereas it prolonged that of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated EPSCs only when the desensitization of AMPARs was reduced by cyclothiazide (CTZ). Furthermore, long-term application of TFB-TBOA induced spontaneous epileptiform discharges with a continuous depolarization shift of membrane potential. These epileptiform activities were mainly attributed to NMDAR activation. Even after pharmacological block of NMDARs, however, TFB-TBOA induced similar changes by activating AMPARs in the presence of CTZ. Thus, the continuous uptake of synaptically released glutamate by glial transporters is indispensable for protecting hippocampal neurons from glutamate receptor-mediated hyperexcitabilities.

Predominant expression of GluR2 among the AMPA receptor subunits in neuronal progenitor cells of the rat hippocampus.

Cells derived from the hippocampus of embryonic day 18 (E18) rats were cultured in B27-supplemented Neurobasal medium without serum. We found the presence of numerous small cells with round or elliptical somata and fine processes in this primary culture. These cells were first detectable on culture day 8 and gradually increased in number that reached the maximum on approximately day 14. They incorporated bromodeoxyuridine (BrdU) and expressed nestin, a marker of stem cells and progenitor cells. Furthermore, nearly a half of these cells also expressed neuron-specific beta tubulin. On the other hand, they did not express O4 and glial fibrillary acid protein (GFAP), markers of oligodendrocytes and astrocytes, respectively. Thus, these small cells are most likely to be neuronal progenitor cells. The whole-cell patch clamp studies revealed that these cells expressed voltage-gated Na+, Ca2+ and K+ channels. With regard to ligand-gated channels, these cells were sensitive to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), but not to N-methyl-D-aspartate (NMDA). The current-voltage relationship of the AMPA-induced current was slightly outwardly rectifying, suggesting that the AMPA receptors contained the GluR2 subunit in their oligomeric assemblies. The single-cell reverse transcription (RT)-PCR analysis revealed that GluR2 is predominant over the other AMPA receptor subunits in these cells. Furthermore, GluR2 was expressed mainly in the flip form.

Contribution of glutamate transporter GLT-1 to removal of synaptically released glutamate at climbing fiber-Purkinje cell synapses.

Rapid removal of synaptically released glutamate from the extracellular space ensures a high signal-to-noise ratio in excitatory neurotransmission. In the cerebellum, glial glutamate transporters, GLAST and GLT-1, are co-localized in the processes of Bergmann glia wrapping excitatory synapses on Purkinje cells (PCs). Although GLAST is expressed six-fold more abundantly than GLT-1, the decay kinetics of climbing fiber-mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(-/-) mice are not different from those in wild-type (WT) mice. This raises a possibility that GLT-1 plays a significant role in clearing glutamate at CF-PC synapses despite its smaller amount of expression. Here, we studied the functions of GLT-1 and GLAST in the clearance of glutamate using GLAST(-/-) mice and GLT-1(-/-) mice. In the presence of cyclothiazide (CTZ) that attenuates the desensitization of AMPA receptors, the decay time constant of CF-EPSCs (tau(w)) in GLT-1(-/-) mice was slower than that in WT mice. However, the degree of this prolongation of tau(w) was less prominent compared to that in GLAST(-/-) mice. The values of tau(w) in GLT-1(-/-) mice and GLAST(-/-) mice were comparable to those estimated in WT mice in the presence of a potent blocker of glial glutamate transporters (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA) at 10 and 100 nM, which reduced the amplitudes of glutamate transporter currents elicited by CF stimulation in Bergmann glia to approximately 81 and approximately 28%, respectively. We conclude that GLT-1 plays a minor role compared to GLAST in clearing synaptically released glutamate at CF-PC synapses.

Roles of glutamate transporters in shaping excitatory synaptic currents in cerebellar Purkinje cells.

Several subtypes of glutamate transporters are abundantly expressed near the excitatory synapses on cerebellar Purkinje cells. We investigated the roles of the glutamate transporters in shaping the excitatory postsynaptic currents (EPSCs) and regulating the levels of extracellular glutamate in the mouse cerebellum using a potent blocker of glutamate transporters, dl-threo-beta-benzyloxyaspartate (dl-TBOA). This drug markedly prolonged AMPA receptor-mediated EPSCs in Purkinje cells evoked by stimulating both parallel fibres and climbing fibres. The decay phase of the prolonged EPSCs was fitted by double exponentials, of which the slower component was preferentially inhibited by a low-affinity competitive antagonist of AMPA receptors, gamma-d-glutamyl-glycine, indicating that the slow component induced by dl-TBOA was the AMPA receptor-mediated current activated by lower concentrations of glutamate than those contributing to the peak of the EPSC. This result suggests that dl-TBOA prolongs the stay of synaptically released glutamate in the synaptic cleft and also induces glutamate spillover to extrasynaptic targets as well as neighbouring synapses. Furthermore, high concentrations of dl-TBOA in the presence of cyclothiazide generated a continuous inward current in Purkinje cells, of which the amplitude reached the peak level of the climbing-fibre EPSC. This continuous inward current was abolished by the blocker of AMPA receptors, indicating that the strong inhibition of glutamate uptake causes the rapid accumulation of glutamate in the extracellular space. These results highlight the importance of glutamate transporters in maintaining the proper glutamatergic transmission in Purkinje cell synapses.

Functional NMDA receptor channels generated by NMDAR2B gene transfer in rat cerebellar Purkinje cells.

The adult cerebellar Purkinje cell is an exceptional neuron in the central nervous system in that it expresses high levels of NMDAR1 (NR1) mRNA without expressing any NMDAR2 (NR2) mRNAs. It has no functional NMDA receptor (NMDAR) channels, although it receives enormous numbers of excitatory inputs. Despite the high level of NR1 mRNA expression, the presence and localization of NR1 protein in mature Purkinje cells are controversial. To examine the presence of NR1 protein and its ability to form functional NMDARs, we expressed the NR2B subunit in rat mature Purkinje neurons by Sindbis viral-mediated gene transfer. The recombinant virus encoding both the NR2B and enhanced green fluorescent protein (GFP) genes (designated as SIN-EG-NR2B) infected Purkinje cells without infecting glial cells. GFP fluorescence was detected in the soma and throughout dendrites of Purkinje cells 18-24 h postinfection. In most of GFP-positive cells, the expression of NR2B protein was detected by immunostaining with NR2B-specific antibodies. In Purkinje cells infected with SIN-EG-NR2B, the iontophoretic application of NMDA induced prominent NMDAR-mediated current responses, indicating that the exogenous NR2B was assembled with endogenous NR1 to form functional NMDARs. Furthermore, NMDAR-mediated synaptic currents were detected at both the climbing fibre and parallel fibre synapses in infected Purkinje cells. Thus, the mature Purkinje cell produces NR1 protein that is ready to combine with NR2 to form functional NMDARs in excitatory synapses.