Masae Iino

Permeation of calcium through excitatory amino acid receptor channels in cultured rat hippocampal neurones.

1. N‐methyl‐D‐aspartate (NMDA)‐, quisqualate‐ and kainate‐induced currents were recorded in cultured rat hippocampal neurones using the whole‐cell voltage‐clamp technique. To isolate the inward currents carried by Ca2+ and other divalent cations (Sr2+, Ba2+, Mn2+ and Mg2+), both Na+ and K+ in the control external solution were replaced with the impermeant cation N‐methylglucamine (NMG). 2. Replacement of Na+, K+ and Ca2+ with NMG abolished NMDA‐, quisqualate‐ and kinate‐induced inward currents. In Na(+)‐, K(+)‐free (abbreviated simply as Na(+)‐free) solution containing 10 mM‐Ca2+ NMDA caused prominent inward currents at ‐60 mV. In this solution with the internal solution containing 165 mM‐Cs+, the reversal potential of the NMDA‐induced current was ‐5.0 +/‐ 0.7 mV (n = 36), indicating a value of PCa/PCs = 6.2 for the ratio of the permeability coefficients of Ca2+ and Cs+ according to the constant‐field equation. 3. NMDA elicited inward current responses at ‐60 mV in Na(+)‐, Ca2(+)‐free solution containing 10 mM‐Sr2+, Ba2+, or Mn2+, but not in Na(+)‐free, 10 mM‐Mg2+ solution. On the basis of reversal potential measurements, the permeability sequence of NMDA receptor channels among the divalent cations was determined to be Ba2+ (1.2) greater than Ca2+ (1.0) greater than Sr2+ (0.8) greater than Mn2+ (0.3) much greater than Mg2+ (less than 0.02). 4. The reversal potential of the quisqualate‐induced current was more negative than ‐80 mV in Na(+)‐free, 10 mM‐Ca2+ solution, indicating a value of PCa/PCs less than 0.18. 5. Kainate‐induced current responses were classified into two types. In the type I response the reversal potential of the kainate‐induced current was more negative than ‐80 mV in Na(+)‐free, 10 mM‐Ca2+ solution, indicating that the Ca2+ permeability of this type of kainate channel is as low as that of the quisqualate channel. In the neurones which showed a type I response, there was a tendency of outward rectification in the current‐voltage plots of the kainate response in control solution. 6. In the type II response kainate caused prominent inward currents at ‐60 mV in Na(+)‐free, 10 mM‐Ca2+ solution. The reversal potential was ‐23.3 +/‐ 5.6 mV (n = 17), indicating a permeability ratio PCa/PCs = 2.3. In the neurones which showed a type II response, a remarkable inward rectification was observed in the current‐voltage plots of the kainate response in control solution. 7. Type II kainate channels showed relatively poor selectivity among divalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)

Subunit composition at the single-cell level explains functional properties of a glutamate-gated channel

The diversity of known glutamate-gated channels has been markedly increased by the discovery of multiple subunits and their spliced and edited variants. These subunits can potentially form different oligomeric complexes with diverging properties. A crucial question is therefore to determine the actual subunit composition of naturally occurring glutamate receptors. We have coupled patch-clamp recordings and reverse transcription followed by PCR amplification to correlate the presence of mRNAs for each subunit and the functional properties of native glutamate receptors at the single-cell level. In a homogeneous population of functionally identified hippocampal neurons (type II) in culture bearing a glutamate receptor of the AMPA subtype with a high calcium permeability, we found that, among the multiple subunits, only two, the flop forms of GluR1 and GluR4, were expressed. In particular, GluR2 was never detected. This composition explains the uncommon properties of AMPA receptors in type II neurons.

Blocking effect of 1-naphthyl acetyl spermine on Ca2+-permeable AMPA receptors in cultured rat hippocampal neurons

Effects of 1-naphthyl acetyl spermine (NASPM), a synthetic analogue of Joro spider toxin (JSTX), on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors were studied in cultured rat hippocampal neurons using the whole-cell patch clamp technique. A population of cultured neurons had AMPA receptors with a strong inward rectification and a high permeability to Ca2+ (type II neurons). Whereas most neurons (type I neurons) had AMPA receptors with a slight outward rectification and little Ca2+ permeability. NASPM selectively suppressed the inwardly rectifying and Ca(2+)-permeable AMPA receptors expressed in type II neurons. It had no effect on AMPA receptors in type I neurons. The blocking effect of NASPM on the Ca(2+)-permeable AMPA receptors was use and voltage-dependent. When the effect of NASPM reached a steady state, current responses induced by ionophoretic applications of kainate, a non-desensitizing agonist of AMPA receptors, in type II neurons were suppressed by NASPM in a dose-dependent manner at -60 mV (IC50 0.33 microM, and Hill coefficient 0.94). The response to kainate recovered partially after washing out NASPM. NASPM did not affect the Ca(2+)-permeable AMPA receptors when the neuronal membrane was held at potentials more positive than +40 mV. Furthermore, the blockade by NASPM which was attained at negative potentials was transiently removed by shifting membrane potential to +60 mV for 5 s together with a single ionophoretic application of kainate. NASPM would be useful as a pharmacological tool for elucidating both physiological and pathological significances of Ca(2+)-permeable AMPA receptors in the CNS.

Distribution of neurones expressing inwardly rectifying and Ca(2+)‐permeable AMPA receptors in rat hippocampal slices.

1. Current‐voltage (I‐V) relationships and Ca2+ permeability of receptor channels activated by bath application of kainate, a non‐desensitizing agonist of alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors, were examined in various types of neurones in hippocampal slices of 5‐ to 13‐day‐old rats by using the tight‐seal patch clamp recording technique. 2. Three types of responses were observed: type I response with outwardly rectifying I‐V relationship, type II response with I‐V relationship of marked inward rectification, and intermediate response with I‐V relationship of weaker inward rectification. Neurones with type I, type II and intermediate I‐V relationships of kainate responses were referred to as type I, type II and intermediate neurones, respectively. 3. Permeability of Ca2+ ions was estimated by the reversal potential of kainate response in the outside‐out patch in Na(+)‐free extracellular solution containing 100 mM Ca2+. The reversal potentials were ‐44.4 +/‐ 14.0 mV (mean +/‐ S.D.) for type I (n = 7), +11.8 +/‐ 3.6 mV for type II (n = 5), and ‐8.7 +/‐ 7.4 mV for the intermediate neurones (n = 7). The values of PCa/PCs, the ratios of the permeability coefficients of Ca2+ and Cs+, estimated according to the constant‐field equation were 0.08 for type I, 1.71 for type II, and 0.50 for the intermediate neurones. 4. Type II and intermediate responses were observed mainly in non‐pyramidal neurones in various areas of the hippocampus, most frequently observed in the stratum molecular of the dentate gyrus and in the stratum radiatum and the stratum lacunosum‐molecular of both the CA1 and CA3 regions. Both type II and intermediate neurones stained with biocytin had round‐ or ellipsoidal‐shaped somata and issued divergent axonal projections to the surrounding structures. 5. Excitatory postsynaptic currents (EPSCs) recorded in type II neurones had 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX)‐sensitive fast and D‐2‐amino‐5‐phosphonovalerate (APV)‐sensitive slow components. The I‐V relationship of the fast component showed a strong inward rectification, indicating that inwardly rectifying AMPA receptors are involved in excitatory synaptic transmission.

Voltage‐dependent blockage of Ca(2+)‐permeable AMPA receptors by joro spider toxin in cultured rat hippocampal neurones.

1. The effect of synthetic joro spider toxin (JSTX‐3) on alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor channels in cultured rat hippocampal neurones was investigated using the whole‐cell patch‐clamp technique. 2. A population of cultured neurones had AMPA receptors with strong inward rectification and substantial Ca2+ permeability (type II neurones), whereas most neurones (type I neurones) had slight outward rectification and little Ca2+ permeability. JSTX‐3 selectively suppressed the inwardly rectifying and Ca(2+)‐permeable AMPA receptors expressed in type II neurones without affecting AMPA receptors in type I neurones. 3. The effect of JSTX‐3 on the Ca(2+)‐permeable AMPA receptors was use and voltage dependent. In the steady state, current responses induced by ionophoretic applications of kainate (a non‐desensitizing agonist of AMPA receptors) were suppressed by the toxin in a dose‐dependent manner at negative potentials (IC50 = 56 nM at ‐60 mV). 4. At the standard membrane potential (‐60 mV), recovery from the blockage by JSTX‐3 was very slow. Even after washout for more than 7 min, the recovery was only partial. However, the blockage was completely removed immediately after application of a +60 mV voltage pulse for 5 s in conjunction with a single ionophoretic application of kainate.

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.

Spermine mediates inward rectification of Ca2+-permeable AMPA receptor channels

AMPA-gated glutamate receptors with an inwardly rectifying current-voltage (I-V) relationship and substantial Ca2+ permeability are expressed in a population of cultured rat hippocampal neurones (type II neurones). The inward rectification of these AMPA receptors was gradually lost in cell-free membrane patches. The I-V relationship of the AMPA receptors displayed a slight outward rectification in most patches 10 min after excision. This loss of inward rectification was not accompanied by a change in the Ca2+ permeability. The inward rectification was maintained by applying physiological concentrations of spermine to the cytoplasmic side of patch membranes. These results indicate that some cytoplasmic factor mediates inward rectification in the Ca(2+)-permeable AMPA receptors, and that a candidate for this substance is spermine.

Three types of voltage-dependent calcium current in cultured rat hippocampal neurons

Voltage-dependent calcium (Ca2+) currents in cultured rat hippocampal neurons were studied with the whole-cell recording mode of the patch-clamp technique. On the basis of the voltage-dependence of activation, kinetics of inactivation and pharmacology, 3 types of Ca2+ currents were distinguished. The low-threshold Ca2+ current (Il) was activated at -60 mV, and completely inactivated during a 100-ms depolarization to -40 mV (time constant: tau = 16 +/- 1 ms). The high-threshold currents (Ih), which were activated at -20 mV, could be separated into two types. The high-threshold, fast inactivating current (Ih,f) decayed quickly during a maintained depolarization (tau = 33 +/- 3 ms at 0 mV), whereas the high-threshold, slowly inactivating current (Ih,s) decayed with a much slower time constant (tau = 505 +/- 42 ms at 0 mV). The inactivations of Ih,f and Ih,s exhibited different time- and voltage-dependencies. Nickel ions (Ni2+, 25 microM) markedly suppressed Il, but little affected Ih. Cadmium ions (Cd2+, 10 microM) almost completely suppressed Ih, but left a small amount of Il. Lanthanum ions (La3+, 10 microM) almost completely suppressed both Il and Ih. Ih,s was sensitive to block by the dihydropyridine antagonist nicardipine (10 microM).

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.

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‐β‐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, γ‐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.