Seiji Ozawa

Glutamate receptors in the mammalian central nervous system.

Glutamate receptors (GluRs) mediate most of the excitatory neurotransmission in the mammalian central nervous system (CNS). In addition, they are involved in plastic changes in synaptic transmission as well as excitotoxic neuronal cell death that occurs in a variety of acute and chronic neurological disorders. The GluRs are divided into two distinct groups, ionotropic and metabotropic receptors. The ionotropic receptors (iGluRs) are further subdivided into three groups: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptor channels. The metabotropic receptors (mGluRs) are coupled to GTP-binding proteins (G-proteins), and regulate the production of intracellular messengers. The application of molecular cloning technology has greatly advanced our understanding of the GluR system. To date, at least 14 cDNAs of subunit proteins constituting iGluRs and 8 cDNAs of proteins constituting mGluRs have been cloned in the mammalian CNS, and the molecular structure, distribution and developmental change in the CNS, functional and pharmacological properties of each receptor subunit have been elucidated. Furthermore, the obtained clones have provided valuable tools for conducting studies to clarify the physiological and pathophysiological significances of each subunit. For example, the generation of gene knockout mice has disclosed critical roles of some GluR subunits in brain functions. In this article, we review recent progress in the research for GluRs with special emphasis on the molecular diversity of the GluR system and its implications for physiology and pathology of the CNS.

Kainate receptor‐mediated presynaptic inhibition at the mouse hippocampal mossy fibre synapse

1 The presynaptic action of kainate (KA) receptor activation at the mossy fibre‐CA3 synapse was examined using fluorescence measurement of presynaptic Ca2+ influx as well as electrophysiological recordings in mouse hippocampal slices. 2 Bath application of a low concentration (0·2 μM) of KA reversibly increased the amplitude of presynaptic volley evoked by stimulation of mossy fibres to 146 ± 6 % of control (n= 6), whereas it reduced the field excitatory postsynaptic potential (EPSPs) to 30 ± 4 %. 3 The potentiating effect of KA on the presynaptic volleys was also observed in Ca2+‐free solution, and was partly antagonized by (2S,4R)‐4‐methylglutamic acid (SYM 2081, 1 μM), which selectively desensitizes KA receptors. 4 The antidromic population spike of dentate granule cells evoked by stimulation of mossy fibres was increased by application of 0·2 μM KA to 160 ± 10 % of control (n= 6). Whole‐cell current‐clamp recordings revealed that the stimulus threshold for generating antidromic spikes recorded from a single granule cell was lowered by KA application. 5 Application of KA (0·2 μM) suppressed presynaptic Ca2+ influx to 78 ± 4 % of control (n= 6), whereas the amplitude of the presynaptic volley was increased. 6 KA at 0·2 μM reversibly suppressed excitatory postsynaptic currents (EPSCs) evoked by mossy fibre simulation to 38 ± 9 % of control (n= 5). 7 These results suggest that KA receptor activation enhances the excitability of mossy fibres, probably via axonal depolarization, and reduces action potential‐induced Ca2+ influx, thereby inhibiting mossy fibre EPSCs presynaptically. This novel presynaptic inhibitory action of KA at the mossy fibre‐CA3 synapse may regulate the excitability of highly interconnected CA3 networks.

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.

Kainate receptor-mediated inhibition of presynaptic Ca2+ influx and EPSP in area CA1 of the rat hippocampus

1 The effect of a low concentration (1 μM) of kainate (kainic acid; KA) on presynaptic calcium (Ca2+) influx at the Schaffer collateral‐commissural (SCC) synapse was examined in rat hippocampal slices. 2 Following selective loading of the presynaptic terminals with the fluorescent Ca2+ indicator rhod‐2 AM, transient increases in the presynaptic Ca2+ concentration (pre[Ca2+]t) and field excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of the SCC pathway were recorded simultaneously. 3 Bath application of 1 μM KA reversibly suppressed field EPSPs and pre[Ca2+]t to 37.7 ± 4.0 % and 72.9 ± 2.4 % of control, respectively. Excitatory postsynaptic currents (EPSCs) recorded with the use of the whole‐cell patch‐clamp technique were also suppressed by 1 μM KA to 42.6 ± 6.3 % of control. A quantitative analysis of the decreases in pre[Ca2+]t and the amplitude of field EPSP during KA application suggests that KA inhibits transmission primarily by reducing the pre[Ca2+]t. 4 Consistent with a presynaptic site for these effects, paired‐pulse facilitation (PPF) was enhanced by 1 μM KA. 5 A substantial KA‐induced suppression of NMDA receptor‐mediated EPSPs was detected when AMPA receptors were blocked by the AMPA receptor‐selective antagonist GYKI 52466 (100 μM). 6 The suppressive effect of KA on field EPSPs and pre[Ca2+]t was antagonized by the KA antagonist NS‐102 (10 μM). 7 These results suggest that the presynaptic inhibitory action of KA at the hippocampal CA1 synapse is primarily due to the inhibition of Ca2+ influx into the presynaptic terminals.

Ca2+-Permeable AMPA Receptors Regulate Growth of Human Glioblastoma via Akt Activation

Evidence has been accumulated that glioblastoma cells release and exploit glutamate for proliferation and migration by autocrine or paracrine loops through Ca2+-permeable AMPA-type glutamate receptors. Here, we show that Ca2+ signaling mediated by AMPA receptor regulates the growth and motility of glioblastoma cells via activation of Akt. Ca2+ supplied through Ca2+-permeable AMPA receptor phosphorylated Akt at Ser-473, thereby facilitating proliferation and mobility. A dominant-negative form of Akt inhibited cell proliferation and migration accelerated by overexpression of Ca2+-permeable AMPA receptor. In contrast, introduction of a constitutively active form of Akt rescued tumor cells from apoptosis induced by the conversion of Ca2+-permeable AMPA receptor to Ca2+-impermeable receptors by the delivery of GluR2 cDNA. Therefore, Akt functions as downstream effectors for Ca2+-signaling mediated by AMPA receptor in glioblastoma cells. The activation of the glutamate-AMPA receptor-Akt pathway may contribute to the high degree of anaplasia and invasive growth of human glioblastoma. This novel pathway might give an alternative therapeutic target.

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.

Acetylcholine-induced membrane depolarization and potential fluctuations in the rat adrenal chromaffin cell

1. The effect of acetylcholine (ACh) on the rat adrenal chromaffin cell membrane was examined in culture by using an intracellular recording technique. ACh was applied with gas pressure through a pipette which had an internal tip diameter of about 3 μm.

Dual mechanism for presynaptic modulation by axonal metabotropic glutamate receptor at the mouse mossy fibre‐CA3 synapse

1 To investigate mechanisms responsible for the presynaptic inhibitory action mediated by the axonal group II metabotropic glutamate receptor (mGluR) at the mossy fibre‐CA3 synapse, we used a quantitative fluorescence measurement of presynaptic Ca2+ in mouse hippocampal slices. 2 Bath application of the group II mGluR‐specific agonist (2S,1′R,2′R,3′R)‐2‐(2,3‐dicarboxycyclopropyl)glycine (DCG‐IV, 1 μM) reversibly suppressed the presynaptic Ca2+ influx (to 55·2 ± 4·6 % of control, n= 5) as well as field EPSPs recorded simultaneously (to 3·1 ± 2·0 %). Presynaptic fibre volley was not affected by 1 μM DCG‐IV. 3 A quantitative analysis of the inhibition of presynaptic Ca2+ influx and field EPSP suggested that DCG‐IV suppressed the field EPSP to a greater extent than would be expected if the suppression were solely due to a decrease in the presynaptic Ca2+ influx. 4 DCG‐IV at 1 μM suppressed the mean frequency (to 73·8 ± 3·9 % of control, n= 11), but not the mean amplitude (to 97·0 ± 3·5 %), of miniature EPSCs recorded from CA3 neurones using the whole‐cell patch‐clamp technique. 5 These results suggest that group II mGluR‐mediated suppression is due both to a reduction of presynaptic Ca2+ influx and downregulation of the subsequent exocytotic machinery.

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.