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ABSTRACT: 1. The role of cystine-glutamate exchange in controlling the extracellular glutamate concentration in the central nervous system was examined by whole-cell clamping neurons in rat brain slices, and using their glutamate receptors as sensors of extracellular glutamate concentration. 2. Applying cystine to cerebellar slices generated a membrane current in Purkinje cells which was abolished by glutamate receptor blockers. Similar cystine-evoked currents were seen in pyramidal cells of frontal cortex slices. 3. Control experiments on non-N-methyl-D-aspartate (non-NMDA) receptors in enzymatically isolated Purkinje cells showed that cystine did not produce a current in slice Purkinje cells by directly activating glutamate receptors, nor by potentiating the action of background levels of glutamate on receptors. Experiments on isolated salamander Muller cells showed that cystine did not block Na+-dependent GLAST glutamate transporters (homologous to the transporters in the Bergmann glia ensheathing the Purkinje cells), nor did it block the current produced by EAAT4 and EAAC1 glutamate transporters in Purkinje cells. Thus the cystine-evoked current in Purkinje cells is not due to a rise in extracellular glutamate concentration caused by block of Na+-dependent uptake. 4. The dependence of cystine-evoked current on cystine concentration in slice Purkinje cells could be fitted by a Michaelis-Menten relation with a Km of 250 microM. The Km predicted from this for cystine activating glutamate efflux is less than 140 microM, because of the non-linear dependence on glutamate concentration of the Purkinje cell current. The current evoked by 1 mM cystine was little affected by removal of extracellular chloride or addition of 1 mM furosemide (frusemide), but was potentiated by 1 mM 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS). 5. These data suggest that external cystine generates a current in slice Purkinje cells by activating cystine-glutamate exchange in cells of the slice, releasing glutamate which activates non-NMDA receptors in the Purkinje cell membrane.
The Journal of Physiology 03/1999; 514 ( Pt 3):783-93. · 4.72 Impact Factor
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Progress in brain research 02/1998; 116:45-57. · 3.04 Impact Factor
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ABSTRACT: We have described how whole-cell clamping of neurons in brain slices has allowed a characterization of postsynaptic transporters, probably a mixture of EAAC1 and EAAT4, in cerebellar Purkinje cells. Similar experiments have been carried out on transporters (mainly GLAST) in cerebellar Bergmann glia, and have revealed an uptake current occurring as these carriers remove glutamate released at the parallel fiber synapses. As more transporters are cloned and their regulation is characterized in heterologous expression systems, it will be increasingly important to use methods similar to those outlined above to investigate to what extent the behavior of the carriers is similar in situ in the nervous system.
Methods in Enzymology 02/1998; 296:608-17. · 2.04 Impact Factor
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ABSTRACT: Glutamate transporters in neurones and glia, four of which have been cloned from mammals, play a crucial role in controlling the extracellular glutamate concentration in the brain. In normal conditions, they remove glutamate from the extracellular space and thereby help to terminate glutamatergic synaptic transmission and to prevent the extracellular glutamate concentration from rising to neurotoxic values. Glutamate transport on these carriers is thought to be driven by the cotransport of Na+, the counter-transport of K+, and either the cotransport of H+ or the counter-transport of OH-. Activating the transporters also activates an anion conductance in their structure, the anion flux through which is not coupled to glutamate movement and varies widely for the different transporters. During hypoxia or ischaemia, glutamate transporters can run backwards, releasing glutamate into the extracellular space, triggering the death of neurones and thus causing mental and physical handicap. The rate of glutamate release by this process is slowed by the acid pH occurring in hypoxia/ischaemia, which may help protect the brain during transient, but not sustained, ischaemia.
Journal of Experimental Biology 02/1997; 200(Pt 2):401-9. · 3.00 Impact Factor
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ABSTRACT: 1. Whole-cell clamp experiments on Purkinje neurons in rat cerebellar slices were used to test whether glutamate transporters, detected immunocytochemically in the somata and dendrites of the cells, are functional in the cell surface membrane, and to investigate their role in terminating synaptic transmission. 2. A membrane current was detected with the pharmacology, voltage and ion dependence of a glutamate uptake current. Part of the current was generated by an anion conductance activated when uptake occurs. 3. With sodium and glutamate inside the cell, raising the external potassium concentration generated an outward current attributable to reversed operation of glutamate transporters. 4. The magnitude of the uptake current suggested that Purkinje cell transporters could help to terminate transmission at the climbing and parallel fibre to Purkinje cell synapses. Reducing postsynaptic glutamate uptake with intracellular D-aspartate prolonged the climbing fibre EPSC. 5. These data establish the existence of functional postsynaptic glutamate transporters, show that they contribute to terminating synaptic transmission, and suggest that they may play a role in the preferential death of Purkinje cells in ischaemia.
The Journal of Physiology 01/1997; 497 ( Pt 2):523-30. · 4.72 Impact Factor
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ABSTRACT: Excitatory postsynaptic currents (EPSCs) at the parallel fiber and climbing fiber to Purkinje cell synapses were studied by whole-cell clamping Purkinje cells in cerebellar slices. Reducing glutamate release with adenosine or GABA decreased the amplitude of the EPSCs, with a larger suppression being produced at the parallel fiber synapse. Reducing glutamate release also speeded the decay of the EPSCs, and this effect was not a series resistance artefact since postsynaptic reduction of the current with CNQX did not speed the EPSC decay. Blocking glutamate uptake slowed the decay of the EPSCs. At the climbing fiber synapse, adenosine had little suppressive effect on the smaller EPSC evoked by the second of a pair of stimuli. Blocking desensitization of postsynaptic AMPA receptors prolonged the EPSC decay, preferentially increased the size of the second EPSC, and resulted in adenosine having a similar suppressive effect on the first and second EPSC. These data suggest that, at these synapses, the fall of glutamate concentration in the synaptic cleft overlaps with the decay of the EPSC, and that the EPSC size and duration are controlled by the amount of glutamate released, the rate of glutamate uptake, and desensitization.
Journal of Neuroscience 09/1995; 15(8):5693-702. · 7.11 Impact Factor
