2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline and 3-hydroxyaspartic-acid

Numerous data indicate that nonsynaptic release of glutamate occurs both in normal and pathophysiological conditions. When reaching receptors in the postsynaptic density (PSD), glutamate (Glu) could affect the synaptic transmission. We have tested this possibility in the hippocampal CA1 synapses of rats, either by applying exogenous Glu to the CA1 neurons or by disruption of Glu transporter activity. L-Glu (400 microM) was directly applied to the hippocampal slices acutely isolated from the rats. It produced a strong inhibition of both ortho- and antidromically elicited action potentials fired by CA1 neurons while the excitatory postsynaptic current (EPSC) measured in these neurons remained totally unaffected. The optical isomer D-Glu which is not transported by the systems of Glu uptake inhibited not only orthodromic and antidromic spikes, but also EPSC. Non-specific glutamate transporter inhibitor DL-threo-beta-hydroxyaspartic acid (THA, 400 microM) mimicked the effects of exogenous Glu and produced strong inhibition of both orthodromic and antidromic spikes, without any influence on the amplitude of EPSCs. Dihydrokainate (DHK, 300 microM), selective inhibitor of GLT-1 subtype of glutamate transporter, exerted a significant inhibitory action on the orthodromically evoked spikes and also on the EPSC. Our results indicate that extrasynaptic and PSD membranes of CA1 neurons form separate compartments differing in the mechanisms and efficiency of external Glu processing: the protection of PSD markedly prevails.

Topics: 4-Aminopyridine; Amino Acid Transport System X-AG; Animals; Animals, Newborn; Aspartic Acid; Dizocilpine Maleate; Drug Interactions; Evoked Potentials; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Hippocampus; In Vitro Techniques; Kainic Acid; Models, Neurological; N-Methylaspartate; Neural Inhibition; Neurons; Potassium Channel Blockers; Quinoxalines; Rats; Rats, Wistar; Synapses

High affinity glutamate transport plays an important role in maintaining a low extracellular glutamate concentration in the CNS. Excitotoxicity due to a loss of glutamate transporter function has been implicated in disease processes such as stroke and amyotrophic lateral sclerosis (ALS). We studied the effects of glutamate transport inhibitors on thalamocortical synapses at developing (postnatal day 3-8) layer IV neurons in the barrel cortex using the thalamocortical slice preparation and whole-cell recordings. Inhibition of glutamate transport by D,L-threo-beta-hydroxyaspartate (THA), a combination of THA and dihydrokainate (DHK), or by L-trans-pyrrolidine-2,4-dicarboxylate (tPDC), caused a reversible blockade of AMPA and kainate receptor-mediated dual component excitatory postsynaptic currents (AMPA/KA EPSCs). This effect was not blocked by cyclothiazide (CTZ) indicating that is was not due to desensitisation of AMPARs. Under conditions in which NMDA receptors were unblocked the transport inhibitors caused the massive activation of NMDA receptors leading to the rapid loss of recordings. Previous studies using these transport inhibitors on brain slices from older animals reported no or only modest effects on synaptic transmission. Therefore the data in the present study suggest that neurons in the developing neocortex are particularly sensitive to glutamate transporter function. Furthermore the effects of transport inhibition are dependent upon whether neurons are sufficiently depolarised to relieve the voltage-dependent block of NMDA receptors.

Topics: Amino Acid Transport System X-AG; Animals; Aspartic Acid; ATP-Binding Cassette Transporters; Benzothiadiazines; Biological Transport; Dicarboxylic Acids; Diuretics; Dose-Response Relationship, Drug; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Glutamic Acid; In Vitro Techniques; Kainic Acid; Neurotransmitter Uptake Inhibitors; Picrotoxin; Pyrrolidines; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Sodium Chloride Symporter Inhibitors; Somatosensory Cortex; Synaptic Transmission; Thalamus

The role of transporters in clearing free glutamate from the synaptic cleft was studied in rat CA1 hippocampal neurons cultured on glial microislands. The time course of free glutamate in the cleft during a synaptic event was estimated by measuring the extent to which the rapidly dissociating AMPA receptor antagonist kynurenate (KYN) was replaced by glutamate during a synaptic response. Dose inhibition of the AMPA receptor EPSC by KYN was less than predicted by the equilibrium affinity of the antagonist, and the rise time of AMPA receptor miniature EPSCs (mEPSCs) was slowed by KYN. Both results indicated that KYN dissociated from AMPA receptors and was replaced by synaptically released transmitter. When transporters were blocked by D,L-threo-beta-hydroxyaspartic acid (THA) or Li+, the mEPSC rise time in the presence of KYN was slowed further, indicating that transporters affect the glutamate concentration in the first few hundred microseconds of the synaptic response. The glutamate transient necessary to cause these effects was determined by developing a detailed kinetic model of the AMPA receptor. The model replicated the effects of KYN on the amplitude and rise time of the synaptic responses when driven by glutamate transients that were similar to previous estimates (; ). The effects of THA were replicated by slowing and enlarging the slower phase of the dual component transient by about 20% or by prolonging the single component by almost 40%. Because transport is too slow to account for these effects, it is concluded that transporters buffer glutamate in the synaptic cleft.

Topics: Animals; Animals, Newborn; Aspartic Acid; Cells, Cultured; Evoked Potentials; Glutamic Acid; Hippocampus; Kinetics; Kynurenic Acid; Lithium; Models, Neurological; Neurons; Quinoxalines; Rats; Receptors, AMPA; Synapses; Synaptic Transmission; Tetrodotoxin; Time Factors