Maintaining glutamate at low extracellular concentrations in the central nervous system is necessary to protect neurons from excitotoxic injury and to ensure a high signal-to-noise ratio for glutamatergic synaptic transmission. We have used DL-threo-␤-benzyloxyaspartate (TBOA), an inhibitor of glutamate uptake, to determine the role of glutamate transporters in the regulation of extracellular glutamate concentration. By using the N-methyl-D-aspartate receptors of patched CA3 hippocampal neurons as ''glutamate sensors,'' we observed that application of TBOA onto organotypic hippocampal slices led to a rapid increase in extracellular glutamate concentration. This increase was Ca 2؉ -independent and was observed in the presence of tetrodotoxin. Moreover, prevention of vesicular glutamate release with clostridial toxins did not affect the accumulation of glutamate when uptake was inhibited. Inhibition of glutamine synthase, however, increased the rate of accumulation of extracellular glutamate, indicating that glial glutamate stores can serve as a source in this process. TBOA blocked synaptically evoked transporter currents in astrocytes without inducing a current mediated by the glutamate transporter. This indicates that this inhibitor is not transportable and does not release glutamate by heteroexchange. These results show that under basal conditions, the activity of glutamate transporters compensates for the continuous, nonvesicular release of glutamate from the intracellular compartment. As a consequence, acute disruption of transporter activity immediately results in significant accumulation of extracellular glutamate.

The hippocampus, a brain structure essential for memory and cognition, is classically represented as a trisynaptic excitatory circuit. Recent findings challenge this view, particularly with regard to the mossy fibre input to CA3, the second synapse in the trisynaptic pathway. Thus, the powerful mossy fibre input to CA3 pyramidal cells might mediate both synaptic excitation and inhibition. Here we show, by recording from connected cell pairs in rat entorhinal-hippocampal slice cultures, that single action potentials in a dentate granule cell evoke a net inhibitory signal in a pyramidal cell. The hyperpolarization is due to disynaptic feedforward inhibition, which overwhelms monosynaptic excitation. Interestingly, this net inhibitory synaptic response changes to an excitatory signal when the frequency of presynaptic action potentials increases. The process responsible for this switch involves the facilitation of monosynaptic excitatory transmission coupled with rapid depression of inhibitory circuits. This ability to immediately switch the polarity of synaptic responses constitutes a novel synaptic mechanism, which might be crucial to the state-dependent processing of information in associative hippocampal networks.

(2, 3). Whereas some studies suggest that the source of accumulating glutamate is vesicular (synaptic) (4-6), others provide evidence for nonvesicular mechanisms (7-11).Previous investigations usually have used biochemical (e.g., microdialysis) or radiolabeling techniques to measure excitatory amino acid concentrations at predefined time points (3, 7-9). These methods, however, provide low spatial and͞or temporal resolution and offer little information on glutamate levels at the synaptic cleft, where the receptors mediating excitotoxicity are located. To detect changes in ambient glutamate levels during energy deprivation (ED) in real time, we have adopted an alternative approach in which the NMDARs of neurons undergoing ED are used as high-affinity glutamate sensors (12). This technique has allowed us to determine the relative contribution of vesicular and nonvesicular release to the increase in [glu] o during ED. Materials and MethodsOrganotypic Hippocampal Slice Preparation. Experiments were performed on rat organotypic hippocampal slice cultures. Tissue slices of 400-m thickness were prepared from 6-day-old rats and cultured by means of the roller-tube technique as described previously (13).Experimental Conditions. After 12-25 days in vitro, cultures were transferred to a recording chamber mounted onto the stage of an upright microscope (Axioscop FS 2; Zeiss), and then they were superfused with external solution (137 mM Na ϩ ͞2.7 mM K ϩ ͞146.2 mM Cl Ϫ ͞2.8 mM Ca 2ϩ ͞0.5 mM Mg 2ϩ ͞11.6 mM HCO 3 Ϫ ͞0.4 mM H 2 PO 4 Ϫ ͞5.6 mM D-glucose) at 29-31°C, pH 7.4. To induce ED, glucose was replaced with 2 mM 2-deoxyglucose, and 5 mM sodium cyanide (NaCN) was added (14).

Electrophysiological and pharmacological properties of glycine receptors were characterized in hippocampal organotypic slice cultures. In the presence of ionotropic glutamate and GABAB receptor antagonists, pressure‐application of glycine onto CA3 pyramidal cells induced a current associated with increased chloride conductance, which was inhibited by strychnine. Similar chloride currents could also be induced with β‐alanine or taurine. Whole‐cell glycine responses were significantly greater in CA3 pyramidal cells than in CA1 pyramidal cells and dentate granule cells, while responses to GABA were similar among these three cell types. Although these results demonstrate the presence of functional glycine receptors in the hippocampus, no evidence for their activation during synaptic stimulation was found. Gabazine, a selective GABAA receptor antagonist, totally blocked evoked IPSCs in CA3 pyramidal cells. Glycine receptor activation is not dependent on transporter‐controlled levels of extracellular glycine, as no chloride current was observed in response to sarcosine, an inhibitor of glycine transporters. In contrast, application of guanidinoethanesulfonic acid, an uptake inhibitor of β‐alanine and taurine, induced strychnine‐sensitive chloride current in the presence of gabazine. These data indicate that modulation of transporters for the endogenous amino acids, β‐alanine and taurine, can regulate tonic activation of glycine receptors, which may function in maintenance of inhibitory tone in the hippocampus.