Introduction
Central glutamate pathways are implicated in stress, substance abuse, schizophrenia, and Parkinson’s disease. Information about the neuronal release of glutamate to the extracellular space is, therefore, of significant interest. The blockade of voltage-dependent sodium channels with tetrodotoxin (TTX) is often used to confirm the neuronal origin of a substance. Small decreases in glutamate levels have been observed during in vivo push-pull perfusion with TTX¹. Basal glutamate levels in microdialysate, however, are reported to be insensitive to TTX². We now describe the use of sensitive and selective electrochemical microsensors for the assessment of the neuronal contribution to basal extracellular glutamate levels in the striatum of chloral hydrate-anesthetized rats.

Materials and Methods
Extracellular glutamate was monitored with electrochemical microsensors (10 mm in diameter, 300-400 mm in length), which were operated at a constant potential of -0.1 V vs. Ag/AgCl. The microsensors were prepared by immobilization of glutamate oxidase, horseradish peroxidase, and ascorbate oxidase onto a carbon fiber microelectrode by means of a cross-linkable redox polymer. A Nafion film was applied prior to in vivo experiments. Microsensors prepared in identical fashion except for the omission of glutamate oxidase were used to obtain glutamate-free background signals in vivo. A glutamate microsensor, a background microsensor and a microinfusion pipet were implanted in the striatum of anesthetized rats. The average distance between each microsensor and the injection pipet was 100 mm. The vehicle for microinfusion of drugs was artificial cerebrospinal fluid (aCSF).

Results
Calibration demonstrated that the glutamate microsensors exhibit sub-micromolar detection limits and a linear response over three decades of concentration. The microsensors are highly selective towards glutamate: they do not respond to and are not interfered with by physiologically relevant concentrations of ascorbate, urate, dopamine, DOPAC, and HVA.

The basal in vivo signal at the glutamate microsensors was consistently larger than that at the background microsensors. By means of post-calibration data, the basal signal in striatum was found to correspond to a glutamate concentration of 29 ± 9 mM (mean ± s.d., n=26; without corrections for in vivo diffusion coefficients or extracellular volume fraction). Figure 1 shows responses recorded simultaneously with glutamate and background microsensors implanted side-by-side in rat striatum during the local infusion of glutamate (10 mM, 200 nl), a glutamate uptake inhibitor, PDC, (4 mM, 1.6 mL), and TTX (100 mM, 200 nL). The signal at the glutamate microsensors, but not at the background microsensors, increased transiently upon the microinfusion of glutamate (n=5) and PDC (n=3). The signal at the glutamate microsensors, but not at the background microsensors, decreased upon the local infusion of TTX (n=9). The decrease in the signal ranged between 25 and 85% of the pre-infusion basal signal. Neither type of microsensor responded significantly to volume-matched microinfusion of the vehicle.

Figure 1 Responses observed at (A) glutamate and (B) background microsensors in rat striatum during microinfusion of glutamate (10 mM, 200 nL), PDC (4 mM, 1.6 mL) or TTX (100 mM, 200 nL). The horizontal bar shows the time period of the microinfusion. The vertical scale bar applies only to the glutamate trace.

Discussion
These findings show that glutamate microsensors respond selectively and sensitively to glutamate levels in the extracellular space of rat striatum. Furthermore, these findings demonstrate that extracellular glutamate is under the combined regulation of glutamate uptake and neuronal release.

The TTX-sensitivity of the basal signal at these implanted glutamate microsensors is considerably greater than that noticed previously by push-pull and microdialysis perfusion methods. We speculate that this difference is attributable to the ability of the microsensors, which are ca. 10,000 times smaller than conventional microdialysis probes (v/v), to gain closer proximity to viable glutamate terminals in brain tissue.

This work was supported by NIH: NS 31442.

References

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2. Lada MW, Vicroy TW and Kennedy RT (1998) Evidence for neuronal origin and metabotropic receptor-mediated regulation of extracellular glutamate and aspartate in rat striatum in vivo following electrical stimulation of the prefrontal cortex. J. Neurochem. 70: 617-625.