Monitoring extracellular glutamate in hippocampal slices with a microsensor.

The direct local assessment of glutamate in brain slices may improve our understanding of glutamatergic neurotransmission significantly. However, an analytical technique that monitors glutamate directly in brain slices is currently not available. Most recording techniques either monitor derivatives of glutamate or detect glutamate that diffuses out of the slice. Microsensors provide a promising solution to fulfill this analytical requirement. In the present study we have implanted a 10 microm diameter hydrogel-coated microsensor in the CA1 area of hippocampal slices to monitor extracellular glutamate levels. The influence of several pharmacological agents, which facilitate glutamate release from neurons or astrocytes, was investigated to explore the applicability of the microsensor. It was observed that KCl, veratradine, alpha-latrotoxine (LTX), DL-threo-beta-benzyloxyaspartate (dl-TBOA) and L-cystine rapidly increased the extracellular glutamate levels. As far as we know this is the first study in which a microsensor is applied to monitor dynamic changes of glutamate in brain slices and in our opinion this type of research may contribute greatly to improve our understanding of the physiology of glutamatergic neurotransmission.

In vivo monitoring of extracellular glutamate in the brain with a microsensor.

Recent discoveries have revealed that glutamatergic neurotransmission in the central nervous system is mediated by a dynamic interplay between neurons and astrocytes. To enhance our understanding of this process, the study of extracellular glutamate is crucial. At present, microdialysis is the most frequently used analytical technique to monitor extracellular glutamate levels directly in the brain. However, the neuronal and physiological origin of the detected glutamate levels is questioned as they do not fulfil the classical release criteria for exocytotic release, such as calcium dependency or response to the sodium channel blocker tetrodotoxine (TTX). It is hypothesized that an analytical technique with a higher spatial and temporal resolution is required. Glutamate microsensors provide a promising analytical solution to meet this requirement. In the present study, we applied a 10 micro m diameter hydrogel-coated glutamate microsensor to monitor extracellular glutamate levels in the striatum of anesthetized rats. To explore the potential of the microsensor, different pharmacological agents were injected in the vicinity of the sensor at an approximate distance of 100 micro m. It was observed that KCl, exogenous glutamate, kainate and the reuptake inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA) increased the extracellular glutamate levels significantly. TTX decreased the basal extracellular glutamate levels approximately 90%, which indicates that the microsensor is capable of detecting neuronally derived glutamate. This is one of the first studies in which a microsensor is applied in vivo on a routine base, and it is concluded that microsensor research can contribute significantly to improve our understanding of the physiology of glutamatergic neurotransmission in the brain.

Improving the reproducibility of hydrogel-coated glutamate microsensors by using an automated dipcoater.

Hydrogel-coated microsensors based on carbon fiber electrodes (CFEs) are promising tools for in vivo analysis of endogeneous compounds such as glutamate. However, their construction generally depends on manual fabrication, which often results in poor reproducibility. The aim of this study was to improve the reproducibility and performance of glutamate microsensors. CFEs (10 microm diameter, 300-500 microm long) were coated with a cross-linked redox-polymer hydrogel containing l-glutamate oxidase, horseradish peroxidase and ascorbate oxidase. Various parameters that are likely to influence the reproducibility of the glutamate microsensors were studied. It appeared that the most crucial step in determining the microsensor performance is the manual hydrogel-application procedure. To control this procedure an automated dipcoater was constructed, which allowed mechanical application of the hydrogel on the CFE under standardized conditions. Significant improvements in performance were seen when the CFEs were dipcoated for 10 min at 37 degrees C. Further improvements were obtained when the automated hydrogel application was combined with other cross-link methods, such as electrodeposition and electrostatic complexation. A crucial factor in determining the microsensor performance is the hydrogel thickness. Microscopic observations revealed that, despite the use of an automated dipcoater, the layer thickness was not constant. By combining the automated dipcoat technique with amperometry, the layer thickness could be indirectly monitored and controlled, which resulted in significant improvements of the reproducibility of the sensors.