Quantitative microdialysis of serotonin and norepinephrine: pharmacological influences on in vivo extraction fraction.

The present study was designed to investigate the potential influence of various neuronal processes including uptake, release and metabolism, on the in vivo microdialysis extraction fraction (Ed) of serotonin (5HT) and norepinephrine (NE). Paroxetine administration decreased the Ed of 5HT in the nucleus accumbens from 24 +/- 3 to 18 +/- 0.2% (p < 0.05). Similarly, desipramine infusion reduced the NE Ed from 35 +/- 2 to 26 +/- 1% (p < 0.05). However, perfusion with pargyline or tetrodotoxin had no effect on the Ed of either 5HT or NE. Perfusion with agonists for the 5HT, alpha-adrenergic, D2 and histamine receptors had no effect on the Ed of 5HT. In the same manner, perfusion with the alpha-adrenergic agonists, methoxamine or clonidine, did not affect the Ed of NE. These data are in agreement with experimental results obtained for dopamine in the nucleus accumbens and the theory of quantitative microdialysis which predicts that only changes in the rate of clearance will change Ed of monoamines. These results suggest that, like DA, changes in the Ed for 5HT or NE are indicative of changes in the reuptake of these neurotransmitters. The results also indicate that pharmacological agents which do not affect uptake have no effect on the extraction fraction.

Capillary biosensor for glutamate.

We have developed and characterized a novel glutamate biosensor which allows biological transduction of glutamate signal during transport of analyte from the sampling site to the detector. This biosensor exploits the high surface-to-volume ratio found in small-diameter fused silica capillaries. Glutamate dehydrogenase (GDH) was attached to the inner surface of a 75 µm i.d. capillary using biotin-avidin chemistry. In the presence of excess nicotinamide adenine dinucleotide (NAD(+)), GDH converts glutamate to α-ketoglutarate while simultaneously reducing NAD(+) to NADH. Detection of NADH was accomplished using laser-induced fluorescence. Perfusion with 30 µM glutamate in the presence of 3 mM NAD(+) resulted in a strong increase in fluorescence, with a response time of 450 ms. This effect was abolished upon exclusion of NAD(+) from the buffer. The limit of detection is 3 µM (S/N = 3), with a linear working range from 3 to 300 µM. Efficiency of the GDH-modified capillary ranged between 20% and 92% and was positively correlated with concentration of glutamate. The effect of linear velocity was also examined and was shown to be indirectly related to efficiency, with maximum response observed at 4.5 cm/min. In summary, we have demonstrated the successful attachment of glutamate dehydrogenase to the inner wall of a small-diameter fused silica capillary while retaining enzymatic activity. The resulting biosensor exhibits characteristics amenable for in vivo applications. Future efforts will be directed toward the incorporation of this biosensor into current technologies, such as capillary electrophoresis and microdialysis.

Effect of tetrodotoxin and potassium infusion on microdialysis extraction fraction and extracellular dopamine in the nucleus accumbens.

The effects of tetrodotoxin (TTX) and K+ perfusion on extracellular DA concentration (DAext) and extraction fraction (also known as relative recovery or Ed) were studied using a method for quantitative microdialysis under transient conditions. TTX administered through the microdialysis probe decreased DAext with no effect on Ed. Perfusion with high K+ increased DAext, with no initial effect on Ed. However, a decrease in Ed was observed under prolonged depolarization. These data provide further evidence that Ed may change over the course of drug administration. This method of quantitative microdialysis is a reliable means for measuring changes in DAext independently of possible changes in microdialysis probe efficiency.