Amperometric glucose biosensors based on the immobilization of glucose oxidase (GOx) on Pt electrodes with electropolymerized o-phenylenediamine (PPD) were implanted in the right striatum of freely-moving rats. Carbon paste electrodes for the simultaneous monitoring of ascorbic acid (AA) and/or tissue O 2 were implanted in the left striatum. A detailed in vivo characterization of the Pt/PPD/GOx signal was carried out using various pharmacological manipulations. Confirmation that the biosensor responded to changing glucose levels in brain extracellular fluid (ECF) was obtained by intraperitoneal (i.p.) injection of insulin that caused a decrease in the Pt/PPD/GOx current, and local administion of glucose (1 mM) via an adjacent microdialysis probe that resulted in an increase in the biosensor current. An insulin induced increase in tissue O 2 in the brain was also observed. Interference studies involved administering AA and subanaesthetic doses of ketamine i.p. Both resulted in increased extracellular AA levels with ketamine also causing an increase in O 2 . No significant change in the Pt/PPD/GOx current was observed in either case indicating that changes in O 2 and AA, the principal endogenous interferents, have minimal effect on the response of these first generation biosensors. Stability tests over a successive 5-day period revealed no significant change in sensitivity. These in vivo results suggest reliable glucose monitoring in brain ECF.
§ Molecular Sensors Unit, Physiology Department, Parks Road, Oxford OXJ 3PT, UK 1. This study is concerned with the supply of metabolic substrates for neuronal metabolism.Experiments were carried out to investigate whether mechanisms demonstrated in cultured astrocytes also occurred in vivo; these were cAMP-mediated breakdown of glycogen and glutamate uptake-stimulated release of lactate. 2. In vivo microdialysis was used in freely moving rats. Lactate and glucose in the dialysate were assayed using enzyme-based on-line assays. Drugs were given locally through the dialysis probe. Regional cerebral blood flow was measured using the hydrogen clearance method. 3. There was an increase in dialysate glucose in response to the fl-adrenoceptor agonist isoprenaline and to 8-bromo-cAMP, an analogue of cAMP, the second messenger of B-adrenoceptor stimulation. The effect of isoprenaline was blocked by the antagonist propranolol. Isoprenaline had no effect on dialysate lactate, which was increased by the glutamate uptake blocker /-D,L-threohydroxyaspartate (THA).4. Physiological stimulation of neuronal activity produced an increase in both lactate and glucose. The increase in lactate was depressed in the presence of THA but was unaffected by propranolol. The increase in glucose was blocked by propranolol. Regional cerebral blood flow was increased by physiological stimulation but was unaffected by propranolol. 5. These results demonstrate that physiologically stimulated increases in glucose and lactate in the brain are mediated by different mechanisms.Neurones show high rates of energy consumption, required for the maintenance of ionic gradients across their membranes by active transport mechanisms. They have, with a few exceptions, no energy stores. The generally accepted view is that their energy requirements, which fluctuate with the level of their activity, are met by variations in local cerebral blood flow and that this supplies glucose directly to the extracellular compartment (ECF) from which it enters neurones by facilitated diffusion.
The effects of mild stress on nonoxidative glucose metabolism were studied in the brain of the freely moving rat. Extracellular lactate levels in the hippocampus and striatum were monitored at 2.5-min intervals with microdialysis coupled with an enzyme-based flow injection analysis system. Ten minutes of restraint stress led to a 235% increase in extracellular lactate levels in the striatum. A 5-min tail pinch caused an increase of 193% in the striatum and 170% in the hippocampus. Local application of tetrodotoxin in the striatum blocked the rise in lactate following tail pinch and inhibited the subsequent clearance of lactate from the extracellular fluid. Local application of the noncompetitive N-methyl-D-aspartate receptor antagonist MK-801 had no effect on the tail pinch-stimulated increase in lactate in the striatum. These results show that mild physiological stimulation can lead to a rapid increase in nonoxidative glucose metabolism in the brain.
Abstract:We have used a glucose oxidase-based sensor implanted in the striatum of freely moving rats to determine the concentration of extracellular glucose in two distinct ways. With a modification of the zero net flux method, in which different concentrations of glucose are infused through a dialysis probe glued to the biosensor, we calculated the concentration at which there was no change in glucose current by regression analysis; this gave a concentration of 0.351 ±0.016 mM. Calculating the concentration from the basal current and the in vitro calibration of the biosensor was not significantly different from this. The basal extracellular glucose concentration determined by either method remained constant over a period of several days. Infusion of 50 1aM veratridine through the adjacent dialysis probe caused a steep decrease in glucose current as soon as the drug reached the brain in contrast to the delayed fall (7.5 mm) seen with microdialysis in previous experiments from this laboratory. These results demonstrate that this biosensor provides a direct, real-time measure of the extracellular concentration of glucose. Key Words: Glucose biosensorBrain glucose concentration-Veratridine-Rat striatum.
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