Marianne Fillenz

Physiological Stimulation Increases Nonoxidative Glucose Metabolism in the Brain of the Freely Moving Rat

Abstract: 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.

Extracellular Brain Glucose Levels Reflect Local Neuronal Activity: A Microdialysis Study in Awake, Freely Moving Rats

Abstract: The relationship between brain extracellular glucose levels and neuronal activity was evaluated using microdialysis in awake, freely moving rats. The sodium channel blocker tetrodotoxin and the depolarizing agent veratridine were administered through the dialysis probe to provoke local changes in neuronal activity. The extracellular glucose content was significantly increased in the presence of tetrodotoxin and decreased sharply following veratridine application. The systemic injection of a general anaesthetic, chloral hydrate, led to a large and prolonged increase in extracellular glucose levels. The brain extracellular glucose concentration was estimated by comparing dialysate glucose efflux over a range of inlet glucose concentrations. A mean value of 0.47 mM was obtained in five animals. The results are discussed in terms of the coupling between brain glucose supply and metabolism. The changes observed in extracellular glucose levels under various conditions suggest that supply and utilization may be less tightly linked in the awake rat than has previously been postulated.

Acquisition and extinction of continuously and partially reinforced running in rats with lesions of the dorsal noradrenergic bundle

Local injection of 6-hydroxydopamine was used to selectively destroy the dorsal ascending noradrenergic bundle (DB) in rats. Two lesion procedures were used, differing in the extent of depletion of forebrain noradrenaline they produced (greater than 90% or 77%). In Experiments 1-3 the rats were run in a straight alley for food reward on continuous (CR) or partial (PR) reinforcement schedules. The smaller lesion reduced and the larger lesion eliminated the partial reinforcement acquisition effect (i.e. the faster start and run speeds produced by PR during training) and the partial reinforcement extinction effect (PREE, i.e. the greater resistance to extinction produced by PR training); these changes were due to altered performance only in the PR condition. Abolition of the PREE by the larger DB lesion occurred with 50 acquisition trials, but with 100 trials the lesion had no effect. In Experiment 4 rats were run in a double runway with food reward on CR in the second goal box, and on CR, PR or without reinforcement in the first. The larger lesion again eliminated the PREE in the first runway, but did not block the frustration effect in the second runway (i.e. the faster speeds observed in the PR condition after non-reward than after reward in the first goal box). These results are consistent with the hypothesis that DB lesions alter behavioural responses to signals of non-reward, but not to non-reward itself. They cannot be predicted from two other hypotheses: that the DB mediates responses to reward or that it subserves selective attention. Since septal and hippocampal, but not amygdalar, lesions have been reported to produced similar behavioural changes, it is proposed that the critical DB projection for the effects observed in these experiments is to the septo-hippocampal system.

Linear sweep voltammetry with carbon paste electrodes in the rat striatum

Voltammetry has been widely used in attempts to measure catecholamine release in vivo. The voltammogram recorded in the rat striatum using carbon paste electrodes and linear sweep voltammetry with semidifferentiation consists of a number of separate peaks; changes in the height of the first of these peaks have been attributed to changes in catecholamine release. We have found that ascorbate, either microinjected into the striatum or injected intraperitoneally, increases the height of the first peak without changing its potential. Microinjection of dopamine or 3,4-dihydroxyphenylacetic acid, or intraperitoneal injection of 3,4-dihydroxyphenylalanine, caused a shift in the potential of peak 1 of 25-50 mV in a positive direction. Amphetamine, administered intraperitoneally to freely moving animals, caused an increase in the height of the first peak but did not change its potential. Oxidation potentials in vitro and the effect of other drugs on the voltammogram obtained in vivo were also measured. Peak 1 is caused by the oxidation of both ascorbate and catechols whose oxidation potentials differ by only 50 mV in vivo; the contribution of catechols in control animals is negligible. Shifts in the potential of peak 1 caused by drugs are not due to changes in the oxidation potentials of the components but to a change in their relative contributions. Therefore changes in the height of peak 1 with no change in position do not represent changes in the extracellular concentration of catechols but are due to changes in ascorbate concentration. Changes in the concentration of catecholamine-related compounds can be detected at potentials some 50 mV greater than that of the first peak.

The mechanisms controlling physiologically stimulated changes in rat brain glucose and lactate: a microdialysis study.

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 beta‐adrenoceptor agonist isoprenaline and to 8‐bromo‐cAMP, an analogue of cAMP, the second messenger of beta‐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 beta‐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.

Comparison of Stress-induced Changes in Noradrenergic and Serotonergic Neurons in the Rat Hippocampus Using Microdialysis

The effects of stress on the serotonergic and noradrenergic projection to the hippocampus were compared in freely moving rats using microdialysis. Stress‐induced changes in 5‐hydroxytryptamine (5‐HT), noradrenaline and their metabolites 5‐hydroxyindoleacetic acid (5‐HIAA) and 3,4‐dihydroxyphenylacetic acid (DOPAC) were measured in the presence of their respective uptake blockers. Local infusion of tetrodotoxin and replacement of Ca2+ with Cd2+ were used to test dependence on impulse traffic. A 5 min tail pinch or 10 min restraint stress increased 5‐HT, 5‐HIAA, noradrenaline and DOPAC levels. A subcutaneous saline injection produced an increase in 5‐HT and DOPAC but not noradrenaline or 5‐HIAA. Although α2 adrenoceptor agonists and antagonists produced changes in the baseline values of noradrenaline and DOPAC, they had little or no effect on stress‐induced changes. Both the abolition of impulse traffic and its enhancement by stress had a greater effect on transmitter than on metabolite levels. Although the responses to stress of the noradrenergic and serotonergic pathway showed many similarities, there was evidence for their activation by separate pathways.

Voltammetrically monitored brain ascorbate as an index of excitatory amino acid release in the unrestrained rat

To discover the significance of changes in the extracellular concentration of brain ascorbate, we used linear sweep voltammetry to monitor the ascorbate signal. Recordings were made with carbon paste electrodes implanted in the striatum and hippocampus of anaesthetised and unanaesthetised rats under a variety of conditions. Intraperitoneal administration of excitatory amino acid transmitters, but not tyrosine or glycine, increased extracellular striatal ascorbate; similarly, microinfusion of L-glutamate beside striatal electrodes enhanced the ascorbate signal. Electrical stimulation of the perforant path increased the extracellular concentration of dentate ascorbate in the unanaesthetised, but not in the anaesthetised, rat. These results support our hypothesis that changes in the extracellular concentration of brain ascorbate monitored by voltammetry reflect the release of excitatory amino acids.

Tail pinch-induced changes in the turnover and release of dopamine and 5-hydroxytryptamine in different brain regions of the rat

Tail pinch was administered through a paper clip attached to the rats tail. The ex vivo changes in the metabolite/transmitter ratio were used as a measure of changes in the turnover of dopamine and 5-hydroxytryptamine. After a 2-min tail pinch dopamine turnover was increased in the striatum but not in the frontal cortex, hypothalamus or olfactory tubercle; 5-hydroxytryptamine turnover was increased in frontal cortex and hippocampus and was unchanged in striatum, hypothalamus and olfactory tubercle. Microdialysis was used to monitor the changes in extracellular neurotransmitter and metabolite concentrations during and after tail pinch. A 5-min tail pinch caused a rapid rise of both dopamine in the striatum and 5-hydroxytryptamine in the hippocampus. There was a smaller increase in the 5-hydroxytryptamine metabolite 5-hydroxyindoleacetic acid and only a non-significant increase in the dopamine metabolite 3,4-dihydroxyphenylacetic acid.

Continuous Monitoring of Extracellular Glucose Concentrations in the Striatum of Freely Moving Rats with an Implanted Glucose Biosensor

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 µM 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 min) 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.

The physiologically induced release of ascorbate in rat brain is dependent on impulse traffic, calcium influx and glutamate uptake

Extracellular brain ascorbate fluctuates with neuronal activity. There is previous evidence that the release of ascorbate is triggered by the re-uptake of neuronally released glutamate. This hypothesis predicts that drugs which block the release and re-uptake of glutamate will also block the release of ascorbate. In the present experiments we have used a novel dialysis electrode which allows continuous monitoring of physiologically induced ascorbate release from the striatum in freely moving rats. An infusion of the enzyme ascorbic acid oxidase abolished the increase in oxidation current in response to tail-pinch, which identified it as an ascorbate current. Perfusion with tetrodotoxin reduced the response to 25% and with CdCl2 to 4% of control. Perfusion with the uptake blocker L-trans-pyrrolidine-2,4-di-carboxylate reduced the response to 24% of control. A neuroprotective function for this coupling of ascorbate and glutamate release is discussed.