Martyn G. Boutelle

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.

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.

Stimulated release of lactate in freely moving rats is dependent on the uptake of glutamate.

1. Physiological stimulation of neuronal activity induces an increase in extracellular lactate. Experiments were designed to determine the role of the reuptake of neuronally released glutamate in lactate delivery to the extracellular compartment. 2. In vivo microdialysis was used in freely moving rats. The lactate concentration in striatal dialysate was assayed using an enzyme‐based on‐line assay at 1 min intervals. Drugs were given locally through the dialysis probe. 3. The extracellular concentration of lactate, determined using the zero net flux method, was 346 +/‐ 21 microM. 4. Induced grooming caused a maximal increase in lactate concentration in striatal dialysate of 58 +/‐ 10%. 5. Administration of 100 microM glutamate caused a transient increase in dialysate lactate concentration of 72 +/‐ 17%. 6. A 20 min infusion of the glutamate uptake blockers beta‐D,L‐threohydroxyaspartate (THA) or pirrolidine‐2‐4‐dicarboxylate (PDC) produced an increase in basal lactate, which was sustained in response to THA and transient in response to PDC. 7. Grooming induced during the infusion of PDC produced no significant increase in lactate. 8. Grooming induced after the infusion of the glutamate uptake blockers gave rise to a reduced increase in lactate. 9. These results support the hypothesis that stimulated release of lactate is dependent on the uptake of glutamate.

The determination of the extracellular concentration of brain glutamate using quantitative microdialysis

Quantitative microdialysis with two enzyme-based assays was used to determine the extracellular concentration of glutamate in the striatum of freely moving rats. From the difference between infused and dialysate glutamate a value of 3.0 +/- 0.6 microM for the extracellular glutamate concentration was computed by regression analysis. The in vivo recovery, derived from the slope of the regression line, was 50%.

The source of physiologically stimulated glutamate efflux from the striatum of conscious rats.

1. Glutamate in the extracellular compartment of the striatum of freely moving rats was monitored at 5 min intervals using microdialysis and an enzyme‐based assay. 2. Basal levels of dialysate glutamate were 3.6 +/‐ 0.5 microM. Local infusion through the dialysis probe of tetrodotoxin (TTX), cadmium chloride or magnesium chloride produced no reduction in basal levels of glutamate; with the latter two there was, instead, an increase. 3. Neuronal activation stimulated by induced grooming was accompanied by an increase in total glutamate efflux of 47.5 +/‐ 25.0% above basal level; this increase was not reduced by local infusion of TTX. 4. We propose that the TTX‐insensitive release of glutamate in response to physiological stimulation is derived from glial cells and is a Ca(2+)‐dependent mechanism triggered by a receptor‐mediated release of Ca2+ from internal stores that spreads through the network of astrocytes.

Rapid changes in extracellular glucose levels and blood flow in the striatum of the freely moving rat

The dynamics of regional cerebral blood flow and brain extracellular glucose were studied in the freely moving rat. These two variables were measured in the striatum during and following both mild tail pinch and restraint stress. Blood flow was monitored using a refinement of the hydrogen clearance technique that allowed repeated measurements at 5-min intervals. A slow stream of hydrogen was directed at the rats snout for 10-20 s through lightweight tubing attached to the animals head and detected at a chronically implanted platinum electrode. Extracellular glucose was monitored with microdialysis in a separate group of animals using an on-line, enzyme-based assay that provided 2.5-min time resolution. Mean striatal blood flow 24 h following implantation was 89.9 +/- 2.5 ml.(100 g)-1.min-1. A 5-min tail pinch caused flow to increase immediately to 169.5 +/- 20 ml.(100 g)-1.min-1. In contrast, there was no change in blood flow during restraint stress, although there was a small increase following the end of the stress. Significant increases in blood flow were also observed in the striatum during periods of eating and grooming. Extracellular glucose levels increased following both forms of stress, to a maximum of 170 +/- 22% of baseline with restraint compared to 110 +/- 2% with tail pinch. In both cases, the increase occurred after the stress had ended and persisted while blood flow returned to basal levels.

A Role for Astrocytes in Glucose Delivery to Neurons

The present paper examines the possible role of astrocytes in the delivery of glycogen-derived glucose for neuronal metabolism. Such a process would require astrocytic expression of glucose-6-phosphatase. The degree and significance of brain expression of glucose-6-phosphatase (EC 3.1.3.9) has been a subject of controversy. Published immunohistochemical data are consistent with expression of glucose-6-phosphatase by astrocytes, both in vivo and in vitro. In this paper additional confirmation of the expression of glucose-6-phosphatase mRNA in rat brain is presented. Although cultured astrocytes demonstrate glucose-6-phosphatase activity in vitro under assay conditions, there is very limited in vitro evidence that this activity confers a glucose-export capacity on astrocytes. Under most conditions in vitro, lactate export predominates, however this may relate to aspects of the in vitro phenotype. Data relating to astrocytic glucose and lactate export are considered in the context of hypotheses of trafficking by astrocytes of substrates for neuronal metabolism, hypotheses that imply and require compartmentation of these substances, in contrast with current formulations of glucose transport into and within brain that imply no glucose compartmentation. Microdialysis studies of the properties of the brain extracellular fluid (ECF) glucose pool in the freely moving rat were performed seeking evidence of glucose compartmentation. Results of these studies do imply compartmentalisation of brain glucose, and are consistent with a model envisaging the majority of glucose reaching the neuron via the astrocytic intracellular space and the ECF. In addition, such studies provide evidence that rises in ECF glucose concentration are not the direct result of local recruitment of cerebral blood flow, but suggest the influence of intermediate, astrocyte-based mechanisms. Astrocytic glucose-6-phosphatase may permit astrocytes to modulate the trans-astrocytic flux of glucose to adjacent neurons in response to signals reflecting increased neuronal demand.

Rapid changes in striatal ascorbate in response to tail-pinch monitored by constant potential voltammetry

The first peak in the voltammogram recorded with linear sweep and a carbon paste electrode implanted in the rat striatum is due to the oxidation of ascorbic acid. When the potential is held at a level slightly positive to this peak a current is recorded which is abolished by the microinjection of ascorbic acid oxidase in the vicinity of the electrode; this suggests that it is due to the oxidation of ascorbate. This current shows the same diurnal variation as the size of the ascorbate peak and its rise and fall coincides with the onset and offset of motor activity. A tail-pinch applied through a paper clip causes an immediate rise in the ascorbate current which begins to fall as soon as the paper clip is removed. Measurement of the ascorbate current at constant potential provides a technique for monitoring rapid changes in extracellular brain ascorbate in response to physiological stimuli.

The role of N-methyl-D-aspartate receptors in the regulation of physiologically released dopamine

In vivo voltammetry was used to measure changes in ascorbate, which are an index of changes in the release of glutamate, and microdialysis was used to measure changes in dopamine in the striatum of freely moving rats. A 5 min tail pinch produced a rapid rise in striatal ascorbate paralleled by an increase in motor activity and a slower, more prolonged rise in dopamine. Systemic administration of ketamine or dizocilpine maleate, non-competitive antagonists of the N-methyl-D-aspartate glutamate receptor, produced an increase in the basal level of ascorbate but not dopamine; however, the tail pinch-evoked rise in both ascorbate and dopamine was completely abolished by these drugs. The rise in dopamine was also abolished by local infusion of dizocilpine maleate into the striatum. Local application of N-methyl-D-aspartate produced a dose-dependent increase in dopamine, which was partially reduced in the presence of tetrodotoxin. The results show that the tail pinch-evoked increase in motor activity involves an increase in the release of striatal dopamine which requires the activation of N-methyl-D-aspartate receptors in the striatum. This suggests that phasic increases in striatal dopamine release are triggered by the action of glutamate on dopaminergic nerve terminals.

In vivo neurochemical effects of tail pinch

Tail pinch in the rat gives rise to a well characterised pattern of behaviour which includes gnawing, licking and eating. We have used both in vivo voltammetry and microdialysis to monitor neurochemical changes which accompany the behavioural response to a 5-min tail pinch. Tail pinch resulted in a increase of extracellular 5-hydroxytryptamine and a smaller and more delayed increase of 5-hydroxyindole acetic acid in the hippocampus. In the striatum there was a rise of both extracellular dopamine and ascorbate. With a recently developed constant potential voltammetric technique we can continuously monitor changes in extracellular ascorbate. Using this technique we found a very rapid rise in ascorbate current during a 5-min tail pinch; the current began to decline as soon as the clip was removed. The high time resolution of the technique also allowed us to record similar ascorbate changes during a 0.5-s tail pinch.