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 m M . 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 µ M 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 ketogenic diet changes metabolite levels in hippocampal extracellular fluid

Despite successful use of the ketogenic diet (KD) for the treatment of drug-resistant epilepsy, its mechanism of action is unclear. After KD-feeding, increased plasma D-beta-hydroxybutyrate (BHB) levels appear to be important for protection against seizures. We hypothesized that the KD leads to metabolic changes in the brain, which are reflected in the hippocampal extracellular fluid (hECF). CD1 mice were fed control or KD for 2-3 weeks since weaning. In vivo microdialysis of hECF was used to measure the levels of glucose, lactate, as well as BHB under basal conditions and during 30 min stimulation with 60 mM K(+), which was retrodialysed. The hECF BHB concentration in KD-fed mice was determined as 43.4±10.1 μM using the zero-flow method and 50.7±5.5 μM based on in vitro recovery. The total BHB concentration in brain homogenate from KD-fed mice was 180 nmol/g. The intracellular BHB concentration is therefore estimated to be about 3-fold higher than the extracellular level, which suggests that BHB in adolescent mouse brains may not be quickly metabolized. The basal hECF glucose concentration was 30% lower in KD-fed mice, indicating that glucose may be less important as an energy source. Lactate levels were similar in control and KD-fed mice. High potassium stimulation elevated lactate by 3-3.5-fold and decreased glucose by 40-50% in both diet groups, consistent with similar anaerobic and aerobic metabolism in both diet groups during high hippocampal activity. Overall, these data (1) defined the BHB concentration in the hippocampal extracellular fluid in KD-fed mice and (2) showed lower glucose metabolism compared to control diet-fed mice. This work will now enable other researchers to mimic the hippocampal extracellular environment in experiments aimed at deciphering the mechanisms of the KD.     

Steady-State Cerebral Glucose Concentrations and Transport in the Human Brain

Abstract: Understanding the mechanism of brain glucose transport across the blood-brain barrier is of importance to understanding brain energy metabolism. The specific kinetics of glucose transport have been generally described using standard Michaelis-Menten kinetics. These models predict that the steady-state glucose concentration approaches an upper limit in the human brain when the plasma glucose level is well above the Michaelis-Menten constant for half-maximal transport, K _t. In experiments where steady-state plasma glucose content was varied from 4 to 30 m M , the brain glucose level was a linear function of plasma glucose concentration. At plasma concentrations nearing 30 m M , the brain glucose level approached 9 m M , which was significantly higher than predicted from the previously reported K _t of ∼4 m M ( p < 0.05). The high brain glucose concentration measured in the human brain suggests that ablumenal brain glucose may compete with lumenal glucose for transport. We developed a model based on a reversible Michaelis-Menten kinetic formulation of unidirectional transport rates. Fitting this model to brain glucose level as a function of plasma glucose level gave a substantially lower K _t of 0.6 ± 2.0 m M , which was consistent with the previously reported millimolar K _m of GLUT-1 in erythrocyte model systems. Previously reported and reanalyzed quantification provided consistent kinetic parameters. We conclude that cerebral glucose transport is most consistently described when using reversible Michaelis-Menten kinetics.     

Correlation of isonicotinic acid hydrazide infiltration across the blood-brain barrier to postnatal development in rats.

Changes in the isonicotinic acid hydrazide (INH) concentration in rat blood and brain were studied in correlation to postnatal development in groups of animals aged 21 and 42 days and 3 months. In the first part of the experiments, INH was administered intravenously to all the age groups in a dose of 100 mg/kg. In the second part, the dose was related to extracellular fluid volume, so that the 3-week-old rats were given 154 mg/kg, the 6-week-old animals 129 mg/kg and the 3-month-old animals 100 mg/kg. After a dose of 100 mg/kg, INH levels in the blood of 21-day-old rats were significantly lower than in 42-day-old and adult animals. The brain INH levels did not differ significantly. On relating the dose to the amount of extracellular fluid, there were no significant differences in the blood INH levels, but the levels in the brain of 21- and 42-day-old rats were significantly higher than in 3-month-old animals. Blood volume related to body weight and brain weight did not differ in the various age groups. The authors conclude that the blood-brain barrier for isonicotinic acid hydrazide alters in rats during postnatal development. In young animals (21- and 42-day-old), more INH infiltrates into the CNS than in adult animals.     

Influence of Perfusate Glucose Concentration on Dialysate Lactate, Pyruvate, Aspartate, and Glutamate Levels Under Basal and Hypoxic Conditions: A Microdialysis Study in Rat Brain

Abstract: The aim of this study was to evaluate the influence of perfusion media with different glucose concentrations on dialysate levels of lactate, pyruvate, aspartate (Asp), and glutamate (Glu) under basal and hypoxic conditions in rat brain neocortex. Intracerebral microdialysis was performed with the rat under general anesthesia using bilateral probes (o.d. 0.3 mm; membrane length, 2 mm) perfused with artificial CSF containing 0.0 and 3.0 m M glucose, respectively. Basal dialysate levels were obtained 2 h after probe implantation in artificially ventilated animals. Dialysate levels of glucose were also measured for the two different perfusion fluids. The mean absolute extracellular concentration of glucose was estimated by a modification of the no-net-flux method to be 3.3 mmol/L, corresponding to an average in vivo recovery of 6% for glucose. Hypoxia was induced by lowering the inspired oxygen concentration to 3%. Hypoxia caused a disturbance of cortical electrical activity, evidenced by slower frequency and lower amplitudes on the electroencephalogram compared with prehypoxic conditions. This was associated with significant elevations of lactate, Asp, and Glu levels. There were no statistically significant differences in dialysate metabolite levels between the two perfusion fluids, during either normal or hypoxic conditions. We conclude that microdialysis with glucose-free perfusion fluid does not drain brain extracellular glucose in anesthetized rats to the extent that the dialysate lactate, pyruvate, Asp, and Glu levels during basal or hypoxic conditions are altered.     

Combination of microdialysis and glucosensor permits continuous (on line) SC glucose monitoring in a patient operated device. II. Evaluation in animals.

The microdialysis technique was used for following the glucose content of the extracellular subcutaneous (SC) fluid under varying blood glucose levels in rats. The glucose content in the microdialysis perfusion fluid was continuously analyzed by means of the measuring flow chamber of an ex vivo glucose monitor. In six ChBB rats blood glucose levels were varied between 40 mg/dl and 575 mg/dl by intravenous (IV) infusion of glucose and by SC injections of insulin, respectively. After a running-in period of about half an hour, the glucose content in the perfusion fluid was closely related to the blood glucose concentration (r > 0.92) up to a time period of 6 hrs. The "relative recovery" rate of glucose by the microdialysis probe in the SC tissue varied within the 6 experimental sessions. The relative recovery rate could be shown to be not dependent on the absolute blood glucose levels in the individual rat within the glucose concentration range tested.     

Deficits in the Activation and Phosphorylation of Hippocampal Tyrosine Hydroxylase in the Aged Fischer 344 Rat Following Intraventricular Administration of 6-Hydroxydopamine

Abstract: Tyrosine hydroxylase activity was measured under optimal and suboptimal assay conditions in hippocampal extracts from young (2 month), mature (12 month), and old (24 month) Fischer 344 male rats 72 h after the infusion of 200 µg of the neurotoxin 6-hydroxydopamine or vehicle into the lateral ventricle. The lesion resulted in a 45–55% decrease of tyrosine hydroxylase activity measured under optimal conditions (pH 6.1, 3.0 m M 6-methyl-5,6,7,8-tetrahydropterin) and an ∼35% decrease in the relative concentration of immunoreactive tyrosine hydroxylase. When measured under suboptimal conditions (pH 6.6, 0.7 m M 6-methyl-5,6,7,8-tetrahydropterin), tyrosine hydroxylase activity in 2- and 12-month-old lesioned animals was twice that measured in vehicle-treated animals. However, in the old lesioned animals, tyrosine hydroxylase activity measured under suboptimal conditions was not different from that measured in age-matched vehicle-treated animals. Isoforms of tyrosine hydroxylase were identified on immunoblots after two-dimensional gel electrophoresis using enhanced chemiluminescence. The relative proportion of lower pl isoforms of tyrosine hydroxylase in the 2-month-old lesioned animals was greater than that observed in vehicle-treated controls. In contrast, no difference was seen in the relative proportion of tyrosine hydroxylase isoforms in the 24-month-old lesioned versus control animals. These data indicate that the ability of locus ceruleus neurons to rapidly respond to and compensate for insult is attenuated in 24-month-old Fischer 344 rats due to a deficit in stimulus-evoked enzyme phosphorylation.     

Brain Lactate Is an Obligatory Aerobic Energy Substrate for Functional Recovery After Hypoxia: Further In Vitro Validation

Abstract: This study used the rat hippocampal slice preparation and the monocarboxylate transporter inhibitor, α-cyano-4-hydroxycinnamate (4-CIN), to assess the obligatory role that lactate plays in fueling the recovery of synaptic function after hypoxia upon reoxygenation. At a concentration of 500 µ M , 4-CIN blocked lactate-supported synaptic function in hippocampal slices under normoxic conditions in 15 min. The inhibitor had no effect on glucose-supported synaptic function. Of control hippocampal slices exposed to 10-min hypoxia, 77.8 ± 6.8% recovered synaptic function after 30-min reoxygenation. Of slices supplemented with 500 µ M 4-CIN, only 15 ± 10.9% recovered synaptic function despite the large amount of lactate formed during the hypoxic period and the abundance of glucose present before, during, and after hypoxia. These results indicate that 4-CIN, when present during hypoxia and reoxygenation, blocks lactate transport from astrocytes, where the bulk of anaerobic lactate is formed, to neurons, where lactate is being utilized aerobically to support recovery of function after hypoxia. These results unequivocally validate that brain lactate is an obligatory aerobic energy substrate for posthypoxia recovery of function.     

Local Cerebral Glucose Utilization During Development and Aging of the Fischer-344 Rat

Abstract: Local cerebral glucose utilization was measured in brain regions of awake Fischer-344 rats. Measurements were taken in 15 regions of 1-month-old rats, and 19 regions of 3-, 12-, 24-, and 34-month-old rats. Between 1 and 3 months, glucose utilization tended to increase in all brain regions; statistically significant increases occurred in seven regions. Between the ages of 3 and 12 months, glucose utilization decreased significantly in 12 regions. The greatest reductions (25% or more) occurred in the striatum, inferior colliculus, and pons, but the hypothalamus and thalamus, nucleus accumbens, and septum showed no statistically significant change. Cerebral glucose utilization did not change between 12 and 24 months or between 24 and 34 months of age. The results demonstrate a rise in cerebral glucose utilization with development from 1 to 3 months, a decline between 3 and 12 months, and a constancy in the second and third years that does not reflect reported senescence-associated neurochemical and morphological cerebral changes.     

Astrocytes are poised for lactate trafficking and release from activated brain and for supply of glucose to neurons

Brain is a highly-oxidative organ, but during activation, glycolytic flux is preferentially up-regulated even though oxygen supply is adequate. The biochemical and cellular basis of metabolic changes during brain activation and the fate of lactate produced within brain are important, unresolved issues central to understanding brain function, brain images, and spectroscopic data. Because in vivo brain imaging studies reveal rapid efflux of labeled glucose metabolites during activation, lactate trafficking among astrocytes and between astrocytes and neurons was examined after devising specific, real-time, sensitive enzymatic fluorescent assays to measure lactate and glucose levels in single cells in adult rat brain slices. Astrocytes have a 2- to 4-fold faster and higher capacity for lactate uptake from extracellular fluid and for lactate dispersal via the astrocytic syncytium compared to neuronal lactate uptake from extracellular fluid or shuttling of lactate to neurons from neighboring astrocytes. Astrocytes can also supply glucose to neurons as well as glucose can be taken up by neurons from extracellular fluid. Astrocytic networks can provide neuronal fuel and quickly remove lactate from activated glycolytic domains, and the lactate can be dispersed widely throughout the syncytium to endfeet along the vasculature for release to blood or other brain regions via perivascular fluid flow.     

Astrocyte Leucine Metabolism: Significance of Branched-Chain Amino Acid Transamination

Abstract: We studied astrocytic metabolism of leucine, which in brain is a major donor of nitrogen for the synthesis of glutamate and glutamine. The uptake of leucine into glia was rapid, with a V _max of 53.6 ± 3.2 nmol/mg of protein/min and a K _m of 449.2 ± 94.9 µ M . Virtually all leucine transport was found to be Na⁺ independent. Astrocytic accumulation of leucine was much greater (3×) in the presence of α-aminooxyacetic acid (5 m M ), an inhibitor of transamination reactions, suggesting that the glia rapidly transaminate leucine to α-ketoisocaproic acid (KIC), which they then release into the extracellular fluid. This inference was confirmed by the direct measurement of KIC release to the medium when astrocytes were incubated with leucine. Approximately 70% of the leucine that the glia cleared from the medium was released as the keto acid. The apparent K _m for leucine conversion to extracellular KIC was a medium [leucine] of 58 µ M with a V _max of ∼2.0 nmol/mg of protein/min. The transamination of leucine is bidirectional (leucine + α-ketoglutarate ↮ KIC + glutamate) in astrocytes, but flux from leucine → glutamate is more active than that from glutamate → leucine. These data underscore the significance of leucine handling to overall brain nitrogen metabolism. The release of KIC from glia to the extracellular fluid may afford a mechanism for the "buffering" of glutamate in neurons, which would consume this neurotransmitter in the course of reaminating KIC to leucine.     

A Temporary Local Energy Pool Coupled to Neuronal Activity: Fluctuations of Extracellular Lactate Levels in Rat Brain Monitored with Rapid-Response Enzyme-Based Sensor

Abstract: A successfully developed enzyme-based lactate microsensor with rapid response time allows the direct and continuous in vivo measurement of lactic acid concentration with high temporal resolution in brain extracellular fluid. The fluctuations coupled to neuronal activity in extracellular lactate concentration were explored in the dentate gyrus of the hippocampus of the rat brain after electrical stimulation of the perforant pathway. Extracellular glucose and oxygen levels were also detected simultaneously by coimplantation of a fast-response glucose sensor and an oxygen electrode, to provide novel information of trafficking of energy substances in real time related to local neuronal activity. The results first give a comprehensive picture of complementary energy supply and use of lactate and glucose in the intact brain tissue. In response to acute neuronal activation, the brain tissue shifts immediately to significant energy supply by lactate. A local temporary fuel "reservoir" is established behind the blood-brain barrier, evidenced by increased extracellular lactate concentration. The pool can be depleted rapidly, up to 28% in 10–12 s, by massive, acute neuronal use after stimulation and can be replenished in ∼20 s. Glutamate-stimulated astrocytic glycolysis and the increase of regional blood flow may regulate the lactate concentration of the pool in different time scales to maintain local energy homeostasis.     

Impairment of GABAergic neurotransmitter system in the amygdala of young rats after 4-week zinc deprivation

On the basis of the evidence that the excitability of hippocampal glutamatergic neurotransmitter system is enhanced by dietary zinc deficiency, the response of amygdalar neurotransmitter system was checked in young rats fed a zinc-deficient diet for 4 weeks. Extracellular zinc concentration in the amygdala, which was measured by the in vivo microdialysis, was almost the same as that in the hippocampus and decreased by zinc deficiency. Extracellular zinc concentration in the amygdala was increased both in the control and zinc-deficient rats by stimulation with 100 mM KCl, suggesting that the increase in extracellular zinc in the amygdala, as well as that in the hippocampus, is linked with neuronal depolarization. In amygdalar extracellular fluid, the basal glutamate concentration was not significantly different between the control and zinc-deficient rats and was increased to almost the same extent between them by stimulation with 100 mM KCl, unlike more increase in extracellular glutamate concentration in the hippocampus in zinc deficiency. On the other hand, the basal GABA concentration in the amygdalar extracellular fluid was significantly lower in zinc-deficient rats and was not increased both in the control and zinc-deficient rats by stimulation with 100 mM KCl. These results suggest that GABAergic neurotransmitter system is critically impaired in the amygdala of young rats after 4-week zinc deprivation.     

Glutamate Uptake Impairment and Neuronal Damage in Young and Aged Rats In Vivo

Abstract: The extracellular concentration of glutamate increases during hypoxia/ischemia probably due to deficient uptake. Glutamate might contribute to neuronal damage associated with this disorder and to neurodegeneration during aging. In the present study, we have tested the effect of two inhibitors of glutamate transport, l - trans -pyrrolidine-2,4-dicarboxylate and dihydrokainate, on the extracellular levels of glutamate and on neuronal damage, which was quantitatively studied by image analysis of histological brain sections. Drugs were administered by microdialysis and glutamate concentration was determined by HPLC in the striatum and the hippocampus of 3-month-old and 22–24-month-old rats. In both regions studied, the basal concentration of extracellular glutamate was higher in aged than in young rats. Pyrrolidine dicarboxylate induced a substantial elevation of extracellular glutamate in both regions, and although this increase was almost twofold higher in old than in young animals, no neuronal damage was observed. In contrast, dihydrokainate had a poor effect on glutamate levels, but induced clear neuronal damage in the striatum and the hippocampus in both groups of rats. The present results suggest that age appears not to be a significant factor in the sensitivity of neurons to the toxic effect of extracellular glutamate increase via blockade of its transport system.     

1H Nuclear Magnetic Resonance Spectroscopy Study of Cerebral Glutamate in an Ex Vivo Brain Preparation of Guinea Pig

Abstract: Cerebral glutamate was monitored in a superfused cerebral cortical preparation by ¹H NMR spectroscopy using a semiselective spin-echo sequence N -acetyl aspartate (NAA) as an internal concentration reference. During controlled metabolic conditions, the cerebral ¹H NMR-detected glutamate-to-NAA ratio was ∼ 20–30% lower than expected from the ratio of neutralized perchloric acid extracts of the preparations. Inhibition of respiration in the presence of glucose did not change the ¹H NMR glutamate-to-NAA ratio in brain slice preparation. In contrast, either complete depletion of ATP during cyanide poisoning together with 0 m M glucose, anoxia in the absence of glucose, or treatment with nigericin or with a protonophore, carbonyl cyanide- m -fluorophenylhydrazone, increased ¹H NMR-detected glutamate/NAA in the cerebral preparations without a change in the relative and absolute concentration ratios determined from the tissue acid extracts. Spin-spin relaxation times of glutamate and NAA peaks in anoxic slices were 749 ± 89 and 729 ± 94 ms, respectively, and thus, the portion of glutamate that could not be detected by ¹H NMR was quantified in absolute terms. It was calculated that an increase in the glutamate-to-NAA ratio from 0.55 ± 0.02 to 0.67 ± 0.02 during aglycemic anoxia corresponded to some 6 mmol/kg of tissue dry weight of glutamate from the total concentration of 28 mmol/kg dry weight. It is suggested that this 22% of total glutamate pool is present in a noncytoplasmic compartment during controlled metabolic state.     

Extracellular Somatostatin Measured by Microdialysis in the Hippocampus of Freely Moving Rats: Evidence for Neuronal Release

Abstract: Intracerebral microdialysis combined with a sensitive and specific radioimmunoassay was used to monitor the neuronal release of somatostatin (somatostatin-like immunoreactivity, SLI) in the dorsal hippocampus of freely moving rats. The sensitivity of the radioimmunoassay was optimized to detect <1 fmol/ml. The basal concentration of SLI in 20-min dialysate fractions (5 μl/min) collected 24 h after probe implantation was stable over at least 200 min. The spontaneous efflux dropped by 54 ± 6.4% ( p < 0.05) when Ca²⁺ was omitted and 1 m M EGTA added to the Krebs-Ringer solution and by 65.5 ± 3.2% ( p < 0.05) in the presence of 1 μ M tetrodotoxin. Depolarizing concentrations of the Na⁺ channel opener veratridine (6.25, 25, 100 μ M ) induced 11 ± 2 ( p < 0.05), 17 ± 2 ( p < 0.05), and 21 ± 5 ( p < 0.01) fold increase in SLI concentration, respectively, during the first 20 min of perfusion. The effect of 100 μ M veratridine was blocked by coperfusion with 5 μ M tetrodotoxin ( p < 0.01) and reduced by 79% ( p < 0.01) in the virtual absence of Ca²⁺. Neuronal depolarization by 20 min of perfusion with Krebs-Ringer solution containing 25 and 50 m M KCl and proportionally lowered Na⁺ increased the dialysate SLI 4.4 ± 1 ( p < 0.05) and 17 ± 3 ( p < 0.01) fold baseline, respectively. Ten micromolar ouabain, a blocker of Na⁺,K⁺-ATPase, increased the dialysate SLI 15-fold baseline, on average ( p < 0.05), during 80 min of perfusion. The results demonstrate the suitability of brain microdialysis for monitoring the neuronal release of SLI and for studying its role in synaptic transmission.     

The age-related attenuation in long-term potentiation is associated with microglial activation

It is well established that inflammatory changes contribute to brain ageing, and an increased concentration of proinflammatory cytokine, interleukin-1beta (IL-1beta), has been reported in the aged brain associated with a deficit in long-term potentiation (LTP) in rat hippocampus. The precise age at which changes are initiated is unclear. In this study, we investigate parallel changes in markers of inflammation and LTP in 3-, 9- and 15-month-old rats. We report evidence of increased hippocampal concentrations of the proinflammatory cytokines IL-1alpha, IL-18 and interferon-gamma (IFNgamma), which are accompanied by deficits in LTP in the older rats. We also show an increase in expression of markers of microglial activation, CD86, CD40 and intercellular adhesion molecules (ICAM). Associated with these changes, we observed a significant impairment of hippocampal LTP in the same rats. The importance of microglial activation in the attenuation of long-term potentiation (LTP) was demonstrated using an inhibitor of microglial activation, minocycline; partial restoration of LTP in 15-month-old rats was observed following administration of minocycline. We propose that signs of neuroinflammation are observed in middle age and that these changes, which are characterized by microglial activation, may be triggered by IL-18.     

Susceptibility to kainate-induced seizures under dietary zinc deficiency

Zinc homeostasis in the brain is altered by dietary zinc deficiency, and its alteration may be associated with the etiology and manifestation of epileptic seizures. In the present study, susceptibility to kainate-induced seizures was enhanced in mice fed a zinc-deficient diet for 4 weeks. When Timm's stain was performed to estimate zinc concentrations in synaptic vesicles, Timm's stain in the brain was attenuated in the zinc-deficient mice. In rats fed the zinc-deficient diet for 4 weeks, susceptibility to kainate-induced seizures was also enhanced. When the release of zinc and neurotransmitters in the hippocampal extracellular fluid of the zinc-deficient rats was studied using in vivo microdialysis, the zinc concentration in the perfusate was less than 50% of that of the control rats and the increased levels of zinc by treatment with kainate were lower than the basal level in control rats, suggesting that vesicular zinc is responsive to dietary zinc deficiency. The levels of glutamate in the perfusate of the zinc-deficient rats were more increased than in the control rats, whereas the levels of GABA in the perfusate were not at all increased in the zinc-deficient rats, unlike in the control rats. The present results demonstrate an enhanced release of glutamate associated with a decrease in GABA concentrations as a possible mechanism for the increased seizure susceptibility under zinc deficiency.     

Effect of Ethanol on Extracellular Dopamine in Rat Striatum by Direct Perfusion with Microdialysis

Abstract: The concentration-related effects of ethanol on extracellular dopamine (DA) in rat striatum were studied by direct perfusion through microdialysis probes in freely moving rats. Two sets of three ethanol concentrations were separately tested using a Latin square experimental design. Potassium stimulation with high potassium (50 m M ) in artificial CSF (ACSF) preceding ethanol treatment confirmed the neuronal function of dopaminergic cells by increasing DA concentrations to 200–1,500% of basal levels. The perfusion with calcium-free ACSF applied at the end of each experiment confirmed the calcium dependency of the basal levels of extracellular DA by decreasing basal DA levels by 70%. The striatal volume measurement to examine the possible brain damage by direct ethanol perfusion suggested that ethanol did not increase the damage caused by the probe implantation at any ethanol concentration tested in this study. The 30-min direct perfusion of 510 and 860 m M ethanol resulted in a significant concentration-related stimulatory effect on the extracellular DA concentration in rat striatum (510 m M , 29% increase, p < 0.05; 860 m M , 66% increase, p < 0.05). However, there was no significant effect of ethanol at low concentrations, ≤170 m M . Considering the effective ethanol concentration in tissue areas in which DA is sampled, the data suggest that concentrations of ethanol associated with moderate intoxication do not directly affect the extracellular concentration of DA in the striatum. Therefore, the systemic effects of ethanol on striatal DA found in previous studies may be caused by the interaction with sites other than the striatum.     

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

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.