Ž .Brain Research 788 1998 144–150

Research report

The effects of glucose, mannose, fructose and lactate on the preservation ofneural activity in the hippocampal slices from the guinea pig

Hiroshi Wada a,b, Yasuhiro Okada a,), Takeshi Uzuo a,1, Hajime Nakamura b

a Department of Physiology, School of Medicine, Kobe UniÕersity, 7-5-2, Kusunokicho, Chuo-ku, Kobe 650, Japanb Department of Pediatrics, School of Medicine, Kobe UniÕersity, 7-5-2, Kusunokicho, Chuo-ku, Kobe 650, Japan

Accepted 16 December 1997

Abstract

Using hippocampal slices from guinea pigs, we investigated the effect of different concentrations of glucose and replacement ofŽ .glucose with mannose, fructose and lactate on neural activity. As an index of neural activity, the population spikes PS were recorded in

Ž .the granule cell layer of the dentate gyrus DG and the pyramidal cell layer of the CA3 area in the hippocampal slices. Lowering theconcentration of glucose from 10 mM to 5, 3, 2, 1 and 0 mM caused a reduction in the PS amplitude. There were differences in the decaytimes of the PS evoked in these two regions. PS evoked in CA3 region decayed faster even at a concentration of 3 mM glucose at whichPS in granule cell layer was well maintained. The decay time of the PS in the CA3 region in the presence of glucose up to a concentrationof 3 mM was shorter than that evoked in the DG. After the replacement of glucose with mannose, fructose or lactate, the PS disappearedwithin 35 min and there were no significant differences between the decay times in the two regions of slices incubated in the same

Ž .medium. ATP, creatine phosphate CrP and lactate levels in each slice were determined. To investigate whether mannose and fructosecould be metabolized or not in the tissue slice, anaerobic production of lactate from glucose, mannose and fructose were measured duringoxygen and glucose deprivation. Under anaerobic conditions for 60 min, the levels of high-energy phosphates decreased to 50% of theinitial level and lactate was produced from glucose, mannose or fructose. However, there were significant differences in the rate of lactateproduction between the DG and CA3 areas during application of 3 mM glucose, 10 mM mannose and 10 mM fructose. These resultsindicate that mannose, fructose and lactate can be metabolized and are available for maintaining the levels of high-energy phosphates butnot for neural activity in the tissue slices and that the presence of glucose is indispensable for the maintenance of neural activity. q 1998Elsevier Science B.V.

Keywords: Guinea pig; Hippocampal slice; Population spike; High energy phosphate; Glucose; Lactate; Mannose; Fructose

1. Introduction

The presence of oxygen and glucose is essential formaintaining energy metabolism and neural activity in braintissue. Glucose, especially, is considered to be indispens-able as a substrate for the production of ATP which is usedto maintain the ionic balance across the cell membrane.The degree of sensitivity to oxygen andror glucose depri-vation in terms of energy metabolism and neural functionof the brain differs among animal species.

) Corresponding author. Fax: q81-78-341-5732.1 Present address: Eizai Pharmaceutical, Koishikawa Bunkyoku, Tokyo,

Japan.

w xThe previous reports 10,19 studying the effect ofglycolytic metabolites and other sugars on maintainingneural activity and a level of high energy phosphatesindicated that glucose is essential for the preservation ofsynaptic activity. However, the correlation between thelevel of high energy phosphate and the maintenance ofneural activity by glucose has not yet been clarified.

In this experiment, we studied the ability of glucose tomaintain the level of high energy compounds and neuralactivity in different areas of hippocampal slices and furtherinvestigated whether or not other sugars such as mannose,fructose as well as lactate could preserve neural activityand high energy phosphate levels during deprivation ofglucose.

0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 97 01532-1

( )H. Wada et al.rBrain Research 788 1998 144–150 145

2. Methods

2.1. Preparation of brain slices

Ž .Adult guinea pigs around 60 days of age of both sexeswere used for the experiment. For the preparation of

Ž .hippocampal slices 300–400 mm thick , the hippocampusŽ .with dentate gyrus DG , subiculum and presubiculum was

removed from the brain and was transversely cut along thelong axis. Details on the preparation of slices are described

w xelsewhere 24 . Before starting the experiment, slices wereŽpre-incubated for 30 min in the standard medium con-

centration in mM: NaCl 125, KCl 5, KH PO 1.24, MgSO2 4 4.1.3, CaCl 2, NaHCO 26 and glucose 10 bubbled with2 3

95% O and 5% CO . The temperature of the medium was2 2

maintained at 35.08C throughout the experiment.

2.2. Recording neural actiÕity

After pre-incubation, each slice was transferred to theobservation chamber under a stereomicroscope. The cham-ber itself contains 1.5 ml volume and was perfused contin-uously with the standard medium at a flow rate of 4mlrmin. As an index of neural activity, population spikesŽ .PS were recorded from the granule cell layer of the DGand the pyramidal cell layer of the CA3 region of thehippocampus. The perforant path was stimulated for therecording of PS from the granule cell layer and the DGŽ .mossy fiber layer was stimulated for activation of pyra-midal neurons in the CA3 region. The strength of stimula-tion was adjusted to maintain half maximum amplitudeelicited by supramaximal stimulation. After assuring thestability of PS for at least 20 min, slices were exposed to:Ž .1 a perfusing medium containing glucose at the various

Ž . Ž .concentrations 0, 1, 2, 3, 5 and 10 mM , and 2 mediumŽ . Ž .containing mannose 10 mM or fructose 10 mM or

Ž .lactate 5 mM instead of glucose.

( )2.3. Determination of ATP, creatine phosphate CrP andlactate

The area of the DG containing the granule cell layerand that of the CA3 and CA4 regions containing thepyramidal cell layer were separately dissected from eachslice. Then they were placed in medium deprived of

Ž .oxygen bubbling with N 95% and CO 5% containing2 2

mannose or fructose instead of glucose for 0, 30 or 60 min.Ž .Na S O 2 mM was added for complete removal of2 2 3

oxygen in the medium. At the end of each experiment,each slice was immediately homogenized with ice-cold 0.5M perchloric acid containing 1 mM ethylenediamine te-

Ž . Ž .traacetic acid EDTA and centrifuged 1800=g for 10min. The supernatant was neutralized with KHCO and3

Ž .recentrifuged 850=g for 5 min. The resulting super-natant was used for the determination of ATP, CrP andlactate. ATP, CrP and lactate, both in the tissue slice and

in the medium that surrounded the slide, were determinedenzymatically and fluometrically by measuring the produc-

w xtion of NADPH 21 . The precipitate of the tissue ho-mogenate was used for the determination of protein whichwas determined by the method of Lowry and Passonneauw x13 .

2.4. Determination at the rate of energy use in the granule( )cell layer and pyramidal cell layer CA3 area of hip-

pocampal slices

To determine the rate of energy use in the granule celllayer of the DG and pyramidal cell layer of the CA3 lesionof the hippocampus, ATP, CrP and lactate levels in eachlayer were determined using an enzymatic cycling methodwith NADPH–NADP and NADH–NAD. Hippocampalslices before and after deprivation of glucose and oxygenfor 3 min were frozen in Fneon-12 in liquid nitrogen. In acryotome at y208C, 20 mm thick sections of each slicewere cut and were freeze-dried at y358C under vacuum.The individual layers were dissected out under a micro-scope using a fine knife. The weight of the samples wasestimated with a quartz fiber balance, weighing in therange of 0.5–1.5 mg. The ATP and CrP content of thesamples were determined using the oil well technique andenzymatic cycling method with NADP–NADPH. Lactatein the layer was measured by enzymatic cycling methodwith NAD–NADH. The details of the procedure of theATP and CrP microassays are described elsewherew x22,23,25 . From the initial reduction of ATP and CrP andthe subsequent increase in lactate, the rate of energy useŽ .;P was calculated according to the formula: 2=DATP

w xqDCrPq1.3= lactate 21 .

3. Results

3.1. Neural actiÕity and high energy phosphate leÕelsunder different concentration of glucose

To test the ability of glucose to maintain neural activityat different concentrations, PS were elicited in the granulecell layer of the DG and in the pyramidal cell layer of theCA3 region of the hippocampus. The standard mediumwas replaced with medium containing glucose at concen-

Ž .trations of 0, 1, 2, 3, 5 and 10 mM Fig. 1 . The PS elicitedin the granule cell layer was well maintained for at least 60min in a medium containing glucose in a concentration ofat least 3 mM. However, with 2 mM glucose, there was areduction in the amplitude of the PS and in the mediumcontaining no glucose and 1 mM glucose, the PS wascompletely extinguished within 20 min and 35 min, respec-

Ž .tively Fig. 1B-a . In the case of the PS evoked in the CA3region, however, glucose at concentrations below 5 mMcould not maintain the PS which were extinguished in 15,

( )H. Wada et al.rBrain Research 788 1998 144–150146

Fig. 1. Effect of glucose at different concentrations on synaptic transmis-Ž .sion in the DG and the CA3 area of hippocampal slices. A shows the

typical trace of the PS evoked in the pyramidal cell layer of the CA3Ž . Ž . Ž .region; a response in the standard medium glucose 10 mM , and b 20

Ž .min after deprivation of glucose. B shows the time course of the changeŽ .in the amplitude of the PS responses recorded from: a the granule cell

Ž .layer of the DG, and b the pyramidal cell layer of the CA3 region. TheŽchanges in the PS amplitude the height from the baseline to the negative

.peak of the field potential are expressed as the percentage of the initialvalue measured prior to lowering the concentration of glucose. The

Ž .vertical bars indicate the S.E.M. ns4 to 7 .

20, 35 and 40 min for 0, 1, 2 and 3 mM glucose,Ž .respectively Fig. 1B-b .

Thus, the neural activity of pyramidal neurons decayedearlier than that of granule cells when glucose concentra-tions were reduced in the perfusion medium, indicating

Fig. 2. The time-course of the levels of ATP and CrP in the regions of theDG and the CA3 during application of different concentrations of glu-cose. The changes are expressed as the percentage of the initial value.Initial values of ATP and CrP were 4.8"0.4 mmolrkg dry weight,17.0"1.0 mmolrkg dry weight in the DG area and 4.3"0.4 mmolrkgdry weight, 12.0"1.5 mmolrkg dry weight in the CA3 region, respec-tively.

that pyramidal neurons may be more sensitive than granulecells to low concentrations of glucose.

The concentrations of ATP and CrP in the DG and theCA3–CA4 region were determined after each slice wasincubated for 30 and 60 min in the medium containing

Ž .glucose at 0, 3 and 10 mM Fig. 2 . At 10 mM glucose, thelevels of ATP and CrP in both area were well preservedeven for 60 min but at 3 mM, the levels diminished slowly

Table 1Energy use rate in granule cell layer and in pyramidal cell layer of the hippocampus calculated from the initial reduction in ATP and CrP levels and theincrease in lactate in those layers

Period of deprivation 0 min 3 min Initial energy use rateŽ Ž . Ž .of oxygen and glucose mmolrkg dry weight mmolrkg dry weight mmolrkg dry weightrmin

Granule cell layer ATP 4.78"0.41 1.34"0.17 4.65"0.89CrP 17.03"0.96 13.75"0.90Lactate 50.06"2.96 80.13"6.65

Pyramidal cell layer ATP 4.31"0.36 1.84"0.14 5.90"2.08CrP 11.95"1.52 9.39"0.53Lactate 4.31"5.05 80.93"9.53

( )H. Wada et al.rBrain Research 788 1998 144–150 147

and they were 70–80% of original level at 60 min. Duringcomplete deprivation of glucose, the ATP and CrP levelsdecayed more rapidly and reduced to 50% of their initialconcentrations in 60 min. However, there were no signifi-cant differences in the pattern of decay of high energyphosphates in the CA3–CA4 area of the hippocampus andin the DG.

To investigate the difference in the rate of energy usebetween the dentate and the CA3–CA4 region, the rateswere determined after complete deprivation of oxygen andglucose. The granule cell layer and pyramidal cell layerwere carefully dissected from freeze-dried tissue of eachslice. From the initial reduction in ATP and CrP levels andthe increase in lactate in those layers, the rate of energy

Ž .use ;P was calculated as shown in Table 1. There wereno significant differences in the energy use rate betweengranule cell layer and pyramidal cell layer.

3.2. Application of mannose, fructose and lactate insteadof glucose

To investigate the ability of mannose, fructose andlactate to maintain neural activity, PS were elicited in the

Fig. 3. Effects of mannose, fructose and lactate on the preservation ofsynaptic transmission in the DG. The left column shows the changes inthe levels of ATP and CrP and the right column shows the time-course ofthe reduction of the amplitude of PS recorded from the granule cell layer

Ž . Ž .during application of mannose 10 mM top , fructose 10 mM middleŽ .and lactate 5 mM bottom instead of glucose.

Fig. 4. Effect of mannose, fructose and lactate on the preservation ofsynaptic transmission in the CA3 area. The left column shows thechanges in the level of ATP and CrP, and the right column shows thetime-course of the reduction of the amplitude of the PS recorded from thepyramidal cell layer of the CA3 region during application of mannose 10

Ž . Ž . Ž .mM top , fructose 10 mM middle and lactate 5 mM bottom instead ofglucose.

granule cell layer of the DG and the pyramidal cell layerof CA3 region of the hippocampus and the levels of high

Ž .energy phosphates were also determined Figs. 3 and 4 .In media containing mannose, fructose or lactate instead

of glucose, the PS recorded from the granular cell layer ineach case disappeared within 20 to 35 min after replace-ment, although in all cases, the levels of high-energyphosphates were well maintained even for 60 min. The PSrecorded from pyramidal cell layer from the CA3 regiondisappeared in 10 to 15 min after replacement, although inall cases, the levels of high-energy phosphates were wellmaintained even for 60 min as in the DG. The decay timeof the PS of each slice was shorter in the CA3 region thanin the DG.

3.3. Lactate accumulation

To investigate whether mannose and fructose could bemetabolized as a substrate or not in the two regions of thehippocampus, a production of lactate was determined afterthe addition of mannose or fructose. Lactate produced

( )H. Wada et al.rBrain Research 788 1998 144–150148

Ž . Ž .Fig. 5. The production of lactate in the DG I and the CA3 B area in the presence of glucose, mannose or fructose during deprivation of oxygen for 30Ž . Ž .min left figure and 60 min right figure .

from substrates such as mannose or fructose in the pres-ence of oxygen or during deprivation of oxygen is shownin Fig. 5. There was no significant difference between theinitial levels of lactate of the two regions of the slices:70.6"4.0 mmolrkg protein in the DG area and 72.5"4.3mmolrkg protein in the CA3 area. During deprivation ofoxygen and glucose for 60 min, there was no significantchange in each level of lactate. During deprivation ofoxygen, lactate was generated from mannose and fructose.However, there were significant differences in the lactateproduction between DG and CA3 area after application of10 mM mannose, 10 mM fructose and 3 mM glucose for60 min, although this difference was not distinctly ob-served in the shorter application of these substrates for 30min.

4. Discussion

There have been many reports showing the relationshipbetween energy metabolism and the neural activity in brain

w xtissue 1–7,12,13,26 . The observation of neural activityand excitability is possible using hippocampal slices. In thepresent experiment, we studied the correlation of neuralactivity and the level of high energy phosphate such asATP and CrP in the CA3 region including the pyramidalcell layer and the DG including the granule cell layerduring exposure of slices to low concentrations of glucoseand replacement of glucose with mannose, fructose andlactate.

w xLipton and Whittingham 12 showed a good correlationbetween the decrease in ATP levels in tissue and thereduction of the neural activity of hippocampal slices

w x w xduring hypoxia. Hertz et al. 9 and Yager et al. 30investigated the neural damage during deprivation of glu-cose, hypoxia or ischemia in astrocytes and indicated that

cell death does not occur until the level of ATP wasreduced to 15% of the original level. On the other hand,

w xOkada 24 has reported that there is a discrepancy be-tween the time course of the reduction of ATP levels andthe decrease in the PS amplitude during deprivation ofoxygen and glucose. This correlation has been studiedunder conditions of combined oxygen and glucose depriva-tion. However, the correlation between neural activity andthe level of high energy phosphates has not been exten-sively studied during deprivation of only glucose. Thepresent experiments show that the glucose requirement forneural activity such as synaptic transmission differs in thegranule cells and pyramidal cells of the CA3 area despitethe fact that the levels of high energy phosphates in theseareas were not significantly different during hypoglycemia.This difference in the glucose requirement of these twoareas cannot be explained by a reduction in high energyphosphate levels.

In the present experiments, we homogenized the tissueslices from the DG and the CA3 area in which neurons andglia cells were included together, even though the pyrami-dal cell layer of the CA3 area and granule cell layer of theDG were separately dissected out. ATP could be depletedearlier in neurons than in glia during neural activity orthere might be differences in the rate of production of highenergy phosphates between neuron and glia. Mercer and

w xDunham 14 showed that a glycolytic enzyme binds to thecell membrane and that membrane-bound ATP producedthrough the glycolytic pathway fuels a NarK pump inerythrocytes. So there might be difference in the rate ofATP consumption among the neural cells.

We examined the neural activity and high energy phos-phates during application of mannose, fructose and lactateinstead of glucose. Fructose and mannose have been re-ported to be good substrates for ATP production in brain

w x w xtissue 3,11,20,27,32 . McIlwain and Bachelard 15 showed

( )H. Wada et al.rBrain Research 788 1998 144–150 149

that sugars such as galactose, fructose, mannose andmetabolites such as pyruvate and lactate can also besubstrates for energy metabolism in isolated cortical tis-sues although the rate of utilization of lactate, fructose andmannose were much lower than that of glucose. However,

w xTakata and Okada 29 showed, using an intracellularrecording technique, that lactate cannot replace glucose for

w xthe maintenance of neural activity. Kanatani et al. 10reported that the presence of glucose is essential for thepreservation of synaptic activity in addition to its main roleas the substrate for energy production to maintain the

w xlevels of high energy phosphates. Cox 5 showed thatmannose and high concentrations of fructose can replaceglucose for the maintenance of neural activity. Bachelard

w x w x w xet al. 2 , Cox and Bachelard 7 , Fowler 8 and Schurr etw xal. 28 , using rat hippocampal slices, have reported that

lactate instead of glucose could maintain neural functionalthough the levels of ATP and CrP in each slice were notdetermined. The present experiments, using guinea pighippocampal slices, indicate that glucose should be essen-tial for the preservation of synaptic activity in the twodifferent regions of the DG and the CA3 area of thehippocampus although the sensitivity for glucose is differ-ent in these two regions and that mannose, fructose andlactate could not preserve synaptic potential, although thelevels of high energy phosphate were well preserved bythese substrates. This incompatibility might be resulted inthe difference of the pups, recording areas or other parts ofthe system including incubation time, the flow rate of thechamber and so on.

The pyramidal cells of the CA1, CA3 and CA4 regionsw xare well developed at 20 days of postnatal age 16–18 .

Granule cells in the DG, however, are not matured even atw xthis stage. In a previous study 31 , we reported that in the

immature hippocampal slices, ATP synthesized by aerobicmetabolism using lactate could maintain neural activitywhile this did not occur in the matured rat. The differencein the glucose sensitivity of the two regions might dependon the difference in the properties of the early developedand later matured neurons.

There were no significant differences in the rate oflactate production in the anaerobic state for 3 min in themedium containing 3 mM glucose, 10 mM mannose or 10

Ž .mM fructose Fig. 5 . After 60 min of anoxia, however,the DG and the CA3 region of the hippocampus showedsignificant differences in the production of lactate in thepresence of glucose at the concentration of 3 mM and inthe presence of mannose or fructose at 10 mM. Rate oflactate synthesis in the DG is significantly higher than thatin the CA3 region. These results indicate that the rate ofglycolysis might be different in these two regions, al-though there were no significant differences in the energyuse rate between the two regions. Involvement of theseenzymes in regulating the rate of glycolysis in the dentatearea and the CA3 region of the hippocampus are thusfurther studied.

5. Conclusion

There is a difference in glucose requirement for main-taining neural activity and lactate production between theDG and the CA3 areas of hippocampal slices, although therate of energy use and the production of high-energyphosphates in these two regions are not significantly differ-ent. Mannose and fructose can be metabolized and canmaintain the levels of high-energy phosphates but not theneural activity in tissue slices. Glucose is an absoluterequirement for neural activity.

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