Ž .Developmental Brain Research 101 1997 1–7

Research report

The effects of lactate and b-hydroxybutyrate on the energy metabolismand neural activity of hippocampal slices from adult and immature rat

Hiroshi Wada a,b, Yasuhiro Okada a,), Makoto Nabetani a,b, 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 3 December 1996

Abstract

We investigated the correlation between energy metabolism and neural activity during glucose deprivation and during replacement ofŽ .glucose with lactate and b-hydroxybutyrate OHBA in neural tissue from rats of different ages. Hippocampal slices were prepared from

Ž .4-, 7-, 10-, 13- and 16-day-old and adult rats. The population spikes PS were recorded in the pyramidal cell layer of the CA3 area as theŽ .index of neural activity. ATP and creatine-phosphate CrP levels in each slice were determined during glucose deprivation and during

replacement of glucose with lactate or OHBA. After deprivation of glucose, the PS of the slices from 4-, 7- and 10-day-old and adult ratsdecayed and extinguished in 30 min and the decay time was shortened according to the age of the rat. The levels of ATP and CrP in the

Ž .slices also decreased, but to a lesser extent than the amplitudes of PS. After substitution of lactate or b-hydroxybutyrate OHBA forglucose, PS of the adult rat disappeared as was the case with glucose deprivation, although the levels of high energy phosphates were wellmaintained. In the case of the immature rat, however, PS decayed more slowly. Especially in the case of 4-day-old rat, ATP and CrP inthe slices were maintained as high as those under the initial concentrations and PS amplitude showed no decay even after 60 min. Theseresults indicate that the presence of glucose is essential for neural activity in the adult rat, and lactate or OHBA cannot replace it for themaintenance of neural activity. In the immature rat, glucose metabolites such as lactate and OHBA are available for both neural activityas well as maintaining the levels of high-energy phosphates in the tissue slice.

Keywords: Mature and immature rat; Hippocampal slice; Population spike; High energy phosphate; Glucose deprivation; Lactate; b-Hydroxy-butyrateŽ .OHBA

1. Introduction

The presence of glucose is essential for maintainingenergy metabolism and neural activity of brain tissue. Thedegree of resistance to oxygen or glucose deprivation interms of energy metabolism and neural function of thebrain differs in immature and mature animals. Indeed,neonates from different species are much more resistant to

w xischemia than adults of the same species 8,10,17 . Thereason for this greater resistance is that the brain tissue ofimmature animals has lower energy demands and also

w xproduces energy partly through anaerobic glycolysis 29 ,in contrast to mature animals which utilize energy pro-

w xduced mainly through aerobic metabolism 9,15 .

) Ž .Corresponding author. Fax: q81 78 341-5732.

Many investigations on the difference in resistance toischemia between the immature and mature brain havebeen conducted in intact animals with in vivo analysisw x7,12,29,30 . However, these studies of intact animals invivo do not discriminate precisely between changes inneural activity and in energy metabolism during glucosedeprivation. Measurement of neural function and energymetabolism using in vivo brains is hampered by edema ofthe tissue and post-ischemic circulatory disturbances suchas no-reflow phenomenon and delayed hypoperfusion.

On the contrary, brain slices eliminate post-ischemiccirculatory disturbances and can be easily controlled bysupplying oxygen and glucose through the perfusionmedium. Nabetani et al. studied the neural activity and thelevels of high energy phosphates of immature and adult

w xrats 21 , and showed that neural activity of both immatureand mature rats ceased rapidly during deprivation of glu-cose although the levels of ATP were preserved and

0165-3806r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0165-3806 97 00007-2

( )H. Wada et al.rDeÕelopmental Brain Research 101 1997 1–72

indicated that glucose plays an important role in the preser-vation of neural activity. Immature rats, however, are lessvulnerable for glucose deprivation.

In the present study we investigated the effects of theaddition of lactate and OHBA on the neural activity andenergy metabolism during deprivation of glucose usinghippocampal slices from immature and adult rats anddisclosed that the presence of glucose was essential formaintaining neural activity and that the replacement ofglucose with lactate or OHBA was unable to maintain PSin the adult rat, but it was able in the immature rat.

2. Methods

2.1. Preparation of brain slices

We used 4-, 7-, 10-, 13- and 16-day-old rat pups andŽ .adult rats around 120 days in age of both sexes. For the

preservation of hippocampal slices, the hippocampus withdentate gyrus, subiculum and presubiculum was removed

Ž .from the brain and was transversely 300–400 mm thickcut along the long axis. The detail of the preservation of

w xslices was described elsewhere 25 . Before starting theexperiment, slices were pre-incubated for 30 min in the

Žstandard medium concentration in mM : NaCl 125, KCl 5,KH PO 1.24, MgSO 1.3, CaCl 2, NaHCO 26 and2 4 4 2 3

.glucose 10 bubbled with 95% O and 5% CO . The2 2

temperature of the medium was maintained at 35.08Cthroughout the experiment.

2.2. Recording neural actiÕity

After pre-incubation, each slice was transferred to theobservation chamber under a stereomicroscope. The cham-ber was perfused continuously with the standard mediumat a flow rate of 4 mlrmin. For the index of neural

Ž .activity, population spikes PS were recorded from thepyramidal cell layer of the CA3 region of the hippocampus

Žfollowing stimulation of the dentate gyrus mossy fiber.layer and antidromic spike potentials were recorded after

Ž .stimulation to the fimbria Fig. 1 . After assuring thestability of PS for at least 30 min, slices were exposed to aperfusing medium devoid of glucose or medium containinglactate at the concentration of 5 mM or b-hydroxy-butyrateŽ .OHBA of 10 mM instead of glucose.

2.3. Biochemical analysis

Each slice was placed in the medium deprived ofglucose or containing lactate or OHBA instead of glucosefor 0, 30 or 60 min and was immediately homogenizedwith ice-cold 0.5 M perchloric acid containing 1 mM

Fig. 1. Schematic drawing of the experimental arrangement for recordingneural activity of hippocampal slices. a: population spikes were recordedin the CA3 region of pyramidal cell layer after electrical stimulation tothe granule cell layer and mossy fiber layer and antidromic potentialswere elicited by activation of fimbria. b: antidromic and c: orthodromicresponses of 4-day-, 7-day-, 10-day-old and adult rat.

Ž .ethylenediamine-tetra-acetic acid EDTA and centrifugedŽ .3000 rpm for 10 min. The supernatant was neutralized

Ž .with KHCO and recentrifuged 2000 rpm for 5 min. The3

resulting supernatant was used for the assay of adenosineŽ . Ž .triphosphate ATP and creatine-phosphate CrP . The pre-

cipitate of the tissue homogenate was used for the assay ofprotein which was determined by the method of Lowry etal. ATP and CrP were determined enzymatically and fluo-

w xrometrically by measuring the production of NADPH 24 .

3. Results

Fig. 1 shows traces of typical examples of PS elicitedfrom the pyramidal cell layer of the CA3 region of hip-pocampal slices of 4-, 7-, 10-day-old and adult rat. In theslices from the adult rat, the PS amplitude was larger andthe latency of the PS was shorter than that recorded fromimmature animals.

The right column in Fig. 2 shows the time-course of thereduction of the amplitude of the antidromic and ortho-dromic response during deprivation and reintroduction ofglucose. The left column indicates the changes in thelevels of ATP and CrP of the hippocampal slices of

( )H. Wada et al.rDeÕelopmental Brain Research 101 1997 1–7 3

different aged-rats during deprivation of glucose. DuringŽdeprivation of glucose, the PS decay time the interval

.from the onset of deprivation to the cessation of PS of theslices from 4-, 7- and 10-day-old and adult rats were

Ž20.4"2.7, 25.0"2.0, 20.7"1.4, 12.7"0.7 mean"

.S.E.M. , respectively, and the decay time was significantlyshortened according to the development of age, althoughthe decay time of PS in 4- and 7-day-old rat were notsignificantly different. The decay time was shortened ac-cording to the aging of the rat.

Ž .After the application of lactate Fig. 3 instead ofglucose, PS of the adult rat disappeared similar to thatduring glucose deprivation, although in all cases the levelsof high-energy phosphates were well maintained even 60min after glucose deprivation. PS amplitude, however,reduced slowly after replacement of glucose by lactate inthe case of immature rats. In the slice of 7-day-old rat,decay time was significantly prolonged compared with thatof the adult. In the case of 4-day-old rats, ATP and CrPlevels in the slices were maintained at the levels before

Ž . Ž .Fig. 2. The time-course of the levels of high-energy phosphates such as ATP and CrP left column and the neural activity right column of ratŽ .hippocampal slices of different ages. Each point represents the average value of four to seven slices the open square: ATP and the closed circle: CrP . The

content of ATP in the control slices of 4, 7-, 10-day-old and adult animals were 24.5"4.8, 22.5"3.6, 21.5"3.5, 16.3"3.8 mmolrg proteinŽ .mean"S.E.M. , respectively. The content of CrP in the control slices of 4-, 7-, 10-day-old and adult animals were 42.7"7.8, 40.2"6.9, 33.8"6.7,

Ž .30.5"5.8 mmolrg protein mean"S.E.M. , respectively. Right column indicates the time-course of the change in the amplitude of antidromic andŽ .orthodromic responses. The changes in the PS amplitude the height from the baseline to the negative peak of the field potential are expressed as the

Žpercentage of the initial value measured prior to the deprivation or replacement the open square: orthodromic response and the closed circle: antidromic. Ž .response. In G, x indicates the addition of glucose . The vertical bars indicate the S.E.M. ns4–7 .

( )H. Wada et al.rDeÕelopmental Brain Research 101 1997 1–74

Fig. 3. Time course of the changes in the levels of ATP and CrP and PS amplitude during addition of lactate instead of glucose.

glucose deprivation and PS amplitude did not decay evenafter 60 min replacement.

Ž .After the application of OHBA Fig. 4 instead ofglucose, PS of the adult rat also disappeared similar to thatduring glucose deprivation, however, the decay times of 7-

Ž .and 10-day-old rats were significantly P-0.0001 pro-longed in an age-dependent manner. PS amplitude of theslice from 4-day-old rat remained at 88% of the originallevel after 60 min replacement. The concentrations of ATPand CrP in the slices of all cases after addition of OHBAwere also well preserved. A small decrease in ATP andCrP observed in the adult slices was not statisticallysignificant.

Fig. 5 shows the correlation between ATP levels and PSamplitude of slices from developing brain after 60 mindeprivation of glucose or replacement by lactate or OHBA.In 7- and 10-day-old and adult animal the PS was not

maintained even though the level of ATP was well main-tained. It is to be noted, however, that in the slice from4-day-old rat both the synaptic activity and ATP levelswere well maintained during the addition of lactate orOHBA.

4. Discussion

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

w xtissue 2–6,18,26 . Using hippocampal slices, Lipton et al.w x18 showed a good correlation between the decrease inATP levels in tissue and the reduction of the neural

w xactivity during hypoxia. Yager et al. 14,32 investigatedthe neural damage under deprivation of glucose, hypoxiaor ischemia in astrocytes and indicated that the cell death

( )H. Wada et al.rDeÕelopmental Brain Research 101 1997 1–7 5

does not occur until the level of ATP was reduced to 15%w xof the original level. However, Okada 25 found that there

is a discrepancy between the time course of the reductionof ATP levels and the decrease in the PS amplitude duringdeprivation of oxygen or glucose. ATP and CrP levels inthe hippocampal slices decreased in a similar mannerduring either deprivation of O or glucose whereas the PS2

amplitude diminished and extinguished much faster duringglucose deprivation than that during O deprivation.2

w xKanatani et al. 16 found that presence of glucose isessential for the preservation of synaptic activity in addi-tion to its main role as the substrate for energy productionto maintain the levels of high energy phosphates in thehippocampal slices of adult guinea pig.

Monitoring PS as the index of neural function, wedetermined the concentration of ATP and CrP in eachtissue slice. The present experiment showed that in the

adult rat, replacement of glucose with lactate or OHBAmaintained the levels of ATP and CrP in the tissue slices,although they failed to maintain synaptic function over 30

w xmin. Takata et al. 28 showed, using an intracellularrecording technique, that lactate cannot substitute for glu-

w xcose for neural activity. Arakawa et al. 1 also showedthat OHBA can be a substrate for the production ofhigh-energy phosphate but cannot maintain the neural ac-tivity in the hippocampus of the adult guinea pig. In thepresent experiment, however, in the immature 4-day-oldrat, lactate and OHBA are available for neural activity aswell as for the maintenance of ATP and CrP levels.

w xCox et al. 4 also reported that replacement of 10 mMglucose with 20 mM fructose maintained ATP levels in thetissue but the amplitude of the PS decreased to 70% of the

w xoriginal level. They also showed 2,6 that neural activityin the dentate gyrus was attenuated by lactate although

Ž .Fig. 4. Time course of the changes in the levels of ATP and CrP and PS amplitude during addition of b-hydroxybutyrate OHBA instead of glucose.

( )H. Wada et al.rDeÕelopmental Brain Research 101 1997 1–76

Fig. 5. The ATP levels and PS amplitude in the slices of different agesafter 60 min deprivation of glucose or addition of lactate or OHBAinstead of glucose. The histograms were obtained from Fig. 2. In thefigure, open bars indicate ATP levels of slices and black bars the changesin the PS amplitude. Each value is given as means"S.E.M.

they did not determine the level of ATP in the hippocam-w xpal slices. On the other hand Shurr et al. 27 and Fowler

w x11 reported that lactate supported synaptic transmissionof the Schaffer’s collateral-CA1 connection in the hip-pocampal slices although they did not determine the levelsof ATP and CrP in each slice. Thus the correlation be-tween ATP level and neural activity in the brain is stillcontroversial. Although it is still unclear how glucose isneeded for the maintenance of neural activity, it can bespeculated that in the immature rat ATP synthesized byaerobic metabolism may maintain neural activity while thisdoes not occur in the mature rat brain.

In the 4-day-old rat, lactate and OHBA can be metabo-lized to maintain neural activity but not 7-, 10-day-old andadult rats. Probably in immature animals, ATP producedby aerobic and anaerobic process can both be utilized forthe maintenance of the synaptic potentials, whereas in themature brain the utilization of ATP synthesized by aerobicor by non-aerobic process may be differentiated.

Lactate and ketone bodies can enter the brain andbecome energy substrates in the immature ratw x13,19,22,23,31 . In these studies, however, the correlationbetween energy level and neural function has not been

w xdocumented. Mercer et al. 20 showed that it has beenreported that a glycolytic enzyme binds to the cell mem-

brane and that membrane-bound ATP fuels NarK pump inerythrocytes. This can be true for neuron although furtherexamination should be needed.

5. Conclusion

The presence of glucose is essential for maintainingsynaptic activity and the replacement of glucose withlactate or OHBA is unable to maintain PS in the adult rat,but it is able in the immature rat.

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