Altered mitochondrial oxidative phosphorylation in ... - Semantic Scholar

Brain Research 826 Ž1999. 236–242

Research report

Altered mitochondrial oxidative phosphorylation in hippocampal slices of kainate-treated rats Wolfram S. Kunz ) , Ivan V. Goussakov, Heinz Beck, Christian E. Elger Department of Epileptology, UniÕersity of Bonn Medical Center, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany Accepted 16 February 1999

Abstract Mitochondria provide the main neuronal energy supply and are important organelles for the sequestration of intracellular Ca2q. This indicates a possible important role for mitochondria in modulating neuronal excitability in normal function as well as in disease. Therefore, we have investigated mitochondrial oxidative phosphorylation in the kainate model of epilepsy. We measured the oxygen consumption of single 400-mm rat hippocampal slices applying high resolution respirometry and determined mitochondrial NADŽP.H autofluorescence signal changes in single slices by laser-excited fluorescence spectroscopy. We observed an about 2-fold higher Ž p - 0.001. basal glucose oxidation rate in slices from kainate-treated animals. This increased endogenous energy consumption was found to be unrelated to spontaneous activity since it was not sensitive to the inhibitors of the sodium–potassium ATPase ouabain and of the mitochondrial adenine nucleotide translocator atractyloside. This finding suggested an increased mitochondrial energy turnover in kainate-induced epilepsy. Furthermore, the uncoupler-stimulated oxygen consumption of the slices was approximately 1.3-fold higher Ž p - 0.01. in the kainate model. In accordance with the respirometric data, fluorescence spectroscopy showed decreased reduction levels of the mitochondrial NAD-system in glucose oxidizing slices from kainate-treated rats. The preincubation of epileptic hippocampal slices with either BAPTA AM, ruthenium red or TPPq increased the atractyloside sensitivity of glucose oxidation to about 1.4-fold Ž p - 0.01.. These observations indicate that the increased mitochondrial energy turnover in hippocampal slices from kainate-treated rats is most possibly caused by futile Ca2q-cycling. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Kainate-treated rat; Epilepsy model; Hippocampal slice; Oxidative phosphorylation; Calcium cycling

1. Introduction Mitochondrial oxidative phosphorylation provides the major source of ATP in neurons. Adequate levels of ATP are essential to maintain the neuronal plasma membrane potential via the sodium–potassium ATPase which consumes about 40% of the energy w1x. In addition, mitochondria are an important intracellular Ca2q sequestration system w15,30x. Due to both features, mitochondria can modulate neuronal excitability and synaptic transmission w4,28x. Under pathophysiological conditions, adequate supply of energy-rich substrates as well as mechanisms regulating intracellular Ca2q levels become increasingly important. In epilepsy, the energy consumption as well as the Ca2q load of neuronal cells increases during epileptiform activity. Furthermore, chronic changes can be observed that affect different Ca2q buffering and sequestration systems. For ) Corresponding author. [email protected]

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instance, loss of the Ca2q-binding protein calbindin-D28K has been observed in hippocampal granule cells following kindling epileptogenesis w2x. In addition, an up-regulation of Ca2q influx through voltage-dependent Ca2q channels in the kindling and kainate model w3,9,19,33x and changes in the regulation of NMDA receptors have been discussed w18,20x. Very recently, the involvement of mitochondria in Ca2q sequestration after kainate application in vitro on cultured neurons was shown w17x. However, until now, little is known about possible changes in mitochondrial function in chronic epilepsy. Therefore, we have studied possible changes in oxidative phosphorylation in the hippocampus of the kainate model of temporal lobe epilepsy w6x. This model replicates key features of human temporal lobe epilepsy, i.e., segmental neuronal loss in the Ammon’s horn, severe astrogliosis and axonal reorganization. In this model, we have characterized mitochondrial function in single hippocampal slices using high-resolution respirometry and laser-excited fluorescence spectroscopy. Our results sug-

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 2 7 9 - 2

W.S. Kunz et al.r Brain Research 826 (1999) 236–242

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2. Materials and methods

PC-supported Oroboros high resolution oxygraph w14x. For determination of the rates of oxygen consumption of single hippocampal slices, the first derivative of the digitally acquired oxygen concentration trace was used. The consumption rate was calculated as the average of datapoints of the first derivative trace following steady state until the next addition.

2.1. Materials

2.4. Protein content

gest a markedly increased basal glucose oxidation rate in the hippocampus of kainate-treated rats which seems to be related to futile calcium cycling at the mitochondrial inner membrane.

The salts, atractyloside, pyruvate, ruthenium red and glucose were purchased from Sigma ŽDeisenhofen, Germany.. Tetraphenylphosphonium bromide ŽTPPq. was from Aldrich ŽSteinheim, Germany. and glycine, N, N Xw1,2 - ethanediylbisŽoxy - 2,1-phenylene.xbisw N-w2-wŽacetyloxy . methoxy x-2-oxoethyl xx-,bis wŽacetyloxy .methyl xester ŽBAPTA AM, cell permeant. was from Molecular Probes ŽEugene, OR.. Ouabain and tetrodotoxin were from Boehringer ŽMannheim, Germany.. The uncoupler TTFB Ž4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole. is a kind gift from Dr. B. Beechey ŽAberystwyth, UK.. 2.2. Kainate treatment of animals Young adult male Sprague–Dawley rats Ž26–33 days; 60–100 g. were injected twice with kainic acid Ž12.5 mgrkg i.p.. on two consecutive days. Kainate-treated rats showed an onset of seizure activity 15–20 min after kainic acid administration. The seizures were characterized by recurrent ‘wet dog shakes’ and rearing, followed by bilateral forelimb cloni and occasional falling and jumping. All animals showed bouts of generalized tonic–clonic seizures after the second injection lasting for 1–2 h. After survival periods of 34–44 days, spontaneous seizures were observed in all rats used for the experiments. These animals were anaesthetized by chloroform and killed by decapitation. The brains were immediately removed and placed in ice-cold medium containing 90 mM NaCl, 3 mM KCl, 2 mM MgSO4 , 2 mM CaCl 2 , 1 mM sodium pyruvate, 10 mM glucose, 105 mM sucrose and 10 mM 4-Ž2-hydroxyethyl.-1-piperazine-2-ethanesulfonic acid ŽHEPES-NaOH; pH s 7.4.. Transverse sections Ž400 mm. were prepared with a vibratome ŽLeica, Germany. according to standard methods. The slices were then transferred to a holding chamber where they were stored at 258C in a standard carbogen-gassed Ringer solution, containing 10 mM glucose, until further use. Before starting the experiments, the slices were preequilibrated for at least 60 min in the holding chamber. When indicated, the slices were loaded for 2 h at 258C with 10 mM BAPTA AM. 2.3. Respiration The oxygen consumption of single slices was determined at 308C in a medium consisting of 125 mM NaCl, 3 mM KCl, 2 mM CaCl 2 , 1.25 mM sodium phosphate, 2 mM MgCl 2 and 20 mM HEPES-NaOH ŽpH s 7.4. with a

The hippocampal or cortical slices were removed with 1.5 ml medium from the oxygraph chamber and homogenized with an ultra-turrax homogenizer T25 ŽIKA, Staufen, Germany.. The protein content was determined using a protein assay kit based on Peterson’s modification of the micro-Lowry method according to the instructions of the manufacturer ŽSigma.. 2.5. Fluorescence spectroscopy The NADŽP.H fluorescence changes in single hippocampal slices were determined with a laser fluorometer w22x. The slices were immobilized by attachment to glass wool and perfused at 3 mlrmin with an identical medium as that used for respiration measurements. The slices were deprived from the glucose containing storage solution ŽRinger with 10 mM glucose. by 5 min preperfusion with a glucose-free medium. The NADŽP.H fluorescence was excited with the 8 mW beam of an OMI-2056 HeCd laser ŽOmnichrome, Chino, CA. at an excitation wavelength of 325 nm. The fluorescence emission light was conducted with light guides to a Shimadzu RF-5001PC spectrofluorometer and registered at 450 nm. The fluorescence signals were calibrated using the uncoupler-oxidized Ždetermined in separate experiments. and the cyanide-reduced signals as 0% and 100% NADŽP.H reduction reference states, respectively. 2.6. Data analysis Statistical significance was shown with a Student’s two-tailed t-test with the significance level set to 0.05.

3. Results We applied high-resolution respirometry w14x to investigate differences in mitochondrial oxidative phosphorylation in single 400-mm hippocampal slices of kainate-treated and control rats. In Fig. 1, representative oxygen consumption measurements of single hippocampal slices from control rats ŽA. and kainate-treated rats ŽB. are shown. The indicated additions were made sequentially to the oxygraph chamber. Addition of 10 mM glucose caused a considerable stimulation of oxygen consumption in slices from kainate-treated animals ŽB. while it was almost with-

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Fig. 1. Oxygen consumption of hippocampal slices from control ŽA. and kainate-treated rats ŽB.. Single 400-mm hippocampal slices ŽA—455 mg, B—501 mg protein. were incubated at 308C in the chamber of the high resolution respirometer in 1.5 ml of the medium for oxygraphic determinations. Conditions of measurement as described in Section 2. Additions: Glucose ŽGLU. —10 mM, KCl—10 mM, ouabain—100 mM, TTFB—1.3 mM Žtwo times., pyruvate ŽPYR. —10 mM. The given rates Žcalculated from the first derivative of the oxygen trace. are expressed in nmol O 2 rmin.

out effect in control slices ŽA.. The depolarization of the slices with 10 mM potassium chloride led to an increase in respiration due to the depolarization-induced stimulation of

sodium–potassium ATPase in both experimental groups w11,16x. Since the basal respiration was lower in control slices, the stimulation induced by addition of potassium was much more pronounced in the control group. The addition of the sodium–potassium ATPase inhibitor ouabain caused a considerable inhibition only in slices from control animals while the respiration of slices from kainate-treated rats remained high. The addition of the uncoupler of oxidative phosphorylation TTFB stimulated the glucose-supported respiration of slices to the maximal level in both groups. A further increase in respiration could be obtained by addition of pyruvate, which improves the supply of reducing equivalents to the respiratory chain. The quantitative data from a series of respiration experiments with hippocampal slices are summarized in Table 1. It is remarkable that not only the glucose oxidation rate of control slices is considerably lower Žalmost 2-fold. but also the maximal Žuncoupled. rate of glucose- or pyruvate-supported respiration Žabout 1.3-fold.. These results point to a possible intracellular energy wasting process in hippocampal slices from kainate-treated rats. To localize the compartment in which the putative intracellular energy wasting process occurs, we added the inhibitor of the mitochondrial adenine nucleotide translocase, atractyloside, subsequent to the addition of ouabain. In the control slices, 200 mM atractyloside caused a minor suppression of glucose respiration. Very surprisingly, in the kainate slices, atractyloside even stimulates the glucose-supported respiration Žcf. also original trace in Fig. 3, trace 1.. This finding excludes an extramitochondrial energy utilizing process and makes a mitochondrial one very likely. This conclusion is supported by a separate series of experiments in which addition of 10 mM tetrodotoxin to the medium in order to block spontaneous activity failed to influence glucose-stimulated oxygen consumption Ž n s 3; data not shown.. To evaluate if the effects of kainate treatment on mitochondrial oxidative phosphorylation are only restricted to the hippocampus, we additionally determined the oxygen consumption of slices from the adjacent entorhinal cortex. The quantitative data from a serious of experiments are summarized in Table 2. Again, a clear-cut 1.4-fold higher oxygen consumption of slices was observed in kainate-

Table 1 Respiration rates of hippocampal slices from control and kainate-treated rats Addition

Control slices Ž N s 16., Vresp Žnmol O 2rminrmg.

Kainate slices Ž N s 23., Vresp Žnmol O 2 rminrmg.

Significance

10 mM glucose 10 mM KCl 100 mM ouabain 200 mM atractyloside 2.6 mM TTFB 10 mM pyruvate

5.27 " 1.91 9.49 " 2.30 7.84 " 1.70 7.33 " 1.06 a 11.75 " 1.93 13.88 " 2.89

9.57 " 3.43 11.65 " 4.38 9.74 " 4.12 11.17 " 4.58 a 14.77 " 4.93 16.72 " 5.12

p - 0.001 p - 0.05 p - 0.05 p - 0.05 p - 0.01 p - 0.05

Conditions of the experiments as described in Fig. 1. a Atractyloside was not added in all experiments, number of control experiments N s 11 and number of experiments with slices from kainate-treated rats N s 15.


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Table 2 Respiration rates of cortical slices from control and kainate-treated rats Addition

Control slices Ž N s 13., Vresp Žnmol O 2rminrmg.

Kainate slices Ž N s 19., Vresp Žnmol O 2 rminrmg.

Significance

10 mM glucose 10 mM KCl 100 mM ouabain 200 mM atractyloside 2.6 mM TTFB 10 mM pyruvate

8.12 " 2.54 10.04 " 1.69 8.35 " 1.44 8.36 " 1.37 a 11.73 " 2.06 13.68 " 2.15

13.68 " 4.24 14.57 " 3.62 12.03 " 5.23 12.26 " 5.01a 16.93 " 5.31 18.85 " 4.33

p - 0.01 p - 0.01 NS NS p - 0.05 p - 0.05

Conditions of the experiments as described in Fig. 1. a Atractyloside was not added in all experiments, number of control experiments N s 9 and number of experiments with slices from kainate-treated rats N s 11.

treated rats under all conditions investigated. This can be interpreted to be caused by an increase in the content of mitochondria. On the other hand, the observed differences in the basal oxygen consumption and the atractyloside-inhibited oxygen consumption in hippocampal slices are much less pronounced in the cortical slices. In order to verify these findings with an independent method, we determined the NADŽP.H autofluorescence changes in single hippocampal slices by laser-excited fluorescence spectroscopy. In hippocampal slices, this fluorescence signal seems to originate mainly from mitochondrial NADH w27x. This method allows to quantify the redox state of the mitochondrial NAD-system w22,23x which is a resultant of input of reducing equivalents by dehydrogenases and of efflux of reducing equivalents via the respiratory chain. Typical fluorescence traces of glass wool-immobilized hippocampal slices are shown in Fig. 2. The addition of glucose to glucose-deprived control slices caused an increase of the NADŽP.H fluorescence to about 16% " 6% Ž n s 7.. Interestingly, in the slices from the kainate-treated animals glucose addition was almost without effect ŽNADŽP.H reduction 5 " 7%, n s 3.. Potassium chloride led in control slices to a slight decrease of NADŽP.H fluorescence. This is obviously due to the depolarizationinduced stimulation of mitochondrial oxidative phosphorylation. In the slices from kainate-treated rats we could not observe any effect of potassium chloride ŽFig. 2B.. In the control slices, the effect of the potassium chloride addition could be reversed by ouabain, leading to a NADŽP.H reduction of 30 " 6% Ž n s 7., while in the kainate slices the ouabain addition had only a small effect on the NADŽP.H fluorescence ŽNADŽP.H reduction—6 " 5%, n s 3.. The maximal reduction of the mitochondrial NADsystem can be adjusted by the addition of the inhibitor of cytochrome c oxidase cyanide Ž100% reduction reference state.. The observation that glucose addition did not cause any measurable NADŽP. reduction in the kainate slices further supports the idea of an increased basal energy turnover in hippocampal slices from kainate-treated animals since the higher oxidation velocity of the respiratory chain in the epileptic slices would cause an elevated efflux

of reducing equivalents from the NAD-system. This would render the mitochondrial NAD-system mostly oxidized under all experimental conditions.

Fig. 2. NADH fluorescence changes of immobilized hippocampal slices from control ŽA. and kainate-treated rats ŽB.. Single 400-mm hippocampal slices were immobilized by attachment to glass wool and perfused at 3 mlrmin with the medium for oxygraphic determinations. Conditions of measurements as described in Section 2. Additions: Glucose ŽGLU. —10 mM, KCl—10 mM, ouabain—100 mM, KCN—4 mM. The signals were calibrated using the uncoupled state Ž2.6 mM TTFB present, determined in separate experiments. as the 0% and the KCN-inhibited state as the 100% NADŽP.H reduction reference states, respectively.


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leak of the mitochondrial inner membrane, an intramitochondrial energy consuming process or ion cycling at the mitochondrial inner membrane. Numerous previous studies on epilepsy models and human epilepsy suggest substantial changes in factors influencing intracellular Ca2q homeostasis w3,9,10,33x. Therefore, we have tested if mitochondrial futile Ca2q cycling might occur in kainate-treated rats. To this end, we preincubated the epileptic slices with either 10 mM BAPTA AM or 0.5 mM ruthenium red. BAPTA AM is a cell membrane permeable calcium chelator w5x and ruthenium red is a specific inhibitor of the mitochondrial Ca2q uniport w12,13x. The latter, however, also blocks Ca2q channels at the plasma membrane and the endoplasmic reticulum w29x. To overcome the known membrane permeability problems of this polycationic dye w24,28x, we equilibrated the slices for 10 min at a concentration of 0.5 mM ruthenium red. This procedure seemed not to cause secondary effects on mitochondrial oxidative phosphorylation since it did not affect the potassium chloride-stimulated oxidation rates Ž14.2 " 2.9 vs. 13.3 " 4.5 nmol O 2rminrmg protein. or the uncoupled oxidation rates Ž17.9 " 1.6 vs. 16.6 " 5.1 nmol O 2rminrmg protein. of the hippocampal slices from the kainate-treated rats. As shown with the original oxygen traces in Fig. 3, the preincubation with either ruthenium red Žtrace 2. or BAPTA AM Žtrace 3. almost restored the ‘control’ behavior of oxidative phosphorylation in hippocampal slices from kainate-treated animals: it increased the atractyloside sensitivity and restored the uncoupler stimulation of glucose oxidation Žfrom about 1.2-fold Žtrace 1. to about 1.7 fold.. Since both BAPTA AM and ruthenium red may act via unspecific reduction of internal Ca2q we tested in addition the effects of 10 mM tetraphenylphosphonium bromide known to block rather selectively the mitochondrial sodiumrcalcium antiport w12,13,28x. As shown in Fig. 3, trace 4, atractyloside did not cause a stimulation of slice respiration and TTFB substantially stimulated glucose oxidation rate. The quantitative data from a series of these experiments are summarized in Table 3. Taken together, these results indicate that the increased energy turnover in hippocampal slices from kainate-treated rats is most probably related to Ca2q cycling activity of mitochondria.

Fig. 3. Effects of atractyloside and uncoupler on oxygen consumption of glucose oxidizing hippocampal slices from kainate-treated rats Ž1. and from kainate-treated rats in the presence of 0.5 mM ruthenium red Ž2., preloaded with 10 mM BAPTA AM Ž3. or in the presence of 10 mM TPPq Ž4.. Single 400-mm hippocampal slices Ž1—501 mg protein, 2—520 mg protein, 3—491 mg protein and 4—537 mg protein. were incubated at 308C in the chamber of the high resolution respirometer in 1.5 ml of the medium for oxygraphic determinations containing additionally 10 mM glucose. Additions: Atractyloside—200 mM, TTFB—2.6 mM, pyruvate ŽPYR. —10 mM. In Expt. 3, the slices were loaded 2 h with 10 mM BAPTA AM in the storage chamber.

In further experiments we tried to elucidate the cause for the increased basal energy turnover in hippocampus slices from kainate-treated animals. Since our experiments with ouabain and atractyloside suggest a mitochondrial process, the most interesting candidate mechanisms for the increased oxidative metabolism are an increased proton

Table 3 The effects of ruthenium red, BAPTA AM and tetraphenylphosphonium iodide on the oxygen consumption of hippocampal slices from kainate-treated rats Controls Ž N s 11. Glucose KCl Ouabain Atractyloside TTFB

38 " 11 69 " 13 57 " 12 55 " 11 84 " 14

Kainate Ž N s 15.

Kainateq RR Ž N s 8. U

60 " 13 p - 0.001 71 " 14 NSU 62 " 17 NSU 73 " 9 p - 0.001U 91 " 12 NSU

UU

49 " 15 NS 76 " 11 NSUU 65 " 12 NSUU 55 " 8 p - 0.001UU 99 " 10 NSUU

Kainateq BAPTA AM Ž N s 5. UU

42 " 14 p - 0.05 62 " 11 NSUU 52 " 7 NSUU 56 " 8 p - 0.001UU 79 " 9 NSUU

Kainateq TPPq Ž N s 6. 44 " 9 p - 0.05UU 61 " 8 NSUU 47 " 10 NSUU 46 " 6 p - 0.001UU 80 " 9 NSUU

Conditions of the experiment as described in Fig. 1. The relative rates are expressed in percentage of the maximal uncoupled respiration rate with 10 mM pyruvate as substrate. Additions: Glucose—10 mM, KCl—10 mM, ouabain—100 mM, atractyloside—200 mM, TTFB—2.6 mM. U Significance tested between the kainate and control group. UU Significance tested between the kainate group in absence or presence of either 0.5 mM ruthenium red, 10 mM TPPq or 2 h preincubation with 10 mM BAPTA AM.


4. Discussion Administration of kainic acid, an acidic pyrrolidine isolated from the seaweed Digenea simplex, causes chronic epileptic seizures in rats after a latency period of 7 to 30 days w6x. The histopathological changes in the rat hippocampus closely resemble the features of human temporal lobe epilepsy— Ži. segmental neuronal cell loss in the CA3 subfield of the Ammon’s horn with a relative sparing of dentate granule cells, Žii. recurrent sprouting of mossy fibers into the inner molecular layer of the dentate gyrus, and Žiii. severe reactive astrogliosis. In this model of epilepsy, we applied high-resolution respirometry w14x to elucidate possible changes in mitochondrial oxidative phosphorylation in single 400-mm hippocampal and cortical slices. It is well-established that the measurement of oxygen consumption in brain slices allows to monitor changes in mitochondrial energy metabolism, caused by plasma membrane depolarization w11,16x or the action of excitatory amino acids w25,26x. The application of high resolution respirometry w14x enabled us to perform this study on single 400-mm thick rat hippocampal microslices. Our results demonstrate severe alterations of mitochondrial oxidative phosphorylation in the hippocampus of kainate-treated rats. These alterations include Ži. an approximately 2-fold increased basal energy turnover with glucose as substrate and Žii. an about 1.3-fold higher uncoupled rate of respiration. The increased basal energy turnover was proven to be not related to the spontaneous activity of the epileptic slices since it could not be diminished by the sodium channel blocker tetrodotoxin and the inhibitor of the sodium–potassium ATPase ouabain. In order to determine if this increased basal energy turnover is caused by an intracellular ATP-consuming process, we applied the inhibitor of the mitochondrial adenine nucleotide translocase atractyloside w32x. Again we observed no inhibition, but even a small stimulation. Taken together, these results implicate an intrinsic mitochondrial energy utilizing process or a changed effectiveness of the energy converting machinery. An increased proton leak of the mitochondrial inner membrane, an intramitochondrial ATP-splitting process or a futile ion cycling are possible candidates. A multitude of experimental results indicate changes in intracellular Ca2q buffering w2x and sequestration systems w3,9,20,33x in chronic epilepsy. In addition, numerous changes in the expression of Ca2q conducting ion channels such as voltage-dependent Ca2q channels or ionotropic glutamate receptors have been described w3,18,20x. Thus, it seemed likely to us that changes in mitochondrial Ca2q handling either secondary to or contributing to impaired Ca2q homeostasis might occur in epilepsy. We have applied BAPTA AM, ruthenium red and tetraphenylphosphonium bromide in oxygraphic experiments to clarify this question. BAPTA AM is a cell membrane permanent Ca2q chelator w5x. Ruthenium red is well-known to inhibit the

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mitochondrial Ca2q import w12,13x, and is known to protect neurons from impairment of Ca2q homeostasis w7,8x. To exclude possible additional effects of ruthenium red on the ryanodine receptor-mediated Ca2q efflux from endoplasmic reticulum w24x and plasma membrane calcium influx through voltage-sensitive Ca2q channels w13,31x related to its poor membrane permeability w24x, we have applied additionally tetraphenylphosphonium bromide. This lipophilic cation is membrane-permeable and known to inhibit selectively the mitochondrial calciumrsodium antiporter w12,13,28x. In our hands, the preincubation of epileptic hippocampal slices with either 10 mM BAPTA AM, 0.5 mM ruthenium red or 10 mM TPPq caused a depression of the glucose oxidation rate and restored the atractyloside sensitivity of glucose oxidation. These results support the idea that the increased basal glucose oxidation rate in hippocampal slices from kainate-treated rats is most possibly due to Ca2q cycling at the mitochondrial inner membrane. This futile, energy-consuming mitochondrial Ca2q cycling occurs at elevated intracellular Ca2q concentrations as result of the combined action of the membrane potential driven Ca2q uniport and the NaqrCa2q antiport w21x. In addition to this intramitochondrial energy wasting process which seems to be present rather selectively in hippocampal slices, the approximately 1.3 to 1.4-fold higher uncoupled oxidation rates with both glucose or pyruvate suggest either an increased number of mitochondria or altered activities of mitochondrial respiratory chain in both hippocampal and cortical slices of kainate-treated rats. Interestingly, this phenomenon occurs in spite of the neuronal cell loss present in the hippocampal specimens. It is possible to speculate that the increased number of synapses due to aberrant sprouting and neuronal reorganization may contain additional amounts of possibly synaptosomal mitochondria. In summary, our results indicate pronounced alterations in the activity and the intrinsic properties of mitochondria in an experimental model of temporal lobe epilepsy. The observed specific alterations of mitochondrial function seem to be related to futile Ca2q cycling, resulting in markedly increased endogenous rate of mitochondrial oxidative phosphorylation. These changes may have profound effects on neuronal excitability and the survival of neurons in chronic epilepsy.

Acknowledgements The excellent technical assistance of Paul Rausch and Daniel Langendorfer is gratefully acknowledged. WSK is ¨ supported by the BONFOR program of the University of Bonn Medical Center. IVG is supported by the graduate program of the University of Bonn Medical Center. HB is supported by the SFB 400 and DFG.

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