RAPID COMMUNICATION
A Relative Energy Failure Is Associated With Low-Mg 2/ But Not
With 4-Aminopyridine Induced Seizure-Like Events in Entorhinal
Cortex
S. SCHUCHMANN, 1 K. BUCHHEIM, 2 H. MEIERKORD, 2 AND U. HEINEMANN 1
1
Institut für Neurophysiologie and 2Neurologische Klinik und Poliklinik, Universitätsklinikum Charité, HumboldtUniversität Berlin, D-10117 Berlin, Germany
INTRODUCTION
In the early phases of status epilepticus, this potentially
deleterious condition is treatable in the majority of cases with
standard anticonvulsants. However, pharmacological control
becomes increasingly difficult the longer the condition has
endured (Shorvon 1994). An energetic failure of the epileptic neurons has been suggested to be a major factor in the
transition from treatable to refractory status epilepticus
(Treimann et al. 1989).
Depolarization of neurons during epileptiform activity
promotes Ca 2/ influx through voltage-activated and
N-methyl-D-aspartate(NMDA)-gated ion channels, raising
the [Ca 2/ ]i (Uematsu et al. 1990; van den Pol et al. 1996).
Such elevations in [Ca 2/ ]i result in a depolarization of mitochondrial membranes and in subsequent increases in intramitochondrial Ca 2/ concentration (Duchen 1992). As a consequence, respiration is increased and the concentration of
reactive oxygen species elevated (Dugan et al. 1995). Furthermore, increased intramitochondrial Ca 2/ concentration
activates a set of enzymes of the citrate cycle including
pyruvate dehydrogenase, NAD / -isocitrate dehydrogenase,
and a-ketoglutarate dehydrogenase (Moreno-Sánchez and
Hansford 1988; Richter and Kass 1991), which results in
an increase in NAD(P)H/NAD(P) / ratio (Duchen et al.
1993). In their reduced states, pyridine nucleotides, i.e.,
NADH and NADPH, exhibit an autofluorescence at a maximum wavelength of 460 nm (Aubin 1979).
In entorhinal-cortex, seizure-like events (SLEs) can easily
be induced by Mg 2/ -free artifical cerebrospinal fluid
(ACSF) or 4-aminopyridine (4-AP) ACSF. However, the
two models differ strikingly in that SLEs of the first model
regularly evolve to late recurrent discharges that are resistant
to currently used anticonvulsants (Zhang et al. 1995),
whereas in the second model such late activity is seen exceedingly rarely (Lopantsev and Avoli 1998; Perreault and
Avoli 1991).
In the current study, we have tested the hypothesis that an
energetic failure is associated with epileptiform discharges in
the two different in vitro models. We have employed the
NAD(P)H autofluorescence signal to monitor alteration in
the NAD(P)H/NAD(P) / ratio to gain insight into the
mechanisms that lead to the transition to refractory discharges in the low-Mg 2/ model. Part of these results have
been communicated at the ENA meeting 1998 in Berlin in
abstract form (Schuchmann et al. 1998).
METHODS
Subjects
The experiments were performed as described previously
(Meierkord et al. 1997). Briefly, brain slices (400 mm) containing
the temporal cortex area 3, the perirhinal cortex, the entorhinal
cortex, the subiculum, the dentate gyrus, and the ventral hippocampus were prepared in a nearly horizontal plane from Wistar rats
(150–200 g) after decapitation under deep ether anesthesia. The
slices were stored at room temperature in oxygenated (95%
O2-5% CO2 ) ACSF, which contained (in mM) 124 NaCl, 3 KCl,
1 .8 MgSO2 , 1.6 CaCl2, 1 .25 NaH2PO4 , 26 NaHCO3, and 10
glucose (pH 7.4). Slices were individually transferred to a homemade recording chamber and submerged in rapidly flowing ACSF.
The production of spontaneous epileptiform activity under submerged conditions requires a flow rate of about 4.5 { 0.5 (SE)
ml/min and a temperature of Ç36 { 17C.
Experimental methods
The fluorescence measurements were carried out using a system
based on an upright microscope (Axioskop; Zeiss, Jena, Germany)
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Schuchmann, S., K. Buchheim, H. Meierkord, and U. Heinemann. A relative energy failure is associated with low-Mg 2/ but
not with 4-aminopyridine induced seizure-like events in entorhinal
cortex. J. Neurophysiol. 81: 399–403, 1999. During seizure-like
events (SLEs), intracellular Ca 2/ concentration ([Ca 2/ ]i ) increases causing depolarization of the mitochondrial membrane and
subsequent intramitochondrial accumulation of Ca 2/ . Mitochondrial depolarization results in an interruption of oxidative phosphorylation and increase in reactive oxygen species. Calcium activates enzymes of the citrate cycle. A characteristic feature of the
low-Mg 2/ –induced SLEs is that they are transformed to a late
activity refractory to anticonvulsant drugs, which may be regarded
as a model system of difficult to treat status epilepticus. In contrast,
4-aminopyridine (4-AP) –induced activity rarely evolves to such
late activity. The autofluorescence of NAD(P)H was used to monitor changes in cellular energy metabolism in the entorhinal cortex
in two in vitro models of focal epilepsy. During repetitive 4-AP–
induced SLEs there was a short decrease followed by a longlasting overshoot of the NAD(P)H signal. This sequence remained
unaltered during recurring SLEs. In contrast, during recurrent lowMg 2/ –induced SLEs, the brief initial NADH signal reduction was
unchanged but the following overshoot of NADH displayed a continuous decrease. This indicates a relative energy failure, which
may contribute to the transformation to late activity in the lowMg 2/ model.
400
SCHUCHMANN, BUCHHEIM, MEIERKORD, AND HEINEMANN
RESULTS
In 19 of 19 slices spontaneous series of SLEs could be
recorded in the medial entorhinal cortex. The frequency of
low-Mg 2/ –induced SLEs was 0.47 { 0.14 min 01 ; their
duration was 26 { 1.3 s (n Å 10). During SLEs, extracellular potassium concentration [K / ]0 increased to 6.9 { 0.1
mM (Fig. 1A). Late, recurrent discharges (not shown) occurred 2 h (110–130 min) after the first SLE. After exposure
to 250 mM 4-AP, SLEs were observed at a frequency of
0.32 { 0.1 min 01 ; the duration was 19 { 1.5 s, and during
SLEs [K / ]0 reached 7.3 { 0.1 mM (n Å 9; Fig. 1B). Late,
recurrent discharges were not observed in 9 out of 9 slices
after a maximal exposure time of ú3 h.
The NAD(P)H autofluorescence signal was recorded
from an area of 0.8–1.5 mm2 in the medial entorhinal cortex.
In the absence of epileptiform activity, the NAD(P)H autofluorescence signal was stable in both low-Mg 2/ – and
4-AP–containing perfusion solution. During SLEs the
NAD(P)H autofluorescence displayed a characteristic time
course in both models: a short initial decrease was followed
by a long-lasting increase (see Fig. 1). The signal decrease
occurred simultaneously with the electrical onset of SLE.
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The duration of this NAD(P)H decrease during SLEs occurred during low-Mg 2/ –induced SLEs 2.77 { 0.67 s and
during 4-AP–induced SLEs 3.18 { 0.51 s, and showed no
significant difference between the two models (P ú 0.2;
Fig. 2B) and the number of SLEs. The subsequent elevation
in NAD(P)H autofluorescence developed during SLE and
lasted considerably longer than the SLE. Depending on the
time after onset of the first SLE, the amount of NAD(P)H
increase during SLEs showed significant differences in
low-Mg 2/ - and the 4-AP–containing ACSF. In Mg 2/ -free
medium the SLEs induced overshoot in NAD(P)H
autofluorescence showed a reduction with time. Even after
40 repetitive 4-AP–induced SLEs the changes in NAD(P)H
autofluorescence remained unaltered (see Fig. 2A). Moreover, the amount of NAD(P)H increase during 4-AP–induced SLE when compared with the early SLEs of the lowMg 2/ model was significantly smaller. In the low-Mg 2/
model, elevation in NAD(P)H autofluorescence declined
during the first 50 SLEs from 4.58 { 0.48 to 0.79 { 0.3.
The increase in NAD(P)H signal during SLE diminished
significantly after the first 4 SLE in the low-Mg 2/ model
(P õ 0.05). Changes in NAD(P)H autofluorescence signal
during the first 50 4-AP–induced SLE amounted to 2.1 {
0.08 (range 1.94–2.22). Neither the SLE-induced rises in
[K / ]0 nor the field potentials showed significant alterations
in the low-Mg 2/ model or in the 4-AP model.
DISCUSSION
As seen in previous experiments (Meierkord et al. 1997),
SLEs changed their appearance under submerged conditions
in that there was a reduction in amplitude and in duration
and an increase in frequency compared with interface conditions. In addition, under submerged conditions the transformation to late recurrent discharges was delayed (2 h after
the first SLE), which appeared under interface conditions
after 1–2 h in Mg 2/ -free ACSF (Zhang et al. 1995), respectively 50 min after the first seizure (Pfeiffer et al. 1996). It
has to be emphasized that the current investigation only
covers the energetic changes during recurrent SLEs.
The results of the current study demonstrate significant
differences in the cellular energy metabolism associated with
SLEs of the low-Mg 2/ and the 4-AP model of epileptiform
activity. The principal feature of initial brief reduction and
long-lasting overshoot of the NAD(P)H signal during a
single SLE was comparable in the two models. This is in
line with previous studies in which, calculated from metabolites in frozen sections, a decrease was seen in the cytoplasmic-free NAD / /NADH ratio, during electroconvulsive
seizures (Merrill and Guynn 1976), fluorothyl induced seizures (Folbergová et al. 1985), and bicuculline induced status epilepticus (Fujikawa et al. 1988) . The pyridine nucleotide NAD / is predominantly oxidized under normal aerobic
conditions in most tissues (Thurmann and Lemasters 1988).
Therefore effects, even a slight rise in NADH, caused a clear
decrease in NAD / /NADH ratio (Merrill and Guynn 1982).
In contrast to our study, these investigations only covered
the time course after one event, a method that does not allow
for the study of dynamic changes following series of SLEs.
Furthermore, the temporal resolution in such biochemical
approaches fails to monitor early changes at the onset of an
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with a 110 water-immersion objective (numerical aperture 0.3;
Zeiss, Jena, Germany), a xenon light source with a monochromator
(Photon Technology Instruments, Wedel, Germany), and a photomultiplier (Seefelder Messtechnik, Seefeld, Germany). Autofluorescence of NAD(P)H was excited at 360 nm and measured
above 400 nm using a 390 nm dichroic mirror and a 400 nmlong pass filter. The autofluorescence signal measured under these
conditions is derived from both mitochondrial and cytosolic NADH
and NADPH, whose autofluorescence spectra overlap. We therefore refer to NAD(P)H, indicating the signal derived from either
NADH or NADPH or both. Under these conditions, an increase in
autofluorescence signal indicates an increase in the reduced state
of the pyridine nucleotide, i.e., NAD(P)H, and a decrease in autofluorescence signal an increased oxidation to NAD(P) / .
Changes in NAD(P)H autofluorescence are given in reference to
the outset NAD(P)H signal as DF/F0 . Except for NAD(P)H,
autofluorescence in mammalian cells is mainly influenced by the
flavoprotein fluorescence of a-ketoglutarate and pyruvate dehydrogenase (excitation/emission maximum Ç450/515 nm) and the absorption by cytochrome oxidase (415–550 nm; (Chance et al.
1979). However, the clearly different excitation maximum from
NAD(P)H versus flavoproteins and cytochrome oxidase enables
a sufficient separation of the NAD(P)H autofluorescence signal
with the use of a suitable optical filter combination (Benson et al.
1979).
Ion-selective microelectrodes were prepared using the method
described by Lux and Neher (1973). Double-barrelled glass was
filled on one side with 154 mM NaCl operating as the reference.
The silanized ion sensitive barrel (5% trimethyl-1-chlorosilane in
95% CCl4 ) was filled with a potassium ionophore I cocktail
A60031 Fluka. A modified Nernst equation was employed to obtain
molar K / concentrations from recorded potential values (mV).
The viability of the slices was also tested and monitored by observing the extracellular field potential in the medial entorhinal cortex
layer III/IV following stimulation of the lateral entorhinal cortex.
Epileptiform activity was induced by omitting Mg 2/ from or adding 250 mM 4-AP (Sigma) to the perfusion solution. Data were
stored on chart recorder or computer hard disc and subsequently
analyzed off-line. All values are given as means { SE. Statistical
differences were assessed by analysis of variance (ANOVA) and
Bonferroni-Dunn contrast.
ENERGY FAILURE IN LOW MG 2/ BUT NOT 4-AP INDUCED SLEs
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2/
FIG . 1. A: low-Mg
–induced epileptiform activity. Left: time course of changes in NAD(P)H autofluorescence (first
line), extracellular K / concentration (second line), and field potentials (f.p., third line) during 4 recurrent SLEs. Right:
expanded time scale for the 1st and 40th SLE. Note the decline of NAD(P)H autofluorescence overshoot, which appears
already within the first 4 SLEs. B: 4-AP–induced epileptiform activity. Left: similar presentation of NAD(P)H autofluorescence (first line), extracellular K / concentration (second line), and field potentials (f.p., third line) during 4 recurrent SLEs.
Right: expanded time scale demonstrates that there is no change of the NAD(P)H autofluorescence overshoot even after
40 SLEs.
SLE; this may explain why the initial brief decrease of the
signal has not been demonstrated before.
The initial reduction in NAD(P)H signal represents the
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Ca 2/ induced depolarization of the mitochondrial membrane
and therefore increased activity of mitochondrial respiratory
chain (Duchen 1992). The subsequent elevation in NAD(P)H
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SCHUCHMANN, BUCHHEIM, MEIERKORD, AND HEINEMANN
signal indicates an activation of Ca 2/ -dependent enzymes of
the citrate cycle (Duchen et al. 1993). Accordingly, more electrons reach the mitochondrial respiratory chain via transport of
NADH and were used here to drive mitochondrial proton
pumps. Therefore the potential difference across the inner mitochondrial membrane increases and induces an augmentation in
mitochondrial ATP production. The enlarged neuronal energy
requirement during epileptic activity results in an elevated consumption of ATP (Fujikawa et al. 1988). To cover the increased use of ATP, an elevated energy production is required.
Monitoring the NAD(P)H autofluorescence signal thus gives
insight into the changes of intracellular energy metabolism
during SLEs. Changes in energy metabolism induced by SLEs
with the use of 4-AP and indicated by NAD(P)H autofluorescence signal were stable throughout the experiments ( ¢40
SLE). This implies that neurons could compensate for the
increased energy requirement during 4-AP-induced epileptiform activity. They may therefore only rarely display late recurrent discharges.
In contrast, a significant higher but gradual decline of the
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overshoot of the NAD(P)H autofluorescence signal was already observed during the first 10–15 low-Mg 2/ –induced
SLEs. After 35–40 recurrent SLEs, the overshoot had almost
vanished. Our findings correspond well with hypotheses that
attribute the transition into late activity in the low-Mg 2/ model
to an energetic failure. A gradual decrease afterhyperpolarization following each SLE in the low-Mg 2/ model was demonstrated with intracellular recordings (Schmitz et al. 1997). It
is suggested that this decreased hyperpolarizations could result
from a rundown of the electrogenic Na / -K / ATPase. The
pump-induced hyperpolarizations would inactivate voltage dependent Na / and Ca 2/ inward currents and NMDA gated channels (Fukuda and Prince 1992a,b). The gradual decrease in
the overshoot of the NAD(P)H autofluorescence after each
SLE in the low-Mg 2/ model could result in decreased activity
of the electrogenic Na / -K / -ATPase following gradual reductions of intracellular ATP concentrations.
Furthermore, an observed decrease in GABAA receptormediated inhibition in the low-Mg 2/ model may result from
a loss of ATP and a consequent partial dephosphorylation
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FIG . 2. A: effects of low-Mg
–induced (filled symbols) and 4-AP–induced (open symbols) epileptiform activity on
the NAD(P)H autofluorescence overshoot during 40 SLE. NAD(P)H autofluorescence signal was exciting at 360 nm and
measured above 400 nm. Maximum of NAD(P)H autofluorescence overshoot is given in DF/F0 as function of number of
SLE per slice. Whereas the NAD(P)H autofluorescence of 4-AP–induced SLEs was stable over the time, there was a
significant difference to the NAD(P)H overshoot associated with low-Mg 2/ –induced SLEs (low-Mg 2/ vs. 4-AP: * P õ
0.05, *** P õ 0.001). Decrease of the NAD(P)H signal overshoot was significant after the 4th, compared with the 1st,
low-Mg 2/ –induced SLE (low-Mg 2/ : 1. SLE vs. 4. SLE # P õ 0.05; 1. SLE vs. 10. SLE ## P õ 0.001). B: initial decrease
in NAD(P)H autofluorescence signal induced by SLEs showed no significant difference in the 2 models over time. Symbols
as in Fig. 2A.
ENERGY FAILURE IN LOW MG 2/ BUT NOT 4-AP INDUCED SLEs
of GABAA receptor (Whittington et al. 1995). The increased
consumption of MgATP in the absence of intracellular Mg 2/
may result in a decrease in MgATP level and furthermore,
in a loss in compensation of increased energy requirement.
This group showed that the erosion of GABAergic inhibition
could be reversed by the application of MgATP, but not by
an increased [Mg 2/ ]i only (Whittington et al. 1995).
Our data underline that the relative energy failure may
contribute to the drug resistance of the late recurrent discharges in the low-Mg 2/ model and after long periods of
status epilepticus in humans.
Received 16 June 1998; accepted in final form 25 September 1998.
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We thank D. Schmitz for critical discussion of the study and reading of
the manuscript. We thank U. Dirnagl and A. Strong for helpful comments.
This work was supported by the Deutsche Forschungsgemeinschaft Klinische Forschergruppe Neue Methoden zur nichtinvasiven Funktions-Diagnostik des ZNS to H. Meierkord and U. Heinemann.
Address for reprints requests: S. Schuchmann, Institut für Neurophysiologie, Universitätsklinikum Charité, Humboldt-Universität Berlin, Tucholskystr. 2, D-10117 Berlin, Germany.
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