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Fast activity at seizure onset is mediated by inhibitory circuits in the entorhinal cortex in vitro.

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Fast Activity at Seizure Onset Is Mediated
by Inhibitory Circuits in the Entorhinal
Cortex In Vitro
Vadym Gnatkovsky, MD, PhD, Laura Librizzi, PhD, Federica Trombin, PhD, and Marco de Curtis, MD
Objective: Network mechanisms responsible for focal seizure initiation are still largely unknown. One of the prevalent seizure
patterns observed during diagnostic intracranial recordings performed in patients with mesial temporal lobe epilepsy is characterized by fast activity at 20 to 30Hz. We reproduced 20 to 30Hz oscillations at seizure onset in the temporal lobe of the in vitro
isolated guinea pig brain to study cellular and network mechanisms involved in its generation.
Methods: Seizure-like activity was induced in the isolated brain by 3-minute arterial perfusion of 50␮M bicuculline. Intracellular, extracellular, and ion-selective electrophysiological recordings were performed simultaneously in the entorhinal cortex (EC)
during interictal-ictal transition.
Results: Principal neurons in deep and superficial layers of the EC did not generate action potentials during fast activity at ictal
onset, whereas sustained firing was observed in putative interneurons. Within 5 to 10 seconds from seizure initiation, principal
neurons generated a prominent firing that correlated with the appearance of extracellular hypersynchronous bursting discharges.
In superficial neurons, fast activity correlated with rhythmic IPSPs that progressively decreased in amplitude during the development of a slow depolarization associated with an increase in extracellular potassium.
Interpretation: We conclude that in an acute model of temporal lobe ictogenesis, sustained inhibition without firing of EC
principal neurons correlates with the onset of a focal seizure. The progression of the ictal discharge is contributed by a potassiumdependent change in reversal potential of inhibitory postsynaptic potentials. These findings demonstrate a prominent role of
inhibitory networks during the transition to seizure in the EC.
Ann Neurol 2008;64:674 – 686
The cure of epilepsies depends on how effectively available treatments control seizure onset and propagation.
In 30% of focal epilepsies, seizures are resistant to antiepileptic drugs. New strategies to treat pharmacoresistant epilepsies will benefit from the identification of
the mechanisms involved in the transition from the interictal to the ictal state, which, despite many studies,
remain elusive. The most common surface electroencephalographic correlate of seizure onset in human focal epilepsies originating from the temporal lobe is the
occurrence of small-amplitude fast activity (electroencephalographic flattening) in the temporal region.1,2
Such activity may evolve into large-amplitude, rhythmic discharges that secondarily diffuse to adjacent cortical areas. Invasive presurgical studies with intracranial
depth electrodes have been utilized to circumscribe the
epileptogenic zone in patients suffering from mesial
temporal lobe epilepsy resistant to pharmacological
treatment.3,4 Such invasive diagnostic procedures dem-
onstrated that the ictal discharge associated with a temporal lobe seizure most often initiates with a sequence
of fast activity at 20 to 30Hz in the hippocampus and
in the parahippocampal region.1,5–12 Even though alternative ictal onset patterns have been described,8,13,14
it has been proposed that fast 20 to 30Hz activity has
the greatest localizing value for the identification of the
epileptogenic region.6,11,15
Network and cellular mechanisms associated with
the generation of fast, small-amplitude cortical activity
in the beta-gamma range that initiates a seizure in the
mesial temporal lobe are not yet clearly identified.
Seizure-like fast ictal discharges in mesial temporal lobe
structures can be experimentally reproduced in animal
models of seizures.16 Ictogenesis can be reliably induced in the temporal lobe of the in vitro isolated
guinea pig brain preparation17,18 by acute, partial, and
transient disinhibition with the GABAA receptor antagonist bicuculline.19,20 This procedure was utilized to
From the Unit of Experimental Epileptology and Neurophysiology,
Fondazione Istituto Neurologico Carlo Besta, Milan, Italy.
Potential conflict of interest: Nothing to report.
Received Nov 21, 2007, and in revised form Jul 3. Accepted for
publication Jul 8, 2008.
Published in Wiley InterScience (
DOI: 10.1002/ana.21519
Address correspondance to Dr de Curtis, Unit of Experimental
Epileptology and Neurophysiology, Fondazione Istituto Neurologico Carlo Besta, via Celoria 11, 20133 Milano, Italy.
© 2008 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
study network mechanisms that regulate the generation
of 20 to 30Hz activity (also designated as beta-gamma
activity) at seizure onset in the entorhinal cortex (EC)
of the in vitro isolated guinea pig brain. We focused on
the EC because this temporal lobe region is primarily
involved in seizure generation in humans,9,10 and in
experimental models of temporal lobe epilepsy and seizures.19,21–24
In mammals, the EC is reciprocally connected with
the hippocampus proper.25–27 The network interactions within the EC and between this region and the
hippocampus have been analyzed extensively.28 –32
Neurons in superficial EC layers II and III project to
the dentate gyrus and to the CA1 field, and deep EC
neurons receive a feedback projection from CA1/subiculum. A recently demonstrated direct feed-forward
projection from these areas to superficial EC interneurons33,34 may play a crucial role in controlling EC excitability by preventing reentrant activation of the EChippocampal-EC circuit.34 Studies on temporal lobe
ictogenesis and epileptogenesis identified enhanced network interactions and altered intrinsic neuronal properties in superficial and deep EC neurons.16,24,35,36
This study demonstrates for the first time that seizure
onset in the EC is associated with a complete interruption of neuronal firing in principal neurons and is supported by the activation of an inhibitory network.
Materials and Methods
Guinea pig brains were isolated in vitro according to the previously described procedure.17,18 After administration of barbiturate anesthesia (80mg/kg sodium thiopental intraperitoneally), intracardiac perfusion with cold (15°C) saline
solution (see later) was performed for 3 minutes to reduce
brain temperature during dissection. The entire brain was
isolated and transferred to a perfusion chamber. A poliethylene cannula was inserted in the basilar artery to restore brain
perfusion with a solution composed of 126mM NaCl, 3mM
KCl, 1.2mM KH2PO4, 1.3mM MgSO4, 2.4mM CaCl2,
26mM NaHCO3, 15mM glucose, and 3% dextran molecular weight 70,000, oxygenated with a 95% O2/5% CO2 gas
mixture (pH 7.3). Experiments were performed at 32°C.
The experimental protocol was reviewed and approved by
the Committee on Animal Care and Use and by the Ethics
Committee of the Istituto Nazionale Neurologico, in accordance with national and international guidelines on care and
use of laboratory animals.
Extracellular recordings were performed with glass pipettes
filled with 0.9M NaCl (2–5M⍀ resistance). Intracellular recordings were performed with sharp electrodes filled with
3M potassium acetate and 2% biocytine (60 –120M⍀ input
resistance). Electrophysiological signals were amplified via a
multichannel differential amplifier (Biomedical Engineering,
Thornwood, NY) and an intracellular amplifier (Neurodata,
New York, NY). Data were acquired and analyzed utilizing
software (ELPHO) that Dr Vadym Gnatkovsky developed in
our laboratory. Joint time-frequency analysis was applied to
show how the frequency content of the seizures evolves over
Fig 1. (A, left) Schematic drawing of the positions of the recording electrodes in PC and in medial and lateral EC, and
of the stimulating LOT electrode. (A, right) Pairing test in
the PC: (a) PC response to a single stimulus, characterized by
a monosynaptic (asterisk) and a disynaptic potential (solid
circle); (b) PC response to a paired test with a 25-millisecond
interstimulus interval; (b-a) subtracted traces enhance the inhibition of the disynaptic response. (B) Efficacy of inhibition
measurement in sampled traced recorded in PC (left) and lateral EC (right). (C) Time course of the average inhibition
efficacy values in the PC (left) and in the lateral EC (right)
during the arterial perfusion of bicuculline (gray shaded
area). Pairing tests were performed every 10 seconds. Seizure
onset is marked by the arrows. Time values were normalized
between experiments with reference to the time of seizure onset
recorded in the medial EC.
time (see Figs 2A, 2B, 3A). Cross-correlation coefficient was
calculated in sequences of 100-millisecond time windows
and represented as correlogram over time (see Fig 3B).
Ictal discharges in the EC were induced by brief (3minute) arterial perfusions with 50␮M of the GABAA receptor antagonist bicuculline methiodide19,20,37,38 (SigmaAldrich, St. Louis, MO). Pairing of the responses evoked by
stimulation of the lateral olfactory tract (LOT) in the piriform and entorhinal cortices was performed to evaluate the
reduction of inhibition efficacy during perfusion with bicuculline. As discussed elsewhere,39 recurrent GABAergic inhibition is responsible for the depression of the disynaptic po-
Gnatkovsky et al: Inhibition at Seizure Onset
In brief, ion-selective electrodes (tip diameter, 3–5␮m) were
filled with the potassium ionophore I cocktail A (Fluka
60031, Hamburg, Germany). Absolute [K⫹]o values were
calculated by solving the equation y ⫽ a ⫹ b logx, where x
is the [K⫹]o, y is the measured voltage reading induced by
the changes in [K⫹ ]o, and a ⫹ b is the slope coefficient
derived from the calibration curve performed for each K⫹sensitive electrode (calibration solutions with K⫹ concentrations of 1, 2.5, 6, 12.5, and 48mM). Only electrodes with a
response of 30 to 40mV for 10mM K⫹ were utilized. Ionselective signals were amplified with a high-input impedance
head-stage amplifier (Biomedical Engineering, Thornwood,
NY), and field potential values were subtracted.
Principal neurons in layers II to III (n ⫽ 24), layers V to
VI (n ⫽ 6), and putative interneurons located in the superficial 500␮m (n ⫽ 10) were identified from their response to
the hippocampal input driven by stimulation of the olfactory
area.41 Thirteen of 30 principal cells were further identified
morphologically as stellate or pyramidal cells. At the end of
the electrophysiological experiment, brains were fixed in 4%
paraformaldheide, and the standard horseradish peroxidase
protocol (ABC kit; Vector Laboratories, Burlingame, CA)
was utilized to show neurons injected with biocytine. Sections were counterstained with thionine to identify cortical
Fig 2. Extracellular recording of a typical ictal discharge in
the medial (EC). Ictal onset was characterized by fast activity
(in the expansion below) consistently followed by irregular
spiking (small stars) that progressively evolved into a regular
bursting pattern. (inset) Postictal depression is shown. Average
frequency content from 5-second recordings just before seizure
and after the termination of rhythmic bursting (n ⫽ 7 experiments) show a marked reduction of the activity in all frequency ranges after the seizure (black shading) in comparison
with preictal activity (gray shading). The fast activity at seizure onset is shown in the expanded trace. The relative spectrogram highlights the power of fast activity at 20-30 Hz
(and subharmonics). (B) Averaged spectrogram of seizure onset
(n ⫽ 7).
tential in the conditioned response evoked within 30
milliseconds after the conditioning stimulus. The ratio between the amplitude of the disynaptic components of the
potentials evoked by a conditioning and a conditioned LOT
stimuli separated by 25 milliseconds was utilized to monitor
changes in inhibition efficacy (see Results). Statistical T test
was used to evaluate changes of inhibition efficacy.
Recording of extracellular potassium concentration
([K⫹]o) in the EC was conducted as described previously.40
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Seizure-like Activity Correlates with Transient and
Partial Entorhinal Cortex Disinhibition
Seizure-like, ictal activity was reliably induced in the
medial EC of the in vitro isolated guinea pig brain by
a brief arterial perfusion with bicuculline methiodide
(50␮M). Three-minute perfusions of bicuculline induced transient and partial disinhibition of the isolated
brain, as demonstrated by paired-pulse test42 (Fig 1).
The depression of the conditioned responses evoked by
paired stimulation of the LOT were analyzed in the
piriform cortex (PC) and in the lateral EC, a region
that receives a direct olfactory input19 bordering the
seizure onset area in the medial EC (see scheme in Fig
1A). In control solutions, pairing with a 25-millisecond
interstimulus interval determined a 56.3 ⫾ 0.8 and
67.1 ⫾ 1.9% (mean ⫾ standard error of the mean;
n ⫽ 10) reduction of the conditioned disynaptic responses recorded in both PC and lateral EC (see Figs
1A, B, filled circles). This parameter of inhibition efficacy was measured as the difference between the amplitude of the disynaptic response to a single stimulus
(see Fig 1A, part a) and the amplitude of the subtracted disynaptic paired response (see Figs 1A, part
b-a, and 1B; n ⫽ 12). Responses in which control/
conditioned monosynaptic potentials (see Figs 1A, B,
asterisks) did not match in amplitude were excluded
from the analysis. The pairing test was repeated every
10 seconds, and the time course of the changes in inhibition efficacy were evaluated during bicuculline arterial
perfusion (see Fig 1C). Sample point values were syn-
Fig 3. (A) Simultaneous extracellular and intracellular recordings from a principal neuron of the superficial layers of the medial EC
(top trace) during the transition into seizure-like discharge. Recording segments outlined by boxes a, b, and c are expanded in (B).
Resting membrane potential (rmp) was ⫺64mV. (B) At the onset of the seizure, the membrane potential of the neuron was depolarized by 5mV, to enhance the abrupt hyperpolarization that correlates with seizure onset (arrowhead). Cross-correlation coefficient
for intracellular and extracellular recording was calculated in sequences of 100-millisecond time windows and represented as correlogram over time. Phases of the seizure identified as irregular firing (b) and regular bursting (c) are illustrated, respectively. (C)
Power content of intracellular (solid line) and extracellular (dotted line) signals during the seizure shown in (A). High-pass filter
was set at 10Hz. Bottom graph shows the average power content of intracellular (solid line) and extracellular (dotted line) fast
activities recorded at seizure onset in 17 superficial layer neurons.
chronized between experiments (n ⫽ 12) with reference
to the seizure onset in the medial EC. At the time of
seizure initiation (see Fig 1C, arrows), measurements of
inhibition efficacy were reduced by 3.9 and 33.5% in
the PC and lateral EC, respectively ( p ⬍ 0.02). We conclude from the pairing test that the efficacy of inhibition
is only partially reduced in the isolated brain preparation
when EC seizures start. An abrupt and more robust reduction of inhibition efficacy was observed during the
bursting phase of the ictal discharge and after the sei-
zure. In the lateral EC, paired-pulse inhibition reverted
into excitation 30 seconds after seizure onset, and inhibition efficacy could not be measured after this time.
The reduction of inhibition efficacy could still be evaluated in the PC, a brain region usually not involved in
seizure discharge.20
Only experiments in which ictal events initiated
with fast activity at 20 to 30Hz (27/32 tests) were
selected for this study (Figs 2A, B). Two to 5 seconds
after fast ictal onset, bursts of high-amplitude poten-
Gnatkovsky et al: Inhibition at Seizure Onset
Fig 4. Reversal of inhibitory postsynaptic potentials (IPSPs) evoked by LOT stimulation (a; arrowhead) and fast activity (b) in superficial EC neurons. Simultaneous extracellular potentials are shown for each recording (extra). (A) The membrane of the neuron
was hyperpolarized from resting potential (⫺63mV; dotted line) by injection of a steady negative current. The reversal potentials of
the LOT-evoked IPSP (a) and the intracellular correlate of the fast IPSP activity (b) are illustrated; the arrow points at the intracellular correlate of the spike that initiates the seizure. (left) Two different traces recorded at ⫺47 and ⫺80mV are illustrated to
show the reversal potential of the LOT-evoked responses. In a different neuron in (B), a positive current was injected to depolarize
the membrane potential during the LOT-evoked response (a) and during the ictal-onset IPSPs (b; rmp ⫺61mV). Arrowhead in (a)
mark the LOT stimulation. Arrow points to the onset of the seizure-like discharge. (C) Correlation between membrane potential
depolarization observed during seizure onset and the amplitude changes of IPSPs in five experiments, identified by the different symbols. Linear regression lines were calculated and plotted for each experiment. The absolute changes in membrane potential depolarization (MP) were utilized to plot IPSP changes, regardless of the initial resting potential. (D) Average of extrapolated linear regression plots represent changes in IPSPs during membrane potential depolarization (n ⫽ 5, mean ⫾ SD).
tials appeared and progressively increased with time in
amplitude, regularity, and duration19,20 (see Fig 2A;
see also Figs 3–5 and 8). Seizure-like events (mean
duration ⫾ standard deviation, 8.4 ⫾ 1.4 minutes;
n ⫽ 22) were typically followed by postictal depression, characterized by a decrease in the global activity
content measured by signal frequency analysis (see Fig
2A, top). As demonstrated previously,19 interictal
spikes could be observed ahead of an ictal discharge
(see Figs 2A, 4, and 7). In this study, we focus on the
characterization of specific networks that generated
fast 20 to 30Hz activity by recording different types
of neurons in the medial EC.
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Cellular Correlates of Fast Activity at Ictal Onset:
Superfial Entorhinal Cortex Neurons
Stellate and pyramidal neurons in the superficial layers
II and III of the medial EC were characterized by electrophysiological and morphological features, as reported previously.34,41 In correlation with the appearance of fast activity at seizure onset, superficial layers
principal cells did not generate action potential firing.
When the neuron was depolarized above firing threshold before seizure onset, as shown in Figures 3A and B,
action potential generation ceased and inhibitory
rhythmic potentials appeared during the extracellular
fast oscillation (see Fig 3B, thin arrows). The specto-
gram in the lower part of Figure 3A shows the frequency content of the extracellular signal during the
seizure. Small-amplitude intracellular potentials were
correlated with extracellular fast activity, as demonstrated by the correlogram in the bottom of Figure 3B
(part a). Mean correlation value calculated between
pairs of intracellular and extracellular recordings during
fast activity was 0.63 ⫾ 0.11 (n ⫽ 17). The bottom
part of Figure 3C also shows the average frequency
power of fast activity in superficial neurons (see Fig
3C, continuous line) and the corresponding extracellular signals (see Fig 3C, dotted line) (n ⫽ 17).
During fast activity, membrane potential showed an
abrupt hyperpolarization (see Fig 3B, part a, arrowhead) followed by a slow depolarization (see later). In
coincidence with the fragmentation of fast activity, erratic firing resumed in superficial neurons (see Fig 3B,
part b, thick arrow) in parallel with the emergence of
irregular bursting in the extracellular trace (see Figs 3A,
3B, part b). Within a few seconds, bursts of action potentials superimposed on paroxysmal depolarizing shift
appeared in coincidence with the occurrence of regular
bursts in the extracellular trace (see Fig 3A). These
bursts and their extracellular correlates became progressively more robust and less frequent (see Fig 3A and
3B, part c), and were followed by postictal depression.
To better analyze the intracellular correlates of fast
activity at seizure onset, we induced seizures when the
membrane potential of neurons was hyperpolarized/depolarized by injection of steady current via the intracellular recording electrode. When the membrane potential of the recorded neuron was hyperpolarized, no
spontaneous firing was observed (see Fig 4A, part b).
In this condition, the fast rhythms observed at seizure
onset correlated with a depolarizaion of the membrane
potential and often triggered a field event (see Figs 4A,
part b, and 4B, part b, arrows) similar to that observed
during a preictal spike (see Figs 4A, part a, and 7). The
amplitude of the intracellular fast activity progressively
decreased, whereas membrane potential slowly depolarized (see Fig 4A, part b). In other experiments in
which membrane potential was depolarized to values
more positive than ⫺55mV, fast potentials at seizure
onset were hyperpolarizing (see Fig 4B, part b); rebound spikes could be generated at the break of each
fast hyperpolarizing potential. The polarity of the fast
activity deflection matched the polarity of the inhibitory postsynaptic potential (IPSP) evoked by LOT
stimulation34 recorded just before seizure onset (n ⫽
20; see Figs 4A, part a, and 4B, part a); it was depolarizing for membrane potentials more negative than
⫺65mV (n ⫽ 5) and hyperpolarizing when the membrane potential was depolarized to values positive to
⫺60mV (n ⫽ 15). In all experiments, the ictal discharge was preceeded by interictal spikes (see Fig 4A,
arrow). Preictal spikes correlated with a membrane po-
tential deflection that showed the same reversal of
LOT-evoked IPSPs (see Figs 4A, part b, and 7). Based
on this evidence, we conclude that the intracellular correlates of both preictal spikes and repetitive smallamplitude potentials coupled with the extracellular fast
activity are IPSPs. These data also confirmed that synaptic inhibition is preserved at the onset of seizures induced by brief bicuculline perfusion (see Discussion).
A gradual reduction of the fast IPSPs was consistently observed in parallel with the spontaneous membrane depolarization that occurred at the onset of the
seizure. The changes of fast IPSP amplitude during
membrane depolarization is shown in Figure 4C. Each
experiment (n ⫽ 5) is identified by a different symbol.
The progressive reduction of fast IPSPs with membrane depolarization is further illustrated in the averaged plot in Figure 4D.
Cellular Correlates of Fast Activity at Ictal Onset:
Deep Entorhinal Cortex Neurons
In principle, ictal fast activity could be generated by
neurons located in deep layers of the medial EC. Intracellular recordings from layer V and VI principal
cells identified from previously described electrophysiological features34,41 demonstrated that these neurons
do not generate neuronal firing at seizure onset. During extracellular fast activity, they showed an abrupt
steplike membrane hyperpolarization (see Fig 5A), followed by a slow depolarization that lasted 5 to 10 seconds. To demonstrate the inhibitory correlates, we depolarized the membrane potential of the deep neurons
by a steady positive current injected via the intracellular pipette, as shown in the representative neuron illustrated in Figure 5. Unlike superficial neurons, no fast
IPSPs were observed in deep principal cells (n ⫽ 6).
Neuronal firing reappeared in deep neurons during the
extracellular burst activity that developed later on during the seizure (see Fig 5B, C; n ⫽ 6).
We conclude that, as for superficial principal neurons, firing activity was dampened in deep-layer EC
principal cells, and membrane potential was transiently
hyperpolarized in temporal correlation with the appearance of fast activity at seizure onset.
Cellular Correlates of Fast Activity at Ictal Onset:
Putative Entorhinal Cortex Interneurons
Finally, we recorded from 10 cells in superficial layers
(superficial 600␮m of the medial EC) identified as putative interneurons from the previoulsy described electrophysiological criteria.41 They generated fast-burst
firing in response to LOT stimulation and generated
nonadapting firing at more than 100Hz during spontaneous or stimulus-induced depolarizing events. Three
of these putative interneurons were recorded for 10
minutes before bicuculline application and during the
initiation of a seizure. As illustrated in Figure 6, puta-
Gnatkovsky et al: Inhibition at Seizure Onset
Fig 5. Intracellular correlate of seizure activity in a principal neuron of the deep layers of the medial entorhinal cortex (EC). Simultaneous extracellular recording (bottom trace, extra) and intracellular recording from a principal neuron of the deep layers of
the medial EC (top trace) during the transition into seizure-like discharge. Parts a, b, and c, outlined by the squares, are expanded
in the bottom part of the figure. Arrow marks the abrupt hyperpolarization at the onset of seizure, characterized by fast activity in
the extracellular recording. The resting membrane potential (rmp) of the neuron (⫺67mV) is outlined by the dotted line.
tive interneurons generated a barrage of high-frequency
action potentials at more than 150Hz either at the onset (see Fig 6A) or few milliseconds ahead of the seizure (see Fig 6B). Continuous firing gradually evolved
into phasic bursting that gradually became time locked
with the extracellular discharge pattern (see Fig 6A,
part b).
Preictal Spikes in Different Types of Entorhinal
Cortex Neurons
Overall, the intracellular findings demonstrate that putative interneurons generate firing during fast activity
at seizure onset, whereas principal neurons in all EC
layers are inhibited and silent. To further analyze the
role of inhibitory EC circuits during the transition to
the ictal state, we evaluated the intracellular correlates
of the preictal spikes that occur 30 seconds ahead of
the initiation of the seizure-like discharge. Figure 7
shows representative examples of intracellular recordings from principal neurons in superficial and deep layers, and from putative interneurons during the preictal
spikes (right traces) and during responses evoked by
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LOT stimulation in the preictal state34 (left traces). Superficial principal cells (n ⫽ 17) generated a direct
IPSP in response to LOT stimulation (see Fig 7, top
panel) and a negative potential with time course and
membrane reversal similar to the evoked IPSP in correspondence to a preictal spike. Also, deep principal
neurons (n ⫽ 6) generated a pronounced hyperpolarizing potential both during the preictal spike and in
response to LOT stimulation (see Fig 7, middle panel).
Unlike principal neurons, the correlate of preictal
spikes in the three putative interneurons was a marked
bursting discharge (see Fig 7, bottom right traces). A
similar bursting response was also observed in response
to LOT stimulation (see Fig 7, bottom left traces).
These findings confirm that, in the medial EC, inhibition is preserved in the preictal state, just ahead of seizure discharge.
Extracellular Potassium Changes during Fast Activity
at Ictal Onset
Finally, we investigated the mechanisms that promote
the gradual switch from fast activity to burst discharges
Fig 6. Firing features of putative interneurons during seizure-like activity induced by bicuculline. (A) Simultaneous extracellular
(bottom traces, extra) and intracellular recordings from a putative interneuron of the entorhinal cortex (EC) superficial layers (top
trace). Expansions of parts a and b are shown in the bottom part of the panel. (B) The firing of another putative interneuron is
illustrated. As shown in the expanded trace (a) on the right, the cell firing started before the onset of the fast oscillatory activity that
initiates the seizure. rmp ⫽ resting membrane potential.
during the ictal event. Extracellular field recordings and
intracellular recordings from superficial principal neurons were performed in close proximity to a two-barrel
electrode positioned in layers II to III that recorded extracellular potassium changes, [K⫹]o, during a seizure,
close to the fast activity generators (Fig 8). [K⫹]o gradually increased with the appearance of fast 20 to 30Hz
activity, whereas the superficial cell was still silent. The
[K⫹]o increase was closely paralleled by membrane potential depolarization of the neuron and correlated with
the slow downward shift in the extracellular trace.
When both [K⫹]o and slow membrane depolarization
reached a plateau value, irregular firing initiated both
in the intracellular and extracellular recordings (see Fig
8A, top panel). The time course of the membrane potential depolarization, the [K⫹]o increase, and the gradual decrease in amplitude of the fast IPSPs are shown
in the expanded traces in the bottom panel of Figure
8A. The IPSPs associated with the fast activity progressively decreased in amplitude in parallel with membrane depolarization and [K⫹]o increase (see Fig 8,
traces a, b, and c in the right panel). Correlation data
obtained in three experiments performed with the same
protocol are illustrated in Figure 8B. Finally, the plot
of the IPSP amplitude as a function of the changes in
[K⫹]o is illustrated for the three neurons in Figure 8C.
We demonstrate that transient and partial disinhibition
of an in vitro isolated guinea pig brain induces in the
medial EC seizure-like discharges that initiate with 20to 30Hz oscillations. Such fast activity correlated with
the following conditions: (1) no firing in principal neurons of superficial and deep layers, (2) sustained firing
in putative interneurons, (3) fast IPSPs at 20 to 30Hz
in superficial principal neurons, and (4) a slow [K⫹]o
increase associated with a progressive decrease of fast
IPSPs in superficial neurons. A few seconds after the
onset of fast activity, the ictal discharge procedes with
the appearance of erratic bursting that becomes more
regular and robust with time, and gradually ceases
within 10 minutes. As expected after a seizure, postictal
depression was consistently demonstrated by signal fre-
Gnatkovsky et al: Inhibition at Seizure Onset
Fig 7. Intracellular-extracellular correlates of preictal spikes
(right panels) compared with the potentials evoked by stimulation of the lateral olfactory tract (LOT; arrowheads in left
panels) recorded in superficial (top traces) and deep (middle
traces) principal cells, and in putative interneurons (bottom
traces) of the medial entorhinal cortex (EC). Resting membrane potentials (rmp) were ⫺57mV for the superficial neuron, ⫺58mV for the deep neuron, and ⫺56mV for the putative interneuron. In the superficial cell, the LOT-evoked
intracellular traces were recorded at resting membrane potential (top trace) and during steady membrane hyperpolarization
to ⫺82mV. The inset on the right in the middle panel shows
a longer extract of the deep neuron response during the preictal spike.
quency analysis. The mechanisms of ictal onset generation is the main focus of this study.
Simulation studies based on intracranial recordings
from human hippocampus and EC proposed that epileptic fast activity at 20 to 30Hz can be explained by a
transient and partial GABAergic impairment.15,43 This
hypothesis was recently confirmed by reproducing in a
computer model of the EC the seizure patterns observed in the isolated guinea pig brain with the experimental protocol utilized in this study.44 We demonstrate now that, in this model, 3-minute arterial
perfusion of bicuculline induced a moderate reduction
of GABAergic transmission that results in a paradoxical
transitory reinforcement of inhibitory networks. By
evaluating paired-pulse depression of disynaptic activity, we observed a reduction of 3.9 and 33.5% in the
efficacy of inhibition at the time of seizure onset in the
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EC and in the PC, respectively, where depression of
disynaptic inhibitory recurrent circuits could be analyzed accurately. Such a measure could not be performed in the medial EC because this region does not
receive a direct input from LOT.45 Local stimulation
into the medial EC to study inhibition efficacy was not
performed to avoid stimulus-induced changes in local
Intracellular recordings demonstrated that inhibition
prevails when seizures initiate and in the preictal state.
In principal neurons of the EC, seizures start out with
an abrupt membrane potential hyperpolarization that
coincides with robust firing in putative interneurons.
Moreover, we observed that interictal spikes recorded
during the 30 seconds that precede a seizure (preictal
spikes) correlate with an inhibitory potential in superficial neurons and a burst discharge in putative interneurons. Because these responses were similar to those
responsible for the generation of GABAA receptor–dependent IPSPs evoked in normal excitability conditions
by LOT stimulation,34 we propose that the intracellular correlates of the preictal spikes and the abrupt hyperpolarization at seizure onset are mediated by
GABAergic inhibition. We conclude that the transition
from interictal to ictal in EC is promoted by synchronous inhibitory events. Avoli and coworkers46 – 48 demonstrated that, in hippocampal and EC slices in vitro,
the initiation of an ictal discharge induced by either
4-aminopyridine or low-magnesium solution correlates
with the generation of a large-amplitude, depolarizing,
GABAA receptor–mediated potential that lasts approximately 1 second.49 The inhibitory responses observed
in our experiments retain the classic features of IPSPs
(short duration and hyperpolarizing in nature), suggesting that the mechanisms of ictal generation in the
4-aminopyridine and low-Mg2⫹ slice models may be
different. Moreover, unlike EC slice studies, principal
EC neurons in our experiments do not generate firing
in the early phases of the ictal discharge and, therefore,
cannot be responsible for seizure onset. The sustained
firing observed in the imminence of a seizure in the
limited number of putative interneurons recorded in
our close-to-in-vivo condition suggest that a paradoxical reinforcement of inhibition associated with seizure
initiation may be enforced by a prevalent reciprocal release of inhibition between inhibitory neurons induced
by bicuculline.
Long sequences (approximately 5–10 seconds) of fast
activity at 20 to 30Hz were consistently observed in
superficial EC neurons (but not in deep neurons) during the initial phase of the ictal discharge. These oscillations showed a membrane potential reversal similar to
the LOT-evoked IPSP (see also Gnatkovsky and Curtis’s article34) and should, therefore, be considered fast
inhibitory synaptic potentials. What is the source of
such activity? We recently demonstrated that the hip-
Fig 8. Changes in the extracellular potassium ([K⫹]o) during seizure onset in the medial entorhinal cortex (EC). (A) Simultaneous
recordings of a principal neuron in superficial layers (top trace), extracellular potential (middle trace), and changes in [K⫹]o (bottom trace) during an ictal event induced by 3-minute perfusion of bicuculline. The tract outlined by the box is expanded in the
lower left part in (A). Resting membrane potential (rmp) was ⫺59mV. Expanded sweeps of fast activity sequences marked by a, b,
and c are illustrated in the right. Values of potassium concentration are reported for each trace. (B) Average values (⫾ standard
error) of the changes in inhibitory postsynaptic potential (IPSP) amplitude (solid squares; left scale) and potassium concentration
(filled circle; right scale) during seizure onsets recorded in three experiments. The time after the onset of the fast activity is reported
in the abscissae. (C) The dependence of the IPSP reduction on the changes in [K⫹]o are illustrated with different symbols for the
three experiments.
pocampal output generates direct IPSPs into the superfical neurons of the medial EC, via a feedforward inhibition mediated by EC interneurons located in
superficial layers.34 According to this observation, we
propose that the 20- to 30Hz oscillations in superficial
neurons are generated by feedforward inhibition derived from fast activity that originates in the hippocampus, possibly in CA1 and subiculum. A more detailed
simultaneous intracellular analysis of hippocampal and
superficial medial EC neurons will allow to test this
hypothesis. If this will prove to be the case, seizure activity in the medial EC may be considered to be secondary to hippocampal activation. Yet, the mechanisms by which seizures are generated within the
medial EC rely on the reinforcement of activity in local
inhibitory circuits. Our findings suggest the possibility
that both EC and hippocampus may be necessary to
produce seizures in the temporal lobe, possibly through
different local network mechanisms.
Fast activities at 20 to 80Hz recorded in vivo50,51 or
in vitro either by tetanic stimulation52,53 or by pharmacological manipulation in the hippocampus54 –56
and in the EC57,58 are sustained by reciprocal interactions between inhibitory and excitatory networks, with
a prevalent role played by the synchronous activation
of networks of interconnected interneurons.54 Fast oscillations in the beta-gamma band were also observed
during seizure-like activities induced in the hippocampus in vitro by either tetanic stimulation59,60 or by
double-pulse stimulation in EC slices of chronic epileptic rats.61 It has been proposed that these oscillations may arise from excitatory GABAergic depolarizing potentials59 mediated by hypersynchronization of
GABAergic interneuronal networks.60,62 Brief runs of
ultrafast activity (200 – 600Hz), denominated fast ripples, were observed in the hippocampus and in the
parahippocampal cortex of patients affected by temporal lobe epilepsy either in coincidence with an interictal
Gnatkovsky et al: Inhibition at Seizure Onset
spike or in isolation63,64 but were never observed during a seizure. Fast ripples can be reproduced in chronic
animal models of temporal lobe epilepsy65 and were
proposed to be generated by the synchronous activation of clusters of highly interconnected neurons capable of overcoming interneuron feedback inhibition. No
direct relation between fast ripples and the 20- to
30Hz epileptiform activity has been reported. Therefore, these two patterns of fast activity associated with
temporal lobe epileptic conditions are probably mediated by different mechanisms.
If interneuron activation is the prevalent event at seizure onset, what are the mechanisms that promote the
transition from seizure onset into the massive and
highly synchronous bursting typically observed during
the advanced phase of an ictal event? Gradual inactivation of IPSPs mediated by extracellular ion changes
may be the main factor.66 In our experimental conditions, interneurons may contribute to ictal transition
by generating pronounced bursting just ahead of and at
the onset of seizures. This interneuronal hyperactivity
occurs in the absence of principal neuron activation
and is probably responsible for the large extracellular
potassium changes observed in our experiments. At the
onset of a seizure-like event, the increase in [K⫹]o correlated with membrane potential depolarization and a
gradual decrease in fast IPSP amplitude in layer II to
III principal neurons. IPSP amplitude should increase
when the membrane depolarizes above reversal potential for GABAA-mediated chloride current, because of
the increase of ionic drive. Interestingly, we observed
that fast IPSPs decreased in amplitude during the
membrane depolarization associated with seizure onset
and potassium increase. This suggests the possibility
that the reversal potential of the current associated with
the fast IPSPs may gradually change because of the potassium changes. A depolarizing shift in the reversal potential of the GABAA receptor–mediated chloride current is expected, indeed, when [K⫹]o increases.67
When GABAA reversal depolarizes above resting membrane potential, the inhibitory efficacy diminishes,
chloride conductance becomes inward, and GABAA receptor activation may turn into excitatory.43 This
sequence of events could restore neuronal firing in
principal neurons and may favor neuronal hypersynchronization, therefore promoting synchronous bursting discharges and the progression of seizure activity.
High correlation between IPSPs and field potential oscillations at seizures onset indicates strong synchronization of interneuronal activity. Decrease in interneuronal synchrony with the progression of the seizure in
addition to changes in excitability related to potassium
increase could also contribute to the observed IPSP
amplitude reduction.
Annals of Neurology
Vol 64
No 6
December 2008
This study was supported by the Italian Health Ministry (Ricerca
Corrente e Ricerca Finalizzata RF 64) and by the Mariani Foundation (R06-50) (V. G., L. L., F. T., M. D.).
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