Laboratoire de Neurophysiologie, Faculté de Médicine, Université Laval, Quebec, Canada G1K 7P4
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ABSTRACT |
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Steriade, Mircea and Florin Amzica. Intracellular Study of Excitability in the Seizure-Prone Neocortex In Vivo. J. Neurophysiol. 82: 3108-3122, 1999. The excitability of neocortical neurons from cat association areas 5-7 was investigated during spontaneously occurring seizures with spike-wave (SW) complexes at 2-3 Hz. We tested the antidromic and orthodromic responsiveness of neocortical neurons during the "spike" and "wave" components of SW complexes, and we placed emphasis on the dynamics of excitability changes from sleeplike patterns to seizures. At the resting membrane potential, an overwhelming majority of neurons displayed seizures over a depolarizing envelope. Cortical as well as thalamic stimuli triggered isolated paroxysmal depolarizing shifts (PDSs) that eventually developed into SW seizures. PDSs could also be elicited by cortical or thalamic volleys during the wave-related hyperpolarization of neurons, but not during the spike-related depolarization. The latencies of evoked excitatory postsynaptic potentials (EPSPs) progressively decreased, and their slope and depolarization surface increased, from the control period preceding the seizure to the climax of paroxysm. Before the occurrence of full-blown seizures, thalamic stimuli evoked PDSs arising from the postinhibitory rebound excitation, whereas cortical stimuli triggered PDSs immediately after the early EPSP. These data shed light on the differential excitability of cortical neurons during the spike and wave components of SW seizures, and on the differential effects of cortical and thalamic volleys leading to such paroxysms. We conclude that the wave-related hyperpolarization does not represent GABA-mediated inhibitory postsynaptic potentials (IPSPs), and we suggest that it is a mixture of disfacilitation and Ca2+-dependent K+ currents, similar to the prolonged hyperpolarization of the slow sleep oscillation.
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INTRODUCTION |
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Repetitive sensory volleys or synchronous stimuli
applied to central pathways can trigger epileptic seizures in
susceptible animals and humans. Initially focal seizures can be
elicited by different types of stimuli, such as stroboscopic
flash-lights, repeated sounds, cerebral scars, and electrical stimuli
to cortex or thalamus, all acting on a hyperexcitable cortex. Studies
on focal epilepsy have recently been performed in brain slices
maintained in vitro, and, because of the high incidence of temporal
seizures in humans, the investigations have mainly focused on ionic
mechanisms and network operations in the hippocampus, entorhinal and
piriform cortices (De Curtis et al. 1998;
Pelletier and Carlen 1996
; Traub et al.
1993
, 1996
; Wheal et al. 1998
).
The intrinsic cellular properties, synaptic mechanisms, and spread of
seizure activity have also been studied in neocortical slices,
generally after application of GABAA receptor
antagonists, 4-aminopyridine, or other epileptogenic drugs (Aram
et al. 1991
; Barkai et al. 1995
; Chagnac-Amitai and Connors 1989
; Chervin et al.
1988
; Gutnick et al. 1982
; Sutor et al.
1994
).
In this and the companion paper (Amzica and Steriade
1999) in vivo studies, we asked whether the two main components
of spike-and-wave (SW) seizures, associated with
opposite features of membrane polarization, are characterized by a
differential excitability in neocortical neurons, whether precursor
changes in the responsiveness of neocortical neurons can be detected
before gross electrographic signs of seizures, and how do central
stimuli modulate the timing of such paroxysmal episodes. In particular,
as the "wave"-related hyperpolarization of cortical neurons was
repeatedly regarded as produced by GABA-mediated inhibitory
postsynaptic potentials (IPSPs) (Destexhe 1998
;
Giaretta et al. 1987
; Pollen 1964
), we
tested the responsiveness of cortical neurons to antidromic and
orthodromic volleys during both ("spike" and "wave") components
of SW complexes. We used a model of seizures consisting of SW or
polyspike-wave (PSW) complexes at 2-3 Hz, often associated with fast
runs at 10-20 Hz (Steriade et al. 1998a
). Such seizures
appear spontaneously, develop without discontinuity from the slow
oscillation (<1 Hz) which characterizes the state of anesthesia
(Steriade et al. 1993b
) as well as natural slow-wave sleep (Acherman and Borbély 1997
; Amzica
and Steriade 1997
) in animals and humans, and can also be
triggered by electrical stimulation of the neocortex or thalamus. These
paroxysms originate in the neocortex as their appearance in the
thalamus lags by a few seconds the initiation in neocortex
(Neckelmann et al. 1998
; Steriade and Amzica
1994
), and they can occur after ipsilateral thalamectomy (Steriade and Contreras 1998
). The neocortical origin of
continuous SW discharges, and the absence of parallel metabolic
alterations in the thalamus, was also shown by means of positron
emission tomography (PET) studies in children (Maquet et al.
1995
). The relatively high incidence of spontaneous SW/PSW
seizures in our animal experiments, in the absence of
GABAA receptors antagonists or other convulsive
substances and without deliberate electrical stimulation, may be
explained by the widespread corticothalamic coherence of low-frequency
oscillations during natural sleep, the hypersynchronizing action of
ketamine-xylazine anesthesia in acute experiments, the large numbers of
stimulating/recordings electrodes, and the repeated stimuli used for
neuronal identification (Steriade et al. 1998a
). It is
indeed known that cortical SW seizures preferentially occur in the
state of drowsiness and resting sleep in behaving monkeys
(Steriade 1974
) and in humans (Kellaway
1985
; Maquet et al. 1995
). Rhythmic or sustained
electrical stimuli are constantly used to produce models of complex
seizures in limbic and thalamocortical systems (Goddard
1967
; Rafiq et al. 1993
, 1995
;
Shouse and Ryan 1984
; Steriade and Yossif
1974
).
We investigated the dynamics of neocortical responsiveness during the transition from sleep patterns to seizure with emphasis on the differences between the spike and wave components of SW/PSW complexes, and we hypothesized that precursor signs in neuronal excitability will be observed in advance of full-blown seizures at the electroencephalographic (EEG) level.
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METHODS |
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Experiments were conducted on 32 adult cats of either sex, anesthetized with ketamine and xylazine (10-15 mg/kg and 2-3 mg/kg im, respectively). In addition, all tissues to be excised and pressure points were repeatedly infiltrated with lidocaine (2%). When the EEG displayed sleeplike patterns, a muscle relaxant (gallamine triethiodide) was administered, the animal was placed in the stereotaxic apparatus and artificially ventilated with the control of end-tidal CO2 between 3.5 and 3.8%. The heart beat was kept constant (90-110/min) and the body temperature maintained at 37-39°C. To compensate for fluid loss, 20-30 ml iv saline was administered during the experiment. The depth of anesthesia was continuously monitored by EEG recording, and additional doses of the general anesthetic were given at the slightest tendency toward an activated EEG (fast and low-amplitude waves). The stability of intracellular recordings was ensured by hip suspension, drainage of cisterna magna, bilateral pneumothorax, and covering the hole made in the skull with a warm solution of agar (4% in 1% saline). At the end of experiments, the animals were given an intravenous lethal dose of pentobarbital sodium.
Recordings and stimulation
Field potential and intracellular activities were simultaneously
recorded from suprasylvian association areas 5 and 7. 1) For
field potentials we used coaxial electrodes, with the ring placed at
the cortical surface and the tip inserted in deep cortical layers
(0.8-1 mm). Small adjustments of macroelectrodes were made so that the
reversal of depth-to-surface signals typical for the slow oscillation
is obtained (Contreras and Steriade 1995). In all
figures, we illustrate monopolar recordings from the cortical depth
(the indifferent electrode was placed on neck muscles), with the
relative positivity up, as for intracellular recordings. 2)
Intracellular activity was recorded by means of glass micropipettes filled with 3 M potassium acetate (DC resistance, 25-50 M
). A high-impedance amplifier with active bridge circuitry was used to
record the membrane potential (Vm) and
inject current into the neurons. Intracellular signals were recorded,
together with field potential activity, on an eight-channel tape with a
band-pass of 0-9 kHz. 3) Stimulation was applied in the
vicinity of the recording micropipette [within the same cortical area,
or in the adjacent suprasylvian area (i.e., area 7 when recording was
made from area 5)] or to appropriate dorsal thalamic nuclei [lateral posterior (LP) or rostral intralaminar central lateral (CL) that have
reciprocal projections to and from areas 5 and 7; (see Jones 1985
)]. We used bipolar stimulation through coaxial
electrodes, similar to those used for recording the field potential
activity from cortex.
Analyses
1) For the calculation of the latency of synaptically evoked responses, maximal and minimal values of the prestimulus period were calculated over a time span of at least twice the maximum expected latency. These limits represent a voltage range within which the Vm may vary spontaneously even after the delivery of the stimulus. The first point crossing the maximal threshold in the depolarizing sense was considered as the latency of the excitatory component of the response. The point where the excitatory potential was crossing again the upper threshold was considered to mark the end of the excitation and served to the calculation of the duration of excitatory postsynaptic potentials (EPSPs). 2) The amount of excitation produced by a stimulus was quantified by the surface area lying below the excitatory response and above the threshold. The surface depolarization area was calculated as the integral of the response over its duration.
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RESULTS |
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Database and neuronal identification
We analyzed data from 166 regular-spiking neurons (mainly slow-adapting, but some fast-adapting) that were recorded during SW/PSW seizures which occurred 1) as episodic paroxysms, developing without apparent discontinuity from the sleeplike patterns of the slow oscillation (e.g., Figs. 1 and 10); or 2) as one of the numerous spontaneous seizures that appeared during the same experiment and were separated from preceding and succeeding paroxysms by long periods of postictal depression (e.g., Figs. 7 and 8). No epileptogenic substances were used in the present experiments.
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All 166 neurons were recorded for at least 15-20 min (but up to 90 min), had Vms more negative than 60
mV and overshooting action potentials. Antidromically evoked spikes
were differentiated from orthodromically evoked ones by take-off
directly from the baseline, fixed latency, and collision with
spontaneous action potentials at proper time intervals.
General pattern of spontaneously occurring seizures
At the intracellular level and at a resting Vm, most numerous seizures (158 of 166 neurons) consisted of SW/PSW complexes, generally at 2-3 Hz, associated or not with fast runs at 10-20 Hz, developing over a depolarizing envelope that lasted as long as the seizure (generally 10-20 s) and accompanied by a reduction in the amplitude of action potentials (Fig. 1). The onset of EEG seizures was considered at the moment where the acceleration of the slow oscillation (<1 Hz) crossed 1 Hz, frequency known to mark the upper limit of this sleeplike oscillation under ketamine-xylazine anesthesia; this time was also associated with increased amplitude of EEG waves (see rightward arrows in Fig. 1, A and B). The sustained, prolonged neuronal depolarization started a few seconds before the seizure was visible at the macroscopic EEG level (Figs. 1 and 2). As a rule, such seizures started with isolated paroxysmal depolarizing shifts (PDSs; Fig. 1A1) that increased their incidence and eventually became rhythmic SW or PSW complexes at 2-3 Hz. Although the generalized SW seizures are known to not be associated with postictal depression at the EEG level, intracellular recordings showed a short period of hyperpolarization (3-5 s) after cessation of seizure (Fig. 1A).
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Synaptic and antidromic responses to cortical stimuli
Orthodromic responses, tested during control (pre- and postseizure) and seizure epochs consistently showed a progressively decreased latency, increased amplitude, and faster slope at the EPSP onset, during the transition from the slow oscillation (or isolated, interictal PDSs) to SW/PSW paroxysms (n = 52). In Fig. 2, the cortically evoked EPSPs decreased their latency from 4-4.5 ms to 2-3 ms, and the duration of the depolarization increased roughly by 60% (from ~40 ms to ~65 ms) during the 10 s approaching the seizure (compare traces a to f during the preseizure epoch). Stimuli with the same parameters were applied throughout seizures, and contrasting results were obtained during the two main components of SW paroxysms, as follows. 1) During the hyperpolarization following each PDSs and related to the wave component of EEG SW complexes, cortical stimuli reliably elicited PDSs that started at ~3 ms, quite similarly to the EPSP latency before seizure. 2) When falling during the PDSs associated with the spike component of SW complexes, the same stimuli were completely ineffective in eliciting an overt response (Fig. 2C2).
The comparison between antidromic and orthodromic responses in the same
cortical neuron provided evidence for the dependency of both responses
on the Vm and network activity. The
unusually long latency of antidromic response (13 ms) in the neuron
illustrated in Fig. 3 is attributable to
the low-intensity stimulation of a thin axonal collateral.
1) The antidromic response was elicited during the wave
component of SW complexes only on steady depolarization, bringing the
Vm to 65 mV (Fig. 3B1),
but failed at the hyperpolarized level of the resting
Vm (
80 mV). During the spike
component, antidromic action potentials were only elicited during the
declining, repolarizing phase of PDSs (Fig. 3C1), but were
absent if stimuli fell during the middle of PDSs. This was not due to
Vm because at the same
Vm (
50 mV), but during a different
phase, the antidromic spike was elicited (Fig. 3C1).
Collision with orthodromic spikes is also precluded because stimuli
were delivered during an epoch with no spontaneous discharges (Fig.
3C2). 2) Synaptically elicited PDSs were present
during the wave, after the antidromic spike (Fig. 3B1) or in
isolation, at a more hyperpolarized Vm
(Fig. 3B2). At the hyperpolarized level (
80 mV), when the
antidromic response failed, the onset of PDSs constantly revealed
small-amplitude EPSPs that, by summation, gave rise to the giant
synaptic potential crowned by high-frequency spike bursts (Fig.
3B2). An embryonic PDS could follow the antidromic response
toward the end of the spike component, when the
Vm repolarized (Fig. 3C1),
but was absent when the stimulus was delivered during the PDSs (Fig.
3C2).
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The analysis of dynamic responsiveness throughout SW seizures in
neurons displaying both antidromic and synaptic responses to cortical
stimuli (n = 14) is illustrated in Fig.
4 with a neuron from area 7 that was
antidromically as well as synaptically driven by stimuli applied to
area 5. This is a typical example of the reciprocal relations between
the two association areas in cat, demonstrated morphologically
(Grüner et al. 1974) and electrophysiologically (Amzica and Steriade 1995
).
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As shown in Fig. 4A, the first four cortical stimuli
triggered PDSs that opened the scene for a prolonged (~60 s) seizure. As in the preceding figure, synaptic activation elicited PDSs only
during the wave component of SW complexes, whereas antidromic invasion
was effective during certain periods of the spike component. 1) The latency of the EPSP during preseizure epochs was
1.5-1.7 ms (Fig. 4Ba) and decreased to 1.0-1.4 ms from the
beginning of the seizure to its climax (Fig. 4, Bb and
Bc), to resume control values after the postictal depression
(Fig. 4, Bd and Be). This progression was
accompanied by a similar evolution of the slope of EPSP onset, which
became steeper toward the middle of the seizure, was sluggish during
postictal depression, and returned later to control values. In a sample
of eight neurons, the EPSPs latency dropped from 1.8 ± 0.1 (SD) ms to 1.3 ± 0.1 ms (28%), and the slope increased
by 74% (the slope was measured as the tangent of the segment between
the take-off of the depolarization and the initiation of the spike).
2) The antidromic response appeared during the spike
component but only at Vms more negative than 45 mV, at a time when the PDSs repolarized toward the wave (Fig. 4C). The absence of antidromic response at more positive
Vms was not due to collision
with spontaneous action potentials.
The duration and overall spread of synaptically evoked PDSs depended on the state of network activity. The largest PDSs were triggered at some time distance (>500 ms) from previous PDSs (Fig. 5A). When preceding, spontaneously occurring PDSs ended at time intervals of ~100 ms before the tested PDSs, they produced a shortening of ~50-70% in the evoked PDSs (Fig. 5B). Further reduction in the evoked PDSs was observed when they were preceded at shorter time intervals by spontaneously occurring paroxysmal events (Fig. 5C). Figure 6 quantifies the relation between the size of PDSs and the time interval from the preceding PDS, as shown in Fig. 5. It shows that the depolarizing surface of the PDS was most sensitive to short time lags and that it saturated for longer time intervals. It also emphasizes that PDSs are not all-or-none events in the classical sense, but rather display a graded size. The time constant of the best exponential fits was 152 ± 12 ms (mean ± SD) for spontaneous seizures (Fig. 6A) and 409 ± 54 ms for seizures elicited by electrical stimulation (Fig. 6B). This time constant represents the time lag (between the offset of a PDS and the onset of the next one) after which the excitatory surface area increases by 63%.
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Synaptic responses to thalamic stimuli
Similarly to the induction of seizures by cortical stimuli (see Fig. 4), thalamic volleys triggered isolated PDSs that eventually developed into seizures with SW/PSW complexes and fast runs (Figs. 7A and 8A; n = 38). After a series of thalamic volleys, the evoked EPSPs in related cortical neurons became gradually ampler (Fig. 7B1), and the postinhibitory rebound excitation developed into PDSs (Fig. 7B2). Occasionally, however, the PDSs developed directly from the early EPSPs, the large hyperpolarizations that usually follow PDSs were suppressed, and the PDSs were followed by fast runs at 10-20 Hz over a depolarizing plateau (Fig. 7B3). Eventually, the seizure became self-sustained and, depending on the "time-distance" from the previous PDSs (see Figs. 5 and 6), thalamic stimuli evoked longer or shorter PDSs, followed by sequences of fast runs (Fig. 7B4).
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We analyzed the evolution, throughout thalamically evoked seizures, the latencies and surface areas of EPSPs' and postinhibitory rebound excitations (n = 16). The results matched those obtained by the analysis of cortically elicited EPSPs during the preseizure epoch and during the wave component of SW complexes (see Fig. 4). A typical example of the shortened latency and increased surface area of EPSPs is shown in Fig. 8. Throughout the seizure, the EPSPs' latencies decreased from ~5 ms (during the control, preseizure epoch) to 3.7 ms. The depolarization surface area of EPSPs increased only slowly during the first part of the seizure (Fig. 8, inset between arrows in B) but, starting with the time when the early EPSPs merged with the rebound depolarizations to constitute giant PDSs (see trace 33 in D), the surface area of EPSPs displayed a huge increase (part corresponding to stimulus 33 in B). A similar increase was observed in the surface area of the postinhibitory rebound (C). In fact, the surface of EPSPs almost tripled (it increased by 273%), and the depolarization surface of the postinhibitory rebound increased ~17 times (1,724%).
The basic difference between the mechanisms underlying seizures generated by cortical and thalamic stimuli, i.e., the occurrence of isolated PDSs from the early EPSPs in the former case and from the postinhibitory rebound in the latter, is illustrated in Fig. 9 for the same neuron tested with both types of stimuli. The early excitation elicited by local cortical (area 7) stimulation was slightly more synchronous and occurred at a shorter latency than that evoked by thalamic (LP) stimulation: four action potentials at a latency of 1 ms in the former case, three action potentials at 1.6 ms in the latter. However, this slight difference was associated with a dramatic change in the evoked PDS that appeared immediately after the early, cortically evoked excitation, whereas it was generated after the thalamically evoked postinhibitory rebound, at a latency of ~230 ms (see DISCUSSION). It should be emphasized that this contrasting aspect only took place during isolated PDSs, but not with full-blown seizures when the thalamically evoked PDSs merged with the early EPSPs in cortical neurons (see again traces 11 and 33 in Fig. 8D).
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Responses to depolarizing current pulses
The cell's excitability was further tested with depolarizing
current pulses. This procedure provided a comparison of neuronal excitability to impulses reaching the dendritic arbor with the readiness of the soma to fire action potentials in response to direct
somatic depolarizations (the recording pipette is generally located in
the soma). We injected direct depolarizing pulses during control
periods (slow sleep oscillation) and SW seizures. As known, SW seizures
may develop without discontinuity from the slow oscillation, and the
spikes and waves of SW complexes are an exaggeration of the
depolarizing and hyperpolarizing components of the slow oscillation (see Fig. 4 in Steriade et al. 1998a). On one hand, the
hyperpolarizing phase of the slow oscillation was compared with the
wave phase of the SW seizure, and, on the other, the depolarizing phase
of the slow oscillation was compared with the spike component of the SW seizure.
The results from SW seizures developing over a depolarizing envelope
are depicted in Fig. 10. 1)
The intensity of the depolarizing pulses was adjusted in relation with
the Vm during the hyperpolarizing phase of the slow oscillation, to generate a few action potentials (Fig. 10, Slow oscillation 1). During the seizure, the
Vm during the wave component was more
depolarized (due to the depolarizing envelope), whereas the deflection
induced by the same current pulse was reduced when compared with the
control conditions (Fig. 10, Seizure 1). The reduction was
of in the range of 60-70% in 12 tested neurons. No average value can
be provided because there were variations within the same seizure as a
function of its development. 2) Depolarizing pulses applied
during the excitatory phase of the slow oscillation produced smaller
deflections (Fig. 10, Slow oscillation 2) when compared with
their equivalents during the hyperpolarizing phase. The following
results are, however, to be considered with caution because the
amplitude of the depolarization was limited by the firing of the
somatic action potentials. In the cases where an equivalent number of
action potentials was triggered as in the control situation, there was
a reduction of the depolarizing deflection by 20-30%. The changes
from the depolarizing phase of the slow oscillation to the spike
component were much more drastic. Figure 10, Seizure 2,
displays a rather conservative case in which a decreased voltage
deflection of ~25% was seen. Generally, during the PDS
depolarization reaching a Vm of 40 mV, intrasomatic current pulses were unable to produce any voltage deflections, and no additional action potentials were triggered.
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DISCUSSION |
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The main results of this investigation are 1) the differential excitability of neocortical neurons during the wave and spike components of the SW/PSW complexes, i.e., the possibility of triggering PDSs during the hyperpolarizations associated with waves, but the ineffectiveness of incoming signals to elicit such paroxysmal discharges during the spikes; 2) the precursor signs announcing the occurrence of full-blown seizures, consisting of progressively increased amplitude, slope, and duration of the evoked EPSPs, before any paroxysmal sign was visible at the gross EEG level; and 3) the difference between the effects of local cortical and thalamic stimulation, the former triggering PDSs immediately after the early EPSPs, whereas the latter producing paroxysmal discharges as a consequence of the postinhibitory rebound excitation.
Constituent elements of SW seizures
At the resting Vm, the
overwhelming majority of SW/PSW seizures developed over a depolarizing
envelope (see Fig. 1). Intra- and extracellular recordings in
anesthetized (Pollen 1964) and behaving (Steriade
1974
) animals showed that the EEG spike is accompanied by
depolarization and spike trains or spike bursts, whereas the EEG wave
is associated with hyperpolarization and silenced firing.
1) The PDSs that form the spikes can be produced by systemic
or local application of substances blocking inhibitory processes, such
as penicillin (Prince and Farrell 1969) or bicuculline
(Steriade and Contreras 1998
), but neuronal events
similar to those produced by bicuculline are generated spontaneously or
after electrical stimulation (Steriade et al. 1998a
).
The spike patterns that were described during the penicillin model of
generalized SW seizures (Giaretta et al. 1987
;
Gloor et al. 1977
) were probably less ample than the
PDSs in the present experiments. The depolarization underlying the PDS
is a large synaptic potential in CA1-CA3 hippocampal neurons (Johnston and Brown 1981
, 1984
) and in
neocortex (Ayala et al. 1973
). This view is supported by
small, repetitive EPSPs that build up the onset of PDSs during seizures
(see present Figs. 3 and 5). In addition to EPSPs, the spike component
of SW seizures also contains an important inhibitory component. Indeed,
recording with Cl
-filled pipettes revealed
depolarizing shifts by 10-30 mV during the spike component, and
conventional fast-spiking inhibitory interneurons (some of them
intracellularly stained and showing aspiny dendrites and locally
arborizing axons) discharge at very high rates (500-600 Hz) during the
spike component of SW seizures (Steriade et al. 1998b
).
In view of data resulting from the extracellular measures of
[K+]o (Dichter et
al. 1972; Lux and Neher 1973
), it can be
suggested that the giant depolarization recorded intracellularly during the PDS also reflects the increase in
[K+]o, which produces a
positive shift of the Nernst equilibrium potential. Some intrinsic
properties of neocortical neurons, activated by the synaptic
depolarization, may be a supplementary factor for the generation of
this epileptic event. Therefore the synaptic origin and the altered
intrinsic membrane properties of "epileptic neurons"
(Gutnick et al. 1982
; Prince 1967
) are
not incompatible notions. Interestingly, a peculiar class of
neocortical cells, that fire rhythmic, fast, high-frequency spike
bursts in response to depolarizing current pulses, have been recently
disclosed to play an important role in the induction of SW/PSW seizures
at the level of association areas 7 and 5 (see Fig. 15 in
Steriade et al. 1998a
).
The neocortical origin of PDSs during SW seizures in intact-brain
animals was demonstrated by their presence after thalamectomy (Steriade and Contreras 1998). Also, in developing
thalamocortical slices in vitro, PDSs are present in cortex isolated
from thalamus, and connections between deep cortical layers are
sufficient for their synchronization (Golshani and Jones
1999
). Even in those models of SW seizures in which the
paroxysms were regarded as developing from thalamically generated
spindle waves, a selective decrease in cortical excitability, produced
by topical application of KCl to cortex, caused the SW discharges to
revert to spindles (Avoli and Gloor 1982
). Moreover,
more than half of thalamocortical neurons are tonically hyperpolarized
throughout such cortically generated seizures and exhibit phasic IPSPs
during the paroxysmal discharges of cortical neurons, due to the
synaptic engagement of GABAergic thalamic reticular neurons, but do not
display postinhibitory rebound spike bursts (Lytton et al.
1997
; Steriade and Contreras 1995
). The
unexpected finding of hyperpolarization in thalamocortical neurons
during SW seizures and the precursor activities in cortical neurons
have been corroborated by data from experiments on animals with genetic
absence epilepsy (Pinault et al. 1998
;
Seidenberger et al. 1998
).
2) The mechanisms of the hyperpolarization during the wave
component are debated. It was initially assumed that this is an inhibitory phase (Pollen 1964), presumably elicited by
synaptic activation of local GABAergic interneurons. In keeping with
this idea, some suggested that the first part of the hyperpolarization during the wave is constituted by a GABAA
receptor-mediated IPSP (Giaretta et al. 1987
), whereas
a biophysical modeling study predicted the genesis of this component by
a slow, GABAB receptor-mediated inhibition due
to a K+ current (Destexhe 1998
).
Our data show that 1) with Cl
-filled
pipettes, the wave-related hyperpolarization is not significantly altered (Steriade et al. 1998b
); 2) with
pipettes filled with QX-314, the wave-related hyperpolarization is also
unaffected (I. Timofeev, F. Grenier, and M. Steriade, unpublished
data); QX-314 promotes Na+ conductance
inactivation, but also blocks GABAB IPSPs
(Nathan et al. 1990
) and G-proteins (Andrade
1991
) that are required to activate K+
channels underlying GABAB IPSPs (Destexhe
and Sejnowski 1995
); 3) the wave-related
hyperpolarization is obliterated in recordings with
Cs+-filled pipettes (unpublished data);
4) measurements of Rin,
using short hyperpolarizing pulses, reveal that, during PDSs, the
Rin is many times lower than during
the wave-related hyperpolarization, because PDSs comprise numerous
IPSPs (Steriade et al. 1998b
); and
Rin decreases by only 20-30% during
the wave-related hyperpolarization, compared with the hyperpolarizing
phase of the slow sleep oscillation (Neckelmann et al.
1999
), much less than what would be expected if this component
of SW complexes were a GABA-mediated IPSP. In sum, we propose that the
wave-related hyperpolarization is a mixture of
Ca2+-dependent K+ currents
(Schwindt et al. 1988
, 1992
) and
disfacilitation, quite similar to the mechanism of the prolonged
hyperpolarization of the slow sleep oscillation (Contreras et
al. 1996
; Steriade et al. 1993a
).
Responsiveness of cortical neurons during the spike and the wave, dynamics of excitability changes, and differential effects of cortical and thalamic stimuli
The absence of stimulus-evoked PDSs during the spike-related
giant depolarization (20-25 mV) is explained by shunting IPSPs and a
large increase in conductance during this component. However, PDSs
could easily be evoked during the wave, thus corroborating the only
moderate decrease in Rin during the
hyperpolarizing component of SW seizures and further disproving the
prevalent GABAergic origin of this phase. The preserved
excitability during the wave-related hyperpolarization of SW seizures
is in line with the increased EPSP of neocortical neurons in response
to cortical stimulation during the hyperpolarization of the slow
rhythm, as compared with the depolarizing phase of this sleep
oscillation (Timofeev et al. 1996). In fact, these two
(normal and pathological) events, sleep and seizures, are related, and
the relations between intracellular events and field potentials are
virtually identical in both cases (Steriade et al.
1998a
).
The induction of PDSs during the wave-related hyperpolarization
is ascribable to a network phenomenon in which repetitive EPSPs are
summated and eventually reach the threshold of the giant depolarizing,
epileptic event (see Figs. 3B2 and 5). This process is
similar to the increased frequency of EPSPs, just before the occurrence
of PDSs, in CA3 neurons from hippocampal slices (see Fig. 1 in
Chamberlin et al. 1990). The shape and duration of PDSs depend on the history of the network as well as on synaptically activated intrinsic conductances, because such evoked paroxysmal events
are largely diminished when they follow by short time distances spontaneously occurring PDSs (Figs. 5 and 6). This relative
refractoriness may constitute a self-protective mechanism and could
explain the fact that PDSs' frequencies do not generally exceed 4 Hz
during SW seizures. As the seizure accelerates (and this is the common signature of the presently described SW paroxysms that start with isolated PDSs, continue with low-frequency SW complexes at 1-2 Hz, and
eventually reach higher frequency SW complexes), the increased rate of
excitatory drives from the network is counteracted by the rather long
refractory period of PDSs. Shorter refractory phases may be present in
the genetic absence of epilepsy and other types of rat seizures in
which SW complexes appear at higher frequencies, 7-9 Hz
(Danober et al. 1998
; Kandel and Buzsáki
1997
). In slices treated with bicuculline, PDSs often fail to
occur at a frequency 0.1 Hz or higher (Hwa and Avoli
1991
).
The network origin of PDSs is further indicated by the
progressive increase in amplitude and decrease in latency of cortically evoked EPSPs that precede by a few seconds the appearance of seizure at
the global EEG level (see Figs. 2, 4, 7, and 8). Although these seizures are initiated and generated within the cortex even in the
absence of thalamus, once entrained in paroxysms thalamocortical neurons may contribute to the development of seizures. However, whereas
cortically evoked PDSs originate immediately after the early
excitation, thalamic stimulation produces isolated PDSs only after the
postinhibitory rebound excitation. This is probably due to the fact
that, whereas the initial thalamic stimulus set into action a
restricted pool of thalamic neurons, the postinhibitory rebound was
more widely distributed (due to thalamic reticular projections) and
contained many more action potentials (see trace 11 in
Figs. 8D and 9). The role of the thalamic postinhibitory rebound in promoting an increased cortical excitability was
demonstrated in dual intracellular recordings (Grenier et al.
1998). This justifies the assumption that, even in processes
initiated and generated in given structures, i.e., the neocortex in the
present case, the intact connectivity with related subsystems
contributes to the full development of various events.
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ACKNOWLEDGMENTS |
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We thank P. Giguère and D. Drolet for technical assistance.
This work was supported by Grant MT-3689 from the Medical Research Council of Canada and Grant RG-81/96 from the Human Frontier Science Program to M. Steriade. F. Amzica was partially supported by the Fonds de recherche en santé du Québec.
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FOOTNOTES |
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Address reprint requests to M. Steriade.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 23 April 1999; accepted in final form 23 August 1999.
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REFERENCES |
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