Suppression of pilocarpine-induced ictal oscillations in the

Suppression of pilocarpine-induced ictal oscillations in the

Epilepsy Research 49 (2002) 61 – 71
www.elsevier.com/locate/epilepsyres
Suppression of pilocarpine-induced ictal oscillations in the
hippocampal slice
Eldad J. Hadar b, Yili Yang a, Umit Sayin a, Paul A. Rutecki a,b,c,d,*
a
Department of Neurology, Uni6ersity of Wisconsin, 2500 O6erlook Tr., Madison, WI 53705, USA
Department of Neurosurgery, Uni6ersity of Wisconsin, 2500 O6erlook Tr., Madison, WI 53705, USA
c
Department of Neuroscience Training Program, Uni6ersity of Wisconsin, 2500 O6erlook Tr., Madison, WI 53705, USA
d
William Middleton VA Hospital, Uni6ersity of Wisconsin, 2500 O6erlook Tr., Madison, WI 53705, USA
b
Received 29 October 2001; received in revised form 21 January 2002; accepted 28 January 2002
Abstract
Activation of muscarinic cholinergic receptors produces oscillations in the hippocampal slice that resemble the theta
rhythm, but also may produce abnormal synchronous activity that is more characteristic of epileptiform activity. We
used pilocarpine, a muscarinic agonist and convulsant, and an elevation in extracellular potassium (5 – 7.5 mM) to
produce synchronous neuronal activity that was prolonged ( \ 2 s) and mimicked synchronization noted during
seizures in vivo (ictal activity). In the CA3 region of adult rat hippocampal slices, prolonged ictal oscillations
consisted of rhythmic field potentials occurring at 4–10 Hz for up to 30 s (ictal duration) that occurred in a regular
periodic pattern every 12–166 s (ictal interval). The duration and interval between ictal oscillations were measured
before and after application of drugs to define determinants of ictal occurrence. High threshold calcium channel
antagonists (nifedipine and verapamil) blocked ictal activity. Release of calcium from intracellular stores also
appeared to be important for ictal synchronization because ictal activity was blocked by dantrolene, an inhibitor of
calcium release from intracellular stores, and by thapsigargin which blocks the ATPase that maintains intracellular
calcium stores. These suppressive effects appeared to be postsynaptic because nifedipine, dantrolene, and thapsigargin
had no effect on evoked fEPSPs. Enhancement of presynaptic inhibition by activation of GABAB or adenosine A1
receptors suppressed ictal activity and depressed the amplitude of evoked population synaptic potentials. The results
point to an important role for high threshold calcium channels and release of calcium from intracellular stores in
addition to strength of synaptic connections in generation of prolonged oscillations that underlie seizure activity.
© 2002 Elsevier Science B.V. All rights reserved.
Keywords: L-type calcium channel; Dantrolene; Thapsigargin; GABAB; Adenosine; CA3
1. Introduction
* Corresponding author. Tel.: +1-608-280-7057; fax: + 1608-263-0412.
E-mail address: [email protected] (P.A. Rutecki).
Neuronal synchronization manifest as rhythmic
activity recorded extracellularly occurs during
normal and abnormal conditions. The hippocampus demonstrates a variety of normal oscillatory
0920-1211/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 0 - 1 2 1 1 ( 0 2 ) 0 0 0 1 6 - 5
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E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71
activity (Bland, 1986; Fisahn et al., 1998; Traub et
al., 1998), but is susceptible to abnormal synchronization that is epileptiform in character (Traub et
al., 1992; Williams and Kauer, 1997; Rutecki and
Yang, 1998). Interictal epileptiform activity consists of brief (B500 ms) recurrent discharges that
underlie the interictal spike or sharp wave
recorded in the EEG. More prolonged synchronous activity characterizes a seizure or ictal
discharge. The transition from a pattern of interictal epileptiform activity to an ictal pattern is the
critical determinant of seizure occurrence.
Studies of epileptiform activity in the hippocampal slice support the hypothesis that the interictal
discharge is driven by synchronous synaptic transmission (Johnston and Brown, 1982; Miles and
Wong, 1983; Rutecki et al., 1985). Synchronous
synaptic input underlies prolonged ictal-like activity produced by pilocarpine and elevated [K+]o
(Traynelis and Dingledine, 1988; Rutecki and
Yang, 1998). Fewer studies have defined factors
that control the transition from an interictal to an
ictal pattern of epileptiform activity (Anderson et
al., 1986; Swartzwelder et al., 1987; Traynelis and
Dingledine, 1988; Swann et al., 1993; Nagao et al.,
1996; Barbarosie and Avoli, 1997; Rutecki and
Yang, 1998). In the hippocampal slice, ictal patterns depend on the concentration of extracellular
potassium ([K+]o) (Traynelis and Dingledine,
1988; Rutecki and Yang, 1998), a reduction in
extracellular space (Traynelis and Dingledine,
1989; Rutecki and Yang, 1998) or other nonsynaptic
mechanisms
of
synchronization
(Schweitzer et al., 1992; Jefferys, 1995), the occurrence of prolonged synchronized synaptic input
(Swann et al., 1993; Rutecki and Yang, 1998), and
in some cases GABAergic synaptic transmission
(Avoli et al., 1996; Williams and Kauer, 1997;
Kohling et al., 2000).
Pilocarpine, a muscarinic agonist and convulsant, activates phosphotidyl inositol metabolism
and the production of inositol triphosphate (IP3)
that can cause release of calcium from intracellular
stores (Irving and Collingridge, 1998; Nakamura
et al., 1999). Calcium may also be released from
intracellular stores by calcium-dependent mechanisms (Simpson et al., 1995; Emptage et al., 1999).
Release from intracellular stores can induce cal-
cium oscillations (Charles et al., 1993; Simpson et
al., 1995; Berridge, 1998; Irving and Collingridge,
1998) and the potential role of calcium release
from intracellular stores in synchronizing a network of neurons is not clear (Berridge, 1998).
Modeling studies have suggested that regenerative calcium currents and calcium influx in dendrites of CA3 neurons favor more prolonged ictal
patterns of activity (Traub et al., 1993, 1996).
Furthermore, non-inactivating dendritic calcium
current was used in modeling to create ictal discharges (Traub et al., 1993). Voltage-dependent
calcium channels exist in the dendrites of CA3
neurons (Wong and Prince, 1979) and may be
activated by synaptic potentials or back-propagating action potentials (Jaffe et al., 1992; Avery and
Johnston, 1996; Magee et al., 1996).
In the present study, we evaluated the effects of
calcium channel blockade and manipulations that
alter the release of calcium from intracellular
stores. These effects are compared to those produced by activation of presynaptic receptors that
inhibit synaptic transmission (GABAB and
adenosine A1). Our findings point to the important
role of synaptic transmission as well activation of
high threshold L-type calcium channels and release
of calcium from intracellular stores in the generation of recurring ictal oscillations produced by
pilocarpine and elevated [K+]o.
2. Methods
2.1. Slice preparation
Hippocampal slices were prepared from young
adult male Sprague–Dawley rats that weighed
125–300 g. Rats were anesthetized with ether or
pentobarbital (60–75 mg/kg given i.p.), decapitated, and the hippocampus was removed and
transferred to iced artificial cerebrospinal fluid
(ACSF). Transverse slices (400–500 m thick) were
prepared with either a McIllwain tissue chopper or
vibratome (Technical Products International) and
transferred to an interface chamber.
Slices were incubated for 1 h while perfused
(0.3–0.5 ml/min) with ACSF that contained (in
mM): NaCl 124, KCl 5, NaH2PO4 1.25, CaCl2 2,
E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71
63
MgSO4 2, NaHCO3 26, glucose 10. The bathing
solution was then changed to one containing 10
mM pilocarpine and an extracellular potassium
concentration ([K+]o) of either 5 or 7.5 mM.
Most (85 of 90 slices) of the ictal recordings were
made in 7.5 mM [K+]o, a concentration that
favors ictal activity (Rutecki and Yang, 1998),
and all interictal recordings were made in 7.5 mM
[K+]o ACSF. Recordings began after a 1-h exposure to the pilocarpine containing saline.
2.2. Recording techniques, characterization, and
quantification of epileptiform acti6ity
Extracellular recordings were made from the
CA3b or CA3c subfield using either a Getting
amplifier (Model 5A) or an Axoclamp 2 amplifier
in current clamp mode. Glass microelectrodes of
2– 10 MV resistance filled with 2 mM NaCl were
positioned in stratum pyramidale and adjusted to
obtain a maximal amplitude signal. Spontaneously occurring activity was monitored and
characterized as desynchronized unit activity or
epileptiform activity. The epileptiform activity
was characterized as either recurrent, brief (B 500
ms duration) interictal or ictal discharges that
consisted of recurrent, prolonged oscillations that
lasted greater than 2 s occurring between 4 and 10
Hz (Rutecki and Yang, 1998) (Fig. 1). Some slices
displayed a combination of interictal and ictal
activity but were classified as ictal (Fig. 1).
Slices with epileptiform activity were recorded
from before and after ascending concentrations of
drugs were added to the bathing solution. Each
concentration (between 2 and 4) was applied for
30– 60 min before the next higher concentration
was applied. Multiple slices were monitored and
care was taken to record from the same area of
CA3 for any given slice. This required movement
of the recording electrode. Although the amplitude often changed, the pattern of activity did not
appear to differ between multiple recordings from
the same slice bathed in the same solution.
Three–five minute epochs of activity were digitized using a Digidata 1200 interface (Axon Instruments). The duration of ictal discharges and
the interval between ictal discharges were measured from digital records (see Fig. 1). A conver-
Fig. 1. High threshold calcium channel blockade suppressed
ictal activity. (A) Top trace shows the ictal pattern of epileptiform activity produced by 10 mM pilocarpine in 7.5 mM
[K+]o. The recording was made in CA3 stratum pyramidale
and demonstrates population activity occurring at 4 – 6 Hz.
The second trace shows that ictal activity and its recurring
pattern. The solid line demonstrates the ictal duration measurement, and the dashed line the ictal interval measurement.
At 0.1 mM, nifedipine suppressed the occurrence of ictal
discharges and at 1 mM stopped their occurrence. (B) Both
nifedipine and verapamil decreased the occurrence of ictal
discharges in a dose-dependent fashion. At higher concentrations both high threshold calcium channel blockers reduced
the number of slices demonstrating ictal patterns and hence
the large SEMs. The changes were significant by Kruskal –
Wallis (P =0.0011 for nifedipine, P= 0.0038 for verapamil).
(C) The ictal duration was also reduced in a dose-dependent
manner (PB 0.05 post hoc comparisons for all concentrations
of nifedipine and concentrations of 10 mM or greater for
verapamil).
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E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71
sion of an ictal pattern to an exclusively interictal
pattern was noted and the ‘ictal duration’ was
considered to be zero. The rate of interictal discharges in slices only displaying interictal activity
were measured and compared to the rate after a
drug application.
To assess synaptic transmission, association/
commissural fibers were stimulated in stratum
radiatum and the resultant field excitatory synaptic response (fEPSP) was measured in stratum
radiatum in the CA3b subfield. Stimulation was
delivered through a bipolar electrode with a pulse
duration of 50 ms. The stimulus intensity was
adjusted to just below population spike threshold
as judged by contamination of a population spike
in the synaptic sink waveform. The position of the
field and stimulating electrode were maintained
before and after solution changes. Stimulation
was given at 0.1 Hz, and five responses were
averaged every 5 min. After a 15-min baseline was
obtained, the solution was changed to one containing the effective concentration of a agent that
altered ictal activity. Comparisons were made at
20–30 min following bath change and in the case
of thapsigargin following a 30-min wash to pilocarpine and high [K+]o.
2.3. Chemicals and solutions
All drugs were obtained from Sigma except for
thapsigargin that was obtained from Calbiochem;
nifedipine,
N6-cyclopentyladenosine
(CPA),
dantrolene, and verapamil from RBI; and 2-hydroxysaclofen from Tocris. All drugs were made
as a stock solution, frozen, thawed, and then
added to a larger volume of ASCF to achieve the
desired concentration. Nifedipine, dantrolene, and
thapsigargin were prepared in a DMSO vehicle,
and in these cases DMSO was added to the
control ACSF containing pilocarpine.
2.4. Statistics
Parametric testing (paired t-test or ANOVA)
was used when the data were normally distributed,
and non-parametric testing (Mann– Whitney,
Krushkal Wallace) when the distribution was not
normal. Significance was set at less than 0.05.
3. Results
3.1. High threshold calcium channel antagonists
block ictal discharges
Two L-type calcium channel blockers were evaluated for effects on the pattern of interictal or
ictal discharges produced by elevated [K+]o and
pilocarpine. Nifedipine, a dihyropyridine derivative, or verapamil, a phenylalkylamine compound,
both suppressed the occurrence of ictal discharges
in a dose-dependent manner. In the presence of
0.3 mM nifedipine, 10 of 16 slices that initially
demonstrated ictal activity converted to an interictal pattern of activity. At 1 mM, nifedipine converted four of the six slices with residual ictal
discharges to only interictal patterns of activity
(Fig. 1A). Verapamil (30 mM) changed an ictal
pattern to an interictal pattern in 11 of 14 slices.
The interval between ictal discharges was more
than doubled at lower concentrations of nifedipine
(0.3 mM) and verapamil (10 mM) (Fig. 1B). Both
compounds also decreased the duration of the
ictal discharge in a dose-dependent manner (Fig.
1C). In slices that only displayed interictal discharges initially, nifedipine did not significantly
change the rate of interictal discharges (control
rate of 0.309 0.02 and 0.249 0.02 Hz for 1 mM
nifedipine, n= 17).
The suppressant effect of L-type calcium channel blockade could be explained by a depression in
synaptic transmission; however, nifedipine at a
concentration 10-fold greater than that which
blocked ictal activity did not reduce the amplitude
of the fEPSP (see below, Table 1).
3.2. Role of intracellular calcium release and ictal
acti6ity
To assess the potential role of intracellular calcium release on the production of ictal activity, we
evaluated the effects of two compounds that depress intracellular calcium release, dantrolene and
thapsigargin. Dantrolene prevents the release of
calcium from intracellular stores (Frandsen and
Schousboe, 1991; Charles et al., 1993). At 30 mM,
dantrolene prolonged the interval between ictal
discharges by more than 100% (Fig. 2). At 100
E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71
65
Table 1
Effect of drugs that suppress ictal activity on evoked population EPSPs
% Control evoked population
EPSP9 SEM
Baclofen 1 mM
CPA 100 nM
Nifedipine 10 mM
Dantrolene 100 mM
Thapsigargin 5 mM
Acute
Wash
52.49 6.1a
56.29 8.5a
1059 9.2
1069 3.8
10894.1
10694.9
All measurements were compared to control; measurements
made 30 min after drug exposure except for thapsigargin,
which included acute 30 min exposure, followed by a 30 min
wash. CPA refers to N6-cyclopentylodenosine.
a
Significantly different from other experiments, F = 13.3,
PB0.0001, ANOVA and PB0.05 by post hoc analysis. N= 5
slices for all experiments.
mM, dantrolene converted the pattern of epileptiform activity from ictal to interictal in seven of 11
slices. The effects on interval between discharges
and ictal duration were dose-dependent (Fig. 2B
and C).
Thapsigargin (1 mM), an irreversible blocker of
the ATP-dependent calcium pump that creates the
concentration gradient for endoplasmic reticulum
calcium storage (Irving and Collingridge, 1998),
converted the ictal pattern of activity to an interictal pattern in three of 12 slices and caused one
slice to stop having spontaneous epileptiform activity. In the eight slices that continued to demonstrate ictal patterns, the duration of the ictal
discharge was decreased significantly (16.99 2.5 –
7.3 90.8 s) and the interval between discharges
was shortened (from 42.997.3 to 23.69 1.8 s,
P B0.05, Fig. 3). After a 30– 40 min exposure,
50% of all slices stopped having spontaneous activity and five of eight slices with ictal activity
converted to an interictal pattern. Changing the
solution back to pilocarpine and 7.5 mM [K+]o
did not reverse the effect. At a concentration of 5
mM, thapsigargin converted the ictal pattern observed in five slices to an interictal pattern in two
slices and spontaneous activity stopped in another
slice. With prolonged exposure (\ 30 min) fol-
Fig. 2. Dantrolene effects on ictal activity. (A) The top trace
represents control ictal activity in the presence of pilocarpine
and elevated potassium. Dantrolene (30 mM) suppressed ictal
occurrence and interictal discharges were observed. (B)
Dantrolene increased the interval between ictal discharges in a
dose-dependent fashion (P= 0.003, Kruskal – Wallis, significant for a concentration of 100 mM by Dunn’s method of
comparisons). (C) Dantrolene also reduced the ictal duration
as a function of concentration (P =0.015, Kruskal – Wallis,
significant for 100 mM by Dunn’s method).
lowed by return to pilocarpine and 7.5 mM [K+]o,
only one slice displayed spontaneously occurring
interictal discharges.
Fig. 3. Thapsigargin alters ictal discharges in a time-dependent
manner. Top trace shows ictal activity produced by pilocarpine and high [K+]o. The ictal discharges became more
frequent but shorter in the presence of 1 mM thapsigargin. The
ictal activity was replaced by interictal activity that continued
despite wash to control solution (10 mM pilocarpine and 7.5
mM [K+]o).
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E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71
nM concentration of CPA resulted in ictal suppression in four of the six slices that continued to
display ictal discharges in 30 nM CPA.
3.4. Effects on fEPSP
Fig. 4. GABAA or adenosine A1 receptor activation block ictal
discharges. (A) Baclofen at a concentration of 1 mM suppressed ictal discharges. (B) The dose-dependent effects of
CPA on ictal discharges. At a concentration of 10 nM, CPA
had a minimal effect on ictal discharges, but 30 nM totally
suppressed ictal discharges.
3.3. Enhanced presynaptic inhibition suppressed
ictal discharges
Baclofen, a GABAB agonist reduces synaptic
transmission at both GABA and glutamate containing synapses and activates a potassium channel conductance that creates a long lasting
synaptic inhibition (Bowery et al., 1980; Newberry
and Nicoll, 1984; Thompson and Gahwiler, 1992).
Baclofen (0.3–1 mM) converted the ictal pattern
of activity produced by pilocarpine to an interictal
pattern in six of seven slices (Fig. 4A). In five
slices that displayed an interictal pattern of activity initially, baclofen slowed the rate of interictal
discharges (0.2890.06 – 0.05 90.02 Hz, P B 0.005
paired t-test).
The GABAB antagonist 2-hydroxysaclofen did
not alter the pattern of pilocarpine-induced ictal
activity. In pilocarpine control saline, 10 of 13
slices demonstrated ictal activity, and nine of 13
slices still demonstrated ictal activity in the presence of 300 mM 2-hydroxysaclofen. No significant
change in the duration (6.89 0.6 vs. 6.390.5 s)
or interval between discharges (55.49 14.7 vs.
43.6 913.7 s) was noted.
CPA is an adenosine A1 agonist and results in
presynaptic inhibition and postsynaptic potassium
conductance activated by a G protein (Dragunow,
1988; Yoon and Rothman, 1991). Like baclofen,
CPA suppressed ictal discharges in a dose-dependent manner (Fig. 4B). At a concentration of 30
nM, CPA suppressed the occurrence of ictal discharges in eight of 14 slices. An increase to 100
The effects of baclofen and CPA may be due to
presynaptic inhibition and reduction in the
strength of recurrent synaptic connections. We
assessed the effects of CPA and baclofen on the
fEPSP evoked by stimulating stratum radiatum.
Both compounds reduced the fEPSP by about
50% (Table 1).
The other manipulations that resulted in suppression of ictal activity were evaluated for effects
on the evoked population EPSP. We found that
nifedipine (10 mM) did not change the amplitude
of the fEPSP recorded in stratum radiatum of
CA3 that was evoked by stratum radiatum stimulation (Table 1). Dantrolene and thapsigargin, at
concentrations that inhibit ictal discharges, also
had no effect on the population EPSP (Table 1).
4. Discussion
4.1. High threshold calcium channel blockers
inhibit ictal patterns
The dihydropyridine high threshold L-type calcium channel blocker nifedipine, and verapamil,
another L-type channel blocker with different
molecular characteristics from nifedipine, depressed ictal discharges in a dose-dependent manner. Nifedipine, at a concentration of 10 mM, did
not change the population EPSP and others have
not found a significant effect of L-type calcium
channel blockers on synaptic transmission in the
hippocampus (Wheeler et al., 1994). Instead the
effect is more likely on dendritic or somatic high
threshold calcium channels. The role of dendritic
calcium spikes in generation of epileptiform activity has been postulated since the 1960s (Purpura
et al., 1966; Wong and Prince, 1979;
Schwartzkroin and Wyler, 1980). Others have described antiepileptic effects of high threshold calcium channel blockers on interictal epileptiform
activity in the slice (Vezzani et al., 1988; Bing-
E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71
mann and Speckmann, 1989; Aicardi and
Schwartzkroin, 1990), and some anticonvulsants
block calcium currents at high therapeutic concentrations (Macdonald and Kelly, 1993). High
threshold calcium channels blockers may inhibit
ictal patterns of activity by reducing calcium
influx at dendritic calcium channels activated by
either synaptic depolarization or back-propagation of action potentials.
A persistent dendritic calcium current has been
used in modeling studies to produce ictal-like
discharges and a long-lasting calcium current has
been described in CA3 neurons (Traub et al.,
1993; Avery and Johnston, 1996). Additionally,
muscarinic activation produces a sustained inward
current blocked by L-type channel blockade
(Fraser and MacVicar, 1996) and such a current
could add to the sustained activity that occurs
during prolonged ictal discharges. High threshold
calcium channel blockade inhibits pilocarpine-induced seizures in vivo and reduces associated
cellular loss (Marinho et al., 1997).
4.2. Releasable intracellular calcium stores
Our results also point to an important role of
calcium release from intracellular stores in the
occurrence of ictal discharges. A variety of cells,
including neurons and glia are capable of generating intracellular calcium oscillations that are dependent on the release of calcium from
intracellular stores. These stores include those released by interaction of IP3 and its receptor or
calcium with the ryanodine receptor (Simpson et
al., 1995; Berridge, 1998). Our results do not
differentiate between these receptors.
Thapsigargin produced an irreversible depression of ictal discharges and no change in the
evoked population EPSP. The effects of thapsigargin were initially to enhance the frequency of
occurrence of ictal discharges, albeit with an associated decrease in ictal duration. This initial enhancement may relate to a decrease in the
endoplasmic buffering capability produced by
blockade of the ATPase. With time the shortening
and eventual suppression of ictal discharges probably results from a loss of releasable calcium from
intracellular stores.
67
Dantrolene decreases the release of calcium
from intracellular stores in a variety of preparations including calcium release produced by carbachol in acinar cells (Zhang and Melvin, 1993).
Dantrolene depresses calcium oscillations in glia
(Charles et al., 1993) and can protect against
neuronal death following ischemia (Zhang et al.,
1993) or high intensity electrical stimulation (Pelletier et al., 1999). In addition, dantrolene has
been reported to suppress seizures produced by
metabotropic glutamate receptor activation (McDonald et al., 1993), and protects against epileptiform activity that follows trauma in neocortical
slices (Yang and Benardo, 1997). The fEPSP was
not affected by dantrolene or thapsigargin so the
effects do not appear to be related to dampening
of synaptic transmission but rather to action at
calcium release sites in the soma or dendrites.
Calcium entry from the extracellular space has
also been hypothesized to replenish intracellular
calcium stores (Simpson et al., 1995; Berridge,
1998; Irving and Collingridge, 1998). High
threshold calcium channel blockade may have an
indirect effect by limiting a source of calcium to
replenish stores. Arguing against this hypothesis is
that blockade of NMDA channel opening and
another potential source of extracellular calcium
influx had no effect on ictal patterns (Rutecki and
Yang, 1998). Alternatively, calcium entering from
the extracellular space through high-threshold
channels could lead to calcium-dependent calcium
release.
Muscarinic receptor activation has been shown
to result in an increase in dendritic calcium
(Muller and Connor, 1991; Irving and
Collingridge, 1998). Although an enhancement in
release of calcium mediated by IP3 is expected
following application of pilocarpine, calcium entering from high threshold plasma membrane
channels may also participate in calcium-dependent calcium release or enhance IP3-mediated calcium release (Irving and Collingridge, 1998;
Nakamura et al., 1999). The synchronous oscillation that comprises the ictal discharge should
have an associated calcium flux from voltage- and
synaptic-dependent mechanisms.
Depressing the release of calcium from intracellular stores by dantrolene or thapsigargin and the
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E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71
resultant suppression of ictal activity point to a
role of oscillations of intracellular calcium as a
mechanism that supports neuronal synchronization that underlies ictal activity. Calcium oscillations typically have a time course of seconds and
hence are more likely to influence the duration of
ictal events and the period between them (HarrisWhite et al., 1998; Irving and Collingridge, 1998).
We hypothesize that the period between ictal discharges is controlled by activation of calcium-dependent potassium currents. Our results suggest
that calcium release form intracellular stores may
entrain a network of neurons connected synaptically. One possible mechanism for this effect is the
activation of a calcium-dependent non-specific
cationic current that maintains depolarization
above action potential threshold (Fraser and
MacVicar, 1996).
4.3. Presynaptic inhibition, role of synaptic dri6e
Both interictal and ictal patterns of epileptiform
activity are driven by synchronous synaptic drive
(Johnston and Brown, 1982; Miles and Wong,
1983; Swann et al., 1993; Rutecki and Yang,
1998) and some convulsants appear to act by
enhancing both excitatory and inhibitory synaptic
transmission (Rutecki et al., 1987). Transmitters
that inhibit synaptic transmission presynaptically
should depress synaptic synchronization. The
GABAB agonist baclofen was effective in stopping
ictal discharges at concentrations that reduced
evoked EPSPs. The A1 adenosine agonist CPA
has similar properties and adenosine has been
considered to be an endogenous anticonvulsant
(Dragunow, 1988). These receptors also mediate
slower postsynaptic inhibition and such action
should also be anticonvulsant. The concentrations
of transmitter agonists that inhibited ictal discharges also depressed synaptic transmission measured at a population level. This is in contrast to
the action of L-type channel blockers or inhibition of release of calcium from intracellular
stores.
Paradoxically, muscarinic activation inhibits
synaptic transmission in the CA3 region
(Valentino and Dingledine, 1981; Williams and
Johnston, 1990; Sayin and Rutecki, 1997). Recent
studies hypothesize that epileptiform activity in
the CA3 region may be limited in duration by the
availability of releasable transmitter stores (Staley
et al., 1998). In the low magnesium model of
epileptiform discharges, baclofen enhances the occurrence of ictal discharges (Swartzwelder et al.,
1987), and this may be explained by presynaptic
inhibition that limits release so that a long duration pattern of epileptiform activity may occur
(Staley et al., 1998). The degree of presynaptic
inhibition required for a longer duration discharge may be produced by exposure to pilocarpine and further inhibition by GABAB or
adenosine A1 activation results in loss of ictal and
interictal activity.
In the present study, antagonism of the GABAB
receptor with 2-hydroxysaclofen did not alter the
pattern of epileptiform activity, suggesting that
endogenous activation of GABAB receptors does
not influence the generation or termination of
ictal discharges. In rhythmic slow activity produced by carbachol, neither GABAA nor GABAB
synaptic transmission appeared to be a critical
determinant of synchronization (Macvicar and
Tse, 1989; Traub et al., 1992, but see Williams
and Kauer, 1997).
Action potentials generated from ectopic sites
including axon terminals may contribute to
hippocampal synchronization (Stasheff et al.,
1992; Traub et al., 1995, 1996). Activation of
presynaptic receptors that act through G-proteins
to decrease calcium currents, increase potassium
currents, or create a shunt would be expected to
decrease ectopic action potential generation near
the terminal. GABAB and adenosine A1 receptor
activation could depress ictal transitions by this
mechanism in combination with depression of
synaptic transmission.
4.4. Determinants of ictal epileptiform acti6ity
Previous studies have demonstrated a relationship between extracellular potassium concentration, extracellular space, and the pattern of
epileptiform activity (Traynelis and Dingledine,
1988, 1989; Schweitzer et al., 1992; Rutecki and
Yang, 1998). In this study, the role for L-type
calcium channel activation and release of calcium
E.J. Hadar et al. / Epilepsy Research 49 (2002) 61–71
from intracellular stores appear to be critical variables involved in ictal oscillations. The possible
role of changes in intracellular calcium concentrations in glia elements could also effect network
synchronization and our experiments do not distinguish between effects on glia or neurons.
Calcium release from intracellular stores appears to be necessary for the sustained ictal oscillation, but the mechanism by which such stores
synchronize a synaptic network is not obvious.
Synaptic transmission has been shown to cause
calcium release from postsynaptic ryanodine-sensitive intracellular stores (Emptage et al., 1999),
and postsynaptic calcium increases enhance
synaptic strength (Yeckel et al., 1999).
Pilocarpine and activation of group I metabotropic glutamate receptors both result in IP3-mediated calcium release from intracellular stores
and result in ictal patterns of epileptiform activity
in the hippocampal slice (Bianchi and Wong,
1994; Jaffe and Brown, 1994; Taylor et al., 1995;
Irving and Collingridge, 1998; Bianchi et al.,
1999). Muscarinic activation plays a role in generation of theta oscillations and activates a set of
second messenger systems. Similar second messengers are activated by glutamate acting at the
group I class of metabotropic glutamate receptors. Group I metabotropic receptor activation
produces faster frequency oscillations (\20 Hz)
and also produces ictal discharges in the
hippocampal slice (Taylor et al., 1995; Traub et
al., 1998). Activation of either muscarinic or
group I metabotropic glutamate receptors results
in a significant increase in intracellular calcium
that may result from release from intracellular
stores or by depolarization (Muller and Connor,
1991; Bianchi and Wong, 1994; Jaffe and Brown,
1994; Taylor et al., 1995; Irving and Collingridge,
1998; Bianchi et al., 1999).
Depression of L-type high threshold calcium
channels and release of calcium from intracellular
stores both appear to be targets for antiepileptic
drug development. Furthermore, blocking L-type
high threshold calcium channels or release from
intracellular stores may alter the neuronal loss
and plasticity associated with pilocarpine-induced
status epilepticus. We hypothesize that oscillations of dendritic calcium levels sustain synchro-
69
nization driven by recurrent excitatory synaptic
connections. Normal mechanisms of neuronal oscillation may subserve the pathological oscillations associated with epileptiform ictal discharges.
Acknowledgements
The authors thank Tom Sutula and Bob Pearce
for comments on an earlier draft of this paper and
Kathleen Kleckner for help with figure preparation. This work was supported by a Merit Review
grant through VA research to PAR.
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