Inhibition of calcium?calmodulin kinase II alpha
subunit expression results in epileptiform
activity in cultured hippocampal neurons
Severn B. Churn*, Sompong Sombati*, Emma R. Jakoi*, Lawrence Severt†, and Robert J. DeLorenzo*‡§¶
Departments of *Neurology,†Anatomy and Neurosciences,‡Pharmacology and Toxicology, and§Biochemistry and Biophysics, Virginia Commonwealth
University, Medical College of Virginia, Richmond, VA 23298
Communicated by Philip Siekevitz, The Rockefeller University, New York, NY, February 17, 2000 (received for review October 4, 1999)
Several models that develop epileptiform discharges and epilepsy
have been associated with a decrease in the activity of calmodu-
lin-dependent kinase II. However, none of these studies has dem-
onstrated a causal relationship between a decrease in calcium?cal-
modulin kinase II activity and the development of seizure activity.
The present study was conducted to determine the effect of
directly reducing calcium?calmodulin-dependent kinase activity on
the development of epileptiform discharges in hippocampal neu-
rons in culture. Complimentary oligonucleotides specific for the ?
subunit of the calcium?calmodulin kinase were used to decrease
the expression of the enzyme. Reduction in kinase expression was
confirmed by Western analysis, immunocytochemistry, and exog-
enous substrate phosphorylation. Increased neuronal excitability
reduction in calmodulin kinase II expression. The epileptiform
activity was a synchronous event and was not caused by random
neuronal firing. Furthermore, the magnitude of decreased kinase
expression correlated with the increased neuronal excitability. The
data suggest that decreased calmodulin kinase II activity may play
a role in epileptogenesis and the long-term plasticity changes
associated with the development of pathological seizure activity
lation of neurons, unprovoked by a known proximal cause (1, 2).
Epilepsy is the second most common neurological pathology in
the United States, behind only stroke (2). Symptomatic epilepsy
recurrent seizures in previously normal brain. Depending on the
study criteria used to estimate epilepsy prevalence, symptomatic
epilepsies account for one- to two-thirds of new cases (2).
Epileptogenesis is the process responsible for the transforma-
tion of normal neuronal populations into neurons that display
neurophysiological behaviors associated with epilepsy. Although
the processes underlying epileptogenesis are complex, alter-
ations in Ca2?homeostasis have been suggested as a cellular
mechanism in the development of seizure activity and epilepsy
(3). In particular, alteration in the function of calcium and
calmodulin-dependent kinase II (CaM kinase II), a neuronally
enriched Ca2?-dependent second messenger system (4–6), has
been associated with a number of models of epilepsy. Wasterlain
and Farber (7) initially showed a decrease in CaM kinase II
activity was associated with the kindling model of epilepsy.
Subsequently, decreased CaM kinase II activity has been dem-
onstrated in numerous models of epileptogenesis and epilepsy
(8–10). These findings suggest that decreasing CaM kinase II
activity in brain may produce spontaneous epileptiform activity.
Evidence for a direct role in decreased CaM kinase II activity
causing epileptiform discharges came from the observation that
transgenic mice having a null mutation for the ? subunit of CaM
kinase II developed epileptiform activity and epileptic seizures
from limbic structures (11). Although this observation strongly
implicates decreased CaM kinase II activity in the production of
pilepsy is a common neurological condition characterized by
spontaneous, recurrent synchronous discharges of a popu-
epileptiform activity, it does not exclude the possibility that
secondary developmental mechanisms, caused by decreased
CaM kinase II expression, are involved in the development of
limbic seizures. Thus, it is important to further evaluate the
effect of decreased CaM kinase II activity on neuronal excit-
ability. This study was designed to directly determine the effect
of decreasing ? CaM kinase II subunit expression on the
production of epileptiform activity in hippocampal neurons in
culture. The findings demonstrated that inhibition of the expres-
sion of the ? subunit of CaM kinase II resulted in increased
neuronal excitability and epileptiform discharges.
Materials and Methods
Materials. All materials are reagent grade and obtained from
Sigma or Fisher Scientific unless specifically noted. Antisense
and missense oligonucleotides were obtained from Operon
Technologies (Alameda, CA). Radiolabeled flunitrazepam was
purchased from DuPont?NEN. The monoclonal anti-CaM ki-
nase II ? subunit was purchased from Chemicon.
Hippocampal Neuronal Culture. Primary hippocampal cultures
were prepared by a modification of the method of Banker and
were prepared from 2-day-old postnatal rats (Harlan Breeders,
Indianapolis) and grown on a confluent hippocampal astroglial
feeder layer. Astrocytes were prepared from 2-day-old pups by
the methods of Abney et al. (14). The glial cultures were
maintained for 2 weeks in 60-mm dishes (Costar) and fed twice
weekly with MEM?10% FBS?2 mM L-glutamine?10 mM glu-
day before neuronal plating, the glial feed was replaced with
N3-supplemented neuronal feed. The N3supplement contained
25 mM Hepes (pH 7.4), 2 mM glutamine, 5 ?g?ml insulin,
100 ?g?ml transferrin, 100 ?M putrescine, 30 nM sodium
selenite, 20 nM progesterone, 1 mM sodium pyruvate, 0.1%
ovalbumin, 20 ng?ml T3, and 40 ng?ml corticosterone.
Hippocampal cells were plated at a density of 7.5 ? 105cells per
60-mm culture dish onto a confluent glial bed. Cultures were
maintained at 37°C under 5% CO2?95% air. Cultures were fed
three feedings per week (1?2 half media change) with
N3-supplemented Earle’s salts containing MEM. The glutamine,
MEM, and Hepes buffer were obtained from GIBCO.
Inhibition of CaM Kinase II Expression. Inhibition of CaM kinase II
? subunit expression was executed by the addition of antisense
Abbreviation: CaM kinase II, calcium- and calmodulin-dependent kinase II.
¶To whom reprint requests should be addressed. E-mail: RDeLorenzo@HSC.VCU.EDU.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073?pnas.080071697.
Article and publication date are at www.pnas.org?cgi?doi?10.1073?pnas.080071697
May 9, 2000 ?
vol. 97 ?
oligonucleotides for 3 days, unless otherwise noted. Antisense
(3 ?M 5?-GGTAGCCATCCTGGCACT-3?) or missense control
(3 ?M 5?-GGTCGCCATCAGGTCACT-3?) were added directly
to the culture media. The antisense oligonucleotide was com-
plimentary to rat mRNA at nucleotides 33–50 (E value ? 0.001;
National Center for Biotechnology Information, BLAST data-
base). Except for a complimentary match of rat DNA for the ?
subunit of CaM kinase II, there were no other matches for the
antisense oligonucleotide found in primary neuronal cultures
from the rat (National Center for Biotechnology Information,
BLAST database). The missense nucleotide, comprised of a
scrambled sequence of the antisense nucleotide, contained the
same content of nucleic acids, and the identical molecular weight
and chemical properties to the antisense oligonucleotide se-
quence. The missense control did not significantly align to any
known rat RNA or DNA sequences contained in the National
Center for Biotechnology Information BLAST database. In ad-
dition, the missense oligonucleotide did not match any known
RNA sequences found in other species with an E value ? 4.5
(National Center for Biotechnology Information, BLAST data-
base). Other control oligonucleotides tested included sense
oligonucleotide for the CaM kinase II ? subunit, and missense
oligonucleotides for ?-aminobutyric acid type A ?2 receptor
subunit (5?-CAACCGTCGTGGTGGGTCCAC-3?), transcrip-
tion factors including ?fos B (5?-AGATCTTCTCAAGTGCTT-
3?), and serum response factor (5?-TGAGCTACGTCG-
TAGCGC-3?). These additional missense oligonucleotides were
to Western analysis as described (13, 15). Immunoreactivity
against the ? subunit of CaM kinase II was measured by using
computer-assisted densitometry (Loats Associates, Westmin-
ster, MD). To ensure that changes in subunit expression were
within the linear range of visualization, immunoreactivity of
CaM kinase II expression was compared with a standard regres-
sion curve as described (13, 15, 16). Changes in CaM kinase II
subunit expression were expressed as percent of control expres-
sion. To control for the selectivity of the CaM kinase II ? subunit
antisense treatment, we evaluated the effect of antisense treat-
ment on the expression of similar molecular weight proteins and
proteins with similar turnover rates. CaM kinase II ? subunit
blots as described in detail (16). The structural proteins tubulin,
microtubule-associated protein 2, and synapsin were determined
by densitometric quantification of SDS?PAGE protein patterns
as described (13, 15, 16). The antisense oligonucleotide directed
against the CaM kinase II ? subunit did not affect the expression
of the CaM kinase II ? subunit, or any of the structural proteins
Determination of CaM Kinase II Activity. Determination of CaM
kinase II substrate phosphorylation was performed exactly as
described (15). Standard phosphorylation reaction solutions
contained 41 ?g of protein, 60 ?M Syntide II (Sigma), 10 mM
and ? 1 ?g of calmodulin. Standard reactions were performed
in a shaking water bath at 30°C. The phosphorylation reaction
was initiated by the addition of Ca2?, allowed to continue for 1
min, and stopped by the addition of 20 ?M EDTA. Assay
solution (10 ?l) was immediately blotted onto phosphocellulose
filter paper, P-81 (Whatman), as described (15). Each reaction
was quantitated in triplicate. P-81 filter paper was then washed
three times in 50 mM phosphoric acid, rinsed with acetone, and
allowed to air dry. Radioactive phosphate was quantitated by
scintillation counting as described (15).
Electrophysiological Recording. Whole-cell voltage clamp analysis
was used to determine the effects of decreased CaM kinase II ?
subunit expression on neuronal excitability (17). For voltage-
clamp analysis, cultures were placed on the stage of an inverted
microscope (Nikon) and continuously perfused with base-
recording solution containing 1 mM tetrodotoxin, 25 mM 2-ami-
2,3-dione, 145 mM NaCl, 2.5 mM KCl, 10 mM Hepes (pH 7.3),
1 mM MgCl2, 2 mM CaCl2, and 10 mM glucose. The osmolarity
was adjusted to 325 mOsm with sucrose. Patch electrodes (2–4
M? resistance) with pipette solution containing 140 mM CsCl,
1 mM MgCl2, 10 mM Hepes (pH 7.2), and 1.1 mM EGTA were
used and the solution was adjusted to 310 mOsm with sucrose.
After the patch was established, the membrane potential was
clamped at ?50 mV and neuronal recording was performed with
an Axopatch 1D amplifier.
Inhibition of CaM Kinase II Expression. Hippocampal neurons in
culture were exposed to either antisense oligonucleotide specific
for the mRNA for the CaM kinase II ? subunit or missense
(scrambled antisense) oligonucleotide. To determine the level of
CaM kinase II ? subunit expression, Western analysis of ho-
mogenates isolated from cultures after specific periods of time
for exposure to either control or antisense oligonucleotides was
performed (Fig. 1). In agreement with reports (13, 18), the brain
maintains a high level of CaM kinase II protein level expression
(Fig. 1A). Treatment of neuronal cultures with antisense oligo-
nucleotide directed against the CaM kinase II ? subunit resulted
in a significant decrease (53.4 ? 6.0%, n ? 4) in ? subunit
expression after 3 days of exposure (Fig. 1 A and B). In addition,
inhibition of CaM kinase II ? subunit expression resulted in a
35.4 ? 12.3% decrease in calcium-dependent substrate phos-
phorylation activity when compared with missense control (Fig.
1C; n ? 4, P ? 0.001, Student’s t test).
To determine the specificity of the antisense oligonucleotide
treatment, we evaluated the effect of antisense oligonucleotide
exposure on other proteins. Under the conditions used to inhibit
CaM kinase II ? subunit expression, we did not observe signif-
icant inhibition in protein expression of the CaM kinase II ?
subunit, nor to structural proteins including tubulin, microtu-
bule-associated protein 2, or synapsin (see Materials and Meth-
ods). The data demonstrate a selective inhibition in CaM kinase
II ? subunit expression with antisense oligonucleotide exposure.
To test for nonspecific effects of control oligonucleotide expo-
sure, sense oligonucleotide to CaM kinase II ? subunit and
missense oligonucleotides to other protein sequences were
tested. None of the missense oligonucleotides tested resulted in
significant effects on CaM kinase II ? subunit expression.
To determine the cellular location of the decrease in ? subunit
expression, immunocytochemical analysis was performed (Fig.
2). Immunoreactivity with a mAb directed against the ? subunit
demonstrated a high neuronal level of CaM kinase II expression
and a low to nondetectable glial expression of the enzyme (Fig.
2A) (13). Expression of the ? CaM kinase II subunit was
observed in the neuronal soma, dendrites, dendritic spines, and
nuclear regions. There was also a punctate level of expression
along neuronal dendrites which most likely represented the
location of synaptic contacts between neurons. Treatment with
antisense CaM kinase II oligonucleotides resulted in a substan-
tial decrease in ? subunit immunoreactivity (Fig. 2B) that was
observed throughout the neuronal soma and in the dendritic
processes. The decrease in CaM kinase II ? subunit expression
observed in the neuronal dendrites demonstrated that the inhi-
bition of kinase expression was observed in areas closely asso-
ciated with synaptic contacts.
Churn et al.
May 9, 2000 ?
vol. 97 ?
no. 10 ?
Effect of Inhibition of CaM Kinase II ? Subunit Expression on Neuronal
Excitability. To test the effect of decreased CaM kinase II ?
subunit expression on neuronal activity, intracellular recordings
were obtained in hippocampal cultures subjected to naive (no
treatment), missense control, or antisense oligonucleotides. Re-
cordings from naive or missense control cultures revealed a
resting potential of ??60 mV (n ? 10). Both naive and missense
control neurons displayed normal synaptic activity, with inter-
mittent action potentials (?0.4 Hz) (Fig. 3A). The synaptic
activity level observed in control cultures was not significantly
different between missense-treated sham and naive cultures
(17). To further evaluate the selectivity of oligonucleotide
exposure to hippocampal neurons in culture, the sense sequence
for the ? subunit of CaM kinase II and missense sequences for
other proteins including the ?-aminobutyric acid type A ?2
receptor subunit, and transcription factors including ?fos B and
SRF were tested with no significant effect on neuronal survival
or membrane excitability (see Materials and Methods).
In contrast to cultures treated with missense control oligonu-
cleotides, neuronal cultures treated with antisense oligonucleo-
tides against the CaM kinase II ? subunit showed increased
neuronal activity and epileptiform discharges (Fig. 3B). The
frequency of neuronal firing increased to a maximum level
averaging 15.8 ? 0.44 Hz for intermittent periods of time during
epileptiform discharges in the antisense-treated cultures. These
epileptiform discharges manifested a neuronal firing greater
than 3 Hz and ranged in duration from 20 s to 1–2 min. Neuronal
than 20 s was consistent with the definition of electrographic
seizure activity. The increased neuronal excitability associated
with inhibition of CaM kinase II ? subunit expression was also
associated with the development of paroxysmal depolarization
shifts (21 ? 3 mV, range 15–28 mV) and multiple spike firing.
decreased ? CaM kinase II activity and subunit protein expression. Hip-
pocampal neurons in culture were exposed to missense (scrambled anti-
sense) or antisense oligonucleotides complementary to the mRNA to the ?
subunit of CaM kinase II for 3 days. Cultures were then harvested, homog-
enized, and resolved on SDS?PAGE (see Materials and Methods). (A)
Representative Western analysis with a mAb directed against the ? subunit
shows a high level of expression under missense (control) treatment.
Exposure to antisense oligonucleotides resulted in a significant decrease in
CaM kinase ? subunit expression. (B) Densitometry quantification of CaM
kinase II ? subunit expression. Western blots were digitized and compared
with a standard CaM kinase II expression curve (see Materials and Meth-
ods). Western analysis showed that treatment with antisense oligonucle-
otides resulted in a significant decrease in subunit protein expression.
Antisense oligonucleotide exposure for 3 days resulted in a 53.4 ? 6.0%
inhibition in CaM kinase II ? subunit expression when compared with
missense-treated control cultures. (C) Inhibition of CaM kinase II ? subunit
expression resulted in decreased substrate phosphorylation. CaM kinase
II-dependent phosphorylation of exogenously added Syntide II was signif-
icantly reduced (35.4 ? 12.3% compared with a missense control) after
treatment with antisense oligonucleotide when compared with missense-
treated control.**, P ? 0.001, Student’s t test, n ? 4.
Inhibition of CaM kinase II ? subunit mRNA translation resulted in
subunit expression. Neuronal cells in culture were treated with missense
control or antisense oligonucleotides for 3 days. Indirect immunocytochem-
istry was performed with a mAb directed against the CaM kinase II ? subunit
(see Materials and Methods). Exposure to antisense oligonucleotides resulted
in a substantial decrease in CaM kinase II ? subunit immunocytochemical
staining in cell soma, dendrites, dendritic spines, and nuclear regions. The
figure shown was representative of 15 different experiments. Although im-
munocytochemical studies are not best suited for quantitative determina-
tions, exposure of neurons in culture to antisense oligonucleotides produced
a decrease in the ? subunit of CaM kinase II protein expression in all cultures
Immunocytochemical evaluation of the decreased CaM kinase II ?
www.pnas.orgChurn et al.
The paroxysmal depolarization shifts were not observed in naive
and missense-treated control cultures.
Inhibition of CaM Kinase II ? Subunit Expression Correlates with
Increased Neuronal Excitability. A significant decrease in CaM
24 h of antisense oligonucleotide exposure (Fig. 4A). After 24 h
of oligonucleotide exposure, the CaM kinase II ? subunit
enzyme expression level was 90 ? 4.0% of control values (n ?
was not observed until 2 days of antisense oligonucleotide
exposure (65 ? 10.8% control, n ? 4). Maximal inhibition of
CaM kinase II ? subunit enzyme expression was not observed
until after 60 h of antisense oligonucleotide exposure (46.6 ?
3.2% control). Approximately 53.4% inhibition of CaM kinase
II ? subunit enzyme expression was observed at all time points
measured after 2.5 days of exposure. No significant decrease in
? subunit expression was observed between missense (control)-
treated and naive cultures.
To determine whether or not inhibition of the CaM kinase II
? subunit expression correlated with increased neuronal excit-
ability, subunit expression was compared with multiple measures
of neuronal excitability. Epileptiform activity did not appear
until day 2 of antisense oligonucleotide exposure. Once ob-
served, burst duration averaged 2.5 ? 0.2 s per episode. How-
ever, spike frequency during an epileptiform episode increased
from no epileptiform discharges (day 0) to a maximal frequency
of 10.62 spikes?s after 3 days of oligonucleotide exposure (Fig.
4A). The percent inhibition of CaM kinase II ? subunit expres-
sion correlated with increased spike frequency with an r2? 0.945
To further correlate the inhibition of CaM kinase II ? subunit
activity in hippocampal neurons in culture. (A) Representative intracellular
recording from a missense-treated (control) neuron displaying background
spontaneous synaptic activity (n ? 10). An expanded record of spontaneous
spike activity shows a single spike and no alteration of baseline polarity. (B)
Representative intracellular recording from a neuron exposed to antisense
oligonucleotides demonstrated epileptiform discharges and a high level of
spontaneous synaptic activity (n ? 10). An expanded record of three bursts
shows numerous spikes superimposed on a large paroxysmal depolarization
sweep); and vertical bar represents 40 mV.]
increased neuronal excitability. (A) CaM kinase II ? subunit antisense oligo-
nucleotide exposure resulted in inhibition of CaM kinase II ? subunit expres-
sion and increased spike frequency. Both spike frequency (F) and decreased
immunoreactivity of CaM kinase II ? subunit protein (Œ) were measured as a
in the same cultures. A significant increase in spike frequency and inhibition
of CaM kinase II ? subunit expression was not observed until 2 days of
antisense oligonucleotide exposure, and maximal inhibition of kinase expres-
sion and spike frequency were observed after 2.5–3 days of oligonucleotide
exposure. (*, P ? 0.001 different from missense control, n ? 4, one-way
ANOVA). (B) Correlation between inhibition of CaM kinase II ? subunit en-
zyme expression and increased neuronal excitability. Linear regression anal-
spike frequency was performed. Both inhibition of CaM kinase II ? subunit
expression and spike frequency were measured in the same cultures (data are
expressed as means ? SEM for an n ? 4). Inhibition of CaM kinase II ? subunit
expression correlated with increased spike frequency with an r2? 0.945.
Inhibition of CaM kinase II ? subunit expression correlates with
Churn et al.
May 9, 2000 ?
vol. 97 ?
no. 10 ?
expression with increased neuronal activity, the CaM kinase II
? subunit was compared with percent seizure duration and
frequency of seizure episodes. The total epileptiform activity
observed per unit time and the frequency of seizure episodes
increased as increased inhibition of the CaM kinase II ? subunit
expression was observed (r2? 0.837, each). Percent seizure
duration of recorded time increased from 4% (day 1 of oligo-
nucleotide exposure) to a maximum of 37.3% (day 2.5 of
oligonucleotide exposure). After 3 days of antisense oligonucle-
otide exposure, percent seizure time was 34.9. The frequency of
observed seizure episodes increased as the increased inhibition
of CaM kinase II ? subunit expression was observed. Seizure
frequency increased from 1.2–1.4 seizure burst?min after 1–2
days of oligonucleotide exposure to 8–10 seizure burst?min after
2.5 days of oligonucleotide treatment.
Inhibition of CaM Kinase II ? Subunit Expression Produces Synchro-
nous Epileptiform Activity. To demonstrate whether decreased
CaM kinase II ? subunit expression produced synchronous
epileptiform discharges, multiple, simultaneous recordings from
two individual pyramidal neurons were performed. Whole-cell
voltage clamp recording was performed simultaneously on two
pyramidal neurons in the field of vision (?1 mm). In missense
control cultures, background intrinsic activity analogous to naive
neurons in culture was observed in both neurons (Fig. 5A). In
addition, neuronal activity was random. Inhibition of CaM
kinase II ? subunit expression resulted in synchronous, epilep-
tiform activity in numerous recordings (n ? 15). Two-cell
recordings demonstrated multiple, seizure-like events similar to
that observed in single-cell recordings (Fig. 5B). However, the
seizure-like events and synchronized spike firing were found to
occur simultaneously in both pyramidal neurons demonstrating
a network effect of inhibition of CaM kinase II ? subunit
expression-dependent increased neuronal excitability (Fig. 5C).
Inhibition of CaM kinase II ? subunit expression resulted in
increased synchronous epileptiform discharges analogous to
epileptiform seizure activity characteristic of models of epilepsy.
The studies presented in this manuscript describe the net effect
of inhibition of CaM kinase II ? subunit expression on neuronal
excitability. CaM kinase II activity was reduced by inhibition of
protein expression of the ? subunit. Treatment with oligonucle-
otides directed against the CaM kinase II ? subunit was selective
and did not affect the expression of either the CaM kinase II ?
subunit or other similar molecular weight proteins studied. Once
neuronal excitability increased. The increased neuronal excit-
ability was characterized by repeated, synchronous epileptiform
activity. Furthermore, the magnitude of inhibition of CaM
kinase II ? subunit expression correlated with three measures of
increased neuronal excitability. Missense oligonucleotide expo-
sure did not have any effect on neuronal excitability at any point
studied. Thus, selectively decreasing CaM kinase II activity
resulted in increased neuronal excitability and spontaneous,
recurrent epileptiform discharges.
The data presented in this study demonstrate that exposure of
neurons in culture to oligonucleotides complimentary to the
CaM kinase II ? subunit mRNA (at nucleotides 33–50) resulted
in inhibition of the subunit expression. The decreased expression
of CaM kinase II was observed immunocytochemically in the
soma, neuronal processes, and synaptic regions (Fig. 2). CaM
kinase II is highly expressed in neurons relative to glial cells (4,
19, 20). The ? subunit of CaM kinase II is highly enriched also
in the synaptic regions of excitatory neurons, and is homologous
to the major postsynaptic density protein (21–23). Thus, CaM
kinase II is highly expressed in synaptic regions, in close vicinity
to neurotransmitter receptors. To establish such a high level of
dendritic expression, it has been shown that neurons synthesize
the ? subunit of CaM kinase II in the dendritic processes in situ
by inhibition of mRNA translation would be expected to be
II activity in this region of high neurotransmitter receptor
expression would be expected to significantly alter neuronal
Transgenic mice that express a null mutation for the ? subunit
of CaM kinase II, and manifest decreased enzyme activity,
demonstrate a decreased level of long-term potentiation and
decreased performance on behavioral learning models (26, 27).
The original observations described a decrease in synaptic
potentiation and spatial learning in genetically mutated mice
when compared with nonmutated mice. These mice also exhib-
ited an aberrantly high neuronal excitation level and displayed
recordings from two neurons. Whole-cell voltage-clamp recordings were
obtained from two neurons by dual electrodes. (A) Representative simulta-
neous recordings from missense oligonucleotide-treated neurons in culture
displayed nonsynchronous background neuronal activity (n ? 10). (B) Repre-
10). (C) Faster sweep speed of simultaneous recordings from B revealed a
prolonged duration of synchronous discharge activity, lasting between 1–3 s
and demonstrating a high degree of synchronization. N-1, neuron 1; and N-2,
(fast sweep); and vertical bar represents 20 mV.)
Synchronous epileptiform discharges in simultaneous voltage-clamp
www.pnas.org Churn et al.