Prolonged Bursts Occur in Normal Calcium in

Hippocampal Slices After Raising Excitability and

Blocking Synaptic Transmission
Zhi-Qi Xiong and Janet L. Stringer
J Neurophysiol 86:2625-2628, 2001.

You might find this additional info useful...

This article cites 11 articles, 4 of which can be accessed free at:

http://jn.physiology.org/content/86/5/2625.full.html#ref-list-1

This article has been cited by 3 other HighWire hosted articles

New Type of Synaptically Mediated Epileptiform Activity Independent of Known
Glutamate and GABA Receptors
Jane Skov, Steen Nedergaard and Mogens Andreasen
J Neurophysiol, April 1, 2005; 93 (4): 1845-1856.
[Abstract] [Full Text] [PDF]

Stimulus and Potassium-Induced Epileptiform Activity in the Human Dentate Gyrus from
Patients with and without Hippocampal Sclerosis

Downloaded from jn.physiology.org on April 27, 2012

Siegrun Gabriel, Marleisje Njunting, Joern K. Pomper, Martin Merschhemke, Emilio R. G.
Sanabria, Alexander Eilers, Anatol Kivi, Melanie Zeller, Heinz-Joachim Meencke, Esper A.
Cavalheiro, Uwe Heinemann and Thomas-Nicolas Lehmann
J. Neurosci., November 17, 2004; 24 (46): 10416-10430.
[Abstract] [Full Text] [PDF]

Factors Underlying Bursting Behavior in a Network of Cultured Hippocampal Neurons

Exposed to Zero Magnesium
Patrick S. Mangan and Jaideep Kapur
J Neurophysiol, February 1, 2004; 91 (2): 946-957.
[Abstract] [Full Text] [PDF]

Updated information and services including high resolution figures, can be found at:
http://jn.physiology.org/content/86/5/2625.full.html

Additional material and information about Journal of Neurophysiology can be found at:
http://www.the-aps.org/publications/jn

This information is current as of April 27, 2012.

Journal of Neurophysiology publishes original articles on the function of the nervous system. It is published 12 times a year
(monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2001 by the
American Physiological Society. ISSN: 0022-3077, ESSN: 1522-1598. Visit our website at http://www.the-aps.org/.

Prolonged Bursts Occur in Normal Calcium in Hippocampal Slices
After Raising Excitability and Blocking Synaptic Transmission
ZHI-QI XIONG AND JANET L. STRINGER
Department of Pharmacology and Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030
Received 12 March 2001; accepted in final form 3 July 2001
Xiong, Zhi-Qi and Janet L. Stringer. Prolonged bursts occur in
normal calcium in hippocampal slices after raising excitability and
blocking synaptic transmission. J Neurophysiol 86: 2625–2628, 2001.
This study examined the conditions that are required for the appear-
ance of the long-duration seizure-like activity that can be recorded in
hippocampal slices. Spontaneous interictal activity was induced in
CA1 and CA3 by perfusing hippocampal slices with high potassium,
cesium, 4-aminopyridine, or tetraethylammonium chloride, in normal
levels of calcium. Synaptic transmission was then blocked by the
addition of neurotransmitter receptor blockers (6-cyano-7-nitroqui-
noxaline-2,3-dione, D,L-2-amino-5-phosphonopentanoic acid, and bicu-
culline) or the calcium channel blocker cadmium, resulting in com-
plete blockade of the interictal discharges and the appearance of
spontaneous seizure-like events (ictal-like discharges) primarily in
CA1 and the dentate gyrus. Blocking synaptic transmission in normal
artificial cerebrospinal fluid did not induce ictal-like discharges in
any region. The results demonstrate that ictal-like discharges can
appear in normal levels of extracellular calcium when chemical
synaptic transmission is blocked pharmacologically. The results
suggest that an increase in neuronal excitability and absence of
interictal activity promote the appearance of the longer ictal-like
discharges.
INTRODUCTION
Epilepsy is characterized by the periodic and unpredictable
occurrence of seizures. Clinical electroencephalographic ob-
servations show that epileptiform activity is composed of in-
terictal discharges, ictal events (seizures), and periods of post-
ictal depression. Although the distinction between an interictal
discharge and an ictal event can be debated, in general, the
interictal discharges are characterized by a brief burst of syn-
chronized paroxysmal activity lasting ⬍500 ms (usually 50 –
100 ms). Ictal events have an overall duration of seconds to
tens of seconds. Efforts to understand the mechanisms under-
lying epileptiform activity have focused primarily on in vitro
models of interictal discharges. Although physiological abnor-
malities may be identified in the interictal state, an understand-
ing of the mechanisms underlying the longer ictal events is also
important.
A major obstacle in studying seizure (ictal) activity has been
the lack of an in vitro model that closely mimics the in vivo
situation. Ictal-like discharges, which can last a few seconds up
to tens of seconds, were first described in the CA1 region
(Jefferys and Haas 1982; Taylor and Dudek 1982) and were
Address for reprint requests: J. L. Stringer, Dept. of Pharmacology, Baylor
College of Medicine, One Baylor Plaza, Houston, TX 77030 (E-mail:
janets@bcm.tmc.edu).
www.jn.org 0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society
RAPID COMMUNICATION
later observed in the dentate gyrus (Schweitzer et al. 1992)
when perfusing the slices with no added calcium and high
potassium solutions. Although extracellular calcium levels
have been recorded to fall as low as 0.5 mM after many
seconds of seizure activity (Pumain et al. 1985), the role of low
calcium levels in the onset of the synchronized seizure activity
is not known. The goal of this study was to determine whether
the ictal-like discharges could occur in normal calcium levels
Downloaded from jn.physiology.org on April 27, 2012
and, if so, what conditions were required for them to appear.
METHODS
Hippocampal slices were prepared by conventional methods from
Sprague-Dawley rats (100 –180 g, both sexes). After anesthetizing the
rats (ketamine 25 mg/kg, xylazine 5 mg/kg, acepromazine 0.8 mg/kg
ip), the brains were removed. Transverse slices (400 –500 ␮m)
through the hippocampus were cut with a Vibratome (Technical
Products International). Slices were placed in an interface-type cham-
ber and continuously perfused with artificial cerebrospinal fluid
(ACSF) at 32°C under a stream of humidified 95% O2-5% CO2.
Composition of the ACSF was (in mM) 127 NaCl, 3.5 KCl, 1.5 MgCl,
1.1 KH2PO4, 26 NaHCO3, 2 CaCl2, and 10 glucose. All solutions
were bubbled constantly with 95% O2-5% CO2. Slices were allowed
to equilibrate for 1 h before electrophysiological recording was begun.
Recording electrodes were made of microfilament capillary thin-
walled glass (A-M Systems, 0.9 mm ID, 1.2 mm OD) pulled on a
micropipette puller (P-87, Sutter Instruments). Electrodes were filled
with 2 M NaCl and had impedances between 4 and 10 M⍀. Recording
electrodes were placed in the cell body layer of CA1, CA3, and the
dorsal dentate gyrus. Synaptic transmission was blocked in some
experiments by the addition of 6-cyano-7-nitroquinoxaline-2,3-dione
(CNQX, 20 ␮M), D,L-2-amino-5-phosphonopentanoic acid (APV, 100
␮M), and bicuculline (20 ␮M) or perfusion with Cd2⫹ (200 ␮M).
Cesium chloride (Cs⫹), 4-aminopyridine (4-AP), tetraethylammo-
nium chloride (TEA), and cadmium chloride (Cd2⫹) were purchased
from Sigma Chemical (St Louis, MO). CNQX, DL-APV, and bicucul-
line methiodide were purchased from Tocris (Ballwin, MO). All
chemicals were dissolved directly into the perfusing solution. When
cadmium was added to the perfusing solution, the potassium phos-
phate was omitted and replaced with equimolar amounts of potassium
chloride.
RESULTS
The first experiments confirmed that perfusion with cad-
mium (Cd2⫹, 200 ␮M, n ⫽ 6) or neurotransmitter blockers
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.
2625
2626 Z.-Q. XIONG AND J. L. STRINGER

Downloaded from jn.physiology.org on April 27, 2012

FIG. 1. Effect of blocking synaptic transmission on epileptiform activity induced by high potassium. A: simultaneous chart
recordings of extracellular discharges in the CA1 (top), CA3 (middle), and the dentate gyrus (DG, bottom) after perfusion for 1.5 h
with 12 mM potassium. Spontaneous interictal discharges are present in both CA1 and CA3 regions. The small upward spikes in
the dentate gyrus, here and in subsequent figures, are far-field recording of the activity in CA3. The inset in A is a single evoked
response recorded in CA1 after 15 min of perfusion in the high potassium solution. B: same slice as in A. After 50 min of perfusion
with cadmium (Cd2⫹), spontaneous ictal-like discharges is present in CA1 and the dentate gyrus. The inset in B is a single evoked
response recorded in CA1 just after the interictal activity had stopped. The same stimulus intensity was used for the responses in
the insets in A and B. Two minutes later, no response was recorded in response to stimulation. C: spontaneous epileptiform activity
after perfusion for 60 min in 12 mM potassium. D: same slice as in C, addition of bicuculline, 6-cyano-7-nitroquinoxaline-2,3-dione
(CNQX), and D,L-2-amino-5-phosphonopentanoic acid (APV) blocked the interictal discharges in all regions and ictal-like
discharges appeared 1 h later. In A, the interictal activity is shown on the same time scale as the longer ictal-like activity. Elsewhere
the interictal activity is shown on a faster time scale to show the regularity of the discharges.

(CNQX, 20 ␮M; APV, 100 ␮M; and bicuculline, 20 ␮M, n ⫽
6) blocked synaptically evoked responses, and ictal-like dis-
charges did not appear in normal ACSF in any region of the
hippocampus with at least 2 h of perfusion.
Next, neuronal excitability was increased using elevated
extracellular potassium (Fig. 1, Table 1). Bath application of
12 mM potassium resulted in spontaneous interictal dis-
charges in CA1 and CA3 within 30 min (n ⫽ 33). The
dentate gyrus remained silent (Fig. 1, A and C). In eight
slices, the high potassium perfusion was continued for up to
3 h. In these slices, the interictal activity continued, and
ictal-like discharges were never recorded in any hippocam-
pal region. In the remaining 25 slices, synaptic transmission
was blocked by the addition of either cadmium (200 ␮M,
n ⫽ 13) or neurotransmitter blockers (CNQX, APV, and
bicuculline, n ⫽ 12), and the slices were monitored for the
TABLE 1. Ictal-like bursts in each condition and hippocampal region
presence of ictal-like discharges for at least 2 h. Within 20
min after switching to the perfusing solution containing
blockers of synaptic transmission, interictal activity stopped.
After prolonged perfusion (up to 40 min), ictal-like discharges
developed (Fig. 1, B and D). In the presence of cadmium,
ictal-like discharges were seen in CA1 in 40% of the slices and
in the dentate gyrus in 30% of the slices. In the presence of
neurotransmitter receptor blockers, ictal-like discharges devel-
oped in 33% of slices in CA1 and the dentate gyrus after 1 h
of perfusion (Fig. 1D). No spontaneous ictal-like discharges
were recorded in the CA3 region. Lower concentrations of
potassium (8 –10 mM) would induce interictal activity in CA1
and CA3 in all slices and ictal-like activity in CA1 and CA3 in
about one-third of the slices, but the longer ictal-like dis-
charges did not appear in the dentate gyrus, even after blocking
synaptic transmission.

Increase Neuronal Excitability

12 mM Potassium Cesium TEA 4-AP

Block Synaptic
Transmission Cadmium NT blockers Cadmium NT blockers Cadmium NT blockers Cadmium NT blockers

CA1 7/18 5/15 4/20 2/12 3/9 4/10 2/8 1/6

CA3 1/18 0/15 8/20 4/12 0/9 0/10 0/8 0/6
DG 5/18 5/15 11/20 6/12 2/9 2/10 1/8 1/6

Number of neurons in 12 mM Potassium is 33, in Cesium is 32, in TEA is 19, and in 4-AP is 14. TEA, tetraethylammonium; 4-AP, 4-aminopyridine; NT
blockers, neurotransmitter blockers (6-cyano-7-nitroquinoxaline-2,3-dione/D,L-2-amino-5-phosphonopentanoic acid/bicuculline); DG, dentate gyrus.

J Neurophysiol • VOL 86 • NOVEMBER 2001 • www.jn.org

 CONDITIONS REQUIRED FOR ICTAL-LIKE BURSTS 2627

Downloaded from jn.physiology.org on April 27, 2012

⫹
FIG. 2. Effect of blocking synaptic transmission on spontaneous epileptiform activity induced by 5 mM cesium (Cs ). A:
simultaneous chart recordings of extracellular discharges in CA1 (top), CA3 (middle), and the dentate gyrus (DG, bottom) induced
by perfusion for 1 h with 5 mM cesium. B: same slice as in A. After 40 min of perfusion with cadmium (Cd2⫹), spontaneous
ictal-like discharges developed in CA3 and the dentate gyrus. C: spontaneous epileptiform activity after perfusion for 1 h with 5
mM cesium. D: same slice as in C, addition of bicuculline, CNQX, and APV blocked the interictal discharges and ictal-like
discharges appeared after 50 min. The small-amplitude activity in CA1 is a far-field recording of the larger amplitude activity in
CA3.

To determine whether these results are specific for high DISCUSSION

potassium, in additional experiments excitability was increased
by perfusion with the potassium channel blockers, cesium,
TEA, or 4-AP (Table 1). Addition of cesium (5 mM, n ⫽ 32,
Fig. 2, A and C), TEA (5 mM, n ⫽ 19, Fig. 3A), or 4-AP (1
mM, n ⫽ 12) induced spontaneous interictal discharges that
were synchronized in the CA3 and CA1 regions. The dentate
gyrus was silent. Lower concentrations of 4-AP (100 and 500
␮M) would produce interictal activity, but no ictal-like activity
appeared after blocking synaptic transmission. Addition of
cadmium (200 ␮M), to block synaptic transmission, blocked
the interictal activity in both CA1 and CA3 in all slices tested
(n ⫽ 20 in cesium, n ⫽ 9 in TEA, n ⫽ 6 in 4-AP). Spontaneous
ictal-like discharges appeared after 40 – 60 min of perfusion
with cadmium in CA3 (40% of slices in cesium), CA1 (20% of
slices in cesium, 30% in TEA, 25% in 4-AP), and the dentate
gyrus (55% of slices in cesium, 20% in TEA, 12% in 4-AP;
Figs. 2B and 3B). Addition of neurotransmitter receptor block-
ers (CNQX, APV, and bicuculline) also blocked the interictal
activity in all slices (n ⫽ 12 in cesium, n ⫽ 10 in TEA, n ⫽ 6
in 4-AP). Spontaneous ictal-like discharges appeared after
40 – 60 min of perfusion with neurotransmitter receptor block-
ers in CA3 (33% of slices in cesium), CA1 (20% of slices in
cesium, 40% in TEA, 25% in 4-AP), and the dentate gyrus
(50% of slices in cesium, 20% in TEA, 17% in 4-AP; Figs. 2D
and 3C).
In this study, epileptiform activity was induced by perfusing
hippocampal slices with high potassium or potassium channel
blockers until spontaneous interictal discharges developed in
both CA1 and CA3 (in normal levels of calcium). Synaptic
transmission was then blocked by the addition of neurotrans-
mitter receptor blockers (CNQX, APV, and bicuculline) or the
calcium channel blocker cadmium, resulting in complete
blockade of the interictal discharges and the appearance of
ictal-like discharges in some slices. These results demonstrate
that ictal-like discharges can develop in normal levels of ex-
tracellular calcium, which is in agreement with previous stud-
ies (Patrylo et al. 1994). But the present experiments further
define the conditions required for the appearance of the ictal-
like events. The results suggest that before ictal-like events can
occur in the hippocampus there must be an increase in neuronal
excitability, which in these experiments was achieved by in-
creasing the extracellular potassium or adding potassium-chan-
nel blockers, but that this increase in neuronal excitability is
not sufficient. The present experiments suggest that, in addi-
tion, there must be either blockade of synaptic transmission or
elimination of interictal discharges.
Is it a reduction in synaptic transmission that allows the
appearance of the ictal-like bursts, or do frequent interictal
discharges suppress the ictal-like activity? There is evidence in
support of each of these possibilities. The long duration bursts

J Neurophysiol • VOL 86 • NOVEMBER 2001 • www.jn.org

2628 Z.-Q. XIONG AND J. L. STRINGER
FIG. 3. Effect of blocking synaptic transmission on epileptiform activity
induced by TEA. A: chart recordings of extracellular discharges in CA1 and the
dentate gyrus 40 min after beginning perfusion with TEA 5 mM. B: addition
of cadmium (0.2 mM) blocked the interictal discharges, and ictal-like dis-
charges appeared in CA1 and the dentate gyrus in some slices. C: in the
presence of bicuculline, CNQX, and APV, spontaneous interictal discharges
were blocked and ictal-like discharges appeared. Recordings in CA1 in A and
C are from the same slices. Recordings in the dentate gyrus in A and B are from
the same slice.
were first observed in zero-added calcium conditions (Jefferys
and Haas 1982; Schweitzer et al. 1992; Taylor and Dudek
1982), in which there is no synaptic transmission, suggesting
that the absence of synaptic transmission is critical. It is pos-
sible that in the presence of synaptic transmission that there is
sufficient GABA release or activation of inhibitory synapses to
terminate bursting. Reduction, or elimination, of synaptic
transmission may allow longer duration discharges to appear.
However, in bicuculline alone, which would block GABAA
receptors, longer duration discharges have not been observed
(unpublished observations). Although there is some contro-
versy about the interplay between interictal discharges and ictal
events, there is some evidence that frequent short discharges
will suppress longer duration discharges (Wilson and Bragdon
1993; Xiong and Stringer 1999). In the CA3 region of the
hippocampus in zero-added magnesium, it has been shown that
constant interictal-like activity can suppress the longer seizure-
like events (Anderson et al. 1986; Swartzwelder et al. 1987). In
CA1 in high potassium, cadmium has been shown to block the
interictal activity, but have no effect on the longer ictal-like
events (Jensen and Yaari 1988), suggesting different mecha-
nisms for the two different types of epileptiform discharges.
J Neurophysiol • VOL 86 • NOVEMBER 2001 •
Finally, in the dentate gyrus, frequent short bursts appear to
block the longer duration bursts (Xiong and Stringer 1999).
When the long-duration bursts were first observed in zero-
added calcium, in the absence of synaptic transmission, it was
postulated that nonsynaptic mechanisms were sufficient to
synchronize neurons (Jefferys and Haas 1982; Jensen and
Yaari 1988; Taylor and Dudek 1982). Nonsynaptic mecha-
nisms that may result in synchronization of neuronal activity
have been extensively investigated (see reviews by Dudek et
al. 1998; Jefferys 1995). It is generally accepted that nonsyn-
aptic mechanisms are sufficient to synchronize neuronal activ-
ity (at least in some conditions), but whether these mechanisms
operate to synchronize neurons in the presence of synaptic
activity is not known. If one assumes that these long-duration
bursts are only synchronized by nonsynaptic mechanisms, then
the fact that they can be generated in normal levels of extra-
cellular calcium suggests that nonsynaptic mechanisms may
operate in the presence of synaptic transmission.
This work was supported by National Institute of Neurological Disorders
and Stroke Grant NS-39941 to J. L. Stringer.
Downloaded from jn.physiology.org on April 27, 2012
REFERENCES
ANDERSON WW, LEWIS DV, SWARTZWELDER HS, AND WILSON WA. Magne-
sium-free medium activates seizure-like events in the rat hippocampal slice.
Brain Res 398: 215–219, 1986.
DUDEK FE, YASUMURA T, AND RASH JE. ‘Non-synaptic’ mechanisms in sei-
zures and epileptogenesis. Cell Biol Int 22: 793– 805, 1998.
JEFFERYS JG. Nonsynaptic modulation of neuronal activity in the brain: electric
currents and extracellular ions. Physiol Rev 75: 689 –723, 1995.
JEFFERYS JG AND HAAS HL. Synchronized bursting of CA1 hippocampal
pyramidal cells in the absence of synaptic transmission. Nature 300: 448 –
450, 1982.
JENSEN MS AND YAARI Y. The relationship between interictal and ictal parox-
ysms in an in vitro model of focal hippocampal epilepsy. Ann Neurol 24:
591–598, 1988.
PATRYLO PR, SCHWEITZER JS, AND DUDEK FE. Potassium-dependent prolonged
field bursts in the dentate gyrus: effects of extracellular calcium and amino
acid receptor antagonists. Neuroscience 61: 13–19, 1994.
PUMAIN R, MENINI C, HEINEMANN U, LOUVEL J, AND SILVA-BARRAT C. Chem-
ical synaptic transmission is not necessary for epileptic seizures to persist in
the baboon Papio papio. Exp Neurol 89: 250 –258, 1985.
SCHWEITZER JS, PATRYLO PR, AND DUDEK FE. Prolonged field bursts in the
dentate gyrus: dependence on low calcium, high potassium, and nonsynaptic
mechanisms. J Neurophysiol 68: 2016 –2025, 1992.
SWARTZWELDER HS, LEWIS DV, ANDERSON WW, AND WILSON WA. Seizure-
like events in brain slices: suppression by interictal activity. Brain Res 410:
362–366, 1987.
TAYLOR CP AND DUDEK FE. Synchronous neural afterdischarges in rat hip-
pocampal slices without active chemical synapses. Science 218: 810 – 812,
1982.
WILSON WA AND BRAGDON A. Brain slice models for the study of seizures and
interictal spikes. In: Epilepsy: Models, Mechanisms, and Concepts, edited
by Schwartzkroin PA. New York: Cambridge, 1993, p. 371–387.
XIONG Z-Q AND STRINGER JL. Cesium induces spontaneous epileptiform ac-
tivity without changing extracellular potassium regulation in rat hippocam-
pus. J Neurophysiol 82: 3339 –3346, 1999.
www.jn.org