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Research Report
The slow Ca2+-dependent K+-current facilitates
synchronization of hyperexcitable pyramidal neurons
Jane Skov, Steen Nedergaard, Mogens Andreasen
Department of Physiology and Biophysics, Aarhus University, DK-8000 rhus C, Denmark
A R T I C L E I N F O A B S T R A C T
Article history:
Accepted 13 November 2008
Available online 25 November 2008
Studies on in vivo and in vitro epilepsy models have shown that progression and
maintenance of epileptiform activity can be affected by the slow Ca2+-dependent K+
current (IsAHP). This study aimed to investigate the influence of the IsAHP on population
activity and single cell activity during the transition from the interictal- to the ictal-like
phase of an epileptiform field potential induced by Cs+. Extracellular and intracellular
recordings were performed in area CA1 on 400 m thick hippocampal slices from adult male
Wistar rats. During maintained exposure to Cs+, the transition between the two phases
underwent a slow, time-dependent change, where synchronized population activity
gradually disappeared and a plateau-like prolongation of the interictal-like phase
emerged. In parallel, the size of the ictal-like phase increased. These effects could be
ascribed to a gradual block of the IsAHP and were mimicked by the IsAHP antagonistscarbacholine (2 M), isoproterenol (4 M) and Ba2+ (0.2 mM). Cessation of population activity
generally occurred without a concomitant decrease in firing rate of single CA1 pyramidal
neurons, but was accompanied by the disappearance of hyperpolarizing prepotentials,
indicating a shift in the mechanism of action potential initiation. These findings suggest
that the presence of the IsAHP increases the tendency of hyperexcitable neurons to fire in
synchrony, but at the same time serves to dampen the ictal-like activity that follows the
hyperexcitable state. Our data indicate that both effects can be attributed to the influence of
this current on the steady-state membrane potential in the period of the transition from
interictal- to ictal-like activity.
2008 Elsevier B.V. All rights reserved.
Keywords:
Rat
Hippocampus
Afterhyperpolarization
Cesium
Epileptiform
1. Introduction
Epileptic seizures are paroxysmal events in the central
nervous system caused by abnormal, excessive, synchronized
discharges in aggregates of neurons. From electroencephalo-
graphic recordings of patients with focal epilepsies, brief,sharp spikes can be observed between seizures, and are as
such referred to as interictal activity. The relation between
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Corresponding author. Fax: +45 86129065.E-mail address: [email protected] (J. Skov).Abbreviations: 4-AP, 4-aminopyridine; AHP, Afterhyperpolarization; APV, DL-2-amino-5-phosphonopentanoic acid; BIC, Bicuculline
methobromide; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; Cs-FP, Cesium-induced field potential; ImAHP, The Ca2+-dependent K+-
current underlying the medium afterhyperpolarization; IsAHP, The Ca2+-dependent K+-current underlying the slow afterhyperpolarization;
mGLuR, Metabotropic glutamate receptor; LY341495, (2S)-2-amino-2[1S,2S9-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)propanoic acid; SD,Spreading depression
0006-8993/$ see front matter 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.brainres.2008.11.043
a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m
w w w . e l s e v i e r . c o m / l o c a t e / b r a i n r e s
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interictal activity and seizures, also known as ictal activity,
is still a matter of debate, but it seems evident that ictal
activity cannot merely be attributed to a summation of
interictal discharges. The interplay of cellular mechanisms
causing the two different forms of epileptiform activity
appears to be highly complex, and interictal bursts have
been proposed both to precipitate ictal activity (Traynelis and
Dingledine, 1988) and to dampen it (Avoli, 2001).It is well established that extracellular Cs+ (35 mM) can
induce epileptiform activity in neocortical or hippocampal
brain slices (Hwa and Avoli, 1991; D'Ambrosio et al., 1998;
Janigro et al., 1997; Xiong and Stringer, 1999, 2001). Cs+ has
several known, and potentially epileptogenic, effects on
neurons and glia, and has been shown to induce spontaneous
epileptiform activities that depends on both synaptic (Hwa
and Avoli, 1991; Xiong and Stringer, 1999) and non-synaptic
mechanisms (Xiong and Stringer, 2001). In addition,long-term
application of Cs+ promotes an epileptogenic field potential
(Cs-FP) in area CA1of the rathippocampal slice,which requires
stimulation of the Schaffer collateralcommissural fibers but
persists in the presence of ionotropic glutamatergic and
GABAergic receptor antagonists (Skov et al., 2005). The Cs-FP
is biphasic, consisting of an initial positive phase, which seems
to depend on an enhancement of a synaptic signal, as the
response is sensitive to blockade of synaptic transmitter
release, in addition to a general enhancement in excitability
(Skov et al., 2005, 2006). Hitherto, the nature of the synaptic
signal has remained elusive. The positive phase is followed by
a long-lasting negative phase, which likely reflects a transient
increase in extracellular K+, as it is sensitive to alterations in
glial K+ buffering capacity (Andreasen et al., 2007). As the
positive and the negative phase resemble the interictal and
ictal events, respectively, induced by increasing extracellular
K+ (Jensen and Yaari, 1988; Traynelis and Dingledine, 1988),we
have, for descriptive purposes adopted the terms interictal-
like and ictal-like for the positive and negative phase,
respectively.
Preliminary observations have indicated that the transition
from interictal- to ictal-like activity is under the influence of
the Ca2+-dependent K+-current underlying the slow after-
hyperpolarization (IsAHP). Because of its susceptibility toregulation by neurotransmitters and drugs, the potential role
of the IsAHP in the induction and maintenance of ictal activityis of considerable interest. Indeed, the IsAHP has beensuggested to terminate epileptic afterdischarges (Traub and
Jefferys, 1994) andto prevent ictal discharges (Alger andNicoll,
1980). Blockade of the IsAHP has also been put forward as onesource for the progression of epileptiform activity (Martn
et al., 2001; McCormick and Contreras, 2001). A reduced IsAHPappears to be one of the hallmarks of several in vivo models of
epilepsy (Verma-Ahuja et al., 1995;Watts et al., 1993; Wu et al.,
2003) as is indeed found in dentate granula cells from patients
with clinical seizures (Williamson et al., 1993). However,
which specific cellular and network properties that are
influenced by the IsAHP, and how, is still not fully understood.The aim of the present study was therefore to investigate the
possible influences of the IsAHP on the shaping and progressionof the epileptiform activity induced by Cs+, including its
impact on population activity and on discharge of single
pyramidal neurons. By illuminating new aspects of the role of
the IsAHP, this study provides novel insights into the cellularmechanisms important for ictogenesis.
Some of the results have been presented in abstract form.
2. Results
After 4060 min application of Cs+ (5 mM) together with DL-2-amino-5-phosphonopentanoic acid (APV, 50 M), 6-cyano-7-
nitroquinoxaline-2,3-dione (CNQX, 10 M) and bicuculline
methobromide (BIC, 10 M), a fully developed epileptiform
field potential (Cs-FP) was recorded in area CA1 in response to
Schaffer collateralcommissural fiber stimulation. As
described previously (Andreasen et al., 2007; Skov et al., 2005),
when therecording wasmade in stratumpyramidale, theCs-FP
consisted of an initial positive phase followed by a longer
lasting (up to several seconds) negative phase (see Fig. 1).
Synchronous spike activity was present during the positive
phase and continued through the transition period into the
negative phase (Figs.1Aand2A).We monitored theevolvement
of theCs-FP in an 80 minperiod after itsappearance. The initial
burst was maximally expressed in the early stages of the
development of theCs-FP. In thefollowing period, the length of
the burst was gradually reduced. Eventually it became
restricted to the upstroke region of the positive phase. Thus,
after a period of 2060 min, spike activity was abolished in the
transition region between the two phases (Figs. 1A and 2A)
but continued during the initial part of the positive phase
and, usually, during the negative phase. Concomitant with
the alteration in firing, we noted that the decay of the positive
phase became slower, resulting in a progressive broadening
of this part of the Cs-FP to the point where it in some cases
attained the appearance of a plateau (Fig. 1A). Averaged
measurements revealed that this broadening took place
mainly during the later stage of the development of the Cs-
FP (Fig. 1B).
In an attempt to quantify the changes in synchronized
firing during the transition from the positive to the negative
phase of the Cs-FP, we applied a measurement referred to as
the coastline index (Korn et al., 1987; see also Experi-
mental procedures). The limits of the transition period were
tentatively defined as the part of the response stretching
from the peak of the positive phase to 200 ms from the
stimulus (Fig. 2A). As a control measure, we included the
coastline index for the pre-peak region, stretching from the
stimulus to the peak of the positive phase (Fig. 2A). No
significant changes were found in the average coastline
index of the pre-peak region, which amounted to 10814%
of the initial value after 30 min (Fig. 2B, n =7, P =0.57). Incontrast, the coastline index of the transition period showed
a large decrease in all slices (Figs. 2A and B). The decrease
was progressive and reached 846% on average at the end
of the observation period (30 min, Fig. 2B), an effect which
was highly significant (P
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Since population spikes were not generally suppressed
over time (Figs. 2A and B) the decay of spike activity in the
transition period is likely to involve some specific process(es)
related to the evolvement of the Cs-FP.
2.1. A slow Ca2+-dependent K+ current is important for
the stability of the transition period
We hypothesized that the observed changes could reflect an
alteration of a membrane conductance which influences the
spiking for a period corresponding to the transition region. A
plausible candidate could be the Ca2+-dependent K+ current
underlying the slow afterhyperpolarization (IsAHP), becausethe time-course of this current is long enough to affect the
region of the Cs-FP in question. We therefore tested the effect
of blockers of this current. Application of carbacholine (2 M)
led to a rapid increase in the duration of the positive phase
(Fig. 3A). As exemplified in Fig. 3B, this effect was marked and
partly reversible upon wash out. On average, carbacholine
increased the duration of the positive phase from 12414 ms
to 25124 ms within 6 min (n =12, P
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A medium duration Ca2+-dependent afterhyperpolariza-
tion has been described in CA1 pyramidal neurons (Halliwell
and Adams, 1982). To block the current (ImAHP) behind thisafterhyperpolarization we applied apamine (0.11 M). This
treatment had no discernible effects on the Cs-FP (n=4, notillustrated), making it unlikely that ImAHP makes any majorcontribution to this potential.
To test if the presence of Cs+ is needed for the pharmaco-
logical effect on the interictal-like phase, we used 4-amino-
pyridine (4-AP, 0.5 mM) to generate a synaptic field potential
which is equivalent to the initial part of the Cs-FP (Andreasen
et al., 2007; Skov et al., 2005). In all slicestested (n=4), wefoundthatcarbacholine (2 M) gave a markedand reversible increase
in the duration of the field potential, from 82 10 ms to 203
24 ms (P=0.02).
2.2. The negative phase is enhanced concomitantly with
IsAHP blockade
In addition to the effects described above, we observed that
the presence of isoproterenol was associated with an enlarge-
ment of the negative, ictal-like phase of the Cs-FP (Fig. 4A).
Averaged measurements of the negativity at 1000, 1500 and
2000 ms after stimulation showed a significant increase after
8 min perfusion with 4 M isoproterenol (Fig. 4B, n =8,P
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continuous adjustment of the holding current. We found that
the amplitude of the slow AHP decreased considerably, and in
two recordings it was reversed to an afterdepolarization. On
average, the AHP was reduced to 416% of the initial value, a
highly significant change (Fig. 5B, P
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2.4. Change in discharge mechanism accompanies
population activity decay
In spite of a similar frequency, there were marked differences
in the properties of the firing before and after decay of
population activity. First, measurements in each neuron of the
mean transmembrane potential in the transition period (see
Experimental procedures) revealed a markedly larger depo-
larization of neurons recorded after the decay (average values
before: 41.4 2.7 mV, after: 27.6 3.8 mV; n =10). Thisdifference was statistically significant (P< 0.01). Since the
Fig. 5 Effects of Cs+ on the slow afterhyperpolarization and
action potential firing. (A) Membrane potential recordings
from a pyramidal neuron before and after 60 min application
of Cs+. The amplitude of the afterhyperpolarization was
measured 200 ms after (marked by an arrowhead) the end of
a 600 ms depolarizing current pulse (0.9 nA). The cell was
manually clamped to the pre-Cs+ membrane potential. (B)
The average amplitude of the slow afterhyperpolarization as
a function of time in Cs+ (n=5). (C and D) Membrane potential
recordings from two different pyramidal neurons during a
Cs-FP. The records were taken at a time where the
extracellular Cs-FP no longer displayed synchronized firing
in the transition region. Note that both cells fire action
potentials during the period corresponding to the transition
period(marked by a white bar) when themembrane potential
is held at the pre-Cs+ value by hyperpolarizing current
injection (lower panels). When the clamp was removed
(upperpanels) thecell in C displayedenhanced firing activity,
whereas the cell in D no longer fired action potentials in this
region.
Fig. 4 Isoproterenol enhances the negative phase. (A) Cs-FP
recorded before and after 8 min application of isoproterenol
(4M). The early parts (marked by double arrows) of the
responses are shown on an expanded time-scale below.Note
thatin addition to the disappearanceof spiking activityin the
transition region and the prolongation of the positive phase,
the negative phase is prolonged. (B) Averageamplitude of thenegative phase measured at four different time points after
the stimulation in control conditions (white columns, n= 8)
and after application of isoproterenol (black columns).
* denotes a statistically significant difference between control
and isoproterenol.
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contribution from action potentials was approximately equal,
thedifferencebetweenthesevalues mainly reflects a difference
in tonic membrane potential. The average resting membrane
potential was not significantly different in the two groups
(before: 60.52.6 mV; after: 58.72.1 mV; P>0.05). Second,beforethe decay, individual action potentialswere often seen to
be triggered abruptly from a transient hyperpolarizing
prepotential. Such prepotentials coincided with a larger
negative deflection in extracellular voltage (Fig. 6A), which
indicated that these action potentials were triggered by a
depolarization of the transmembrane potential caused by
transfer of current from neighboring neurons (field effects,
see Jefferys, 1995; Taylor and Dudek, 1984). Conversely, after
decay of the population activity, action potentials were usually
preceded by a depolarizing prepotential (Fig. 6B). In order to
obtain a quantitative estimate of this possible change in
discharge property, we counted, in one sweep for each neuron,
the number of action potentials which displayed a hyper-
polarizing prepotential. The prepotential was defined as the
mean potential 02 ms before the action potential minus the
mean potential in the preceding 2 ms, i.e. 24 ms before the
action potential. Application of this analysis in 10 neurons,
recorded before the decay of population activity, revealed that
hyperpolarizing prepotentials were present in 41 out of 62
action potentials (66%). In 8 neurons, recorded after the decay,
only one action potential out of 46 had a hyperpolarizing
prepotential (2%).
2.5. The possible involvement of metabotropic glutamate
receptors in the development of the Cs-FP
Finally, we sought to address the cause of the Cs+-induced
block of the IsAHP. Since activation of metabotropic glutamatereceptors (mGluRs) is known to block the IsAHP (for review, seeAnwyl, 1999), we hypothesized that Cs+-induced tonic release
of glutamate onto mGluRs is the reason for the block of the
IsAHP and hence for the observed changes in the transitionperiod. In support of such a mechanism, we have previously
observed that the nonselective mGluR antagonist (2S)-2-
amino-2[1S,2S9-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)
propanoic acid (LY341495, 100 M) reduces the duration of the
positive phase by 21% on average (Skov et al., 2005). Our
hypothesis therefore predicts that co-application of Cs+ and
LY341495 will prevent the decay of synchronized activity. We
therefore appliedLY341495 (100M) after 20 minperfusionwith
Cs+, i.e. at a time long before theCs-FP had fully developed. We
found that the decrease in coastline index of the transition
period was 7815% in the presence of LY341495 (n=4, not
Fig. 6 Change in neuronal firing properties during decay of population activity. (A and B) Simultaneous intracellular
(upper sweep) and extracellular (lower sweep)recordings of the Cs-FP in its early phase (A) and, in the same neuron, after decay
of population spikes in the transition period (B). The horizontal broken lines indicate 50 mV. The baseline Vm was at resting
potential in both records. Note larger membrane depolarization during the action potential burst in B compared to A. Records
from the periods of action potential firing in the transition period (marked a and b) are shown enlarged in right panels.
Arrowheads in a mark the transient, hyperpolarizing deflections that precede action potentials. These show exact
correspondence in time to negative deflections in the extracellular potential, indicative of field interactions being the trigger
source for the action potentials. After decay of population firing the action potentials were preceded by a depolarizing
prepotential (b). Note lack of coincidence of action potentials with population spikes. Calibrations in B also apply to A. All
voltages are recorded with respect to a distant ground electrode.
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illustrated), a value which is not substantially different from
the 846% decrease observed in the absence of LY341495. An
unpaired t-test assuming equal variances (F-test: P=0.25) didnot show any statistical significance (P=0.42) between theslices exposed to LY341495 and the controls. Similar results
were obtained when Cs+ wasco-appliedwith atropine(n=3,notshown).
3. Discussion
The present study suggests that blockade of the IsAHP i) causesa reduction in population activity specifically during the
transition from interictal- to ictal-like activity, ii) induces a
plateau-like prolongation of the positive phase, and iii) leads
to an enhancement of the negative phase. These changes
occurred rapidly in response to carbacholine, isoproterenol or
Ba2+, which have the IsAHP as common target, but they alsodeveloped gradually during prolonged application of Cs+. The
latter effect seems to be ascribed to a slow antagonizing effect
of Cs+ on the IsAHP.
3.1. How does Cs+ block the IsAHP?
The reduction in AHP amplitude observed in Cs+ is unlikely to
result from a reduced driving force for the underlying K+
current, because the membrane conductance during the AHP
was severely reduced after application of Cs+, which is
strongly indicative of a channel block. The mechanism of
this blocking effect was not pursued in the present study, but
results from the literature and previous studies on our lab,
points to several possible causes.
Blockade of the IsAHP, through activationof mGluRs has beenreported in 4-AP induced epileptiform activity (Martn et al.,
2001). We found that prior blockade of mGluRs or muscarinic
receptorsdid notprevent theeffects of Cs+, indicatingthat these
receptors were not involved. Since the IsAHP is also sensitive tootherneurotransmitters,e.g. noradrenalinand 5-HT(Sah,1996),
it is conceivable that the observed effect results from a
combined activation of several metabotropic receptor types.
Alternatively, intracellularly accumulating Cs+ in pyramidal
neurons could exert a direct block of the Ca2+-dependent K+
channels (de Sevilla et al., 2006) or indirectly affect the channels
through interference with signal transduction mechanisms
(Brette et al., 2003). Such mechanism seems plausible, since it
requires transport of Cs+ across the membrane, which would
explain the slow time course of the block. Intracellular
accumulation of high concentrations of Cs+ has been demon-
strated in other tissues in response to long-term perfusion of
the ion (Schornack et al., 1997), and our previous studies show
that Cs-FP development is sensitive to blockade of the Na/K
pump (Skov et al., 2006), which is a known route for trans-
membrane transport of Cs+ (Akera et al., 1979). Finally, it cannot
be excluded that the epileptiform activity, in itself, somehow
could affect the IsAHP. In favor of the latter explanation is theobservation that wash out of Cs+ leads to a relatively fast
recovery of synchronized activity during the transition period
which occurs in parallel with the disappearance of the ictal-like
negative phase (Andreasen et al., 2007). Further studies will be
needed to fully resolve this issue.
3.2. Influences of IsAHP blockade on neuronal and
population discharge
Our data from intracellular recordings showed that the
majority of neurons (eight out of ten) recorded after block of
population activity, firedat frequencies thatwere not different
from those recorded before the block. These data therefore
point to desynchronization as the major cause of the block ofpopulation spike firing. The mechanism by which the IsAHPcontributes to spike synchronization is obviously different
from its influence on burst synchronization between CA3
pyramidal neurons (de Sevilla et al., 2006), which occurs at a
time-scale compatible with the slow kinetics of the IsAHP.Instead, the effect observed here is likely to be a more indirect
one, related to the effect of the IsAHP on neuronal membranepotential in the period after the initial burst (see below). The
frequency of population spikes was found to be significantly
higher than the discharge frequency of single neurons,
suggesting that it represents the composite activity of several
smaller neuronal aggregates (see Bikson et al., 2003a). Since
most of our recordings showed that during the loss of
synchronized activity there was no concomitant change in
the population spike frequency, it seems unlikely that
desynchronization involves a change in the number of
neuronal aggregates, but rather a gradual reduction of their
size. To explain this result, we suggest that membrane
depolarization following the IsAHP block could increase theprobability that some neurons would begin to fire indepen-
dently (i.e. fall out of phase with their neighbors) and hence be
dismembered from the aggregate. This interpretation is in
accordance withthe observeddifference in average membrane
potential andin the frequencyof prepotentials before andafter
suppression of population activity. It furthermore implies that
thereasonwhy thedischarge of individualcells could be largely
constant through this period, in spite of a pronounced
membrane depolarization, was because it shifted from an
external drive (field effect) to an internal drive (membrane
potential). In addition however, we observed in two out of ten
neurons a transient cessation of firing corresponding to the
transition period. This block was associated with plateau-like
depolarizationpositiveto actionpotentialthreshold,suggesting
that depolarization-induced inactivation of Na+ channels could
be the cause. If a considerable fractionof neurons enters such a
state of depolarization block, this will contribute further to a
reduced population activity, and hence partially explain the
observed run down.
3.3. IsAHP and epileptiform activity
Conclusive evidence for the role of the IsAHP under hyper-excitable conditions is still rather limited, although it has long
been proposed that blockade of this current is a key feature in
transition from interictal to ictal discharges (Alger and Nicoll,
1980; Dichter and Ayala, 1987). IsAHP blockade seems anecessary mechanism for 4-AP induced spontaneous epilepti-
form activity (Martn et al., 2001), and by reducing the slow
AHP, isoproterenol has been shown to increase the rate of
epileptiform discharges induced by either GABAA-blockade or
an increased extracellular concentration of K+ (Rutecki, 1994).
In agreement with these studies, the present results suggest
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that in the Cs+ model of epileptiform activity, blockade of the
IsAHP contributes to the hyperexcitable state that leads to theepileptiform burst activity. Specifically, we observed that the
negative, ictal-like phase of the Cs-FP was amplified in
response to IsAHP blockade. Furthermore, our data indicatethat the size of the ictal-like phase is correlated to the level of
tonic membrane depolarization during the transition period.
Discharge-dependent release of K+ is believed to be a majorcause of non-synaptic seizure induction (Yaari et al., 1986).
During ictal-like activity, however, the extracellular K+
concentration has been shown to remain elevated in periods
of interruptions of neuronal firing (due to a depolarizing
block), and the continued K+ release in that situation is most
likely promoted by persistent inward currents that depolarize
the membrane to maintain the driving force for K+-efflux
(Bikson et al., 2003b). In line with this notion, we propose that
under our experimental conditions (i.e. with action potential
activity preserved), an increase in steady-state membrane
depolarization can still favor such discharge-independent K+-
release and hence contribute to the progression of interictal to
ictal activity. More detailed studies of this particular aspect of
ictogenesis will be needed before its overall significance can be
assessed.
4. Experimental procedures
All experimental protocols were in accordance with university
guidelines for animal research and complied with Danish and
European law on the care and use of laboratory animals.
Experiments were performed on hippocampal slices prepared
from 41 male Wistar rats (250300 g). The rats were anesthe-
tized with isoflurane and decapitated. The brain was quickly
removed and placed in a dissection medium (see below) at
4 C. The hippocampus was dissected free and 400 m thick
slices were cut on a McIlwan tissue chopper. One slice was
immediately transferred to a recording chamber, where it was
placed on a nylon-mesh grid at the interface between warm
(3132 C) standard perfusion medium (see below) and warm
humidified carbogen (95% O2, 5% CO2). Perfusion flow rate was
1 ml/min. The slice was allowed to rest for 1 h before
recordings were started. The remaining slices were kept in a
storage chamber at room temperature.
4.1. Electrophysiology
We used borosilicate glass microelectrodes (1.2 mm o.d,
Harvard Apparatus, Edenbridge, UK) filled with 1 M NaCl (tip
resistance 525 M) for extracellular recordings, or 3 M KCland
0.1 M K+ acetate (tip resistance 5070 M) for intracellular
recordings. In combined recordings, the electrodes were
placed as closely as possible. A bipolar teflon-insulated
platinum electrode (tip diameter 50 m, intertip distance
25 m) placed in stratum radiatum at the border between area
CA3and area CA1was used for orthodromic stimulation of the
Schaffer collateralcommisural fibers with constant-current
pulses (50 s, 0.20.4 mA) at a frequency of 0.05 Hz.
Conventional recording techniques were employed, using a
high input impedance amplifier (Axoclamp 2A, Axon Instru-
ments) with bridge-balance and current-injection facilities.
Signals were digitized on-line using a labmaster A/D converter
and transferred to a PC employing pCLAMP acquisition
software (Axon Instruments). Signal analysis was performed
using pCLAMP analysis software.
Slices were accepted if they, during control conditions,
displayed a normal orthodromic field excitatory postsynaptic
potential with a single population spike (amplitude between 5
and 15 mV) and showed no additional spikes with supra-maximal stimulation.
Application of Cs+ sometimes leads to the occurrence of
spreading depression (Skov et al., 2005; Xiong and Stringer,
1999). After a spreading depression episode (SD), the field
potential was monitored closely and recordings were stopped
if the field potential did not fully return to its pre-SD level.
Recordings were also stopped if more than three SDs occurred.
With those criteria, we found no differences between results
obtained in the absence or presence of SDs, in agreement with
previous findings (Skov et al., 2005). In the data set where the
temporal changes during wash in of Cs+ were evaluated, only
experiments free of SDs were included.
When intracellular recordings were used, cells were
accepted if they had a resting membrane potential 15 M and action potential height >80 mV.
4.2. Data analyses
In extracellular recordings, all amplitudes weremeasured with
respect to the prestimulus baseline on three to eight averaged
traces. Since population spikes occurred during the rising
phase of the Cs-FP,measurements of therate-of-rise could not
be performed. The duration of the Cs-FP was measured from
the onset of the positive phase to the time where the potential
reached or crossed the pre-stimulus baseline level.
We used coastline index as a quantitative approximation
of the amount of synchronized spike activity during the field
potential. The coastline index express the total length of a line
between two points (Korn et al., 1987). We accomplished this
by using the Pythagorean Theorem to calculate the distances
between individual points on the traces digitized at 0.55 kHz.
The sum of the individual distances gives the coastline index
and has the unit:ffiffiffiffiffiffiffiffiffiffi ffiffiffiffiffiffiffiffiffiffiffi ffiffiffiffiffiffims2 +mV2
q. This parameter is influenced
by any perturbation in the recorded voltage; however fast,
repetitive signals, such as population spikes and high
frequency noise have far more impact on coastline index
than an underlying slow waveform. Therefore, for the coast-
line index to selectively reflect theamount of population spike
activity, the contribution from noise needed to be subtracted.
This was done for each experiment by obtaining the coastline
index from a section of the prestimulus baseline voltage,
where no population spikes were present (ie only containing
noise signals), and subtracting this index from the coastline
indexes obtained during the Cs-FPs from the same experiment
(it was assumed that the level of noise was constant during
each experiment). It should be noted that after noise filtration
the coastline index is highly sensitive to changes in both
frequency and amplitude of population spikes, and does not
discriminate between the two. To be able to pool data from
different experiments, it was necessary to compensate for
variation in the coastline index between experiments. This
was accomplished by normalizing the coastline index in each
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experiment with respect to the value recorded at time zero
(see below) set to 100%. All values of coastline index reported
here reflect the average of four individual traces.
When temporal development was considered, time zero
was set to be the time after startingperfusion with Cs+ where a
clear biphasic potential had developed with population spikes
occurring both during and after the positive phase (see Fig. 2).
This procedure was adopted in order to compensate for thedifferences in induction time of the Cs-FP.
The amplitude of the AHP was measured as the difference
between the pre-stimulus membrane potential and the
membrane potential measured 200 ms after the end of a
600 ms depolarizing pulse (0.9 nA).
The transmembrane potential was calculated as the
difference between the extracellular potential, measured by
an electrode placed close to the recorded neuron, and the
intracellular potential.
4.3. Statistical analysis
Values are given as meanSEM unless otherwise indicated.
For statistical evaluation the paired or unpaired t-test wasused as appropriate with a level of significance set at 5% (two-
sided value). Before the unpaired t-test was applied, theassumption of equal variances was checked using the F-test.n denotes the number of slices or cells used. For eachexperimental approach, slices from more than one animal
were used. In intracellular experiments, one cell from each
slice was studied.
4.4. Drugs and solutions
The composition of the dissection medium was (in mM): NaCl,
120; KCl, 2;KH2PO4, 1.25; HEPES acid, 6.6; NaHEPES, 2.6; NaHCO3,
20; D-glucose, 10;CaCl2,2;MgSO4, 2; bubbledwith carbogen. The
composition of the standard perfusion medium was (in mM):
NaCl, 124; KCl, 3.25; NaH2PO4, 1.25; NaHCO3, 20; CaCl2,2;MgSO4,
2; D-glucose, 10; bubbled with carbogen (pH 7.3). In experiments
where BaCl2 was applied,phosphate and sulphate was omitted
from the standard perfusion medium, in order to prevent
precipitation. Unless otherwise noted, the experiments with
Cs+ were all performed in the presence of CNQX (10 M), APV
(50 M) and BIC (10 M).
The pharmacological compounds were made up in stock
solutions of 1001000 times the required final concentration
and diluted in the standard perfusion medium as appropriate.
4-aminopyridine (4-AP), atropine, BIC, carbacholine, CNQX
and isoproterenol were purchased from Sigma, apamine from
Alomone Labs, APV from Ascent Scientific and LY341495 from
Tocris.
Conflict of interest statementNone of the authors has any conflict of interest to disclose.
Acknowledgments
This project was supported by grants from the Lundbeck
Foundation, The Aarhus University Research Foundation and
The Danish Medical Research Council. The authors would like
to express their gratitude to Bertha P. B Mortensen and Neven
Akrawi for excellent technical assistance.
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