Phase-Dependent Effects of Stimuli Locked to Oscillatory Activity
in Cultured Cortical Networks
Jan Stegenga,
*
Joost le Feber, Enrico Marani, and Wim L. C. Rutten
Biomedical Signals and Systems Group, Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands
ABSTRACT
The archetypal activity pattern in cultures of dissociated neurons is spontaneous network-wide bursting. Bursts
may interfere with controlled activation of synaptic plasticity, but can be suppressed by the application of stimuli at a sufficient
rate. We sinusoidally modulated (4 Hz) the pulse rate of random background stimulation (RBS) and found that cultures were
more active, burst less frequently, and expressed oscillatory activity. Next, we studied the effect of phase-locked tetani (four
pulses, 200 s
1) on network activity. Tetani were applied to one electrode at the peak or trough of mRBS stimulation. We found
that when tetani were applied at the peak of modulated RBS (mRBS), a significant potentiation of poststimulus histograms
(PSTHs) occurred. Conversely, tetani applied at the trough resulted in a small but insignificant depression of PSTHs. In addition
to PSTHs, electrode-specific firing rate profiles within spontaneous bursts before and after mRBS were analyzed. Here,
signif-icant changes in firing rate profiles were found only for stimulation at the peak of mRBS. Our study shows that rhythmic activity
in culture is possible, and that the network responds differentially to strong stimuli depending on the phase at which they are
delivered. This suggests that plasticity mechanisms may be differentially accessible in an oscillatory state.
INTRODUCTION
Multi-electrode arrays (MEAs) offer a means to investigate
synaptic plasticity on the small-network scale (
1–3
). Cultures
of dissociated cortical neurons create a monolayer of cells
on the MEA surface that are easily accessible for recoding
and stimulation, and may facilitate learning and memory
studies (
4
). The occurrence of spontaneous, synchronized
bursting in these nonstructured networks has made it difficult
to achieve consistent results on plasticity using dissociated
cultures (
5–10
). Bursts of action potentials, characteristic
for networks of dissociated neurons, resemble the type of
activity that is observed during early development of the
nervous system. This activity subsides as the brain starts to
receive input from sensory neurons (
11–14
); thus, a lack of
afferent external input to cultures may cause bursting. The
fact that bursts can be suppressed by random background
stimulation (RBS) supports this view (
7
). Given the high rate
of action potential firings, and the fact that NMDA receptors
are activated during bursts, it is reasonable to assume that
plasticity mechanisms are activated in an uncontrolled
man-ner (
15,16
). Furthermore, it has been shown that plasticity
mechanisms are more accessible by stimuli when bursts
are suppressed (
17–19
). Thus, the ability to suppress bursts
in cortical cultures may be beneficial for accessing and
assessing synaptic plasticity.
In this study, we took inspiration from the rhythmic
activity observed in the hippocampus, which is known to
be involved in long-term memorization (
20–24
) and learning
tasks (
22,25
). In particular, applying a train of stimuli (four
pulses, 200 s
1) at the peak of hippocampal oscillation
results in long-term potentiation (LTP), whereas the same
train of stimuli applied at the trough results in long-term
depression (LTD) of postsynaptic potentials (
22,23
). One
hypothesis about the mechanism involved is that the
oscilla-tion of inhibitory neurons modulates the excitability of
neu-rons that are postsynaptic to the stimulated neuron and
consequently modulate the direction of change (
25
).
Oscilla-tory activity, regardless of its origin, may thus have a
pro-found effect on the way in which stimuli are processed by
the network.
In this study, we modified the RBS algorithm by using
stimuli that were Poisson-distributed in time (average of 10
stimuli per second) and delivered to a fully randomized
sequence of locations. Inspired by the oscillatory
depen-dency of plasticity in vivo, we sinusoidally (4 Hz) modulated
the Poisson parameter. By doing so, we found that the
appli-cation of rhythmically modulated RBS (mRBS) in culture
serves two purposes. First, it suppresses bursts that would
otherwise obscure induced plasticity, and evokes oscillating
activity in the network. Second, it modulates excitability in
the culture such that phase-locked trains of stimuli have
effects that are phase-specific.
We assessed changes in the network using two different
methods. First, we considered the response to probe stimuli.
We found that changes in the magnitude of responses were
pathway-specific and indeed depended on the phase at which
the tetani were delivered. Second, we analyzed spontaneous
bursting activity. Previous studies have shown that the
spatiotemporal patterns of activity during bursts are stable
over periods of several hours (
26–28
) but can also be
changed by proper electrical stimuli (
29–31
).
Submitted July 28, 2009, and accepted for publication February 16, 2010. *Correspondence:j.stegenga@utwente.nl
Editor: Herbert Levine.
MATERIALS AND METHODS
Culturing
Cells were obtained from the neocortices of newborn Wistar rats. The cells were dissociated mechanically by trituration, and chemically by treatment with trypsin. MEAs produced by Multi Channel Systems (Reutlingen, Germany) were coated with poly-ethyl-imine to promote cell-to-surface adhe-sion. Next, a drop of plating fluid was placed in the center of the MEA and the cells were allowed to attach for 4 h, after which the plating medium was washed and nonadhering cells were removed. The plating concentration was 1 million cells per milliliter, which resulted in a monolayer of cells with a density of ~2500 cells per mm2at 2 days in vitro (DIV). We used Romijn’s chemically defined R12 medium (32) for plating, maintenance, and experimentation. Half of the medium was replaced every 2 days. Cultures were stored in an incubator at 37C, 5% CO2, and near 100% humidity. The
platinum-nitride MEA electrodes were 30 mm in diameter and spaced 200 mm apart in an 88 grid (excluding the corner electrodes). The electrodes were numbered in matrix form (e.g., electrode 45 was located at the fourth column, fifth row). A total of eight different cultures, taken from five different platings, were used. Culture ages ranged from 20 to 89 DIV, with most (13/17) recording sessions performed between 20 and 35 DIV.
Experimental setup
Measurements
We built our measurement setup around a commercially available MEA-recording setup (1060BC preamplifier, STG1002 stimulus generator) from Multi Channel Systems. Data were acquired at a 16 kHz sampling rate with the use of a 6024E card (National Instruments, Austin, TX), and controlled by custom LabView programs (National Instruments). During measurements, the MEAs were sealed with a semipermeable membrane (Multi Channel Systems) and the temperature was controlled at 36C. The CO
2level was
maintained at 5% and the culture chamber was heated from the top by a Peltier element to prevent condensation of the medium. Cultures were left to acclima-tize in the experimental setup for 30 min before experiments were initiated. The stimuli were always monopolar, biphasic (200 ms per phase, positive phase first) current pulses. During mRBS, the same pulse was used for all electrodes. During probing sessions and spontaneous recordings, spikes were detected using a threshold crossing algorithm and validated in real time using an algo-rithm described by Wagenaar et al. (33). During mRBS sessions, continuous traces of all 60 electrodes were saved because spike validation interfered with the timing accuracy of stimuli. These data files were analyzed offline in the same way as would otherwise have been done in real time.
Protocol
All 60 electrodes were randomly probed four times with three different amplitudes (between 8 and 20 mA) at 5 s intervals to determine the subset of electrodes to be used for mRBS. Ten to 13 electrodes whose poststimulus histograms (PSTHs; summed across all electrodes) showed a peak larger than baseline activity were chosen for further use. A single stimulus amplitude was selected for all electrodes and for the entire duration of the measurement. Once the electrodes had been chosen, the experiment consisted of evaluation periods interleaved with short periods of mRBS (Fig. 1). Each evaluation period included 1), a sequence in which a subset of electrodes were probed such that PSTHs could be calculated; 2), a period in which there was only spontaneous activity; and 3) another probing sequence. Probing sequences involved all electrodes that were also used for mRBS in fully randomized order, using fixed amplitudes at 5 s intervals and repeated 15 times.
mRBS stimulation
Stimuli were generated in a probabilistic manner in both time and space. At every stepdt, one randomly selected electrode was allowed to be acti-vated. The probability of activation (i.e., the rate) was time-varying, as:
rðtÞ ¼ r0
þ r1
cosðumtÞ:
(1)
Where the angular velocity of modulation, um¼ 2pfm, was chosen such thatfm¼ 4 Hz. Random activation events with instantaneous probability r(t)
were generated by comparing at each time step a pseudo-random number x, uniformly distributed between 0 and 1, to r(t) $ dt. The resulting time series was Poisson-distributed with a time-varying parameter (i.e.,r(t)). The average rate of stimulation,r0, was set to 10 s1, which was enough
to significantly suppress bursting (as discussed in Results; but see also Wagenaar et al. (7)). The maximal deviation (r1) was set to either 8 or 10 s1.
The time stepdt was set as small as technically possible, which was deter-mined at 20 ms. The sequence of stimulation sites (10–13 sites) was fully randomized. Consecutive stimuli on the same electrode were allowed.
Phase-locked tetani
At particular phases of the modulation frequency of mRBS, tetani consisting of four pulses at a rate of 200 s1were applied to one electrode that was not already being used for RBS. All experiments consisted of at least three mRBS stimulation sessions with phase-locked tetani. The control group was formed by sham stimulation sessions in which no stimuli at all were applied. The parameters used are summarized inTable 1.
We use cosine notation, such that 0corresponds to peak mRBS stimula-tion rate (18 or 20 s1) and 180to the minimal mRBS rate of 0 or 2 s1(see
Fig. 1). In addition, because of setup limitations, mRBS was suspended from 40 ms before tetanus to 40 ms after tetanus. The number of pulses per tetanus was chosen in accordance with Huerta and Lisman (22), who found that four pulses (at 200 s1) applied at the peak or trough of the hippocampal q oscil-lation were enough to induce LTP or LTD (respectively) with the same magnitude as traditional protocols. Subsequent groups used four to five pulses at either 200 or 400 s1(400 s1chosen to limit tetanus duration) (21,23).
Probe all electrodes
Probe session Spontaneous activity m RBS stim ulation 5 1 0 6 6 30 Time [min] 0° sham 180° P1 P2 P3 P4
FIGURE 1 Timeline of a measurement. First, all electrodes were probed with stimuli of three different amplitudes. Based on the array-wide PSTHs, a selection of 10–13 electrodes was made for further use. Next, evaluation periods were interleaved with mRBS stimulation or sham periods of no stim-ulation. An evaluation period consisted of two probe sessions and 30 min of spontaneous recording. Both probe sessions were used, such that P1 and P2 were compared with P3 and P4 in the analysis. At least two different settings were used (0and 180;long arrows in insets indicate tetani) in random order.
PSTH analysis
The network state was evaluated with the use of PSTHs made during eval-uation periods. Electrode-specific PSTHs were made of responses to probe stimuli delivered through each electrode used also for mRBS. A PSTH for each electrode was calculated as the average histogram of activity at 0–500 ms after probe stimuli, using bins of 5 ms. The change in area of the PSTHs was taken as a measure for changes in network responses:
D
i;j¼
area
after i;jarea
before i;jarea
before i;j(2)
Wherei is for every electrode, j denotes stimulus electrodes, and ‘‘before’’ and ‘‘after’’ are with respect to an mRBS intervention (Fig. 1). We disre-garded PSTHs with an areabeforesmaller than 10 spikes to decrease numeric
fluctuations caused by division by a small number. This also vastly reduced the amount of PSTHs that merely contained direct responses (i.e., responses that involved no or very few synapses). A normalized histogram of changes (all Di,jof all experiments) was made to visualize the aggregate results of
the applied interventions. We tested whether histograms originated from two different distributions by using a two-sample, single-sided Kolmo-gorov-Smirnov test (a¼ 0.05). Another measure that is more sensitive to the extremes of histograms is the potentiation ratio (PR), which is calculated as the number of potentiated (D > 20%) connections divided by the number of depressed connections (D < 20%). The PR takes into account the fact that most connections in the network remain unchanged. We derived the PR from the work of Chiappalone et al. (34), who also proposed the threshold of 20%. Although the relation between PSTHs (as described here) and synaptic plasticity is far from direct, several previous studies revealed concomitant changes when MEA activity was measured in parallel with intracellular activity (5,35–38).
Burst profile analysis
In addition to PSTHs, we analyzed bursts during spontaneous sessions using a previously described method (26). In brief, we calculated the array-wide spiking rate by convolving the spike-train with a Gaussian with a resolution of 1 ms. Whenever the resulting signal crossed a threshold, a part of the signal centered to the first local maximum after threshold crossing was selected. We also calculated the spiking rates of individual electrodes by convolving spike trains with a Gaussian. Thresholds were adjusted to the culture’s activity. The standard deviation used to calculate single electrode spiking rates was 5 ms. Selected episodes extended from 200 ms before, to 400 ms after the time of the maximum array-wide spiking rate. This was wide enough to capture the whole burst in all measurements. In accordance with earlier work, we call the Gaussian-smoothed, array-wide firing rate the ‘‘burst profile’’, and the electrode-specific smoothed firing rate the ‘‘phase profile’’. Before conduct-ing further analyses, we calculated the averages of the profiles over 5 min bins. This reduced the variability and computational load. We treated these averaged profiles as if they were individual profiles. Changes in profiles were calculated as the magnitude of the difference:
change
i;j¼ kprofile
jprofile
ik
isj(3)
wherei and j enumerate bursts in a measurement, and profilejis a 601-element
(i.e., 600 ms) vector describing either a burst profile or a phase profile. To assess changes, we created two groups. The between-group consists of changes between profiles that are separated in time by mRBS intervention, and the within-group contains changes between profiles that are not separated in time by mRBS. The within-group represents natural variability of profiles, whereas the between-group represents variability plus a possible effect of intervention. We used Student’st-tests to determine significance (a¼ 0.05). For clarity of presentation, the between-group changes depicted in Fig. 6 were normalized to the mean within-group changes.
RESULTS
mRBS
Fig. 2
gives an overview of mRBS combined with tetani
applied at 0
. The average rate of stimulation (r0
) was 10 s
1,
modulated with a rhythm (fm
) of 4 Hz and deviation (r1
)
of 8 s
1. We observed that activity lagged stimulation by
~13 ms. This corresponds to the typical response time of
the culture to stimuli, which is also reflected in the typical
PSTH. The interspike interval distribution of observed
activ-ity during mRBS strongly resembles that of the stimuli, with
a relative increase at intervals near 0.25 s due to 4 Hz
modu-lation. In contrast, the interspike intervals of spontaneous
activity are more broadly distributed, with the longer lags
(e.g., > 0.5 s) corresponding to the relatively quiet times
between bursts. It is particularly interesting that the number
of intervals smaller than 20 ms is ~5 times lower during
stim-ulation than during spontaneous activity, indicating less
bursting. The right panels in
Fig. 2
illustrate the burst
suppression by mRBS. By investigating rhythmic activity
instead of bursting activity, one can study plasticity in a
more controlled manner.
PSTHs
The PSTHs showed two phases in the responses (
Fig. 3
).
Many electrodes measured activity only at very short
laten-cies (<10 ms). Such early responses were dominated by
direct responses, which are antidromic or orthodromic action
potentials resulting from activation of an axon (
39
). Activity
at longer latencies involves synaptic transfers, and was
usually part of activity patterns that resembled network
bursts. In the majority of cases, the shapes of PSTHs before
and after intervention were similar, whereas the area
under-neath the curve changed.
In most experiments, a mix of increased and decreased
PSTH areas was found, even when one probe electrode
was considered, as shown in
Fig. 3
. The implication is that
changes were pathway-specific rather than stimulus-site
specific. Such an observation argues against the possibility
that the excitability of the stimulated neuron was somehow
changed. Rather, it suggests that the synaptic connections
between stimulated and observed neurons were changed.
Aggregate results of the changes in PSTH area of all
indi-vidual electrodes that were observed for the two different
mRBS settings and control experiments are shown in
TABLE 1 Parameters used for several modes of mRBSstimulation
Intervention Phase Pulses ITI
No. of experiments No. of cultures mRBS (in phase) 0 4 5 s 20 8 mRBS (out of phase) 180 4 5 s 20 8 Control (no stimulation) - - - 13 8 A sham stimulation session of equal duration was taken as control. Tetani were repeated every 5 s during 6 min of mRBS (seeFig. 1). The last two columns of
Fig. 4
. When tetani were delivered in-phase (at 0
) of the
modulation frequency, the PSTH areas increased
signifi-cantly from those observed when no stimulation was applied
(p
¼ 0.047, single-sided, two-sample Kolmogorov-Smirnov
test). Conversely, tetani delivered in antiphase (at 180
)
did not result in a significant decrease in overall PSTH
area (p
¼ 0.061).
The overall trend was toward a decrease in PSTH area.
This is best seen by dividing the area of the curve showing
a >20% increase by the area of the curve showing
a >20% decrease. The PR was 0.62 when no stimulation
11
100 ms 2 sp/ bin81
18
17
FIGURE 3 Example of PSTH changes. PSTHs in response to probe stimuli delivered to electrode 17 (dark shaded) are shown before (gray) and after (black) in-phase (0) mRBS stimulation. In the complete experi-ment, mRBS stimulation was applied through all shaded electrodes, and tet-ani were delivered through electrode 45 (hatched). Twenty-two PSTHs with an area over 10 (spikes) were considered responsive. Of these, the area of 13 PSTHs increased by at least 20%, whereas that of only two PSTHs decreased by at least 20%. The bin width used was 5 ms. Electrodes are numbered in column-row fashion. -100 -80 -60 -40 -20 0 20 40 60 80 100 0 0.5 1 1.5 2 In-phase mRBS Antiphase mRBS Control (sham)
Changes in PSTH area (%)
Fre
que
nc
y of observation (normalized)
FIGURE 4 Aggregate results for changes in PSTH area. Without stimula-tion, the dotted curve was found. The mean change in the absence of mRBS (D) was3.1%, indicating an overall decrease of PSTH area. With tetani applied in-phase (0) with mRBS, a shift toward increases in PSTH area was found (D:þ5.7%; solid line). The shaded area indicates the standard deviation of the histogram. Slight decreases in PSTH area were found for tetani applied at 180of mRBS (D:6.2%). Bin width: 2.5%.
0 10 20 30 40 50 60
0
40
80
120
160
Time [s]
Spikes/bin
Time [s]
Spikes/bin
0 10 20 30 40 50 60
0
40
80
120
160
1
3
2
0.01 0.1
1 10
0.01
0.1
1
10
100
0
0.2 0.4 0.6 0.8
1
0
10
20
30
40
50
60
70
80
90
Rate [/s]
Time [s]
Observations [normalized]
Inter event intervals [s]
1,
3
FIGURE 2 mRBS stimulation and activity. Top left panel: Histograms of stimuli (black) and activity (dashed) with tetani applied at 0. Activity lagged stimuli by ~13 ms and was scaled by a factor 0.2 for clarity. The activity was successfully modulated by 4 Hz mRBS stimulation. Lower left panel: Interevent interval distribution. Curves 1 and 2 show the interspike interval distributions during stimula-tion and spontaneous activity, respectively. Curve 3 shows the interstimulus interval distribution. Under stimulation, long quiescent periods disappeared, and intense short-latency firing decreased (by ~80%). Right panels: Congre-gate spike rate, in bins of 0.25 s, for 1 min of activity without stimulation (top panel) and during mRBS stimula-tion (lower panel). Stimulastimula-tion increased overall activity but also suppressed bursts and general variability in the overall spike rate.
was applied, 0.39 for tetani applied at 180
of the modulated
stimulation, and 1.84 for tetani applied at 0
.
Burst analysis
To examine the effect of our interventions on spontaneous
activity, we recorded 30 min of bursting activity before
and after each intervention (
Fig. 1
). The firing rates of
indi-vidual neurons during a burst for one particular example are
shown in
Fig. 5
. Although there is usually a short period in
which all contributing electrodes synchronize, there are also
differences between contributing electrodes. For example,
electrode 13 was usually early to fire, whereas electrode 86
contributed mainly to the late phase of bursts. The specificity
of electrode contributions (which we call phase profiles) is
caused by the underlying network connectivity and pathways
of activation. Changes in activity during bursts included
a shift in the time of the main phase of firing, and more (or
less) intense firing. In
Fig. 5
, the tendency was toward
broader profiles and higher peak activity, indicating that
the bursts had become more intense as a whole.
Overall, the analysis of bursts shows that in-phase tetani
were able to induce significant changes in activity patterns,
both in the aggregate firing rate (burst profiles,
p
¼ 0.040,
two-sided Student’s
t-test) and in individual electrode
contributions (phase profiles,
p
¼ 0.025). Tetani delivered
in antiphase of the mRBS were unable to induce changes
in either burst profiles (p
¼ 0.182) or phase profiles
(p
¼ 0.134). In fact, there was a tendency toward
stabiliza-tion of existing patterns, as the mean distance between
profiles was slightly smaller when antiphasic stimulation
was applied than in controls in which no stimulation was
applied.
Fig. 6
summarizes the statistical analysis of changes
in spontaneous bursts.
DISCUSSION
In this study, we investigated whether rhythmic activity in
culture could produce phase-dependent plasticity. To this
end, we first required that naturally occurring network bursts
be replaced by rhythmic activity. This was successfully
achieved by modulated and stochastic RBS, which decreased
the percentage of small, interspike intervals that would
nor-mally be found in network bursts, and at the same time
increased overall activity. These small interspike intervals
are most likely to induce a spike-timing-dependent synaptic
plasticity that may otherwise obscure or negate
experimen-tally induced changes in connectivity (
4
).
The PSTH analysis showed a general decrease in
respon-siveness for the no-stimulation controls, which is not
uncommon in regularly stimulated cultured neuronal
net-works (
40,41
). However, when tetani were applied at 0
,
an increase in overall PSTH area was observed. The decrease
in PSTH area by tetani applied at 180
was not significant,
but it may have been partially obscured by the general
decrease of PSTH area found in controls. Nevertheless,
applying mRBS with phase-locked tetani had an effect
in networks of dissociated neocortical neurons, which
depended on the phase at which the tetani were applied.
200 ms
50 /s
11
18
81
FIGURE 5 Examples of phase profiles before (gray) and after (black) mRBS stimulation with tetani at 0. The profiles are averages of the last 10 min of spontaneous recording before mRBS stimulation, and the first 10 min after. Spontaneous recordings from the same experiment as in
Figs. 3 and 4were used.
0.4 0.6 0.8 1.0 1.2 1.4 1.6
Change (normalized)
Control
In-phase mRBS
Antiphase mRBS
Changes in burst profiles
*
0.4 0.6 0.8 1.0 1.2 1.4 1.6Change (normalized)
Control
In-phase mRBS
Antiphase mRBS
Changes in phase profiles
*
FIGURE 6 Changes in burst profiles (left panel) and phase profiles (right panel) for different settings of mRBS stimulation. The bars indicate the mean change after intervention, relative to inherent variability of profiles. The mean changes induced by mRBS stimulation are compared with ongoing changes in the absence of stimuli (control). All profiles from all experiments were included. Error bars denote standard error of mean; *p < 0.05, two-sided Student’s t-test.
The burst analysis showed results similar to those obtained
by PSTHs, with only tetani applied in-phase with mRBS
resulting in a significant increase in distances between
pro-files as compared to controls. We did not test the duration
of these changes, but they extended at least 1 h after
interven-tion. In this study, recordings of spontaneous activity started
15 min after intervention and were stopped 15 min before
the next intervention. Therefore, the results from the burst
analysis may have been negatively influenced, especially
when one considers that probe stimuli were delivered during
these two blocks of 15 min, which may alter activity patterns
by themselves (
31
).
There may be another explanation for the reduced effect of
synaptic depression. It is possible that the average synaptic
efficacy is relatively low, and thus there is less opportunity
for a further decrease. Since most studies have focused on
potentiation using extracellular recordings, there is no
evi-dence to support this. However, Jimbo et al. (
5,36
) confirmed
a PSTH decrease by whole-cell patch-clamp recordings,
demonstrating that depression is possible and that PSTHs
are sensitive to depression.
Overall, our results indicate that tetani applied in-phase
with mRBS had a definite effect, whereas the effect of
anti-phasic tetani was much smaller at best. This difference in
effect suggests the importance of the network-wide firing
rate at the moment the tetani are applied. The efficacy of
tet-ani may be time-varying because mRBS modulates the firing
threshold for neurons that are postsynaptic to the stimulus
site. Because stimuli were generated at a relatively low
average rate in a probabilistic way, and mRBS was
discon-tinued from 40 ms before tetanus to 40 ms afterward, it is
unlikely that such scaling of excitability is a direct result of
stimulation. Rather, mRBS may excite neurons to oscillate
at an intrinsic oscillation frequency near the modulation
fre-quency. Cortical neurons have a resonance frequency near
4 Hz (
42,43
). In this respect, it is interesting that Wagenaar
et al. (
9,10
) previously combined RBS (fixed aggregate
rate of 50 s
1, cyclic electrode switching) with tetanic
stim-ulation (20 trains, 20 pulses/train, 20 s
1, 2 s between trains)
but found no significant changes in responses to test stimuli.
Our study shows that rhythmic activity and the phase of
tetanus delivery are important factors that strongly influence
further processing in a neuronal network.
The authors thank Remy Wiertz for preparation and maintenance of the cultures.
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