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Management of synchronized network activity by highly active neurons

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2008 Phys. Biol. 5 036008

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IOP PUBLISHING PHYSICAL BIOLOGY
Phys. Biol. 5 (2008) 036008 (13pp) doi:10.1088/1478-3975/5/3/036008

Management of synchronized network

activity by highly active neurons
Mark Shein1,2, Vladislav Volman3,4, Nadav Raichman1, Yael Hanein2
and Eshel Ben-Jacob1,3,5
1
School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Sciences,
Tel Aviv University, Tel Aviv 69978, Israel
2
Department of Physical Electronics, School of Electrical Engineering, Tel Aviv University,
Tel Aviv 69978, Israel
3
Center for Theoretical Biological Physics, University of California at San Diego, La Jolla,
CA 92093-0319, USA
4
Computational Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla,
CA 92037, USA
E-mail: eshelbj@gmail.com

Received 19 May 2008

Accepted for publication 11 August 2008
Published 9 September 2008
Online at stacks.iop.org/PhysBio/5/036008

Abstract
Increasing evidence supports the idea that spontaneous brain activity may have an important
functional role. Cultured neuronal networks provide a suitable model system to search for the
mechanisms by which neuronal spontaneous activity is maintained and regulated. This activity
is marked by synchronized bursting events (SBEs)—short time windows (hundreds of
milliseconds) of rapid neuronal firing separated by long quiescent periods (seconds). However,
there exists a special subset of rapidly firing neurons whose activity also persists between
SBEs. It has been proposed that these highly active (HA) neurons play an important role in the
management (i.e. establishment, maintenance and regulation) of the synchronized network
activity. Here, we studied the dynamical properties and the functional role of HA neurons in
homogeneous and engineered networks, during early network development, upon recovery
from chemical inhibition and in response to electrical stimulations. We found that their
sequences of inter-spike intervals (ISI) exhibit long time correlations and a unimodal
distribution. During the network’s development and under intense inhibition, the observed
activity follows a transition period during which mostly HA neurons are active. Studying
networks with engineered geometry, we found that HA neurons are precursors (the first to fire)
of the spontaneous SBEs and are more responsive to electrical stimulations.

Abbreviations PDMS poly-dimethylsiloxane

SBE synchronized bursting event
MEA multi-electrode array
FI fraction of inter-spike intervals between 102
and 104 ms, relative to the total intervals of a Introduction
neuronal spike train
FS fraction of spikes fired by a neuron during the Electrical activity in the brain is comprised of evoked
network’s silent state (between synchronized activity (induced by external stimuli) and spontaneous
bursting events), relative to all the spikes fired (internally generated) activity (Kandel et al 2000). Over
by this neuron the years, considerable effort has been focused on studying
HA neurons highly active neurons the evoked activity (Adrian 1928) in an attempt to reveal the
relationship between cause (stimulus) and effect (responding
5 Corresponding author. activity). Recently, increasing research efforts have been

1478-3975/08/036008+13$30.00 1 © 2008 IOP Publishing Ltd Printed in the UK

Phys. Biol. 5 (2008) 036008
directed towards the investigation of spontaneous activity
and to studying the physiological mechanisms underlying the
generation and regulation of this activity. These studies are
motivated by an accumulating body of experimental evidence,
which suggests that spontaneous activity may play a decisive
role in shaping the structure of neural cell assemblies (Katz
and Shatz 1996, Zhang and Poo 2001, Hua and Smith
2004, Spitzer 2006). Examples include regulation of cell
migration and neurite navigation (Hanson and Landmesser
2004, Komuro and Kumada 2005), modification of dendrite
motility and morphology (Lohmann et al 2002) and the
fine tuning of synaptic strength (Mooney et al 1993). The
latter hints that hidden non-arbitrary spatio-temporal motifs
may exist in spontaneously generated electrical activity. If
so, it consequently implies that some innate, yet unknown,
mechanisms of activity maintenance, initiation and regulation
should exist. For example, according to the Hebbian
learning postulate, the way in which synaptic strength is
modified crucially depends on the relative temporal ordering
(correlation) between pre- and post-synaptic spikes (Hebb
1949, Bi and Poo 2001). At the same time, theoretical
models (van Rossum et al 2000) and experimental evidence
(Turrigiano 1999, Marder and Goaillard 2006) indicate that
neurons use intrinsic homeostatic regulatory mechanisms to
prevent the unconstrained growth of synaptic strength.
Altogether, the emerging picture is that a network level
(rather than a single cell level) approach is necessary in
studying the spatio-temporal patterns of spontaneous activity
and in searching for the mechanisms that initiate and regulate
this activity. Such an approach can be implemented by
utilizing cultured neuronal networks. Cultured networks that
can exhibit spontaneous activity in the absence of any external
electrical stimulations or chemical cues have been serving as
a valuable model system for studying spontaneous activity
(Maeda et al 1995, Segev et al 2002, 2004, Wagenaar et al
2006).
The spontaneous activity in cultured neuronal networks is
often marked by the existence of synchronized bursting events
(SBEs)—relatively short time windows of several hundreds
of milliseconds during which a large fraction of the cells
is engaged in intense firing (Maeda et al 1995, Segev et al
2001, Baruchi and Ben-Jacob 2004, Beggs and Plenz 2004,
Wagenaar et al 2006). The specific characteristics of the SBEs,
such as their duration and frequency, depend on experimental
conditions and on the developmental stage of the network.
Yet, this mode of collective behavior has been observed across
many different brain structures and preparations (Spitzer 1995,
Yuste 1997, Menendez de la Prida et al 1998, Feller 1999, Ben-
Ari 2001, Palva et al 2000, Garaschuk et al 2000, Khazipov
et al 2001). The time series of the SBEs has a non-arbitrary
temporal appearance with long-range temporal correlations
(Segev et al 2002, Hulata et al 2004). In addition, a network
can exhibit several types of SBEs. Each type is characterized
by its own distinct spatio-temporal patterns of neuronal firings,
reflected in a specific organization of inter-neuron correlations
(Segev et al 2004, Beggs and Plenz 2004, Baruchi and Ben-
Jacob 2004, Volman et al 2005, Raichman et al 2006). Recent
studies also reveal that the spatio-temporal pattern of the SBEs
2
M Shein et al
can endure chemical treatments that inhibit activity and even
hypothermia conditions (Rubinsky et al 2007). Even though
SBEs spontaneously emerge in developing networks, they can
also be artificially evoked by targeted electrical (Madhavan
et al 2007) and chemical (Baruchi and Ben-Jacob 2007)
stimulations. Combined with model simulations (Volman
et al 2004a, 2004b, 2007), these observations suggest that
SBEs may serve as a template for information processing and
storage, and can thus represent a new computational principle
utilized by brain networks (Sejnowski and Paulsen 2006).
In the present study, we used multi-electrode arrays
(MEAs) (Potter 2001, Segev et al 2002) in order to monitor
the electrical activity of in vitro cortical cultured networks.
The advantage of cultures grown on MEAs is that they afford
parallel recording of the electrical activity of many neurons for
very long periods of time. Additionally, cultured networks are
susceptible to geometrical manipulations (Sorkin et al 2006,
Segev et al 2002). Imposing such pre-defined morphological
constraints on the network’s structure enables the study of
small isolated networks. Consequently, it is possible to infer
the spatio-temporal patterns of the network’s activity and the
relations between the activities of many different recorded
neurons.
We confirmed previous results showing that while most
of the neurons fire during the SBEs, there is a small subset
of neurons (∼10% in our cortical cultures) whose activity
persists between the SBEs (Mao et al 2001, Streit et al 2001,
Darbon et al 2002, Volman et al 2004a, Sipila et al 2005,
Eytan and Marom 2006). It has been proposed and tested
in numerical simulations of realistic neural network models
(Volman et al 2004a, 2007) that such highly active (HA)
neurons may maintain and regulate the network’s spontaneous
activity. Following this theoretical work, we performed a
detailed statistical and time-series analysis of the special firing
characteristics of the highly active neurons. We found that
the activity patterns of these neurons are marked by distinctive
firing characteristics—unimodal distribution (versus bimodal
distribution of the rest of the neurons) in the inter-spike
intervals and long time correlations—that may be associated
with the regulation of the SBEs.
To further investigate the putative role of HA neurons in
the maintenance and regulation of the network’s spontaneous
activity, we studied the activity of cultured networks during
their development from a collection of isolated cells into
mature wired networks of interconnected neurons that show
intense spontaneous activity. Our results show that the early
network development is marked by a transient period, during
which a large fraction of HA neurons are detected. This
fraction gradually decreases during the course of the network’s
maturation. A similar effect was observed in the recovery of
networks from intense chemical inhibition of glutamatergic
synapses. The fraction of HA neurons gradually increased,
following the complete abolishment of spontaneous activity in
the network.
In addition, we found that HA neurons act as precursors
of the activation of SBEs, in the sense that they are typically
the first to fire during a synchronized bursting event. To
further understand this effect, we investigated the response
Phys. Biol. 5 (2008) 036008
of regular neurons and HA neurons to electrical stimulations.
We found that HA neurons responded with higher fidelity to
electrical stimulations regardless of the stimulation location.
However, no correspondence was found between electrodes
whose stimulation triggered activity in individual neurons or
in the whole network and electrodes on which HA neurons
were identified. These observations may imply that the HA
neurons respond to inputs from a large fraction of neurons and
may contribute to the generation and synchronization of SBEs.
Materials and methods
Preparation and growing of cultured networks
Dissociated cortical cultures were prepared as follows: the
entire cortices of (E18) Sprague Dawley rat embryos were
finely removed. The cortical tissue was digested with 0.065%
trypsin (Biological Industries, Beit Haemek, 03-046-1) in
phosphate buffered saline (Beit Haemek, 02-023-1) for 15 min,
followed by mechanical dissociation by trituration. Cells were
resuspended in a modified essential medium with Eagle’s salts
(Beit Haemek, 01-025-1), 5% horse serum (Beith Haemek
04-004-1), 5 mg ml−1 gentamycin (Beith Haemek 03-035-1),
50 μM glutamine (Beith Haemek 03-020-1) and 0.02 mM
glucose (BDH 101174Y), and plated on a poly-D-lysine (PDL)
(Sigma, catalog no. p-7889) covered electrode array with a
cell density of 3000–4000 cells mm−2 (∼1.5 × 106 cells were
plated per dish). The cultures were maintained at 37 ◦ C with
5% CO2 and 95% humidity using a specially designed life
support chamber. The growth medium was partially replaced
every 3–4 days. Since the medium replacements elicit a
temporary activity transient that lasts up to 2 h, data recorded
during this period were ignored.
Electrical activity recording
We used a commercial micro-electrode array which consists
of 60 substrate-integrated thin film micro-electrodes (MEA
chip, Multi Channel Systems). The electrodes are 30 μm
in diameter and are arranged in a rectangular array with a
500 μm spacing between the electrodes. Therefore, our
recording area covers 15 mm2 out of the 3 cm2 total area
of the dish. Extra-cellular recordings were collected utilizing
a low noise pre-amplifier board (B-MEA-1060, amplifier, gain
X1200 with a band-pass filter 200 Hz–5 kHz, Multi Channel
Systems). The signals recorded from the microelectrodes were
sampled at a 10 kHz sampling rate and stored on a personal
computer equipped with a 128-channel, 12-bit data acquisition
board (MC Card, Multi Channel Systems) and MC Rack data
acquisition software (Multi Channel Systems, version 3.2.20).
The impedance of the electrodes is 100–500 k at 1 kHz and
their bandwidth ranges from 10 Hz to 3 kHz, which permits the
recording of individual action potentials. Potential spikes were
initially identified according to a threshold crossing detection
(set to 4 standard deviation of the noise) and were later
sorted and discriminated from noise using a wavelet packet
decomposition-based algorithm (Hulata et al 2000).
3
M Shein et al
Electrical stimulation
Electrical stimulations were applied to the cultured network by
delivering voltage pulses to specific electrodes using a stimulus
generator (STG1008, Multi Channel Systems).
All pulses were biphasic (positive-then-negative) pulses,
500 mV in amplitude, with each phase lasting 1 ms. Electrodes
that showed neuronal activity were selected, so as to enable the
monitoring of changes in electrical activity at the locations of
the stimulations. For each electrode, a stimulation session
included three sub-sessions separated by 30 s intervals.
Each sub-session included ten stimulations separated by 5 s
intervals.
Patterning
Cultured networks with a defined geometry were constructed
by manually placing thin strips of poly-dimethylsiloxane
(PDMS), with cross-sections of 0.5 mm × 0.5 mm and several
millimeters length, onto the PDL-coated surface, before
placing the cells. PDMS strongly adheres to the MEA’s glass
surface and prevents electrical connection between neurons
on both sides of the PDMS strip. PDMS is a bio-compatible
substance and does not interrupt neuronal growth (Kane
et al 1999). These cultures did not show any significant
differences in comparison to unrestricted networks in terms
of the parameters we measured (also see Segev et al (2002),
Raichman and Ben-Jacob (2008)).
Prolonged chemical inhibition
Chemical inhibition was performed by applying both a-amino-
3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and
N-methyl d-aspartate (NMDA) receptor antagonists (20 μM
b-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 μM
(2R)-amino-5-phosphonovaleric acid (APV)) to the medium.
Due to thermal fluctuations and the small volume of the
medium, homogeneity in concentration was assumed to be
reached within several seconds.
Analysis methods
Identification of synchronized bursting events. The time
series of SBE occurrences (SBE loci) was calculated by a two-
step analysis: identification of all possible events followed
by elimination of false positive events. We first divided the
recording into 10 ms bins and summed over all neuronal spikes
within each bin to calculate the total population activity. Next,
we convoluted the population activity with a Gaussian (40 ms
in width) to achieve a smooth function (figure 1(C)). The exact
occurrence of the SBE was then determined by taking the time
at which the convoluted population activity function reached
local maxima (figure 1(B)). Our second step was to exclude
false positive identification of SBEs by merging maxima less
than 1000 ms apart (corresponding to the SBE refractory
period (Segev et al 2001)) and by eliminating maxima in
which less than N neurons fired simultaneously (N being 10–
20% of the total number of recorded active neurons in the
network). The duration of each SBE was defined as the time
Phys. Biol. 5 (2008) 036008 M Shein et al

1 (A )

Neuron number
*

56 *
(B )
intensity
Firing

(C )

5 10 15 20 25
Sec

Figure 1. Characterization of electrical activity in cultured cortical networks. The generic activity pattern consists of synchronized bursting
events (SBEs) separated by relatively long periods of low-frequency sporadic firing. A raster plot of the network’s activity, shown in (A),
demonstrates that each bursting event has its characteristic spatio-temporal structure (fingerprint). Yet, while most of the neurons are active
only during the SBEs, a small fraction (marked by asterisks on the right) exhibit persistent firing even during the long (seconds) periods of
network quiescence. In the population activity presentation, shown in (B), we plot the number of spikes per second per neuron as a function
of time bin. This presentation reveals that each SBE possesses the characteristic profile of a fast rise followed by a slower decay. We define
the peaks of the population activity as the temporal loci of SBEs. (C) Application of the burst detection algorithm (as explained in the
Methods section) yields temporal loci of SBEs (black vertical lines), along with their corresponding width (gray regions).

interval between the nearest upward threshold crossing before Results

the activity peak and the nearest downward threshold crossing
after the activity peak of the convoluted population activity
function. Consequently, on a coarse scale, we characterized
two states of the network’s activity: (1) the excited state—a
state exhibiting global network excitations and (2) the silent
state—a state during which only sporadic firing was observed
(figure 1(B)). The convolution of the population activity
function increases the SBE duration. Consequently, choosing
a particularly low value for the threshold to distinguish
between the two states (0.1% of the peak activity in each SBE)
reassures us that the active states included all spikes related to
the SBEs, including the first. Nevertheless, the total temporal
fraction of the SBE durations, out of the total recording length,
was less than 1% in all recordings.
Estimation of power spectral density. For long signals prone
to large fluctuations and non-stationary trends, the power
spectrum can be estimated by calculating a count-based
periodogram (Lowen et al 2001). To build the periodogram
of a spike time series, we proceed as follows. First, the
binary vector of the spike time series of each neuron was
partitioned into non-overlapping contiguous segments of equal
length T. Within each segment, a discrete sequence was formed
by further dividing T into M equal bins, and then counting
the number of events within each bin. A periodogram was
then formed for each of the segments according to SW (f ) =
M
1
|W (f )2 |, where W (f ) is a discrete Fourier transform of
length M for each of the segments. Finally, SW (f ) was
averaged over all segments to obtain S(f )—the count-based
periodogram of the sequence. In our analysis, we set T and M
to be 1 h and 2 min respectively.
Characterization of the network’s activity
We recorded the electrical activity of ten mature (>12 days
in vitro) cortical cultures, all of which exhibited spontaneous
collective activity. In figure 1, we show a typical raster plot of
the recorded network activity. Visual inspection of the raster
plot indicates that the network activity can be characterized by
two states. Most of the time, the majority of the recorded
neurons are silent and only sporadic action potentials in
some of the neurons are seen. From time to time, however,
many of the recorded neurons participate in SBEs—relatively
short (several hundreds of milliseconds) time windows
during which most of the recorded neurons are engaged in
intense electrical activity. In order to identify the temporal
occurrences along with the widths of SBEs, we performed
the following analysis. We first constructed the population
activity representation shown in figure 1(B) (as explained
in the Methods section). Observations on the population
activity during the SBEs revealed that it follows a stereotypical
profile—a fast rise (tens of milliseconds) followed by a
slower fall (hundreds of milliseconds)—suggesting that most
of the recorded neurons are rapidly activated at the onset of
synchronized bursting events. Using the population activity
function, we identified the exact temporal loci of the SBEs
(the peak of the activity function) along with their widths
(figure 1(C)), according to the procedure explained in the
Methods section. The above findings are in agreement with
previous reports and suggest that synchronized bursting events
may constitute a generic organizational motif shared by many
neuronal networks.

4
Phys. Biol. 5 (2008) 036008 M Shein et al

0.8

Cumulative probability
0.6

Regular

HA
0.4

0.2

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
FS

Figure 2. Definition of HA neurons in cultured cortical networks. For an individual neuronal spike time series (T = 10 h), we computed
FS—the fraction of action potentials fired by this neuron during the network’s silent state (between SBEs), relative to the total number of
spikes fired by that same neuron. This procedure was repeated for every recorded time series (N = 581 from ten cultures), and all results
were pooled to obtain a probability distribution of FS values across neurons. In the figure we plot the cumulative distribution, i.e. the
fraction of neurons with a value equal to or less than FS. The resulting plot shows that the majority of neurons (>90%) share a FS value that
is less than 0.1, meaning that they fired more than 90% of their spikes during SBEs and less than 10% of their spikes during the network’s
silent states. These neurons were termed regular neurons. The rest of the neurons (those that fired more than 10% of their spikes outside
SBEs) were termed highly active neurons (HA neurons).

Activity patterns of single neurons
Different neurons exhibit different characteristic firing patterns
during the same SBE (as seen in figure 1(A)), yet most of them
conform to the general rule of being strongly active during the
SBE and weakly active inbetween the SBEs. An exception to
this rule is a small subset of recorded neurons (∼10%), which
fire a considerable fraction of their action potentials outside the
SBEs, during the network’s silent state. In figure 1(A), three
such neurons are clearly shown (marked by asterisks on the
right), as their activity exhibits a significant number of spikes
inbetween the network’s synchronized events. The appearance
of such highly active neurons was consistently observed in
all the cortical cultures studied here (N = 10). Hence, we
proceeded by characterizing the special temporal features and
assessing the dynamical significance of the activity patterns of
these HA neurons.
For each recorded neuron, we quantified the extent of
its activity outside SBEs by calculating FS—the fraction of
spikes generated by the neuron during the network’s silent state
(between SBEs), relative to all the action potentials recorded
from this cell (i.e. during both the active and the silent network
states). We used this measure to define ‘regular’ neurons as
neurons for which FS < 0.1 and highly active neurons as those
for which FS > 0.1. In figure 2, we show results of this analysis
for 10 h neuronal spike trains (N = 581 from ten cultures).
Most of the recorded neurons (91%, N = 530) were classified
as ‘regular’ while approximately 9% of the recorded neurons
(N = 51) were classified as highly active neurons. To be
precise, we selected the threshold in FS according to the sharp
decrease in the fraction of neurons as a function of FS (which
occurs at F S ∼ = 0.1). This sharp decrease is reflected by
the change in the slope of the cumulative distribution shown in
figure 2. Such a threshold best distinguishes between ‘regular’
and HA neurons. Below, we show that, in agreement with the
above classification, the activity of HA neurons has additional
distinguished temporal features.
Statistical analysis. Inter-spike interval (ISI) distribution is
often used in the analysis of neuron firing profiles (Rieke
et al 1997, Koch and Segev 1998, Strong et al 1998, Segev
et al 2002, Beggs and Plenz 2003, Ayali et al 2004, Zochowski
and Dzakpasu 2004, Linder 2004) as it provides information
about the instantaneous firing rate of a neuron. As is shown
in figure 3, the ISI distribution of the HA neurons is markedly
distinguished from that of the regular neurons; the inter-
spike interval distribution of the regular neurons is typically
characterized by a bimodal distribution (figure 3(A)). This
structured distribution is associated with the existence of two
typical time scales—the intra-SBE time scale (hundreds of
milliseconds) and the inter-SBE time scale (>10 s). On the
other hand, the ISI distribution of a typical highly active neuron
is centered inbetween the two maxima of the distribution
of the regular neurons—between the inter-SBE times and
the intra-SBE inter-spike intervals (figure 3(B)). Analysis of
pooled data from several recorded neurons suggested that
these are the generic profiles that characterize HA neurons
(figures 3(C), (D)).
The putative role of HA neurons during network development
The activity of HA neurons between SBEs, in the absence
of global network activation, may be particularly relevant
for the development of dissociated cellular assemblies into
active neuronal networks (Marom and Shahaf 2002, Spitzer
2006). Therefore we took advantage of the possibility of using
MEAs for long-term, non-invasive recordings, in order to track

5
Phys. Biol. 5 (2008) 036008 M Shein et al

20 20
(A ) (B )
Regular neuron HA–neuron
15 15
% of intervals
10 10

5 5

0 0 2 4 6
0 0 2 4 6
10 10 10 10 10 10 10 10
20 20
(C ) (D )
% of intervals (Average)

Regular neuron HA–neuron

15 15

10 10

5 5

0 0 2 4 6
0 0 2 4 6
10 10 10 10 10 10 10 10
ISI [ms] ISI [ms]

Figure 3. HA neurons are marked by a distinct spike time series. (A) An inter-spike interval (ISI) distribution of a typical distribution of a
regular neuron has a characteristic bimodal shape. The bimodality reflects the clear separation of intra-SBE (∼100 ms) and inter-SBE
(>10 s) time scales. In contrast, a typical HA neuron exhibits a considerable fraction of its ISIs in the [100 ms, 10 s] regime, as is shown
in (B). The averaged inter-spike interval histograms for regular neurons ((C), N = 530, ten cultures) and for HA neurons ((D), N = 51, ten
cultures) suggest that the different characteristic forms of ISI distributions may be used as a pivot to discriminate between the two types of
cells. Error bars represent the standard deviation of the averaged values.

very small or no electrical activity during this early stage.

Last
Since only a small fraction of the neurons are active during
early development, it is practically impossible to detect the
SBEs at this stage. In light of this, the method used
neurons
Regular

to discriminate between HA neurons and regular neurons

# neuron

during early development cannot depend on SBE detection.

Consequently, we distinguished between the two types of
neurons according to their ISI profiles; regular neurons, in
contrast to the HA neurons, rarely exhibit ISIs in the interval
neurons

of [102, 104] ms (as shown in figure 3). Consequently, we

HA

First
0 5 10
Hours
Figure 4. Initiation of electrical activity in developing cultured
networks. We show here the activity raster plot of 22 neurons taken
at fifth day in vitro. For clarity of presentation, the individual
neuronal traces are rearranged from bottom to top according to the
time of the appearance of their first spike. During the first 5 h,
mostly HA-like neurons (below the red line) were active. The
collective activity emerged later as short time windows of rapid
neuronal firings (the SBEs), which were separated by longer
intervals of the quiescence state.
the correspondence between the activity of HA neurons and
regular neurons during the network’s development.
Visual inspection of a typical raster plot during the
network’s development (figure 4) indicates that HA neurons
are more abundant in the early stages of development. Two
such neurons are clearly shown in figure 4. At the same
time, most of the neurons in the developing network exhibit
defined HA neurons to be those with higher values of FI—the
15 20
fraction of ISIs in the interval [102, 104]. It is worth noting
that due to the variability in the ISI histogram of different
neurons, the transition in FI between regular neurons and HA
neurons occurs in the interval of 0.1–0.2. However, changing
the threshold between 0.1 and 0.2 did not yield qualitative
changes in the results. As a benchmark test for this measure,
we calculated the FI values for 10 h neuronal traces of regular
neurons (N = 530, ten cultures) and HA neurons (N = 51,
ten cultures) which were classified using the FS measure
(FS = 0.1 was used as the classification threshold). The
average FI for regular neurons and HA neurons was 0.05 ±
0.06 and 0.31 ± 0.25 (mean ± STD), respectively, providing
support to the validity of this measure as a tool to differentiate
between regular neurons and HA neurons at the early stages
of network development.
We used FI to analyze the network’s electrical activity
during early development. Cultures were continuously
recorded for several days, starting from the second day in vitro.

6
Phys. Biol. 5 (2008) 036008
100
(A )
80
% HA-neurons
60
40
20
0
0 20 40 60 80
Time [hours]
100
(B )
CNQX Wash
80
+APV
% HA-neurons
60
40
Control
Chem. inhibition
20
Wash
0 network. More specifically, mature networks (>12 days
0 20 40 60 80
Time [hours]
Figure 5. The activity of HA neurons is correlated with the
homeostatic regulation of the network’s activity. (A) In the
developing cultured network, the fraction of HA neurons (blue
circles) is initially high, and decreases gradually during the course
of the network’s development. The black line is the smoothed
average over data from several experiments (N = 7). (B) Expression
of HA-neuron-like activity patterns is enhanced under acute
blockage of glutamateric synaptic transmission. The network’s
activity was completely abolished several seconds after the
application of AMPA and NMDA antagonists. In the following
hours, several neurons gradually began to exhibit firing patterns
which were characterized by high values of FI. This was manifested
as an increase in the observed number of HA neurons. Following
the wash-out of antagonists, the network’s activity began to recover
along with the simultaneous decrease in the fraction of active HA
neurons. The delay between the application of inhibitors and the
increase in HA-neuron activity varied between different cultures;
however, all three examined cultures (12–22 days in vitro) showed
the same trend of HA-neuron activation.
Individual neurons began to generate action potentials after
3 days in vitro, and the number of recorded active neurons
increased with time. In most cases, the first neurons to start
firing also exhibited activity patterns associated with those
of the HA neurons (figure 4). After this initial increase,
the number of HA neurons did not change significantly over
the first several days of activity. In contrast, the number
of neurons showing regular patterns of activity drastically
increased throughout this period. The activity of single
neurons did not exhibit transitions from HA patterns to a
regular pattern or vice versa over this time of measurement.
To quantitatively estimate the probability of a neuron in
the network to express HA patterns of activity during early
development, we first divided the entire recording into non-
overlapping time windows of 1 h each. For every window,
we then calculated FI for all the active neurons in that
same temporal window and classified every neuron as regular
(FI < 0.13) or highly active (FI  0.13). In figure 5(A), we
show the fraction of HA neurons as a function of time starting
from the first detected spike in the network. As can be
M Shein et al
seen, during the first stages of development, the activity is
marked by a relatively high fraction of HA neurons and this
trend gradually decreases along with the intensification of the
network activity as it continues to develop. These observations
suggest that HA neurons may play a role in the initiation of
the network’s activity.
The putative role of HA neurons during recovery from
100 chemical inhibition
Our findings in the previous section suggest that persistent
firing in single HA neurons is evoked when activity levels
in the network are low. This may hint that HA neurons are
recruited by the network when the network fails to sustain
a certain sufficient level of electrical activity. To further
test this hypothesis, we analyzed the activity of networks
during prolonged chemical inhibition of excitatory synapses,
which strongly reduced the total level of activity in the
in vitro) showing normal levels of network activity with
characteristic patterns of synchronized bursting events (as in
figure 1) were recorded for several hours, after which intense
chemical blockage was applied by simultaneous application
of AMPA and NMDA receptor antagonists (20 μM CNQX,
100 μM APV). This process is analogous to disconnecting the
excitatory coupling between the neurons, thus resetting the
network to a state of activity somewhat similar to that found
during early development, when synaptic connections are still
sparse. Immediately (up to 10 s) following the chemical
treatment, we observed a strong decline in the network’s
total activity. In most cultures, the activity was completely
eliminated. Gradually (on a time scale of hours), an increasing
number of neurons began to exhibit firing at an increasing
rate. The firing profiles of these neurons had high values of
FI, characteristic of HA neurons. Most of these neurons were
not identified as HA neurons before the chemical inhibition.
We proceeded to compute the fraction of HA neurons (using
FI > 0.2 to detect HA neurons) in the network before and
after the application of chemical inhibition. As is shown in
figure 5(B), following chemical inhibition, cultured networks
exhibited a high fraction of HA neurons. No synchronization
was observed among individual neurons throughout the whole
inhibition period and the ISI distribution of these neurons
remained unimodal. This suggests that degradation of blockers
was not a major factor in the observed activity patterns during
the inhibition. We also verified that, following the washout
of the synaptic blockers, the activity of other regular neurons
was restored and the fraction of the HA neurons decreased.
However, the neurons which began to exhibit a HA pattern
during intense chemical inhibition continued to show these
patterns after the wash of the blockers.
These findings, together with our previous observations
on the development of networks, suggest that HA neurons may
be recruited by the network to sub-serve in the initiation and
maintenance of proper system-level network activity.
Time-series analysis. We continued to investigate the special
dynamical characteristics of the HA neurons by performing
7
Phys. Biol. 5 (2008) 036008 M Shein et al

(A ) (B ) (C ) HA–neuron
–1 Regular neuron –1 HA–neuron –1
10 10 10 (Chem. inhibition)

PSD

–2 –2 –2
10 10 10

–3 –2 –3 –2 –3 –2
10 10 10 10 10 10
(D ) (E ) (F ) HA–neuron
–1 Regular neuron –1 HA–neuron –1
10 10 10 (Chem. inhibition)
PSD (average)

–2 –2 –2
10 10 10

–3 –2 –3 –2 –3 –2
10 10 10 10 10 10
Frequency [Hz] Frequency [Hz] Frequency [Hz]

Figure 6. Long-term trends in the activity of the HA neurons. Power spectral density (PSD) was estimated using count-based periodograms
(as explained in the Methods section) on long recordings of T > 50 h from early developing cultures (6–12 day in vitro) and networks under
intense chemical inhibition (12–22 day in vitro). As is shown in (A), the PSD of a regular neuron in a developing network attains relatively
low values in the low-frequency regime, as compared to the PSD of HA neurons (shown in (B)). Similar to developing networks, HA
neurons under intense chemical inhibition exhibit high power spectral density at low frequencies (shown in (C)). (D)–(F) The averaged
periodograms for regular neurons ((D), N = 43, five cultures) for HA neurons in developing networks ((E), N = 16, five cultures) and for
HA neurons under intense chemical inhibition ((F), N = 9, two cultures) suggest that the characteristic long-term trends are generic. Error
bars represent the standard deviation of the averaged values.

power spectral analyses of a spike time series for both
regular and HA neurons in developing networks and in
networks under intense chemical inhibition. The analysis was
performed using the count-based periodograms (explained in
the Methods section) for long (>50 h) recordings in which
HA neurons were defined according to FI > 0.13 for the
developing networks and FI > 0.2 for networks under chemical
inhibition. In figure 6, we show that HA neurons (figures 6(B),
(C), (E), (F)) have a higher power spectral density at low
frequencies in comparison with regular neurons (figures 6(A),
(D)). Such preferential concentration of spectral density at low
frequencies is indicative of long-time auto-correlations in the
firing activity of these cells. Hence, it suggests the existence
of some innate mechanisms which may regulate the firing of
the HA neurons.
The activity of HA neurons in networks with engineered
geometry
So far, we have shown that HA neurons are associated with
the intensification of network activity on a long time scale
(hours). To investigate the putative role of HA neurons in
initiation of SBEs, which occurs on a time scale of tens of
milliseconds, we focused on the correspondence between the
time order of neuronal firing within SBEs and the firing of HA
neurons.
In order to locate the initiation sites of SBEs, we
created small-scale networks with engineered geometry. The
networks’ growth was restricted to the area covered by
the electrodes, thus allowing uniform sampling of the entire
network area. As a result, all of the recorded SBEs were
initiated in the area sampled by the electrodes. These isolated
networks were created by surrounding the designated area
for the network with thin strips of PDMS (see the Methods
section), which physically prohibited processes from being
sent through the strip to the outer network. This procedure
was performed in early developing networks (N = 3) up to
10 h after the onset of intense synchronized activity.
Next, we examined the temporal order of neuronal
activation during SBEs for HA neurons and for regular
neurons. We constructed a raster plot showing the neuronal
activation pattern in every SBE (figure 7(A)). A survey of
these activation patterns revealed that in each SBE, different
neurons are the first to fire. In order to identify temporal
order patterns in neuronal activation, we averaged the spike
trains of all the recorded neurons over many SBEs (spike
trains were aligned according to the SBE loci). We found that
some of the neurons exhibited higher probabilities to fire at
the beginning of the SBEs (figure 7(B)) or act as precursors
of SBEs. Next, we calculated the FS for each neuron in order
to identify HA neurons. We found that HA neurons were
typically the first ones to fire during SBEs (figure 7(B)). To
quantify this correspondence, we identified the first neuron
to fire in every SBE (as shown in figure 7(A)) and calculated
the average number of SBEs initiated per HA neuron and per
regular neuron. Figure 7(C) shows that the relative number of
initiations per HA neuron is considerably higher than that of
regular neurons, indicating the preferential appearance of HA
neurons at the beginnings of SBEs.
Our next aim was to rule out an artifact in which HA
neurons will appear to fire first, simply because they fire

8
Phys. Biol. 5 (2008) 036008
1 1 (B )
(A )
# Neuron
HA-
neurons
Regular
17
# Neuron
neurons
0 1.8
Time [sec]
(C ) Regular neurons
% initiation per neuron
HA-neurons
100
50
HA-
neurons
0
24
1 2 3 0
Culture
Figure 7. Correspondence between HA neurons and the order of
neuronal activation during the SBEs. (A) A spatio-temporal firing
pattern of a typical synchronized bursting event reveals that each
SBE is characterized by a distinct pattern of neuronal activation.
The neuron which fired the first action potential is defined as the
precursor of this SBE. The HA neurons in this example (marked by
arrows) clearly fire ahead of the other regular neurons. (B) Activity
profiles of consecutive SBEs (N = 540) from a developing network
were co-aligned and averaged according to the SBE loci, as defined
in figure 2. The neurons were vertically ordered according to their
FS values, so that HA neurons are at the bottom of the plot. This
analysis disclosed that HA neurons (below the red line
corresponding to FS = 0.1) expressed higher probabilities to be the
first to fire in the SBEs. (C) The normalized (see text for details)
percentage of SBEs in which HA neurons (black bars) were the first
to fire is considerably higher than the normalized fraction of SBEs
in which regular neurons (white bars) fired first. The results are
consistent for all three examined cultures (1–3), in which HA
neurons initiate on average 19 times more SBEs than regular
neurons.
more intensely outside the SBEs. To rule out this possibility,
we first created an artificial SBE sequence with the same
average frequency as appears in the original data but with
a different shuffling of the neurons for each SBE (HA neurons
were deleted from this sequence). We then superimposed
the original spike time series of the HA neurons on the
time series of shuffled SBEs and tried to identify initiating
HA neurons in the artificially constructed data set. The
relative fraction of SBEs initiated per HA neurons was much
lower in the shuffled scenarios as compared to the real data
sets (0.94 on average in the original sets versus 0.07 after
shuffling). These results indicate that the correspondence
between HA neurons and the preferential firing at the
beginning of SBEs is not an artifact, and that HA neurons
act as precursors of SBEs. It should be noted that since the
average fraction of HA neurons is small, it is quite likely that
all SBEs are preceded by the firing of some HA neurons, but
that due to their small fraction, these HA neurons are not
recorded.
These results raise the immediate question of whether HA
neurons ignite the SBEs or whether their preferential activation
in the beginning of SBEs is simply due to the fact that they are
the first to respond.
M Shein et al
Investigating the response to electrical stimulations
To answer the above question, we investigated the response
of neurons in the network to external electrical stimulations.
More specifically, we examined the differences in triggering
and response between electrodes recording from HA neurons
to electrodes recording from regular neurons. For this
purpose, we first identified the HA neurons in the network
and afterwards stimulated all the electrodes in which neuronal
activity was recorded (see the Methods section). In general,
some of the stimulations did not elicit any response in the
network, other stimulations triggered a response in individual
neurons and a small fraction of the stimulations activated
a synchronized burst in the network (figure 8(A)). The
responses varied between the different stimulating electrodes
and different stimulated neurons (figures 8(A), (D)). More
Time [sec] 1
specifically, some of the neurons consistently responded to the
stimulations with a high yield while others were unresponsive
(figure 8(B)). Additionally, some of the stimulating electrodes
consistently activated more neurons and were more likely to
trigger SBEs in the network while others had no influence on
the network’s activity (figure 8(C)).
In these tests, we did not identify correspondence
between the electrodes whose stimulation triggered activity
in individual neurons or the whole network and electrodes
on which HA neurons (defined by FS > 0.1) were identified
(figures 8(C), (D)). On the other hand, we found that
HA neurons responded with higher fidelity to electrical
stimulations in comparison to regular neurons (figures 8(B),
(E)). Electrical stimulations using a multi-electrode array may
trigger a response in more than one neuron. Additionally,
passing by axons may also be triggered by the electrical
stimulations (Wagenaar et al 2004). Consequently, the high
responsiveness of HA neurons suggests that they are coupled
to many physical locations in the network and that they are
sensitive enough to be affected by these pathways. This
suggests that the specific HA neurons we measured are not
necessarily the initiators of SBEs, but rather act as precursors
to the activation of collective events in the network.
Discussion
In this work, we investigated the dynamics of HA neurons,
identified in the spontaneous activity of cortical neuronal
networks. We studied their dynamics in mature networks,
in early developing networks, during recovery from intense
chemical inhibition, in networks with engineered geometry
and in response to electrical stimulations.
Our main findings are as follows.
(1) Activity patterns of single neurons in cortical cultures can
be roughly classified as belonging to either one of two
classes—regular patterns relating to neurons which fire
mainly during the SBEs and highly active patterns relating
to a small subset of neurons (HA neurons), which also
show persistent firing during the network’s silent states
(between SBEs).
(2) The firing profiles of the HA neurons have special
dynamical characteristics.
9
Phys. Biol. 5 (2008) 036008 M Shein et al

(A ) (B ) (C )

5 Regular
Neurons
10
HA
Electrode

Neurons
15

20

25
25 50 75 100 125 5 10 10 20
Time [sec] % Responding % Stimulated
electrodes SBEs
(D ) (E )
** * * * * 100
* 0.5
*
5 80
0.4

% response per neuron

Responding electrode

HA

HA
10 60
0.3
Probability
15 40
0.2
*
20 20
0.1

Reg

Reg
*
25 *
0 0
5 10 15 20 25 1 2
Stimulating electrode Culture

Figure 8. HA neurons and electrical stimulation. (A) An example of a raster plot from a network responding to electrical stimulations (blue
lines). Some of the stimulations did not elicit any response while others activated individual neurons or the whole network. In most cases,
SBEs were triggered in the first stimulation of a sub-session (see the Methods section for electrical session protocol). (B) The probability of
response for different electrodes. For each stimulation, electrodes were marked as responding if they were triggered during the first
100 ms after the stimulus. The probability of response was then averaged over all stimulations in all electrodes. HA neurons (black) were
triggered by the electrical stimulations with a higher probability compared to other neurons. The red line marks the average level. (C) The
probability of different electrodes to successfully trigger a SBE in the network. A successful stimulation in an electrode was defined as one
in which an SBE was detected up to 500 ms after stimulus. No significant correspondence was found between the stimulated electrodes
which successfully triggered SBEs and electrodes on which HA neurons were identified (black). The red line marks the average level.
(D) The response matrix, R(i, j ). Electrode i was marked as responding to stimulation in electrode j, if an action potential was recorded in
electrode i during the first 100 ms after the stimulus was applied to electrode j. The response probability of every electrode to all
stimulations was averaged and is presented in a gray-scale color code. Some electrodes consistently triggered responses in the network and
other electrodes consistently responded to stimulations. There was a strong correspondence between electrodes on which HA neurons were
detected (marked by asterisks and outlined by rectangles) and those which responded to stimulations but not those which triggered activity
in the network. (E) The relative percentage of response, per HA neuron and per regular neuron, was calculated from the response matrix for
two stimulated cultures (8–10 days in vitro). The results indicate that HA neurons are ∼4 times more responsive to electrical stimulations
than regular neurons.

(3) The HA neurons seem to play an important role in the
maintenance of spontaneous activity during development
and under inhibition conditions.
(4) The fraction of HA neurons is regulated according to the
context of the network activity.
(5) The HA neurons act as precursors of the SBEs but do not
seem to directly ignite them.
(6) HA neurons respond with higher fidelity to external
electrical stimulations.
Below we discuss in further detail our findings and their
possible implication for understanding the regulation of the
network’s electrical activity.
The temporal structure of a neuronal firing time series can
be affected by many factors, ranging from the morphology
of its dendritic tree (Mainen and Sejnowski 1996) to the
selective regulation of its inward and outward current densities
(Turrigiano et al 1995). In the cortical cultures studied
here, we identified two qualitatively different patterns of
individual neuronal activities. Most neurons were active
almost exclusively during SBEs, firing only a small number
of occasional spikes during the silent network state (i.e. the
long time intervals between the SBEs). Most neurons have
their own firing profile during the SBEs (Segev et al 2004,
Raichman et al 2006), but, in general, the activity of these

10
Phys. Biol. 5 (2008) 036008
neurons correlates well with the overall network activity. In
addition to this class of regular neurons, whose activity is
‘network dependent’, a relatively small subset of recorded cells
also exhibit persistent firing during the network’s silent states
between SBEs. Such generic classification of neurons, based
on the correspondence between the activity of a single neuron
and the activity of the network, is consistent with previous
findings (Mao et al 2001, Darbon et al 2002, Sipila et al
2005).
At present, it is not clear which biophysical mechanisms
are responsible for the appearance of the HA neurons.
A putative mechanism based on the idea of self-synapses
regulated by astrocytes was proposed by Volman et al (2007).
Generally, neuronal activity can be affected by either cell-
extrinsic or cell-intrinsic factors. Two pieces of experimental
evidence stand in favor of the second scenario. First, the HA
neurons are active long before the network is fully connected
and showing global patterns of collective activity. Second, the
firing activity of these neurons persists during the absence of
the network’s activity (the network’s silent states), as is also the
case when the network undergoes intense chemical inhibition.
Therefore, it appears that an input of action potentials from
other neurons is not necessary in order for the HA neurons to
be active, and thus the mechanism seems to be cell-intrinsic.
It should be noted that even though our studies show
that the HA neurons fire given limited or no electrical input
(action potential signals from other cells), it does not mean
that they do not receive chemical input from other cells
such as astrocytes and microglia, which is likely to be the
case. Mechanistically, the appearance of persistent firing with
limited or no electrical input is reminiscent of pacemaker cell
activity (Sipila et al 2005). However, in our opinion, the
observed wide distribution of inter-spike intervals makes it
unlikely that the HA neurons are simple pacemakers. It is
also possible that more biophysically complicated processes
take place, such as intrinsic bursting, driven by dynamical
bi-stability (Loewenstein et al 2005), that may be triggered
by external chemical inputs. Clearly, more detailed neuro-
physiological investigations of the HA neurons are needed in
order to resolve this issue. Such investigations will also lead
to a better understanding of the role that these special neurons
play in the network’s activity.
Our present findings suggest that the activity of the HA
neurons is prominent when the overall network activity is
quite weak (as was the case in early developmental stages
and when the network underwent chemical inhibition). We
also observed a decrease in the fraction of HA neurons as the
network became fully active. These observations lead us to
suggest that the HA neurons may be employed by the network
as a means to maintain activity homeostasis. It is possible that
in the case of early developing networks, HA firing patterns
may appear due to some developmental constraint; however,
our observation that HA firing patterns appear after intense
chemical inhibition in mature networks supports the notion
that these patterns appear due to the lack of activity. Additional
support for this hypothesis is found in an observation of long-
range (minutes to hours) auto-correlations in the activity of
HA neurons. Memory on such long time scales may imply the
involvement of homeostatic processes.
11
M Shein et al
In order for the network to employ the HA neurons, there
should be a mechanism that reduces the activity of the HA
neurons when they receive high electrical input and vice versa.
The mechanism proposed in Volman et al (2007) presents
such a putative mechanism. It was shown that a self-synapse
which is regulated by astrocytes can reduce the auto-transfer
of action potentials when the network is active. From the
single cell perspective, another putative mechanism may be
based on activity-dependent redistribution of voltage-gated
conductance (Desai et al 1999, Moody and Bosma 2005).
Both mechanisms allow the regulated increase in neuronal
excitability as observed for HA neurons. Such an increase
in excitability complies with the observation that HA neurons
are precursors of SBEs and are more responsive to external
electrical stimulations.
We now turn to reflect on the possible mechanisms which
may underlie the spontaneous generation of the network’s
collective activity. This activity is marked by the generation
of synchronized bursting events, which start to appear in the
spontaneous activity as the dissociated cell culture develops
into a fully connected network. Several studies suggest that
SBEs may be driven by a sub-population of neurons (Mao
et al 2001, Voigt et al 2001, Darbon et al 2002, Sipila et al
2005, Hunt et al 2005, Ham et al 2008). In a recent study,
Feinerman et al (2007) showed that in quasi-1D cortical
cultures there exist ‘hot spots’ that reliably generate SBEs.
In addition, focused electrical and chemical stimulation can
reliably induce new SBEs which persist for long periods of
time (Baruchi and Ben-Jacob 2007, Madhavan et al 2007).
These findings suggest that the generation of SBEs may
depend on a sub-network (sub-population) composed of a
small number of neurons. An alternative hypothesis, which is
supported by the recent studies of Eytan and Marom (2006),
posits that spontaneously generated SBEs are an outcome of
population-level interactions rather than being dependent on
specific cells. In this view, neurons which initiate SBEs form
a sub-population which is recruited in response to collective
events, before the rest of the network. According to our
observations, the same set of neurons which show increased
activity when the global activity levels in the network are low
appear to fire first during SBEs. This implies a link between the
long-term homeostatic mechanisms and the short-term activity
propagation in the network. This link is compatible with
both the views of Eytan and Marom (2006) and Feinerman
et al (2007). According to the former view, HA neurons can
be looked upon as highly excitable neurons which are more
sensitive to fluctuations in the network’s activity and whose
sensitivity can be intrinsically regulated. For this reason,
they are persistently triggered between SBEs, are the first
neurons to fire during SBEs, are more responsive to electrical
stimulations and are more active when the network’s activity is
low. According to the latter view, HA neurons can be viewed
as having intrinsic firing properties which ‘boost’ the activity
whenever the network’s activity levels are low. For this reason,
in a quazi-1D network, such as the one presented in Feinerman
et al (2007), HA neurons can create localized initiation zones
from which SBEs are triggered.
Our observations may also support a new possibility
that bridges between the above two views, based on the
Phys. Biol. 5 (2008) 036008
following reasoning: combined experimental and modeling
studies (Segev et al 2004, Baruchi and Ben-Jacob 2004, 2007,
Volman et al 2005, Baruchi et al 2006, 2008, Raichman and
Ben-Jacob 2008) indicate that large (>1 M neurons) cultured
cortical networks can sustain several SBEs, which differ from
one another in their spatio-temporal patterns of neuronal
activation. The ability of a network to exhibit different bursting
events can be accounted for if the network is composed of
overlapping sub-networks (this idea was discussed in Segev
et al (2004), Baruchi and Ben-Jacob (2004)). To embrace these
findings, while taking into account the observations reported
here regarding the HA neurons, we follow the assumption that
cortical cultures may be sub-divided into overlapping sub-
networks and add the idea that the activity of each of the sub-
networks is mainly regulated by its own set of HA neurons.
In this view, spontaneous collective activity (as manifested by
different SBEs) may still be generated by intrinsically active
cells (in accord with experimental observations as in Sipila
et al (2005), Feinerman et al (2007)). At the same time, activity
feedback from a sub-network determines the propensity of its
associated set of highly active cells to exhibit persistent firing.
Therefore, synchronized bursting events may arise due to the
synergistic action of cell assemblies and their associated sets
of HA neurons. This perspective is additionally supported
by the observation (data not shown) that in some cases, the
activity of HA neurons during the network’s silent states
is correlated. Future experiments with patterned cultured
networks will delineate the interplay between highly active
cells and the network’s architecture of synaptic connections.
Meanwhile, the results of our analysis suggest that HA neurons
may constitute an important pathway recruited by a developing
cortical network in order to regulate its spontaneous activity.
Acknowledgments
The authors thank I Baruchi for sharing his views with us and
I Brainis for technical assistance. This research was supported
in part by the NSF-sponsored Center for Theoretical Biological
Physics (grant numbers PHY-0216576 and PHY-0225630), by
the Israeli Science Foundation and by the Tauber fund at Tel-
Aviv University. VV acknowledges the support of the US
National Science Foundation I2CAM International Materials
Institute, grant DMR-0645461.
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