Mechanisms controlling bursting activity induced by disinhibition in spinal cord networks

Mechanisms controlling bursting activity induced bydisinhibition in spinal cord networks

Pascal Darbon, Luke Scicluna, Anne Tscherter and JuÈrg StreitDepartment of Physiology, University of Bern, BuÈhlplatz 5, CH-3012 Bern, Switzerland

Keywords: network, multielectrode array, central pattern generator, locomotion, burst

Abstract

Disinhibition reliably induces regular synchronous bursting in networks of spinal interneurons in culture as well as in the intactspinal cord. We have combined extracellular multisite recording using multielectrode arrays with whole cell recordings to

investigate the mechanisms involved in bursting in organotypic and dissociated cultures from the spinal cords of embryonic rats.

Network bursts induced depolarization and spikes in single neurons, which were mediated by recurrent excitation throughglutamatergic synaptic transmission. When such transmission was blocked, bursting ceased. However, tonic spiking persisted in

some of the neurons. In such neurons intrinsic spiking was suppressed following the bursts and reappeared in the intervals after

several seconds. The suppression of intrinsic spiking could be reproduced when, in the absence of fast synaptic transmission,

bursts were mimicked by the injection of current pulses. Intrinsic spiking was also suppressed by a slight hyperpolarization. Anafterhyperpolarization following the bursts was found in roughly half of the neurons. These afterhyperpolarizations were combined

with a decrease in excitability. No evidence for the involvement of synaptic depletion or receptor desensitization in bursting was

found, because neither the rate nor the size of spontaneous excitatory postsynaptic currents were decreased following the bursts.Extracellular stimuli paced bursts at low frequencies, but failed to induce bursts when applied too soon after the last burst.

Altogether these results suggest that bursting in spinal cultures is mainly based on intrinsic spiking in some neurons, recurrent

excitation of the network and auto-regulation of neuronal excitability.

Introduction

Local neural networks in the spinal cord of vertebrates provide the

rhythmic output for locomotion (Grillner et al., 1998). Studies in

lower vertebrates like the lamprey and the tadpole suggest that such

central pattern generators are composed of local oscillator networks

of spinal interneurons, which are coupled by mutual inhibitory and

excitatory synaptic connections (Grillner & WalleÂn, 1985; Dale &

Kuenzi, 1997). This concept has been successfully tested in computer

models (WalleÂn et al., 1992) and is now believed to be also valid in

the mammalian spinal cord, although less experimental data are

available there (Cazalets et al., 1995; Kiehn & Kjaerulff, 1998; Beato

& Nistri, 1999).

The study of how local oscillator networks function and how they

are activated is compromised by the limited experimental access to

such networks in intact spinal cord preparations. Cultures of spinal

cord slices have therefore been developed to maintain and analyse

isolated oscillator networks in vitro (Braschler et al., 1989; Ballerini

& Galante, 1998). Indeed, rhythmic activity can be induced in spinal

cord slice cultures. The observed frequencies of the rhythmic activity

are comparable to those seen in the intact spinal cord during ®ctive

locomotion, although the range of possible induction protocols varies

in both preparations (Ballerini et al., 1999). By mapping the

spatiotemporal structure of rhythmic activity in the slice cultures,

we have previously shown that rhythm sources are usually present on

both sides of the slices close to the ventral ®ssure. However, the

activity always spreads from these sources to the whole slice, thus

leading to synchrony of the rhythms on both sides (Tscherter et al.,

2001). These ®ndings suggest the presence of local oscillator

networks on both sides of spinal cord slice cultures. However, the

proper coupling of these networks into functional pattern generators

with an alternating output for both sides is not formed.

Rhythmic bursting activity has also been described in randomized

cultures of dissociated neurons of the spinal cord (Ransom et al.,

1977; O'Brien & Fischbach, 1986; Gross et al., 1993a) and the brain

(Maeda et al., 1995; MuÈller & Swandulla, 1995). We have previously

found similar patterns of rhythmic activity in such cultures as in slice

cultures, although with differences in the details and in the induction

protocols (Streit et al., 2001; Tscherter et al., 2001). Nevertheless the

similarities bring up the following hypothesis: local oscillator

networks function through basic mechanisms which are not speci®c

to spinal networks but which are inherent properties of many neural

networks without the requirement of high levels of structure.

Among such inherent properties, synaptic depression (O'Donovan

& Rinzel, 1997; Staley et al., 1998) and activity dependent changes in

neuronal excitability (Maeda et al., 1995; Sanchez-Vives &

McCormick, 2000; Streit et al., 2001) have been proposed to be

critically involved in bursting. In the present work, we have

investigated the contributions of both mechanisms to bursting

induced by disinhibition in dissociated and organotypic spinal cord

cultures. The behaviour of the network was compared to that of

individual neurons by combining multielectrode arrays (MEAs)

recording and stimulation (Gross et al., 1982; Gross et al., 1993b;

Jimbo et al, 1993) with whole cell patch recordings (Hamill et al.,

1981; Vogt et al., 1995). Our ®ndings suggest that bursting is based

Correspondence: Dr JuÈrg Streit, as above.E-mail: [email protected]

Received 7 September 2001, revised 5 December 2001, accepted 3 January2002

European Journal of Neuroscience, Vol. 15, pp. 671±683, 2002 ã Federation of European Neuroscience Societies

on the intrinsic spiking of some neurons, recurrent excitation of the

network and an auto-regulation of neuronal excitability.

Materials and methods

Cultures

All cultures were made from the spinal cord of rats at embryonic age

14. The cultures were prepared in the same way as described

previously (Braschler et al., 1989; Spenger et al., 1991; Streit et al.,

2001; Tscherter et al., 2001). The embryos were delivered by

caesarian section from deeply anaesthetized rats (0.4 mL pentobarbi-

turate i.m.) and killed by decapitation. Following the delivery of the

embryos, the mother rat was killed by intracardiac injection of

pentobarbiturate. Animal care was in accordance with guidelines

approved by Swiss local authorities. The backs of the embryos were

isolated from their limbs and viscera and cut into 225 mm-thick

transverse slices with a tissue chopper. For the dissociated cultures,

slices of all regions of the spinal cord, without dorsal root ganglia,

were exposed to a 0.3% trypsin solution for 3 min at 37 °C. These

slices were then mechanically dissociated by forcing them through

®ne-tipped pipettes several times. The cells were plated on MEAs or

on glass coverslips at a density of 150 000 or 75 000/150 mL,

respectively. MEAs were produced as described previously

(Tscherter et al., 2001) and coated for one hour with diluted

(1 : 50) Matrigelâ (Falcon/Biocoat, Becton Dickinson AG,

Switzerland). The glass coverslips were coated with polylysine

(1 mg/mL overnight at 37 °C). The cells were restricted to an area

around the electrodes (» 50 mm2) using cloning glass cylinders

attached to the MEAs or coverslips by silicone sealant. They were

maintained in culture dishes containing 150 mL of nutrient medium

and incubated in a 5% CO2-containing atmosphere at 36.5 °C for up

to 12 weeks. Serum-free NeurobasalTM medium (Gibco BRL, Life

Technologies AG, Switzerland) supplemented with B27 and

Glutamax (both Gibco BRL) was used for the MEA cultures and

some of the cultures on the glass coverslips. The other cultures were

kept in a MEM Eagle's Medium supplemented with 10% fetal bovine

serum, 0.2% glucose, B27 and Glutamax. Half of the medium was

changed weekly.

For the organotypic cultures, the spinal cord slices with their

attached dorsal root ganglia were ®xed on MEAs using reconstituted

chicken plasma (Cocalico Biologicals, Pennsylvania, USA) coagu-

lated by thrombin (Sigma, Fluka Chemie AG, Switzerland). The

cultures were maintained in sterile plastic tubes containing 3.5 mL of

nutrient medium and incubated in roller drums rotating at 120 rph in

a 5% CO2-containing atmosphere at 36.5 °C. The medium had the

following composition: 79% Dulbeccos MEM with Glutamax, 10%

fetal bovine serum (Life Technologies), 10% H2O and 5 ng/mL 2.5 S

nerve growth factor (Alomone Laboratories Ltd, Jerusalem, Israel).

During the ®rst week of incubation a medium with 10 ng/mL nerve

growth factor was used. Half of the medium was removed and

replaced with fresh medium weekly.

Recordings

Recordings were made in a chamber mounted on an inverted

microscope (Nikon, Japan) from cultures of 1±3 weeks of in vitro age

(slice cultures) and 2±12 weeks (dissociated cultures). The medium

was replaced by an extracellular solution containing (in mM): NaCl,

145; KCl, 4; MgCl2, 1±2; CaCl2, 2; HEPES, 5; Na-pyruvate, 2;

glucose, 5 at pH 7.4. Recordings were either made in the presence of

continuous superfusion at 1 mL/min or with solution changes every

10±15 min. No differences in the patterns of activity were seen

between these two protocols. All recordings were made at room

temperature.

MEA recording and analysis

MEAs contained 68 electrodes, laid out either in the form of a

rectangle (dissociated cultures) or in the form of a hexagon (slice

cultures). Channels showing activity (usually 10±30 for the dissociated

cultures and 20±50 for the slice cultures) were selected by eye and their

recordings digitized, visualized and stored on hard disc using custom

made virtual instruments within Labviewâ (National Instruments,

Switzerland) as described previously (Tscherter et al., 2001).

FIG. 1. Combined recordings of disinhibition-induced bursting in the wholenetwork and in a single neuron of a dissociated culture. The recordingswere made in the presence of bicuculline (20 mM) and strychnine (1 mM).(A) Event raster plot of MEA recordings (see Materials and methods)showing the activity of 20 channels. (B) Network activity plot (seeMaterials and methods) derived from the recordings in A. To visualize theamount of activity in the whole network, the number of detected eventswere counted within a sliding time window of 10 ms. Note the increasinglevels of activity in the intervals. (C) Whole cell recording from a singleneuron in the network. Note that the activity ceases following the bursts dueto an afterhyperpolarization. (D) Superposition of the network activity(grey) and the whole cell activity (black) for one burst at an expanded time-scale. (E) Whole cell recording from the same neuron as shown in Cshowing persisting spiking in the presence of CNQX (10 mM) and APV(50 mM).

672 P. Darbon et al.

ã 2002 Federation of European Neuroscience Societies, European Journal of Neuroscience, 15, 671±683

Detection of the extracellularly recorded action potentials and further

analysis were carried out of¯ine in the software package IGOR

(WaveMetrics Inc., Oregon, USA) as described previously. The

electrical noise of individual channels was very stable. The electrodes

recorded fast voltage transients (< 4 ms), which correspond to single

action potentials in neurons or axons (single-unit activity). These fast

voltage transients often appeared in clusters (multiunit activity)

originating from closely timed action potentials of several neurons or

axons seen by one electrode. During such multiunit activity, individual

fast voltage transients could not be clearly linked to underlying action

potentials. Therefore no attempt was made to sort spikes seen by one

electrode. For the analysis of the spinal network activity a detector was

designed to ®nd fast voltage transients, and its output was de®ned as

event. During multiunit activity the fast voltage transients could not be

attached to individual action potentials, therefore the detection device

was designed in a way to represent multiunit activity by periods at a

de®ned level of activity. This activity detector reliably translated on

the one hand single-unit activity into one event and on the other

multiunit activity into trains of events at 333 Hz (see Tscherter et al.,

2001). The selectivity of event detection was controlled using

recordings obtained in the presence of tetrodotoxin (TTX, 1.5 mM)

as a zero reference. The processed data were visualized in the form of

event raster plots and network activity plots. Event raster plots show

the time-markers of detected activity of each selected channel (e.g.

Fig. 1A). Network activity plots (e.g. Fig. 1B) show the total activity

of all selected channels within a sliding time window of 10 ms, shifted

by 1 ms.

Stimulation by MEA electrodes

Biphasic monopolar voltage pulses with a duration of 0.5 ms and

amplitudes of 0.5±1.25 V were addressed to one of the MEA

electrodes. No recording was possible with the electrode connected to

the stimulator.

Whole cell patch clamp recording and analysis

Intracellular voltage and current measurements were obtained from

individual neurons using the whole cell patch technique (Hamill et al.,

1981) with either an Axopatch 1B or an Axoclamp 2B ampli®er (Axon

Instruments Inc., California, USA). The patch pipettes were ®lled with

a solution containing (in mM): K-gluconate, 100; KCl, 20; HEPES, 10;

Mg-ATP, 4; Na2-GTP, 0.3; Na2-phosphocreatine, 10; pH 7.3 (with

KOH); for voltage clamp recordings only: EGTA 10. The electrodes

had a resistance of 4±5 MW. No series resistance compensation

was used. Control resting membrane potentials were in the range of

±40 mV to ±70 mV (mean: ±48 6 1.1 mV, n = 29). Cells with a

potential less negative than ±40 mV were discarded. Synaptic currents

were recorded at a clamped membrane potential of ±60 mV. Voltage

and current recordings were digitized, visualized and stored on

computer using either pClamp (Axon Instruments inc.) software or

virtual instruments within Labview. They were analyzed of¯ine using

custom made programs in IGOR. The detection of synaptic currents

was based on a threshold set to the ®rst derivative of the original

current recording at three times the noise level. Bursts were de®ned as

the superposition of four or more subsequent currents separated by an

interval < 10±15 ms. The end of such burst was de®ned when the

interval between two subsequent currents exceeded a threshold of 15±

25 ms.

Statistics

Averages are expressed as mean 6 SEM. Statistics are based on

Student's t-test and ANOVA. A correlation was considered signi®cant

when its coef®cient (r) was signi®cantly different from 0 (t-statistics,

P < 0.05).

Chemicals

All agents were applied by exchanging the bath solution for a solution

containing the drugs. The following agents were used: bicuculline,

strychnine, APV, CNQX, TTX (all Sigma).

FIG. 2. Whole cell recording from a single neuron during block of fastsynaptic transmission by bicuculline (20 mM), strychnine (1 mM), CNQX(10 mM) and APV (50 mM) in a dissociated culture. Intrinsic spiking isstopped by changes in the membrane potential and spike rate due toinjected current pulses. (A) Pulses of depolarizing current injections withincreasing amplitudes induce spiking at increasing rates during the pulseswithout spike frequency adaptation. The grey bars visualize the duration(length of the bars) and the strength (thickness of the bars, range: 50±200pA) of the current pulses. Note that the intrinsic ®ring in the intervalsbetween the pulses is eventually suppressed. (B) Spike intervals fordifferent voltage steps induced by current injections as shown in A. Notethat except for the ®rst three spikes there is practically no spike frequencyadaptation. (C) Steady state response curve of the neuron shown in A andB. Spike rate is a linear function of the membrane potential with a thresholdat ±52 mV and a slope of 2.2 Hz/mV. (D) After the stop of currentinjection, intrinsic activity returns following a refractory period ofapproximately 5 s. Note that the intrinsic activity is ®rst phasic and thengradually returns to the initial tonic activity. (E) Hyperpolarization inducedby pulses of negative current injections (grey bars) stop intrinsic spiking.Note that hardly detectable changes in membrane potential are suf®cient tostop spiking (right pulse) without leaving an oscillating membrane potential.

Bursting activity induced by disinhibition 673


Results

Network and single cell activity

The results are based on the investigation of 104 dissociated spinal

cultures and 22 organotypic slice cultures. From the dissociated

cultures 26 were investigated by MEAs (on average 21 channels per

culture), 66 by whole cell patch clamp and 12 by a combination of

both methods. It has been shown previously that disinhibition by a

combination of strychnine (1 mM) and bicuculline (20 mM) induces a

slow and more or less regular bursting of activity in the whole

network of dissociated cultures, in slice cultures and in the intact

spinal cord (Droge et al., 1986; Streit, 1993; Bracci et al, 1996). MEA

recordings in dissociated cultures showed that such bursting was

characterized by periods of high activity in the whole network,

interrupted by intervals of low network activity (Fig. 1A and B). The

network activity abruptly increased at the burst onset, then slowly

decreased during the bursts and ®nally rapidly decreased at the end of

the burst (Fig. 1D). During the onset of the bursts, a wavefront was

spreading from one electrode (source) to the whole network within

50±100 ms. During the intervals between the bursts, activity at low

rates (around 0.1±1 Hz) persisted in some of the channels. In general,

network activity was low immediately following the bursts and it

increased slowly during the intervals (Fig. 1B and C).

Whole cell recordings of spinal neurons showed that the network

bursts caused depolarization, always leading to one or several spikes

at the beginning of the bursts. These initial spikes were followed by

repetitive spiking at rates of 1±20 Hz in 75% of the cells (Fig. 3). In

25% of the neurons a strongly depolarized plateau potential (at

±19 6 1 mV, n = 10, compared to a depolarization to ±37 6 1.8 mV

in the other neurons, n = 29) was seen during the bursts (Fig. 1D).

These neurons had on average higher spike rates during control

activity (in the absence of disinhibition by strychnine and bicuculline)

than those with repetitive spiking (19.1 6 9.3 vs. 5.9 6 1.4 spikes

per s). This suggests that the plateau potentials are caused by strong

synaptic inputs leading to large scale depolarization with inactivation

of sodium channels. In line with this hypothesis, spikes often

reappeared at the end of the bursts when the membrane potential

repolarized (Fig. 1D). In approximately half of the neurons, bursts

were followed by long lasting afterhyperpolarizations. In the intervals

between the bursts, the spike rate was low. Some of the neurons,

however, started to repetitively ®re at increasing rates (1±10 Hz) in

the second half of the intervals (Fig. 1C), when the afterhyperpolar-

izations had disappeared. Such spontaneous ®ring was characterized

by gradually depolarizing membrane potentials between the spikes.

This behaviour of individual neurons explains the increasing network

activity during the intervals observed with MEA recordings.

It has been shown previously that the bursting is based on recurrent

excitation of the network through glutamatergic synapses (Streit,

1993). Blocking such synapses by a combination of APV and CNQX

stopped the bursting in most of the experiments, while asynchronous

low rate activity persisted at approximately half of the electrodes in

MEA recordings (Streit et al., 2001). As previously shown, such

intrinsic neuronal activity is preferentially found at sites where bursts

are frequently initiated (Streit et al., 2001). As shown in Fig. 1E, in

whole cell recordings of single cells, intrinsic spiking was found in 9

out of 22 neurons in the presence of strychnine, bicuculline, CNQX

FIG. 3. Whole cell recordings showing the alterations in neuronal excitability during disinhibition-induced bursting (bicuculline, 20 mM and strychnine, 1 mM)in a dissociated culture. For this analysis burst rate was slowed by 1 mM CNQX. (A) Example of a neuron, in which bursts were followed by anafterhyperpolarization with a decrease in excitability. Action potentials in the intervals were induced by injecting depolarizing current pulses of 50 msduration applied at 1 Hz. The amplitude of the pulses was adjusted to be just above threshold for most of the time. (B) Example of a neuron, in which burstsare followed by an afterdepolarization with an increase in excitability. The current pulses were adjusted to be just below threshold for most of the time. (C)The percentage of failures to induce action potentials for seven consecutive pulses at 1 Hz following a burst and six pulses preceding the next burst (n = lastpulse before the next burst). Each point represents the mean of six experiments (6 SEM) as illustrated in A (®lled points), and seven experiments asindicated in B (open points).



and APV. The mean spike rate in these neurons was 3.7 6 1.2 Hz

(n = 6). The membrane potential between the spikes was gradually

depolarizing from the hyperpolarized level following the spikes, and

it was on average more depolarized than the resting membrane

potential in non±spiking neurons (±41.4 6 1.3 mV, n = 6 vs.

±48.8 6 3.3 mV, n = 4). In summary, combining extracellular

MEA recordings with intracellular recordings from single neurons,

we could con®rm our previous hypothesis: intrinsic ®ring of some

neurons can activate the whole network through recurrent excitation

and cause a burst (Streit et al., 2001). Intrinsic ®ring ceases following

the bursts and slowly recovers during the intervals (Fig. 1C).

Auto-regulation of intrinsic spiking

The modulation of intrinsic ®ring during bursting could be caused by

the synaptic activity during bursts because neurotransmitters and

neuromodulators are known to change neuronal excitability

(Legendre et al., 1989; White et al., 1996). Alternatively, in the

sense of an auto-regulation of neuronal spiking, it could be in¯uenced

by the rate of spiking itself. In order to distinguish between these two

possibilities we mimicked bursting in the absence of synaptic

transmission by current injections into single neurons in the presence

of blockers of synaptic transmission (bicuculline 20 mM, strychnine

1 mM, CNQX 10 mM and APV 50 mM). We found that intrinsic

spiking was highly sensitive to current injections, i.e. depolarizing

currents increased the ®ring rate. The larger the current pulse, the

stronger was the depolarization, the higher was the spike rate and the

smaller and slower were the spikes (Fig. 2A). A linear relationship

was found between the spike rate and the membrane potential

between spike threshold at ±56.3 6 1.6 mV (n = 6 experiments) and

±30 mV (Fig. 2C). The mean slope of this relationship was

2.0 6 0.2 Hz/mV (n = 6 experiments). In the course of strong

depolarization the spikes became smaller and slower and could ®nally

die out, thus leading to the plateau potentials comparable to those

shown in Fig. 1 for the synaptically induced bursts. However, similar

to the ®ndings in interneurons in slice cultures (Ballerini et al., 1999),

practically no increase in spike intervals (spike frequency adaptation)

FIG. 4. Whole cell recordings showing spontaneous excitatory postsynaptic currents (EPSCs) in a voltage clamped neuron under disinhibition (bicuculline,20 mM, strychnine, 1 mM) in a dissociated culture. The holding potential was ±60 mV (A) EPSCs are organized into bursts. The grey line shows the de®nedbursts (lower level) and intervals (upper level). Note the summation of EPSCs during the bursts, especially at the beginning. The small inset shows thesuperposition of two EPSCs occurring during the intervals. Note the small size and the large variability of these EPSCs. (B) Correlation between the durationof the bursts and the preceding interval; r = 0.78. (C) Correlation between the initial peak amplitude of the summated currents during the bursts and thepreceding interval; r = 0.78. (D) Peak amplitudes of EPSCs occurring during the intervals are plotted vs. interval time. Six consecutive intervals aresuperimposed. (E) Rates of EPSCs occurring in the intervals are plotted vs. interval time. Six consecutive intervals are superimposed.



was found in these cells (Fig. 2B). Only the interval between the ®rst

and second spike of a pulse was usually shorter than the following

intervals.

Pulses of strong current injections suppressed the spontaneous

®ring in the intervals between the pulses in all ®ve neurons (Fig. 2A).

When the current pulses were stopped, intrinsic ®ring re-appeared

after a latency of several seconds: ®rst, phasic activity came up, then

the activity gradually turned into the tonic activity present before the

current injections (Fig. 2D). Because the membrane potential was

usually close to the spike threshold potential, intrinsic spiking was

stopped during pulses of hyperpolarizing current injections, even

when the amount of hyperpolarization did not exceed a few millivolts

(Fig. 2E). Under these conditions no subthreshold oscillation in

membrane potential and no fast postsynaptic potentials were seen. All

these ®ndings are consistent with the hypothesis of an auto-regulation

of intrinsic spiking during bursting.

Changes in the threshold of excitation during bursting

The auto-regulation of spiking could be either a property of

intrinsically ®ring neurons or a general property of neurons in the

cultures independent of their intrinsic activity. In order to distinguish

between these two possibilities, we measured, by whole-cell record-

ing, the excitability of neurons during disinhibition-induced bursting

in dissociated cultures. The neurons selected for these experiments

(n = 13) were not intrinsically spiking in the intervals between the

bursts. The excitability was measured by injecting short current

pulses (50 ms long at 1 Hz) which induced a depolarization just at the

threshold for spike initiation. Both slightly subthreshold and supra-

threshold stimuli were tested to measure changes in excitability in

both directions. The percentage of failures to induce spikes for

constant stimuli was used to express excitability. Approximately half

of the cells (7/13) showed an increase in the failure rate following the

bursts indicating a decrease in excitability (Fig. 3), from which the

cells recovered within 5±10 s. This decrease in excitability went in

parallel with a long-lasting afterhyperpolarization. The other half of

the neurons (6/13) showed a decrease in the failure rate following the

bursts indicating an increase in excitability. In these cells excitability

reached a minimum in the middle of the interval between the bursts.

The low failure rate at the end of the bursts was accompanied by a

slight afterdepolarization. These ®ndings show that modulation of

excitability is not only occurring in intrinsically spiking neurons, and

that excitability can be modi®ed in both directions during bursting.

Excitatory synaptic currents underlying bursting

It has been proposed previously that bursts of activity may be

terminated by a depletion of transmitter causing synaptic depression

(Staley et al., 1998; Fedirchuk et al., 1999). In order to investigate

whether such a mechanism may contribute to disinhibition induced

bursting in dissociated spinal cultures, synaptic currents were

recorded from eight neurons clamped at ±60 mV. Following

disinhibition of the network, bursts consisting of superimposed

synaptic currents appeared (Fig. 4A). Usually, summation of indi-

vidual currents caused a large inward current at the beginning of the

bursts, which was followed by considerably smaller currents of which

the amplitudes gradually declined during the bursts. During the

intervals between the bursts, small currents occurred at rates too low

for a summation of currents. Similar to previous ®ndings (Streit et al.,

2001), the duration of the bursts was positively correlated to the

duration of the preceding intervals (r = 0.53 6 0.08, n = 10, signi®-

cant in 6/10 experiments, Fig. 4B). In these experiments, a stronger

correlation was found between the amplitude of the initial peak

currents of the bursts and the duration of the preceding intervals

FIG. 5. Electrical stimulation by MEA electrodes from a dissociated culture. (A) Original recordings from four MEA channels showing the stimulation artifact(S) and the induced spikes in the ®rst 30 ms following the stimulus. Note that the stimulus-evoked activity is concentrated on the ®rst 15 ms. The location ofthe stimulation (arrow) and recording electrodes (number) is shown in C. (B±D) Spatial patterns of activity evoked by stimulation with two differentintensities (B and C) and two different locations (C and D). Several axons are activated by stimulation at one electrode. The size of the black circlesrepresents the average intensity of the evoked activity in the ®rst 10 ms following the stimulus for each active channel (the channels which recorded activity).Stimulus-evoked activity spans a range from 0 (smallest dots) to 3 events/10 ms (largest circles in C. The upper level is the maximum set by the detectionalgorithm (see Materials and methods). The stimulation electrode (arrow) was not used for recording during stimulation.



(r = 0.73 6 0.07, n = 10, signi®cant in 8/10 experiments, Fig. 4C).

Such a correlation suggests that one or several parameters, which

control peak synaptic currents as well as burst duration, are slowly

recovering during the intervals from the previous burst. Because the

peak currents represent a summation of the contribution from several

active presynaptic neurons, such parameters could include the

excitability of the presynaptic neurons, their synaptic ef®cacy, or

both. In order to investigate the changes in synaptic ef®cacy during

the intervals between the bursts, we measured the rate and peak

amplitudes of the synaptic currents occurring spontaneously during

these intervals. The range of peak amplitudes of these currents (10±

60 pA) was comparable to that previously reported for miniature

currents in spinal neurons (Ulrich & LuÈscher, 1993; Vogt et al.,

1995), suggesting that most of these currents were due to spontaneous

release of glutamate at single synapses. During the intervals, no

change in the peak amplitudes of these currents was seen (Fig. 4D).

The difference between the mean peak amplitude of the ®rst 10

currents following a burst and that of the last 10 currents before the

next burst was 0.84 6 0.66% (n = 9 experiments), which was not

signi®cantly different from 0 (P = 0.24). The rate of spontaneous

currents was high following a burst and decreased towards the next

burst within 5±10 s (Fig. 4E). A signi®cant difference (P = 0.0001)

was found between the mean rate of the ®rst 10 currents following a

burst and that of the last 10 currents before the next burst (±39 6 6%,

n = 9 experiments). These ®ndings exclude transmitter depletion and

the desensitization of postsynaptic receptors as parameters involved

in the recovery process in the intervals between the bursts.

Bursts induced by electrical stimulation

Our ®ndings so far suggest that the initiation of bursts requires

intrinsically ®ring cells and the readiness of the network to respond to

their activity. Both intrinsic ®ring and network activation threshold

are modi®ed during the bursts. In order to investigate the relative

contribution of the network activation threshold to the bursting

patterns, we stimulated dissociated cultures under disinhibition.

Single MEA electrodes were addressed as stimulation electrodes and

the response of the network to the stimulation was measured by the

remaining MEA electrodes. The activities evoked in the network by

stimulation were classi®ed on the basis of their latency either in early

activity (latencies < 10 ms), which was not blocked by CNQX and

APV, or in late activity (latencies > 10 ms), which was due to

synaptic transmission, because it disappeared when excitatory

synaptic transmission was blocked. Early activity, which could be

used as a control for the success of stimulation is shown in Fig. 5 for

different stimulus strengths and locations. Usually several electrodes,

which were distributed in a quite large area, responded to the stimuli

with 1±3 events. Only at the threshold of stimulation (0.5 V) could

the responding electrodes be restricted to a small area (Fig. 5) close

to the stimulating electrode. Changing the stimulating electrode

changed the patterns of the responding electrodes. The reason why

several electrodes responded to single stimuli is probably that several

axons cross the stimulating electrode.

In order to evoke a burst, usually stimuli, which were strong enough

to activate several neurons, were required (0.75±1.25 V). However,

even with such strong stimuli, the network was not able to follow the

stimuli when their frequency was too high. On the other hand, when the

frequency of stimulation was too low, spontaneous bursting was

interfering with the stimulation. Figure 6 shows an experiment in

which the rate of spontaneous bursts was very low (» 2 per min). When

this network was stimulated once every 10 s, it reliably followed the

stimuli with long-lasting bursts. When the frequency of stimulation

was increased to one stimulus every 5 s, the network was still able to

reliably follow stimulation, however, with shorter bursts. We found a

positive correlation between the stimulus interval and the burst

duration in dissociated as well as in slice cultures (Fig. 9B; nine

experiments in slice cultures). When the frequency of stimulation was

further increased to one stimulus every 2 s, the network could follow

every second stimulus only. In ®ve experiments in dissociated cultures,

the success rate of stimuli to induce bursting was 49.2 6 3.2% for

stimulus intervals of 2 s and 98.9 6 1.6% for 5 s intervals

(P < 0.001). As reported previously for cortical cultures (Maeda

et al., 1995), stimuli applied during or immediately following a burst

did not usually prolong the bursts or evoke a new burst, respectively. In

dissociated cultures, the frequency of approximately 0.3 Hz seemed to

represent the highest bursting frequency that could be reached by this

network. Even stimulation at 5 Hz or 10 Hz induced bursting at 0.3 Hz

(n = 8). The mean minimum burst period was 5.8 6 0.9 s (range: 3.2±

10.3 s). The period of spontaneous bursting in the absence of

stimulation in the same group of experiments was 9.2 6 4.5 s

(range: 4.7±17.1 s). In all of these experiments, the maximum

frequency of stimulated bursting was faster than spontaneous bursting.

Together, these ®ndings suggest that the readiness of the network to

respond to active neurons is low following the bursts and slowly

increases during the intervals within several seconds. This network

refractory period determines the maximum possible burst frequency,

whereas the actual spontaneous burst frequency is additionally

controlled by the recovery of the intrinsically ®ring neurons.

The low excitability of some neurons and the refractoriness of the

network at the end of the bursts both suggest that the excitability of

the neurons is on average decreasing during the bursts. In order to

investigate the average network excitability during standardized

bursts, high frequency trains of stimuli were applied by one electrode

in the absence of functional synaptic transmission (in the presence of

strychnine, bicuculline, CNQX and APV). As shown before, several

neurons were directly activated by stimulation via one electrode.

Therefore the total amount of the activity in the network within the

®rst 10 ms following a stimulus was taken as a measure of stimulus-

FIG. 6. Network activity plots of stimulus-locked bursting underdisinhibition in a dissociated culture. In the presence of bicuculline (20 mM)and strychnine (1 mM) the network reliably responds to single stimuli withbursts, if the frequency of stimulation is below approximately 0.3 Hz. Notethat the duration of the bursts is decreasing with increasing stimulationfrequency (compare also Fig. 9B). At a frequency of 0.5 Hz every secondstimulus failed to induce bursts, resulting in an evoked burst rate of0.25 Hz. At even higher frequencies (5 Hz) many stimuli failed to inducebursts resulting in an evoked burst frequency of approximately 0.3 Hz. Thespontaneous burst frequency in the absence of stimulation in thisexperiment was low (< 0.1 Hz). The stimulation strength was 1 V.



induced direct (non-synaptic) excitation of the network. Such

network excitation was usually not affected by stimulus frequencies

up to 5±10 Hz (Fig. 7A). At 20 Hz stimulation it decreased during

the trains (Fig. 7A and B). The decrease occurred within several

seconds and seemed to be also dependent on the history of the train,

i.e. the recovery from previous trains. The decrease in excitability

was not equal for all electrodes that measured activity following the

stimuli (Fig. 7B). This ®nding excludes the possibility that it was due

to high frequency-induced changes in the current ¯ow through the

electrodes. In 11 experiments such a decrease in network excitability

was seen during 20 Hz trains, in some experiments it occurred, to a

lesser extent, already in 10 Hz or even 5 Hz trains. Similar to

intracellular current injection (Fig. 2), trains of 20 Hz extracellular

stimulation suppressed the intrinsic activity (measured in the absence

of synaptic transmission by MEAs) at some of those electrodes,

where spiking was directly induced by stimulation (Fig. 7C, arrows).

In summary, these ®ndings con®rm that, at least in some of the

neurons, spiking at high rates decreases excitability, thus leading, at

the end of a burst, to a suppression of intrinsic spiking on the one

hand and of the responsiveness of the network on the other.

Bursting in slice cultures is ruled by similar mechanisms

The previous ®ndings suggest, that in dissociated spinal cultures,

disinhibition-induced bursting is mainly ruled by intrinsic ®ring and

by the modulation of excitability during bursting, whereas synaptic

depletion or receptor desensitization are not involved. Previously,

however, we have proposed that the fast oscillations seen within

bursts in slice cultures are mainly ruled by synaptic depression (Streit,

1993; Senn et al., 1996; Streit, 1996). To investigate, whether the

bursting in slice cultures is ruled by similar mechanisms as in the

dissociated cultures, we looked for evidence for intrinsic ®ring which

initiates the bursts, and for a modulation of excitability during

bursting in slice cultures. In the dissociated cultures, the suppression

of intrinsic ®ring by the bursts leads to the increase in the rate of

network activity in the intervals towards the next burst (Fig. 1A and

B). In the slice cultures, the rate of activity within the intervals was

lower than in the dissociated cultures (Tscherter et al., 2001).

Nevertheless, the bursts in the slice cultures were often preceded by

intrinsic activity in some channels (Fig. 8A). When the intrinsic

activity in the intervals was measured in time windows of 100 ms, the

windows preceding the bursts contained increasing amounts of

activity. Most of the activity was seen in the last 100 ms before the

bursts (Fig. 8B). Sites with high activity before the bursts were

preferentially situated in the ventral parts of the spinal slices around

the ventral ®ssure (Fig. 8C). This roughly corresponded to the

distribution of the burst sources in the slice (Fig. 8D and E and

Tscherter et al., 2001). When the slices were stimulated by one

electrode, bursts were reliably evoked only in a de®ned range of

FIG. 7. MEA recordings from a dissociated culture showing the response of a network to stimulation (1 V) in the absence of fast synaptic transmission(bicuculline 20 mM, strychnine 1 mM, CNQX 10 mM, APV 50 mM). Several axons are activated by stimulation at one electrode. (A) Response during period ofstimulation with various frequencies from 0.5 to 20 Hz. Note the developing decrease in response during consecutive periods of 20 Hz stimulation. (B)Spatial patterns of activity evoked during a period of 10 Hz stimulation and at the end of period of 20 Hz stimulation, when the network response isdecreased. The size of the black circles represents the average intensity of the evoked activity in the ®rst 10 ms following the stimulus for each activechannel. Stimulus-evoked activity spans a range from 0 (smallest dots) to 3 events/10 ms (largest circles, see also Fig. 5). The location of the stimulatingelectrode is shown by an arrow. Note that the activity at 20 Hz is not reduced at all channels and even increased at one channel. (C) Event raster plots forthree channels during three consecutive periods of 20 Hz stimulation. Note the suppression of intrinsic activity (arrows) following the stimulation periods(bars). Longer stimulus trains seem to provoke stronger suppression. Different experiment from A and B.



frequencies. When the frequency was too low, the spontaneous bursts

interfered with the evoked bursts; when the frequency was too high,

stimulation was not reliable any more and some stimuli failed to

evoke bursts (Fig. 9A). Within the range of reliable stimulation

(intervals of 3±35 s, Fig. 9B±E), the duration of the evoked bursts,

the maximum activation of the network during the bursts and the total

activity within the ®rst 10 ms after the stimulus (excitability)

normally increased with increasing intervals between the stimuli,

whereas the time taken to reach maximal activation decreased

(Fig. 9B±E, for nine experiments). This shows that the earlier after

the last burst the network is stimulated, the less neurons can be

activated, the longer it takes to activate the whole network and the

faster the critical threshold for burst termination is reached. All these

®ndings are in line with those previously described in the dissociated

cultures. Therefore they suggest that the same mechanisms are

involved in the control of bursting in slice cultures as in dissociated

cultures.

Discussion

In the present study we investigated the mechanisms involved in

disinhibition-induced bursting in spinal cultures. These ®ndings

con®rm the hypothesis that bursting is due to intrinsic ®ring in a

subset of spinal neurons, which activate the whole network through

recurrent excitation. Such network activation (the burst) is terminated

after one to several seconds mainly due to a decrease in neuronal

excitability. It is followed by a network refractory period (the

interval), which is mainly determined by an auto±regulation of

neuronal excitability and not by synaptic parameters like depletion or

receptor desensitization.

Intrinsic spiking

We have previously shown that at many of the sites, where bursts are

preferentially initiated (burst sources), some low level of activity

persists when fast synaptic transmission is blocked (Streit et al.,

2001). We have suggested that such activity originates from

intrinsically ®ring neurons. Intracellular recordings from such

neurons now show that intrinsic spiking is due to slow depolarizations

between the spikes which can be switched off by a slight

hyperpolarization. Under such conditions, the membrane potential

is ¯at without spontaneous oscillations as seen in motoneurons and

interneurons in the presence of NMDA (Hochman et al., 1994;

Schmidt et al., 1998; Tresch & Kiehn, 2000). The neurons ®red at

rates of 0.3±7 Hz, similar to rates previously reported for mouse

cultures (Latham et al., 2000). However, as the ®ring rate was highly

dependent on the membrane potential, it was variable and could also

cease and reappear spontaneously. While endogenous bursting cells

have been reported in brainstem slices (Koshiya & Smith, 1999) and

their presence is still a matter of debate in mouse cultures (Legendre

et al., 1985; Latham et al., 2000), most of the intrinsically active cells

found in our cultures were tonically active. However, we found

occasional evidence for intrinsic bursters in extracellular MEA

recordings (Streit et al., 2001) and in one whole cell patch recording

(our unpublished observation). It has to be considered that the

intrinsic ®ring patterns may be modulated by the network activity

(Turrigiano et al., 1994).

Auto-regulation of excitability

While intrinsically spiking neurons certainly provided a source of

activity in the network, the patterns of intrinsic active neurons did not

determine its bursting patterns. Instead, bursting was a network

phenomenon based on the recurrent excitation of the network, which

was initiated by the intrinsically ®ring cells. Following the bursts,

intrinsic ®ring is nearly suppressed and recovers in the subsequent

FIG. 8. MEA recordings from an organotypic slice culture showing thatbursts are preceded by an increase in spike rate in the areas of burstinitiation. (A) Event raster plot from 16 channels at the beginning of oneburst. Note the unitary events preceding the burst onset in three channels.(B) Histogram showing the number of events per 100 ms in the networkduring the whole intervals and in the last ®ve bins of 100 ms of the intervalbefore the start of the burst (n = 20). (C) Sites in the slice where the spikerate increased before the bursts. The circles represent the percentage ofbursts with at least one event during the last 100 ms preceding the burst.(D) Sites of burst initiation. The size of the circles shows at whatpercentage bursts started at the location indicated. (E) Average propagationof the wave-front of the bursts in the slice. The lines (isochrones) show howlong it takes for the wavefront of the burst to reach the different electrodes(5.5 ms between 2 lines). The isochrones are extrapolated from measure-ments of the latencies at the electrodes.



interval. Such a behaviour has been predicted previously in models

based on spike frequency adaptation (Latham et al., 2000) or slow

synaptic depression (Tabak et al., 2000). Spike frequency adaptation

has been described for neurons in the lamprey spinal cord (Elmanira

et al., 1994) and for motoneurons in the cat spinal cord (Kernell,

1965). In rat organotypic cultures spike frequency adaptation was

found in motoneurons (Streit et al., 1991), but not however in spinal

interneurons (Ballerini et al., 1999). Furthermore, it is well known

that neuronal excitability can be in¯uenced by neurotransmitters for

example through Ca2+-dependent K+-conductances (White &

Neuman, 1980; WalleÂn et al., 1989; Knopfel et al., 1990) or by

growth factors like BDNF (Desai et al., 1999a). To investigate a

possible role of such mechanisms in the suppression of intrinsic

spiking following bursts, we simulated bursts in the absence of fast

synaptic transmission by current injection in single neurons and by

high frequency trains of stimulation in the network. These experi-

ments suggest that the suppression of intrinsic activity following

bursts is directly caused by the spiking activity during the bursts, in

the sense of an auto-regulation of excitability, and not by the synaptic

activity. Nevertheless, we found neither evidence for spike frequency

adaptation nor for an accumulation of afterhyperpolarization in these

neurons (Fig. 2B), at least not during current injection. Following the

current pulse, there was a brief afterhyperpolarization, which was

much shorter than the suppression of the intrinsic activity. Thus, the

mechanism that underlies the suppression of intrinsic activity

following bursts remains unclear. It may involve a direct effect of

activity on ionic conductances like Na+ and K+ channels as recently

shown for pyramidal neurons (Desai et al., 1999b), however, on a

much longer time-scale, or it may be related to regulation of

excitability by intracellular Ca2+ (Turrigiano et al, 1994). In line with

FIG. 9. Stimulus-induced bursting in slice cultures which were disinhibited by bicuculline (20 mM) and strychnine (1 mM). (A) Network activity plots showingthat bursts reliably follow external stimuli only in a de®ned range of frequencies. At too low a frequency a spontaneous burst intermingled with the stimulus-induced bursts (upper trace). At too high a frequency some stimuli failed to induce bursts (lower trace). Plots of the burst duration (B), the maximumactivation during the burst (C), the time to reach maximum activation (D), and the network excitability (E) vs. the stimulus interval in nine experiments. Thenetwork excitability was determined by counting the events induced by stimulation at all electrodes in the ®rst 10 ms following the stimulus. All data wereobtained in the range of reliable burst induction.



the hypothesis of an auto-regulation of excitability, the recovery of

intrinsic spiking following the cessation of current injections

(Fig. 2D) usually ®rst led to phasic activity after 5±10 s, which

gradually turned into tonic activity (a damped oscillation of the spike

rate, which is characteristic for an unbalanced auto-regulation).

A decrease in excitability following the bursts was not only seen in

intrinsically ®ring neurons, but also in roughly half of the neurons

without intrinsic activity, as indicated by the decrease in response to

depolarizing current pulses (Fig. 3). It went in parallel with a slow

afterhyperpolarization following the bursts (Figs 1 and 3A). A similar

slow afterhyperpolarization following bursts in the hippocampus has

been suggested to be based on Ca2+ -induced K+ conductances (Alger

& Nicoll, 1980; Elmanira et al., 1994; Traub & Jefferys, 1994;

Empson & Jefferys, 2001). However, it may also be related to the

increased activity of the electrogenic Na/K pump following the bursts

(Ballerini et al., 1997). The other half of the cells without intrinsic

activity were depolarized and the response to depolarizing current

pulses was increased at the end of the bursts (Fig. 3B). These ®ndings

are dif®cult to interpret, because the behaviour of the neurons at the

end of the bursts is in¯uenced by their intrinsic properties, as well as

by ongoing activity in the network.

Burst initiation and termination

As previously mentioned, intrinsic spiking must activate the network

through recurrent excitation in order to initiate a burst. When single

or repetitive spikes were evoked in single neurons by current

injection, bursts could not be initiated. However, when several

neurons were activated together by extracellular electrical stimuli,

bursts could be initiated provided the stimuli were not delivered too

soon following the last burst. Besides the frequency of stimulation

(see below) the success rate of stimulation was dependent on the

strength of the stimulus (i.e. the number of stimulated neurons) and

the location of the stimulating electrode (affecting the number and

selection of the stimulated neurons). These ®ndings suggest that in

order to initiate bursts, either several neurons have to be simultan-

eously active or the right neurons (privileged by their connectivity)

must be active. Even when the stimulation site and strength were

optimized to activate the network, burst initiation was frequency

dependent. When the stimulation frequency was too high, some of the

stimuli failed to initiate bursts (Figs 6 and 9). Similar observations

have been made in the intact spinal cord of the neonatal rat for dorsal

root stimulation (Bracci et al., 1997) and in dissociated cortical

cultures (Robinson et al., 1993; Maeda et al., 1995). These ®ndings

suggest, that the bursts are followed by network refractory periods,

which determine the maximum possible burst rate (Maeda et al.,

1995). The duration of these refractory periods correspond well to the

decrease in excitability, suggesting that they are mainly based on the

modulation in excitability. A similar mechanism has been proposed to

underlie the bursting induced by high potassium in hippocampal

slices (Chamberlin et al., 1990; Traub & Dingledine, 1990) and the

slow oscillations in neocortical slices (Sanchez-Vives & McCormick,

2000).

During the bursts there was a slow steady reduction in network

activity (Fig. 1D), which was probably due to a general reduction in

excitability in the network. This ®nally turned into a rapid reduction

which terminated the burst. The turning point probably corresponds

to the time point when the number of activated neurons is not

suf®cient to maintain full network activation (network activation

threshold). In most of the experiments both the burst duration and the

peak activity of the bursts are controlled by the duration of the

preceding interval (Fig. 6). This is most clearly seen in experiments

in which bursting is reliably induced by stimulation at various

frequencies (Fig. 9B). This correlation between burst parameters and

the preceding interval can be explained with the relative network

refractory period. The earlier in this period a new burst is initiated,

the less neurons are initially activated (Fig. 9E) and the sooner the

network activation threshold is reached.

Synaptic depression

Frequency-dependent synaptic depression has been shown to be

prominent in the intact spinal cord of the neonatal rat (Pinco &

Levtov, 1993) as well as in slice cultures of embryonic rat spinal cord

(Streit et al., 1992). In the latter it has been proposed to underlie the

high frequency oscillations, which are often seen within the bursts

(Streit, 1993; Tscherter et al., 2001). In the spinal cord of the chick

embryo as well as in hippocampal slices, refractory periods following

bursts have also been attributed to slowly recovering synaptic

depression (O'Donovan & Rinzel, 1997; Staley et al., 1998; Tabak &

O'Donovan, 1998; Fedirchuk et al., 1999). In dissociated cultures of

mouse spinal cord, however, depression seems to be less prominent in

synapses connecting interneurons (Jia & Nelson, 1986). Synaptic

depression is often attributed to depletion of the readily releasable

pool of vesicles on the presynaptic side (Pieribone et al., 1995;

Dittman & Regehr, 1998; Wu & Borst, 1999), or to the desensitiza-

tion of the postsynaptic receptors (Trussell et al., 1993; Otis et al.,

1996). To investigate a contribution of a slow recovery from synaptic

depression to the network refractory period following the bursts, we

studied the peak amplitudes and the rate of the spontaneous synaptic

currents occurring during the intervals. Such spontaneous currents

have the same range of peak amplitudes as previously reported for

miniature currents, suggesting that most of them are due to

spontaneous release of transmitter in the absence of presynaptic

spikes (Ulrich & LuÈscher, 1993; Vogt et al., 1995). However, we

have shown before, that spikes occur during the intervals, especially

towards the end, suggesting that a small percentage of these currents

could be evoked by presynaptic spikes. In both cases, the rate and

mean amplitude of such currents should be decreased, when the

readily releasable pool of vesicles were depleted following the bursts

(Highstein & Bennett, 1975; Glavinovic, 1995; Zhou et al., 2000).

This was clearly not seen (Fig. 4E), suggesting that depletion was not

involved. In contrast, the rate of currents was increased following the

bursts and decayed during the intervals. This could be explained by

the high Ca2+ concentration in the presynaptic terminals following

the bursts, as it has been shown that high presynaptic Ca2+

concentration increases the rate of miniature potentials (Erulkar &

Rahamimoff, 1978; Llano et al., 2000). On the other hand, if the

postsynaptic receptors are desensitized following the bursts, the

peak amplitudes of the currents in the intervals should be

decreased. Again, this was not the case (Fig. 4D), excluding such

a mechanism. In summary, we found no evidence for a slow

recovery from synaptic depression during the intervals between

the burst.

In summary, our results support the hypothesis, that the generation

and termination of bursts induced by disinhibition in spinal cultures

(dissociated and organotypic) is mainly controlled by intrinsic spiking

in some neurons, recurrent excitation through glutamatergic synaptic

transmission and auto±regulation of neuronal excitability.

Acknowledgements

We would like to thank Ruth Rubli for carrying out the culture work, MarcHeuschkel for producing the arrays, Denis de Limoges, Hans Ruchti, JuÈrg



Burkhalter and Hans-Ueli Schweizer for the electronic and mechanicalequipment of the recording set-up, Nissim Buchs for software support and DrLucinda Davies and Dr David Thurbon for helpful comments on themanuscript.

This work was supported by the Swiss National Science Foundation (SNFgrants no. 31±59080.99 and 3152±053761.98).

Abbreviations

APV, (+/±)-2-amino-5-phosphonopentanoic acid; AMPA, (+/±)-a-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid hydrobromide; CNQX, 6-cyano-7-nitroquinoxaline-2-3-dione; GABA, g-aminobutyric acid; HEPES,4-(2-hydroxyethyl)-piperazin-1-ethansulphonic acid; 5-HT, 5-hydroxytrypta-mine (serotonin); MEA, multielectrode array; NMDA: N-methyl-D-asparticacid; r, correlation coef®cient; TTX, tetrodotoxin.

References

Alger, B.E. & Nicoll, R.A. (1980) Epileptiform burst afterhyperolarization:calcium-dependent potassium potential in hippocampal CA1 pyramidalcells. Science, 210, 1122±1124.

Ballerini, L., Bracci, E.M. & Nistri, A. (1997) Pharmacological block ofelectrogenic sodium pump disrupts rhythmic bursting induced by strychnineand bicuculline in the neonatal rat spinal cord. J. Neurophysiol., 77, 17±23.

Ballerini, L. & Galante, M. (1998) Network bursting by organotypic spinalslice cultures in the presence of bicuculline and/or strychnine isdevelopmentally regulated. Eur. J. Neurosci., 10, 2871±2879.

Ballerini, L., Galante, M., Grandolfo, M. & Nistri, A. (1999) Generation ofrhythmic patterns of activity by ventral interneurones in rat organotypicspinal slice culture. J. Physiol. (Lond.), 517, 459±475.

Beato, M. & Nistri, A. (1999) Interaction between disinhibited bursting and®ctive locomotor patterns in the rat isolated spinal cord. J. Neurophysiol.,82, 2029±2038.

Bracci, E., Ballerini, L. & Nistri, A. (1996) Spontaneous rhythmic burstsinduced by pharmacological block of inhibition in lumbar motoneurons ofthe neonatal rat spinal cord. J. Neurophysiol., 75, 640±647.

Bracci, E., Beato, M. & Nistri, A. (1997) Afferent inputs modulate the activityof a rhythmic burst generator in the rat disinhibited spinal cord in vitro. J.Neurophysiol., 77, 3157±3167.

Braschler, U.F., Iannone, A., Spenger, C., Streit, J. & LuÈscher, H.-R. (1989) Amodi®ed roller tube technique for organotypic cocultures of embryonic ratspinal cord, sensory ganglia and skeletal muscle. J. Neurosci. Meth., 29,121±129.

Cazalets, J.R., Borde, M. & Clarac, F. (1995) Localization and organization ofthe central pattern generator for hindlimb locomotion in newborn rat. J.Neurosci., 15, 4943±4951.

Chamberlin, N.L., Traub, R.D. & Dingledine, R. (1990) Spontaneous EPSPsinitiate burst-®ring in rat hippocampal neurons bathed in high potassium. J.Neurophysiol., 64, 1000±1008.

Dale, N. & Kuenzi, F. (1997) Ionic currents, transmitters and models of motorpattern generators. Curr. Opin. Neurobiol., 7, 790±796.

Desai, N.S., Rutherford, L.C. & Turrigiano, G.G. (1999a) BDNF regulates theintrinsic excitability of cortical neurons. Learn Mem., 6, 284±291.

Desai, N.S., Rutherford, L.C. & Turrigiano, G.G. (1999b) Plasticity in theintrinsic excitability of cortical pyramidal neurons. Nature Neurosci., 2,515±520.

Dittman, J.S. & Regehr, W.G. (1998) Calcium dependence and recoverykinetics of presynaptic depression at the climbing ®ber to Purkinje cellsynapse. J. Neurosci., 18, 6147±6162.

Droge, M.H., Gross, G.W., Hightower, M.H. & Czisny, L.E. (1986)Multielectrode analysis of coordinated, multisite, rhythmic bursting incultured CNS monolayer networks. J. Neurosci., 6, 1583±1592.

Elmanira, A., Tegner, J. & Grillner, S. (1994) Calcium-dependent potassiumchannels play a critical role for burst termination in the locomotor networkin lamprey. J. Neurophysiol., 72, 1852±1861.

Empson, R.M. & Jefferys, J.G. (2001) Ca(2+) entry through L-type Ca(2+)channels helps terminate epileptiform activity by activation of a Ca(2+)dependent afterhyperpolarisation in hippocampal CA3. Neuroscience, 102,297±306.

Erulkar, S.D. & Rahamimoff, R. (1978) The role of calcium ions in tetanic andpost-tetanic increase of miniature end-plate potential frequency. J. Physiol.(Lond.), 278, 501±511.

Fedirchuk, B., Wenner, P., Whelan, P.J., Ho, S., Tabak, J. & O'Donovan, M.J.

(1999) Spontaneous network activity transiently depresses synaptictransmission in the embryonic chick spinal cord. J. Neurosci., 19, 2102±2112.

Glavinovic, M.I. (1995) Decrease of quantal size and quantal content duringtetanic stimulation detected by focal recording. Neuroscience, 69, 271±281.

Grillner, S., Parker, D. & el Manira, A. (1998) Vertebrate locomotion: alamprey perspective. Ann. N Y Acad. Sci., 860, 1±18.

Grillner, S. & WalleÂn, P. (1985) Central pattern generators for locomotion,with special reference to vertebrates. Annu. Rev. Neurosci., 8, 233±261.

Gross, G.W., Rhoades, B.K. & Kowalski, J.M. (1993a) Dynamics of burstpatterns generated by monolayer networks in culture. In Bothe, H.-W.,Samii, M. & Eckmiller, R. (eds), Neurobionics. Elsevier, Amsterdam, pp.89±121.

Gross, G.W., Rhoades, B.K., Reust, D.L. & Schwalm, F.U. (1993b)Stimulation of monolayer networks in culture through thin-®lm indium-tin oxide recording electrodes. J. Neurosci. Meth., 50, 131±143.

Gross, G.W., Williams, A.N. & Lucas, J.H. (1982) Recording of spontaneousactivity with photoetched microelectrode surfaces from mouse spinalneurons in culture. J. Neurosci. Meth., 5, 13±22.

Hamill, O.P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F.J. (1981)Improved patch-clamp techniques for high-resolution current recordingfrom cells and cell-free membrane patches. P¯uÈgers Arch., 391, 85±100.

Highstein, S.M. & Bennett, M.V. (1975) Fatigue and recovery of transmissionat the Mauthner ®ber-giant ®ber synapse of the hatchet®sh. Brain Res., 98,229±242.

Hochman, S., Jordan, L.M. & Schmidt, B.J. (1994) TTX-resistant NMDAreceptor-mediated voltage oscillations in mammalian lumbar motoneurons.J. Neurophysiol., 72, 2559±2562.

Jia, M. & Nelson, P.G. (1986) Calcium currents and transmitter output incultured spinal cord and dorsal root ganglion neurons. J. Neurophysiol., 56,1257±1267.

Jimbo, Y., Robinson, H.P. & Kawana, A. (1993) Simultaneous measurementof intracellular calcium and electrical activity from patterned neuralnetworks in culture. IEEE Trans. Biomed. Eng., 40, 804±810.

Kernell, D. (1965) High-frequency repetitive ®ring of cat lumbosacralmotoneurons stimulated by long-lasting injected currents. Acta Physiol.Scand., 65, 74±86.

Kiehn, O. & Kjaerulff, O. (1998) Distribution of central pattern generators forrhythmic motor outputs in the spinal cord of limbed vertebrates. Ann. N YAcad. Sci., 860, 110±129.

Knopfel, T., Vranesic, I., Gahwiler, B.H. & Brown, D.A. (1990) Muscarinicand beta-adrenergic depression of the slow Ca2+-activated potassiumconductance in hippocampal CA3 pyramidal cells is not mediated by areduction of depolarization-induced cytosolic Ca2+ transients. Proc. Natl.Acad. Sci. USA, 87, 4083±4087.

Koshiya, N. & Smith, J.C. (1999) Neuronal pacemaker for breathingvisualized in vitro. Nature, 400, 360±363.

Latham, P.E., Richmond, B.J., Nirenberg, S. & Nelson, P.G. (2000) Intrinsicdynamics in neuronal networks. II. experiment. J. Neurophysiol., 83, 828±835.

Legendre, P., Guzman, A., Dupouy, B. & Vincent, J.D. (1989) Excitatoryeffect of serotonin on pacemaker neurons in spinal cord cell culture.Neuroscience, 28, 201±209.

Legendre, P., McKenzie, J.S., Dupouy, B. & Vincent, J.D. (1985) Evidence forbursting pacemaker neurones in cultured spinal cord cells. Neuroscience,16, 753±767.

Llano, I., Gonzalez, J., Caputo, C., Lai, F.A., Blayney, L.M., Tan, Y.P. &Marty, A. (2000) Presynaptic calcium stores underlie large-amplitudeminiature IPSCs and spontaneous calcium transients. Nature Neurosci., 3,1256±1265.

Maeda, E., Robinson, H.P.C. & Kawana, A. (1995) The mechanisms ofgeneration and propagation of synchronized bursting in developingnetworks of cortical neurons. J. Neurosci., 15, 6834±6845.

MuÈller, W. & Swandulla, D. (1995) Synaptic feedback excitation hashypothalamic neural networks generate quasirhythmic burst activity. J.Neurophysiol., 73, 855±861.

O'Brien, R.J. & Fischbach, G.D. (1986) Excitatory synaptic transmissionbetween interneurons and motoneurons in chick spinal cord cell cultures. J.Neurosci., 6, 3284±3289.

O'Donovan, M.J. & Rinzel, J. (1997) Synaptic depression: a dynamicregulator of synaptic communication with varied functional roles. TrendsNeurosci., 20, 431±433.

Otis, T., Zhang, S. & Trussell, L.O. (1996) Direct measurement of AMPAreceptor desensitization induced by glutamatergic synaptic transmission. J.Neurosci., 16, 7496±7504.



Pieribone, V.A., Shupliakov, O., Brodin, L., Hil®ker-Rothen¯uh, S., Czernik,A.J. & Greengard, P. (1995) Distinct pools of synaptic vesicles inneurotransmitter release. Nature, 375, 493±497.

Pinco, M. & Levtov, A. (1993) Modulation of monosynaptic excitation in theneonatal rat spinal cord. J. Neurophysiol., 70, 1151±1158.

Ransom, B.R., Christian, C.N., Bullock, P.N. & Nelson, P.G. (1977) Mousespinal cord in cell culture. II. Synaptic activity and circuit behavior. J.Neurophysiol., 40, 1151±1161.

Robinson, H.P., Torimitsu, K., Jimbo, Y., Kuroda, Y. & Kawana, A. (1993)Periodic bursting of cultured cortical neurons in low magnesium: cellularand network mechanisms. Jpn. J. Physiol., 43, S125±S130.

Sanchez-Vives, M.V. & McCormick, D.A. (2000) Cellular and networkmechanisms of rhythmic recurrent activity in neocortex. Nature Neurosci.,3, 1027±1034.

Schmidt, B.J., Hochman, S. & MacLean, J.N. (1998) NMDA receptor-mediated oscillatory properties: potential role in rhythm generation in themammalian spinal cord. Ann. N Y Acad. Sci., 860, 189±202.

Senn, W., Wyler, K., Streit, J., Larkum, M., LuÈscher, H.-R., Mey, H., MuÈller,L., Stainhauser, D., Vogt, K. & Wannier, T. (1996) Dynamics of a randomneural network with synaptic depression. Neural Networks, 9, 575±588.

Spenger, C., Braschler, U.F., Streit, J. & LuÈscher, H.-R. (1991) Anorganotypic spinal cord-dorsal root ganglion-skeletal muscle coculture ofembryonic rat. I. The morphological correlates of the spinal re¯ex arc. Eur.J. Neurosci., 3, 1037±1053.

Staley, K.J., Longacher, M., Bains, J.S. & Yee, A. (1998) Presynapticmodulation of CA3 network activity. Nature Neurosci., 1, 201±209.

Streit, J. (1993) Regular oscillations of synaptic activity in spinal networksin vitro. J. Neurophysiol., 70, 871±878.

Streit, J. (1996) Mechanisms of pattern generation in cocultures of embryonicspinal cord and skeletal muscle. Int. J. Dev. Neurosci., 14, 137±148.

Streit, J., LuÈscher, C. & LuÈscher, H.-R. (1992) Depression of postsynapticpotentials by high frequency stimulation in embryonic motoneurons grownin spinal cord slice cultures. J. Neurophysiol., 68, 1793±1803.

Streit, J., Spenger, C. & LuÈscher, H.-R. (1991) An organotypic spinal cord-dorsal root ganglia-skeletal muscle coculture of embryonic rat. II.Functional evidence for the formation of spinal re¯ex arcs in vitro. Eur.J. Neurosci., 3, 1054±1068.

Streit, J., Tscherter, A., Heuschkel, M.O. & Renaud, P. (2001) The generationof rhythmic activity in dissociated cultures of rat spinal cord. Eur. J.Neurosci., 14, 191±202.

Tabak, J. & O'Donovan, M.J. (1998) Statistical analysis and intersegmentaldelays reveal possible roles of network depression in the generation ofspontaneous activity in the chick embryo spinal cord. Ann. N Y Acad. Sci.,860, 428±431.

Tabak, J., Senn, W., O'Donovan, M.J. & Rinzel, J. (2000) Modeling of

spontaneous activity in developing spinal cord using activity-dependentdepression in an excitatory network. J. Neurosci., 20, 3041±3056.

Traub, R.D. & Dingledine, R. (1990) Model of synchronized epileptiformbursts induced by high potassium in CA3 region of rat hippocampalslilce. Role of spontaneous EPSPs in initiation. J. Neurophysiol., 64,1009±1018.

Traub, R.D. & Jefferys, J.G. (1994) Are there unifying principles underlyingthe generation of epileptic afterdischarges in vitro? Prog. Brain Res., 102,383±394.

Tresch, M.C. & Kiehn, O. (2000) Motor coordination without action potentialsin the mammalian spinal cord. Nature Neurosci., 3, 593±599.

Trussell, L.O., Zhang, S. & Raman, I.M. (1993) Desensitization of AMPAreceptors upon multiquantal neurotransmitter release. Neuron, 10, 1185±1196.

Tscherter, A., Heuschkel, M.O., Renaud, P. & Streit, J. (2001) Spatiotemporalcharacterization of rhythmic activity in spinal cord slice cultures. Eur. J.Neurosci., 14, 179±190.

Turrigiano, G., Abbott, L.F. & Marder, E. (1994) Activity-dependent changesin the intrinsic properties of cultured neurons. Science, 264, 974±977.

Ulrich, D. & LuÈscher, H.-R. (1993) Miniature excitatory synaptic currentscorrected for dendritic cable properties reveal quantal size and variance. J.Neurophysiol., 69, 1769±1773.

Vogt, K., LuÈscher, H.-R. & Streit, J. (1995) Analysis of synaptic transmissionat single identi®ed boutons on rat spinal neurons in culture. P¯uÈgers Arch.,430, 1022±1028.

WalleÂn, P., Christenson, J., Brodin, L., Hill, R., Lansner, A. & Grillner, S.(1989) Mechanisms underlying the serotonergic modulation of the spinalcircuitry for locomotion in lamprey. Prog. Brain Res., 80, 321±327.

WalleÂn, P., Ekeberg, O., Lansner, A., Brodin, L., TraÊveÂn, H. & Grillner, S.(1992) A computer-based model for realistic simulations of neuralnetworks. II. The segmental network generating locomotor rhythmicity inthe lamprey. J. Neurophysiol., 68, 1939±1950.

White, S.R., Fung, S.J., Jackson, D.A. & Imel, K.M. (1996) Serotonin,norepinephrine and associated neuropeptides: effects on somaticmotoneuron excitability. Prog. Brain Res., 107, 183±199.

White, S.R. & Neuman, R.S. (1980) Facilitation of spinal motoneuroneexcitability by 5-hydroxytryptamine and noradrenaline. Brain Res., 188,119±127.

Wu, L.G. & Borst, J.G. (1999) The reduced release probability of releasablevesicles during recovery from short-term synaptic depression. Neuron, 23,821±832.

Zhou, Q., Petersen, C.C. & Nicoll, R.A. (2000) Effects of reduced vesicular®lling on synaptic transmission in rat hippocampal neurones. J. Physiol.(Lond.), 525, 195±206.



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Mechanisms controlling bursting activity induced by disinhibition in spinal cord networks

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