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Neuroscience 140 (2006) 1401–1413

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RANSIENT RHYTHMIC NETWORK ACTIVITY IN THE SOMATOSENSORY

ORTEX EVOKED BY DISTRIBUTED INPUT IN VITRO

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. BERGER,* H.-R. LÜSCHER AND M. GIUGLIANO1

nstitute of Physiology, University of Bern, Bühlplatz 5, CH-3012 Bern,witzerland

bstract—The initiation and maintenance of physiologicalnd pathophysiological oscillatory activity depends on theynaptic interactions within neuronal networks. We studiedhe mechanisms underlying evoked transient network oscil-ation in acute slices of the adolescent rat somatosensoryortex and modeled its underpinning mechanisms. Oscilla-ions were evoked by brief spatially distributed noisy extra-ellular stimulation, delivered via bipolar electrodes. Evokedransient network oscillation was detected with multi-neuronatch-clamp recordings under different pharmacologicalonditions. The observed oscillations are in the frequencyange of 2–5 Hz and consist of 4–12 mV large, 40–150 mside compound synaptic events with rare overlying actionotentials. This evoked transient network oscillation is onlyeakly expressed in the somatosensory cortex and requires

ncreased [K�]o of 6.25 mM and decreased [Ca2�]o of 1.5 mMnd [Mg2�]o of 0.5 mM. A peak in the cross-correlation amongembrane potential in layers II/III, IV and V neurons reflects

he underlying network-driven basis of the evoked transientetwork oscillation. The initiation of the evoked transientetwork oscillation is accompanied by an increased [K�]o

nd can be prevented by the K� channel blocker quinidine. Inddition, a shift of the chloride reversal potential takes placeuring stimulation, resulting in a depolarizing type A GABAGABAA) receptor response. Blockade of �-amino-3-hydroxy--methyl-4-isoxazole-proprionate (AMPA), N-methyl-D-aspar-ate (NMDA), or GABAA receptors as well as gap junctionsrevents evoked transient network oscillation while a reduc-ion of AMPA or GABAA receptor desensitization increasests duration and amplitude. The apparent reversal potential of27 mV of the evoked transient network oscillation, its phar-acological profile, as well as the modeling results suggestmixed contribution of glutamatergic, excitatory GABAergic,nd gap junctional conductances in initiation and mainte-ance of this oscillatory activity. With these properties,voked transient network oscillation resembles epileptic af-erdischarges more than any other form of physiological orathophysiological neocortical oscillatory activity. © 2006

BRO. Published by Elsevier Ltd. All rights reserved.

ey words: rat, oscillation, afterdischarges, glutamate, GABA,ap junctions.

Present address: Laboratory of Neural Microcircuitry, Brain Mindnstitute, Ecole Polytechnique Fédérale de Lausanne (EPFL), Station5, CH-1015 Lausanne, SwitzerlandCorresponding author. Tel: �41-31-631-5257; fax: �41-31-631-4611.-mail address: berger@pyl.unibe.ch (T. Berger).bbreviations: ACSF, artificial cerebrospinal fluid; AMPA, �-amino-3-ydroxy-5-methyl-4-isoxazole-proprionate; CNQX, 6-cyano-7-nitroqui-oxaline-2,3-dione; D-APV, D-2-amino-5-phosphonovalerate; ETNO,

tvoked transient network oscillation; GABAA, type A GABA; mACSF,odified artificial cerebrospinal fluid; NMDA, N-methyl-D-aspartate.

306-4522/06$30.00�0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reseroi:10.1016/j.neuroscience.2006.03.003

1401

etwork oscillations in different frequency ranges (Buzsákind Draguhn, 2004) are ubiquitous in the cerebral cortex andhey are thought to be important in such diverse tasks andtates like sensory and perceptual binding (Gray et al., 1989;oelfsema et al., 1994; Singer and Gray, 1995; Miltner etl., 1999; Rodriguez et al., 1999), motor programmingMurthy and Fetz, 1996), associative learning (Buzsáki,002), sleep (Steriade, 1997), and epileptogenesis (Traubt al., 2001; Grenier et al., 2003). The fact that differentrain areas are able to generate and sustain oscillationsuggests that comparable single-neuron biophysical prop-rties, connectivity, or synaptic dynamics (Douglas andartin, 1990) are responsible throughout the neocortex for

he induction and maintenance of oscillatory activity. Whileome studies suggest a critical role of single cell propertiesor the induction of oscillatory activity (Silva et al., 1991),ther works focus on a network-driven nature of the rhyth-ic activity (Timofeev et al., 2000; Shu et al., 2003). Inrder to gain comprehensive insights on the neocorticalircuits underlying oscillations, it is imperative to assesshe cellular and synaptic bases of oscillatory activity. Inontrast to in vivo techniques, in vitro electrophysiological

nvestigations give the unique possibility to characterize inetail the features of individual neurons and synapticallyonnected networks, under full control of the physico-hemical conditions.

In this study we established an in vitro model of col-ective in vivo activity, by using temporally and spatiallyistributed extracellular electrical stimulation. This proce-ure aims to mimic the physiological recruitment of a largeraction of afferent input fibers (Giugliano and Lüscher,003), allowing investigation of stimulus-evoked activitytates (Fuster, 1997; Zhang et al., 2004). We used slices ofhe rat somatosensory cortex because of the large wealthf information regarding the in vivo performance of thisrea, as well as for the large amount of previous knowl-dge on the cellular electrophysiology, diversity, and con-ectivity (Zhu, 2000; Markram et al., 2004; Kalisman et al.,005). Different forms of physiological and pathophysio-

ogical oscillatory activity have been described in the ro-ent somatosensory cortex. Prior to whisker movementshe thalamus and the primary sensory cortex display os-illations in the 7–12 Hz range (Nicolelis et al., 1995; Wiestnd Nicolelis, 2003). These so-called � oscillations in theodents were related to epileptic absence seizures (Shaw,004), states of disengagement prior to the acquisition ofensory information (Fontanini and Katz, 2005), but also ton attentive preparatory state (Nicolelis et al., 1995; Wiestnd Nicolelis, 2003). A prominent pattern of epileptic ac-

ivity in sensory systems is so-called absence seizuresved.

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T. Berger et al. / Neuroscience 140 (2006) 1401–14131402

epicting spike-wave activity with a low frequency of aboutHz. Spike-wave activity is a pathological thalamocortical

oop phenomenon which is based on the same circuitsenerating physiological spindles (Destexhe and Steriade,001). Another epileptic activity found in the cortex and theippocampus is so-called afterdischarges following high-requency stimulation (Fujiwara-Tsukamoto et al., 2003;ares et al., 2004; Kaneda et al., 2005). Afterdischarges

how comparable frequency properties in comparison topike-wave activity. However, they are lacking the sponta-eous and repetitive recurrence characteristic of truly ep-

leptic events. In an acute slice preparation of the somato-ensory cortex, we could evoke a transient oscillatory ac-ivity in the frequency range of 2–5 Hz. This cortical activityesembles afterdischarges and its initiation and mainte-ance depend on the interaction of excitatory and inhibi-ory networks via glutamatergic, GABAergic and electricalynapses.

EXPERIMENTAL PROCEDURES

lice preparation

ostnatal 13–20 day-old Wistar rats were killed by decapitation,ollowing the guidelines of the veterinary office of the canton Bern,nd their brains were rapidly removed and immersed in ice-coldrtificial cerebrospinal fluid (ACSF). Three hundred micrometer-hick parasagittal slices of the somatosensory cortex were cut on

vibratome (Dosaka DTK-1000, Kyoto, Japan), incubated at4 °C for 30 min, and afterward left at room temperature until theransfer to the recording chamber.

atch-clamp recordings

yramidal cells in layers II to V of the somatosensory cortexere visualized by infrared differential interference contrastideomicroscopy. Current-clamp (Figs. 1– 6) and voltage-clampFig. 7) whole-cell recordings were obtained from the soma ofp to three neurons or from glial cells, simultaneously. ThreeVC-700A amplifiers (Dagan, Minneapolis, MN, USA) weresed. Bridge balance and capacitance neutralization were ap-lied. Liquid junction potentials were left uncorrected. Elec-rodes were made from borosilicate glass tubing with resis-ances of 4 – 6 M� when filled with intracellular solution. Allxperiments were done at �34 °C. Data were low-pass filteredt 3 kHz using the internal filter of the amplifier and sampled at–10 kHz. Signals were digitized and stored on-line usinglampex 9 (Axon Instruments, Union City, CA, USA). Dataere analyzed off-line with Clampfit 9. Pooled data are ex-ressed as mean�standard deviation (S.D.). Raw data wereompared for statistical significance using the ANOVA test withignificance levels of 0.05 or 0.01.

xtracellular electrical stimulation

ontinuous zero-mean noisy voltage waveforms were used forxtracellular stimulation of the slice preparation. This was done viawo bipolar stimulation electrodes (made from insulated nickelhromium wire, conductor diameter 25 �m, type NI055840; Good-ellow, Huntingdon, UK) placed in layers I/II and VI, respectivelyFig. 1A). Waveforms were computer-generated independently forach bipolar stimulation site as an Ornstein-Uhlenbeck stochasticrocess (1 s, 5–10 kHz; Cox and Miller, 1965). Each resultingaveform is a statistically independent realization, characterizedy stationary gauss-distributed amplitude with zero mean, vari-

nce s2 (0.1–2.5 V2) and correlation time-length � (0.1–1 ms).

4

ig. 1. Distributed activation of the somatosensory network evokes aransient network oscillation (ETNO) in layer V pyramidal cell. (A) Twoipolar stimulation electrodes were placed in layers II and VI and theetwork was activated with gauss-distributed voltage changes de-icted beside the stimulation electrodes. (B) Such a gauss-distributedtimulation (1 s duration, 0 V mean, 2.5 V2 variance, 0.5 ms correlationime length, one of two different realizations shown on bottom) resultsn noisy membrane potential deflections during the stimulation in aayer V pyramidal cell in standard ACSF (black trace). After terminationf the stimulation few EPSP-like events constituting the ETNO areeen. Application of the modified ACSF (mACSF) depolarized the cellnd resulted in the prolonged generation of this activity (gray trace).he averaged and normalized power spectra of the activity from 20ells in ACSF and 12 cells in mACSF (see inset) show maxima at

.3 Hz and 2.0 Hz, respectively. Rat P16.

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T. Berger et al. / Neuroscience 140 (2006) 1401–1413 1403

uch a novel approach, as opposed to biphasic pulse stimulation,s expected to stimulate presynaptic fibers leading to an asynchro-ous activation of synaptic terminals, thus recreating in vivo-likeackground synaptic activity in neocortical slices (Giugliano andüscher, 2003).

hemicals and solutions

lices were continuously superfused with an ACSF containing (inM): 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1gCl2, 20 glucose, bubbled with 95% O2 and 5% CO2. In order to

ncrease excitability in the slice preparations, a modified extracel-ular solution (modified artificial cerebrospinal fluid, mACSF) wassed (Silberberg et al., 2004) with the following altered salt con-entrations (in mM): 6.25 KCl, 1.5 CaCl2, 0.5 MgCl2 comparedith 2.5, 2, and 1, respectively. Pipette solution for current-clampxperiments contained (in mM): 135 K-gluconate, 5 KCl, 10GTA, 10 HEPES, 4 Mg-ATP, 0.3 Na2-GTP, 10 Na2-phosphocre-tine, 10 biocytin, pH adjusted to 7.25 with KOH. Pipette solutionor voltage-clamp experiments contained (in mM): 120 Cs-meth-nesulfonate, 5 CsCl, 10 EGTA, 10 HEPES, 20 TEA-acetate, 4-aminopyridine, 10 QX-314, 4 Mg-ATP, 0.3 Na2-GTP, 10 Na2-hosphocreatine, pH adjusted to 7.25 with CsOH. The followingntagonists were added to the ACSF or mACSF to achieve theoncentrations given: D-2-amino-5-phosphonovalerate (D-APV)0 �M, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) 10 �M,abazine 3 �M, and carbenoxolone 200 �M. They were used tolock the NMDA receptor-mediated and the AMPA receptor-me-iated glutamatergic synaptic transmission, GABAA receptors,nd electrical synapses (gap junctions), respectively. 20 �M cy-lothiazide and 100 �M pentobarbital prevented the desensitiza-ion of AMPA receptors and GABAA receptors, respectively. Allhemicals were from Sigma, Tocris, or Merck.

iocytin staining

uring whole-cell recording, cells were filled with biocytin. Slicesere fixed in 4% paraformaldehyde in 0.1 M phosphate-bufferedolution (pH 7.2) and subsequently processed using an avidin–orseradish peroxidase reaction (Vector Laboratories, Burlin-ame, CA, USA; Hsu et al., 1981). Slices were not dehydrated inrder to minimize shrinkage. After embedding in Moviol, thetained cells were photographed.

odeling

ith the aim of summarizing and expressing in a compact formhe experimental observations collected across several experi-ents, we used a system of equations that accounts for theynamics of a large network of cortical neurons. The networkodel describes an ensemble of neurons generating and partici-ating to the evoked oscillatory activity in vitro, explored in thistudy. Considerably simplifying the heterogeneity of cortical cellypes, we assume that the network is composed by two subpopu-ations of cortical neurons, consisting of excitatory pyramidal neu-ons and inhibitory interneurons. The equations, describing thering rates of these populations, have the same form as in Wilsonnd Cowan (1972), Amit and Tsodyks (1991), and Dayan andbbott (2001):

�e

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here E (I) is the firing rate of excitatory (inhibitory) populationsnd g(x) is a single-neuron response function (see Rauch et al.,003; Giugliano et al., 2004) assumed to be threshold-linear for

he sake of simplicity (i.e. g(x)�x, for positive values of x, and E

(x)�0 otherwise). JEE, JEI, JIE and JII denote the absolute meantrength of the synaptic connections within and between the sub-opulations, while �E/I is an external drive to the excitatory/inhib-

tory subpopulation. Following Tsodyks et al. (1998), we furtherntroduce short-term homosynaptic depression at the excitatoryynapses between pyramidal neurons (Markram et al., 1999), thusaking JEE a function of time and of the presynaptic activity E:

dJEE

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JEE�JEE

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here JEE is the resting excitatory synaptic efficacy, while USE andrec are kinetic parameters characterizing the activity-dependentepression of synaptic transmission.

The accumulation of extracellular potassium, which weelieve is a key step for the expression of the evoked delayedctivity, is modeled as a first order kinetics process simplifyinghe approach proposed in Burgi and Grzywacz (1994) andxpressing in arbitrary units the temporal evolution of the ex-ess potassium �K:

�k

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here �K is the effective time constant associated with passivend active mechanisms of restoring physiological concentration

evels (i.e. �K�0), while S(t) represents the distributed electricalxtracellular stimulation. The increase in excitability, due to theccumulation of extracellular potassium is thus included by set-ing:

�E��E · �K �l��l · �K

The inversion of the chloride Nernst equilibrium, due to aransient failure of the chloride/potassium co-transporter, affectshe sign and the strength of the GABAA-mediated synaptic con-ections established by the inhibitory subpopulation JEI:

JEl�JEl��J · �K

Finally, gap junctions between inhibitory interneurons wereot explicitly modeled, assuming that their main effect is synchro-izing the population activity and thus affecting the I state variables a multiplicative factor.

Model parameters employed in the following are: �E�10 ms,I�50 ms, JEE�1.4, JEI�0.9, JIE�0.9, JII�0, �E��10 Hz,I�10 Hz, �K�1000 ms, �J �5–10, USE�0.01, �rec�500 ms.

RESULTS

patially distributed and noisy stimulation evokestransient network-mediated oscillation (ETNO)

n slices of the somatosensory cortex

n order to elicit oscillatory network activity in acute slicesf the somatosensory network, we tested different ap-roaches (single bipolar stimulation with rectangularulses, spatially restricted but noisy stimulation). Whilehese experimental designs failed, an approach wheretimulation was both noisy and distributed in space (Gi-gliano and Lüscher, 2003; Fig. 1A) resulted in a transientelayed activity with EPSP-like events outlasting the stim-lation itself (“evoked transient network oscillation, ETNO”;ig. 1B). Since the ETNO underwent an adaptation pro-ess, we chose 120 s-long inter-stimulation intervals, inrder to reliably evoke the response for each stimulus. The

TNO was more readily observed by the use of a mACSF

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T. Berger et al. / Neuroscience 140 (2006) 1401–14131404

ith altered potassium, calcium, and magnesium concen-rations (see Experimental Procedures). Induction ofTNO was done with two bipolar extracellular electrodeslaced in supra- and infragranular layers (Fig. 1A). The

mmediate membrane potential responses during a 1 stimulation displayed a heterogeneous picture: facilitating,epressing, or bimodal. Such heterogeneity might ariserom the involvement of different conductances, mediatingistinct stages of the evoked network response.

ETNO was induced in 67 cells from 43 slices. Cells hadresting membrane potential of �68.0�4.6 mV and an

pparent input resistance of 42.3�17.6 M�. Activity wastudied either in ACSF (n�27 cells), mACSF (n�30 cells),r consecutively under both conditions (n�10 cells). After1 s long noisy stimulation, the latency for the first ETNO

vent was 1.4�0.8 s (ACSF; n�37 cells) or 1.5�0.5 smACSF; n�40 cells) (P�0.1; Fig. 1B). The ETNO re-ponse was significantly longer in mACSF than in ACSF3.6�1.5 s and 2.7�1.6 s, respectively, P�0.02; Fig. 1B).he amplitude of the ETNO was larger in ACSF than inACSF, possibly due to a reduced driving force of synap-

ic currents under mACSF (smallest events in ACSF:.0�2.6 mV, smallest events in mACSF: 2.8�2.3 mV,�0.016; largest events in ACSF: 12.2�5.8 mV, largestvents in mACSF: 6.5�3.0 mV, P�0.001). The ETNOvents did rarely reach threshold for spiking activity. Theean event frequency was 4.0�1.3 Hz in ACSF and.5�0.8 Hz in mACSF. This low event rate was not con-tant over the entire length of the response but it decayedntil the ETNO completely disappeared (Fig. 1B). Theower spectra of the subthreshold membrane voltage, av-raged from 20 cells in ACSF and 12 cells in mACSF (Fig.B, inset) showed peaks at 4.3 Hz and 2.0 Hz, respec-ively.

In order to test if the ETNO was either related toingle-cell pacemaking properties or whether it was net-ork-driven, we stimulated the neurons intracellularly withonstant amplitude or gauss-distributed currents (Rauch etl., 2003) with appropriate intensities and similar durations.owever we could never induce any ETNO (not shown).hereafter we recorded from several cells simultaneouslyith the patch-clamp technique to estimate a possibleorrelation of the ETNO between the cells following dis-ributed stimulation. Therefore we made either recordingsrom two neurons (n�20 slices) or from three neuronsn�3 slices) simultaneously. In the majority of the record-ngs, we selected layer V pyramidal cells to monitor thectivity in the output cell type of the somatosensory cortex.owever, in eight of 49 recordings also pyramidal cells in

ayers II to IV and stellate cells in layer IV were used. TheTNO was observed in all cell types (Fig. 2A). This activityas highly correlated between the cells simultaneously

ecorded, with respect to both the temporal appearance asell as to the amplitude of the individual depolarizingvents (Fig. 2B, C). The peak of the cross-correlogramsas shifted from zero, suggesting propagation delays of

he ETNO across different cells (see Fig. 2B). In fact, theean propagation delay was 3.7�2.7 ms (range 0–7.8

s; n�11 cell pairs). The high correlation found in the s

ross-correlograms strongly indicates a network-mediatedasis of the ETNO.

TNO is due to the combined activationf excitatory and inhibitory networks

urrent- and voltage-clamp electrophysiology and phar-acological interventions were employed to better charac-

erize the phenomenon discussed here. An increase in thextracellular potassium concentration has been reported toccompany neuronal activity (Heinemann and Lux, 1975).he possibility of an elevated potassium concentration asprerequisite of ETNO was studied by simultaneously

ecording the membrane potential of layer V pyramidalells and individual glial cells in three slices. We couldimultaneously monitor the slow activity in the neuron andse the glial cell as a “potassium-sensitive electrode” as-uming on a first approximation an exclusive potassiumermeability of the glial membrane (Kuffler et al., 1966;utamachi and Pedley, 1976). The time course of theeuronal and glial membrane depolarization during distrib-ted stimulation had similar kinetics and the peak glialembrane potential reflected an increase of the extracel-

ular potassium concentration of 6�2 mM above restingalues (Fig. 3A). The glial depolarization lasted muchonger than the stimulation, suggesting a slow and pro-onged change in the extracellular potassium concentra-ion. During the ETNO detected in the neuron, the glial cellhowed only minute bumps of its membrane potential (Fig.B). The potassium channel blocker quinidine (100 �M)Fishman and Spector, 1981; Doi et al., 1995; Smirnov etl., 1999; but see also the direct effects of quinine anduinidine on gap junctions composed of connexin 36; Srini-as et al., 2001; Uusisaari et al., 2002) resulted in a re-uced depolarization during stimulation and in additionbolished the ETNO (n�8 cells; Fig. 3C). These experi-ents indicate an increase in the extracellular potassium

oncentration, occurring simultaneously and consecutivelyo the electrical stimulation, and thus suggest the involve-ent of a stimulus-induced potassium accumulation in theeneration of the ETNO.

Recurrent excitatory transmission may play a promi-ent role for network phenomena. In order to evaluate the

nvolvement of AMPA and NMDA receptors for the ETNOn the somatosensory cortex, we blocked these receptorsalf-maximally. The AMPA receptor blocker CNQX (1 �M)nd 5 �M of the NMDA receptor blocker D-APV resulted indiminished depolarization during the noisy stimulation

nd a reduction in amplitude and frequency of the ETNOn�2 cells; Fig. 4A). Fully blocking concentrations of0 �M CNQX plus 50 �M D-APV or 50 �M D-APV alone led

o a complete disappearance of the activity (n�5 cells; nothown and n�5 cells; Fig. 4B, respectively). In contrast,he amplitude of individual events constituting the ETNOncreased, when AMPA receptor desensitization was re-uced with 20 �M cyclothiazide (n�5 cells; Fig. 4C). Thus,TNO depends critically on AMPA as well as NMDA re-eptor activation.

GABAergic transmission was described to be an es-

ential factor in different forms of oscillatory network activ-

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T. Berger et al. / Neuroscience 140 (2006) 1401–1413 1405

ty (Whittington and Traub, 2003). Using slices from adoles-ent rats showing hyperpolarizing GABAA receptor re-ponses (Luhmann and Prince, 1991) and a pipette solutionith a physiological chloride concentration of 5 mM in allxperiments, we could see in a minority of the slices biphasicequences of hyperpolarization and depolarization duringtimulation (e.g. Fig. 6A, B). In order to test the involve-ent of GABAA receptors in the initiation and maintenancef ETNO we studied first the activation of GABAA receptorsuring stimulation. We reduced the stimulation intensity in

ig. 2. ETNO is a network phenomenon. (A) Simultaneous triple whoells under study. In contrast to layer IV cells, in the layer V pyramidaot connected. The trace between the two dotted lines is shown enlaells are truncated. (C) The cross-correlogram obtained from a total ofTNO in different cells. Rat P13 (A, B).

uch a way that nearly no action potentials were evoked r

Fig. 5A, upper part). When the cell was depolarized to45 mV by DC injection and the slice was stimulatedgain, hyperpolarizing events were seen intermingled be-ween the spikes (Fig. 5A, lower part). The hyperpolarizingvents seemed to become smaller during the 1 s-longtimulation. Application of 3 �M of the GABAA receptorlocker gabazine abolished these hyperpolarizing eventsFig. 5B). These experiments show the activation ofABAA receptors during stimulation and suggest a de-reasing driving force of chloride-mediated membrane cur-

ordings from pyramidal cells in layers IV and V show the ETNO in allno spikes are seen on top of the activity. The cells were synapticallyart B. (B) Simultaneously recorded ETNO. Spikes in the two layer IVf simultaneously recorded cells shows a high correlation between the

le-cell recl neuronrged in p11 pairs o

ents, occurring during stimulation. To shed some light on

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T. Berger et al. / Neuroscience 140 (2006) 1401–14131406

ig. 3. Gauss-distributed stimulation results in an increase of thextracellular potassium concentration which is essential for the ETNO.A) Simultaneous whole-cell patch recordings from a pyramidal neuronnd a glial cell in layer V. The depolarization of the glia corresponds ton increase of the extracellular potassium concentration of aboutmM calculated with the Nernst equation assuming an exclusive

otassium permeability of the glial membrane. (B) The ETNO is onlyeen in the neuron while the membrane potential of the glial cellollows this network activity with small depolarizing bumps. (C) Bathpplication of 100 �M of the potassium channel blocker quinidinehortens the depolarization during stimulation and prevents the ETNO.n the inset the voltage axis is expanded by a factor of 2. This is also

rue for Figs. 4 and 6. Rat P16. i

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ig. 4. ETNO depends on AMPA and NMDA receptor activation.A) Bath application of half-maximal blocking concentrations for AMPAnd NMDA receptors (1 �M CNQX and 5 �M D-APV, respectively)educed the ETNO significantly but incompletely. (B) A fully blockingoncentration of the NMDA receptor blocker D-APV (50 �M) resulted inhe complete block of the depolarization during stimulation and abol-shed ETNO. (C) Cyclothiazide (20 �M), which prevents AMPA recep-or desensitization, facilitates the ETNO. Same scale bars for figures

–C. Rats P16 (A), P14 (B), and P13 (C).

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T. Berger et al. / Neuroscience 140 (2006) 1401–1413 1407

ossible changes of the chloride reversal potential, wepplied 10 mM GABA with pressure ejection directly onells in another set of experiments (n�3 cells). With a 1-long application, a biphasic hyperpolarizing-depolarizingesponse sequence was observed (Fig. 5C, upper part)hile a shorter 300 ms-long application evoked only ayperpolarization (not shown). When the interval betweenwo successive GABA applications was kept at 10 s, theiphasic response could be induced repetitively (Fig. 5C,pper part), while the hyperpolarizing component was lostith inter-application intervals of 5 s (Fig. 5C, lower part).he evidences collected in such a series of experimentsuggest that during noisy stimulation GABAA receptors areassively activated. On the other hand, this leads to a shiftf the chloride reversal potential to values positive to theesting potential resulting in a depolarizing GABAA recep-or response. In addition such a shift possibly lasts foreconds, a time scale which is comparable to that of thelow change in the extracellular potassium concentrationFig. 3A).

In an additional set of experiments we studied thenvolvement of GABAA receptors and gap junctions in theeneration of the ETNO. During a transient phase, brieflyfter the blockade of GABAA receptors by application in�M gabazine and just before the slices started to show

pontaneously ongoing spike-wave activity, the ETNOould not be evoked any more (n�10 cells; Fig. 6A). Inontrast, the application of 100 �M of pentobarbital, aABAA receptor activator, resulted in the prolongation of

he evoked ETNO (n�10 cells; Fig. 6B). In addition, theesponse became biphasic with a hyperpolarizing re-ponse following an initial short depolarization. The gap

unction blocker carbenoxolone (200 �M; LeBeau et al.,002; D’Antuono et al., 2005) also abolished the ETNOompletely (n�3 cells; Fig. 6C). About 50% of the parval-umin-positive interneurons in the neocortex contain con-exin 36 (Priest et al., 2001) and the alkaloids quinine anduinidine have been shown to block this type of connexinSrinivas et al., 2001; Uusisaari et al., 2002) in addition toheir effect on different potassium channels (Fishman andpector, 1981; Doi et al., 1995). Thus, part of the effect of00 �M quinidine on ETNO generation shown in Fig. 3Cay be due to a direct blocking effect on connexin 36 in

ortical interneuron networks (compare Deans et al.,001).

In order to study the synaptic conductances underlyinghe ETNO, we performed voltage-clamp experiments. At a

ell was depolarized to �45 mV with DC current injection, hyperpo-arizing events are seen which gradually decrease during the stimula-ion (arrow). (B) A fully blocking concentration of the GABAA receptorlocker gabazine (3 �M) results in the complete disappearance of

hese hyperpolarizing events at �45 mV (arrow). (C) GABA at 10 mMas applied to a layer V pyramidal cell with pressure ejection for 1 s

see bar). This results in a biphasic sequence of hyperpolarization andepolarization. This sequence stays unchanged when GABA waspplied with a 10 s interval (upper traces). With a 5 s interval betweenABA pulses, the hyperpolarizing phase is no longer observed with

ig. 5. GABAA receptor activation during stimulation can lead to ad-

itional depolarization. (A) At the resting membrane potential, stimu-

he second pulse (lower traces) Black lines, first GABA pulses; grayines, second GABA pulses. Rat P16.

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T. Berger et al. / Neuroscience 140 (2006) 1401–14131408

lamp potential of �70 mV, distributed extracellular stim-lation resulted in a series of inward currents, reflecting theorresponding synaptic currents of the ETNO (n�7 cells;ig. 7A). When the clamp potential was varied in a ramp-ise fashion from �10 to �90 mV during the occurrence of

he ETNO, its reversal potential could be determined (Fig.B). The sequence from depolarized to hyperpolarizedlamp potentials prevented the initiation of active currentsssociated to action potentials. The measured currentrace was compensated for leak components, by subtract-ng the current under a rampwise change of the clampotential without extracellular stimulation. In seven cells, a

ig. 7. The reversal potential of the ETNO reflects a combinationf glutamatergic, GABAergic and junctional conductances. (A) A

ayer V pyramidal cell was voltage-clamped to �70 mV and theTNO was activated with distributed stimulation (upper traces).his resulted in unclamped action currents during the stimulationnd consecutive inward currents underlying the ETNO potentialhanges. Same scale bars for figures A and B. (B) In order tobtain the reversal potential of the ETNO currents, the clampotential was changed in a rampwise fashion from �10 to �90 mV.his sequence from depolarized to hyperpolarized clamp potentialsrevented action current initiation. In order to get rid of the under-

ying leakage current, a rampwise voltage change without stimula-ion was subtracted. The resulting ETNO currents showed a rever-al (EETNO) at �23 mV in this cell suggesting a combined GABAer-ic and glutamatergic conductance. Rat P16.

ig. 6. ETNO depends on a network interconnected via GABAA re-eptors and gap junctions. (A) A fully blocking concentration of theABAA receptor blocker gabazine (3 �M) results in ictal spiking activitynd the complete disappearance of ETNO. (B) Reducing the GABAA

eceptor desensitization with 100 �M pentobarbital increases the de-ay of ETNO and prolongs it. In addition the ETNO events are curtailedy a strong hyperpolarization. (C) The gap junction blocker carbenox-lone at 200 �M abolishes ETNO. Same scale bars for figures A–C.

ean reversal potential of �27.0�13.3 mV (range �10 to

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T. Berger et al. / Neuroscience 140 (2006) 1401–1413 1409

36 mV) was found, suggesting a mixed glutamatergic/ABAergic contribution to the ETNO.

Finally, we simulated and studied the implications ofhe pharmacological manipulations described so far in theathematical model and detailed in the Experimental Pro-

edures section. Thereby we could confirm the followingeatures of ETNO: i. Network-driven oscillations can arises a consequence of the interplay between excitatory and

nhibitory cortical subpopulations (Fig. 8), in spite of thebsence of intrinsic pacemaker mechanisms. In thisodel, activity in the inhibitory population always follows

he activity in the excitatory population (see also Fig. 6B).i. Recurrent excitation is a key component in the inductionnd transient maintenance of the ETNO and is reproducedy the mathematical model (not shown). iii. The transient

ncrease in the extracellular potassium concentration af-ects the efficiency of the membrane mechanisms thatestore resting intracellular chloride levels. This disruptshe potential for oscillations, as inhibitory synapses turnxcitatory on a first approximation, and in addition keepshe system out of equilibrium for a few seconds after thetimulation (Fig. 8). iv. A transient after-depolarization thatas often observed in the recordings leads to a tonicischarge preceding the ETNO itself. The properties ofxcitatory synaptic transmission and the recovery timeonstants of short-term synaptic depression account for

ig. 8. Phenomenological model of the ETNO. The model descreneration of evoked slow oscillations in the firing probability of gextracellular distributed stimulation (gray shading), the increase in thf GABAergic synapses (i.e. JEI) due to an accumulation of intracelluhe time evolution of the membrane voltage of a single integrate-anomponents. The inset shows a sketch of the architecture of the nere not further considered.

his transient after-depolarization (not shown). W

DISCUSSION

ethodological considerations

etwork-driven activity patterns like oscillatory activity canbviously be best investigated in the intact cortex in vivo.nder these conditions, however, the ability of the inves-

igator to study underlying mechanisms for instance withharmacological tools, is clearly restricted. In contrast,educed preparations like acute neocortical slices enablehe use of modulators, though the re-creation of the con-itions for oscillatory activity might be difficult. In fact, acutelice preparations are characterized by low levels of spon-aneous activity, altered passive membrane properties andonsecutively affected integrative properties of individualeurons, in comparison to the situation in vivo (Bernandert al., 1991; Destexhe and Paré, 1999; Léger et al., 2005).epending on the preparation and animal species consid-red, modifications of the ionic composition of the extra-ellular solution or addition of excitatory transmitters aresed to overcome the deafferentation due to the slicingrocedure and help to restore network activity (for reviewee Traub et al., 2004). These modifications compriseeduction of the extracellular calcium and magnesium con-entration (prefrontal cortex of ferrets; Sanchez-Vives andcCormick, 2000; McCormick et al., 2003), low extracel-

ular magnesium concentrations (somatosensory cortex;

the Experimental Procedures section accounts for the transientitatory and inhibitory neuronal populations (E(t), I(t)). After a briefllular potassium concentration (�K), transiently modify the strengthide. With the only purpose of comparison, the lower trace indicatesodel neuron, driven by the excitatory as well as inhibitory synapticodel. Gap junction connections within the inhibitory cell population

ibed inneric exce extracelar chlord-fire m

u et al., 1999), increase in the extracellular potassium

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T. Berger et al. / Neuroscience 140 (2006) 1401–14131410

oncentration (CA1 or CA3 hippocampus; LeBeau et al.,002), application of nanomolar concentrations of kainater domoate (entorhinal cortex; Cunningham et al., 2003),pplication of NMDA and dopamine D1 receptor agonistssomatosensory cortex slice cultures, Beggs and Plenz,003; prefrontal cortex acute slices, Tseng and O’Donnell,005), or application of carbachol (CA3 hippocampus;isahn et al., 1998). To facilitate excitability of the somato-ensory slices we employed a modified extracellular solu-ion with reduced calcium and magnesium, but increasedotassium concentration (Silberberg et al., 2004). In ordero mimic the physiological input to the somatosensoryortex, extracellular stimulation was then applied. A com-ination of time-varying extracellular stimulation, delivered

n a spatially distributed way via bipolar stimulation elec-rodes (this study) or via multi-electrode arrays (M. Giugli-no and H. R. Lüscher, unpublished data), turned out to bereliable paradigm for the ignition of oscillatory activity in

he somatosensory microcircuits.

TNO is a network phenomenon of theomatosensory cortex

hysiological and pathophysiological oscillations in theeocortex and the hippocampus are based on two largeroups of underlying mechanisms: oscillations in networksoupled via chemical and electrical synapses, and intrinsicscillations of pacemaker cells. In the CA1 region of theippocampus proper and different cortical areas in vitro,igh-frequency stimulation was reported to induce seizure-

ike afterdischarges at a frequency of about 3 Hz (Fujiwara-sukamoto et al., 2003; Kaneda et al., 2005). This electri-al activity resembles very much the ETNO found in thistudy in the somatosensory cortex, although the hip-ocampal activity is easily evoked and outlasts the stimu-

ation much longer. This difference between CA1 and so-atosensory cortex may be due to a lower susceptibility of

he cortical neurons for epileptic events (Steriade, 2003).n CA1, the intense stimulation leads to an intracellularccumulation of chloride ions and a redistribution of bicar-onate resulting in a depolarizing GABAA receptor re-ponse (Staley et al., 1995; Isomura et al., 2003b). Thisesponse is intensified by a concomitant increase in thextracellular potassium concentration which prevents thextrusion of the intracellular chloride via the K�-Cl�-co-ransporter KCC2 (Kaila et al., 1997). Similarly, in theomatosensory cortex, a stimulation-induced increase inhe extracellular potassium concentration and a shift of theeversal potential for chloride are essential factors in thenitiation of the ETNO (this study). These ionic shifts areufficient to bring the somatosensory network transientlyut of its equilibrium resulting in the ETNO. In contrast tour finding, in the CA1 region there is no involvement ofMDA-type and only a minor importance of AMPA-typelutamate receptors in the afterdischarge generation (Kailat al., 1997; Fujiwara-Tsukamoto et al., 2003) while theropagation to the adjacent subiculum is critically depend-

ng on glutamatergic synaptic transmission (Isomura et al.,003a). Such a difference between hippocampus and neo-

ortex is presumably due to differences in the respective t

ytoarchitecture, as in comparison to the hippocampuslutamatergic transmission is essential in the neocortex toctivate long-range postsynaptic cortical targets.

Oscillatory activity in the neocortex has been related tohe intrinsic properties of single pacemaking cells (Silva etl., 1991). Recent studies suggest that the starting point ofhe oscillations may either be intrinsically bursting cellsCunningham et al., 2004) or a local increase in the extra-ellular potassium concentration (Gutnick et al., 1982).ntrinsically bursting cells are found in different densities inifferent preparations. However, it seems that regularlypiking cells can be switched into a bursting mode if exci-atory resting conductances like the persistent sodium cur-ent INa(p) are activated and inhibitory conductances likealcium-dependent potassium currents are blocked (Traubt al., 2003). This intrinsic bursting ability is reinforced byhe membrane resonance of hippocampal and neocorticalyramidal cells (Hu et al., 2002; Ulrich, 2002). Intrinsiconductances like the hyperpolarization-activated current

h play also an essential role when oscillatory activity isnduced due to local increases in extracellular potassiumoncentration (Timofeev et al., 2002; Bazhenov et al.,004). In our experiments, blockade of the Ih resulted in aisappearance of the ETNO and the generation of burstctivity (T. Berger, H. R. Lüscher and M. Giugliano, un-ublished observations). However, these bursts were notorrelated between different cells and seem to reflect theacilitation of dendritic calcium events (Berger et al., 2003;erger and Lüscher, 2003) instead of epileptic activity

Timofeev et al., 2002). Taken together, our electrophysi-logical data and our modeling study showed that network-ased mechanisms are sufficient to evoke ETNO without

ntrinsic pacemaking mechanisms.

orrelation of the ETNO to network oscillationsf the somatosensory cortex in vivo

hen investigating an unknown object or space, rodentsove their posterior whiskers back and fourth rhythmically

n a behavior known as whisking (Welker, 1964). Prior tohisking, rats are in a quiet state, in which they move theirhiskers very rhythmically with small amplitude (“whisker

witching”; Nicolelis and Fanselow, 2002). The thalamusnd the primary sensory cortex display at the same timescillations in the 7–12 Hz range (Nicolelis et al., 1995;iest and Nicolelis, 2003). These oscillations during

witching resemble the � or � activity found in humansSemba et al., 1980; Tiihonen et al., 1989; Nikouline et al.,000). The functional background of the � oscillations in

he rodents is hotly debated. The majority of studies sug-est either a pathophysiological state like an epileptic ab-ence seizure (Shaw, 2004) or a physiological one likeisengagement prior to the acquisition of sensory informa-ion (Fontanini and Katz, 2005). Nicolelis and coworkers1995; Wiest and Nicolelis, 2003) have proposed a thirdossibility, describing the � activity as a sign for an atten-

ive state, preparatory for the sensory input.A prominent pattern of pathophysiological activity in

ensory systems is so-called absence seizures. Originally

hey were thought to be initiated subcortically in the thal-

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T. Berger et al. / Neuroscience 140 (2006) 1401–1413 1411

mus and to spread to the connected cortical areas. Thisould lead to the uncoupling of the sensory cortex from its

ncoming information resulting in the clinical phenomenonf the absence. Recent studies, however, have shown thathe perioral region of the somatosensory cortex can be theource of this epileptic activity which is intensified in aortico-thalamic loop (Steriade and Contreras, 1998; foreview see Timofeev and Steriade, 2004; Meeren et al.,005). Absence seizures show spike-wave activity with a

ow frequency of about 3 Hz in the cortex. While thepike-wave activity seems to be initiated in the cortex, it isenerated in the thalamus. The same circuits betweenxcitatory relay and inhibitory reticular nuclei which gener-te under physiological conditions the spindle oscillations7–14 Hz) are now modified and build up the slower spike-ave activity (3 Hz). This shift is due to a reduced GABAer-ic drive within the cortex leading to the generation ofaroxysmal bursts in the reticular nucleus via corticotha-

amic fiber activation. The prolonged activity pattern of theABAergic reticular cells itself leads to a prolonged GABA

elease on the relay neurons and thereby to the activationf GABAB receptors. These receptors are not activateduring spindle activity due to their low GABA affinity. Theifferent kinetics of GABAA and GABAB receptors resultow in a thalamocortical output with oscillations at aboutHz and no more at about 10 Hz (reviewed in Destexhe

nd Steriade, 2001).The ETNO found in the present study in the somato-

ensory cortex resembles neither � activity nor epilepticpike-wave activity. � Activity is characterized by a higherrequency range and thalamic as well as cortical neuronshow burst activity (for review see Nicolelis and Fanselow,002). Although this bursting activity cannot be seen dur-

ng each cycle in vivo, we saw no bursting activity at all inur in vitro preparation. Single spikes were only rarelyeen in layer V pyramidal cells and the activity consistedrimarily of large EPSP-like events. However, we have tossume that spiking activity in many neurons is needed toustain ETNO. Although the frequency of 2–4 Hz fits veryell with an epileptic activity in the range of 3 Hz, thebsence of bursting activity does not suggest spike-wavectivity at all. In addition, full spike-wave activity needs an

ntact thalamocortical feedback loop which was not presentn our slice preparation. Another difference between spike-ave activity and ETNO is the fact, that spike-wave pat-

erns are activated with a reduced cortical GABAergic driveDestexhe and Steriade, 2001) while ETNO disappearedollowing blockade of the excitatory GABAergic action (thistudy). However, in a preliminary set of experiments in therefrontal cortex, we could induce spike-wave activityhich lasted very long and which needed much less efforts

or induction and maintenance in comparison to the so-atosensory ETNO activity (H. R. Lüscher and M. Giugli-no, unpublished observations).

Afterdischarges follow brain electrical stimulation orepetitive sensory input like stroboscopic flash-lights. Al-hough epileptic discharges are defined by their spontane-us and repetitive appearance, evoked afterdischarges

replay” epileptic mechanisms which are possible in the

timulated network. Thereby, the afterdischarges displayifferent phases which are dependent on the stimulation

ntensity. In an initial phase a “fast run” of spiking activity at0–20 Hz is seen (compare Timofeev et al., 1998, Fig. 3).

n our study we reduced the stimulation intensity to preventhis post-stimulus spiking. Following the fast run a low-requency, high amplitude subthreshold activity can beeen in cortical cells in vivo (Timofeev et al., 1998) as wells in vitro (this study). In contrast to spike-wave activity,fterdischarges are localized events as they can be in-uced in cortical slices and isolated cortical slab prepara-ions in vivo. In addition, afterdischarges are not visible inhe electroencephalogram of neighboring areas (Timofeevt al., 1998). Based on frequency range and underlyingechanisms, the ETNO activity described in this study

esembles afterdischarge activity more than any form ofeocortical oscillatory activity.

cknowledgments—This research was supported by the Swissational Foundation (31.6133.5.00, 3100.107529, to H.-R.L. and.B, respectively) and by the Human Frontier Science ProgramLT00561/2001-B, to M.G.). We are grateful to Drs. A. Rauch and. Fusi, for the many stimulating discussions, Drs. S. Crochet, F.eubauer and D. Ulrich for reading an earlier version of theanuscript, and C. Bichsel for her excellent assistance.

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APPENDIX

upplementary data associated with this article can be found, in

he online version, at doi: 10.1016/j.neuroscience.2006.03.003.

(Accepted 2 March 2006)(Available online 24 April 2006)