Thalamocortical oscillations in a genetic model of absence seizures

Thalamocortical oscillations in a genetic model of absenceseizures

Giovanna D'Arcangelo,1,2 Margherita D'Antuono,3 Giuseppe Biagini,4 Richard Warren,5 Virginia Tancredi2 andMassimo Avoli1,3

1Montreal Neurological Institute and Department of Neurology & Neurosurgery, McGill University, 3801, University St.,

MontreÂal, QC, H3A 2B4, Canada2Dipartimento di Neuroscienze, UniversitaÁ degli Studi di Roma `Tor Vergata', 00173 Roma, Italy3IRCCS Neuromed, 86077, Pozzilli (Isernia), Italy4Dipartimento di Scienze Biomediche, UniversitaÁ degli Studi di Modena e Reggio Emilia, 41100 Modena, Italy5DeÂpartement de Psychiatrie, UniversiteÂ de MontreÂal and Centre de Recherche Fernand-Seguin, MontreÂal, QC, H1N 3V2,

Canada

Keywords: 4-aminopyridine, epileptiform activity, extracellular potassium, GABA

Abstract

We used ®eld potential recordings in an in vitro thalamocortical slice preparation to compare the rhythmic oscillations generatedby reciprocally connected networks of the thalamus and cerebral cortex obtained from epileptic (> 160 days old) WAG/Rij and

age-matched, nonepileptic control (NEC) rats. To increase neuronal excitability, and thus to elicit spontaneous ®eld potential

activity in vitro, we applied medium containing: (i) zero [Mg2+]; (ii) high [K+] (8.25 mM); or (iii) low concentrations of the K+

channel blocker 4-aminopyridine (4AP, 0.5±1 mM). Of these procedures, only the last was effective in triggering oscillatory activitythat depended on the type of tissue. Thus, during 4AP application: (i) sequences of fast (intraburst frequency 9.5±16.1 Hz) and

slower (5±8.9 Hz) ®eld potential oscillations (FPOs) were recorded in WAG/Rij slices (n = 23), but (ii) only fast FPOs were seen

in NEC slices (n = 7). Slower FPOs in WAG/Rij slices re¯ected a larger degree of thalamocortical synchronization than fastFPOs, and disappeared after surgical separation of cortex and thalamus (n = 5); under these conditions fast FPOs continued to

occur in thalamus only. In addition, fast and slower FPOs disappeared in all areas of the WAG/Rij slice during thalamic

application of the excitatory amino acid receptor antagonist kynurenic acid (n = 3), while fast FPOs continued to occur inthalamus when kynurenic acid was applied to the cortex (n = 4). Bath application of the N-methyl-D-aspartic acid (NMDA)

receptor antagonist 3,3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonate (CPP) abolished slower FPOs in WAG/Rij cortex and

thalamus (n = 6) without infuencing fast FPOs recorded in WAG/Rij (n = 6) or NEC slices (n = 4). Moreover, cortical application

of CPP (n = 6) abated slower FPOs although they persisted following CPP application to the thalamus (n = 7). Our datademonstrate that highly synchronized, slower FPOs can occur during 4AP application in WAG/Rij but not in NEC slices. This

activity, which may represent an in vitro hallmark of thalamocortical epileptogenicity, requires the function of reciprocally

connected thalamic and cortical networks and depends on cortical NMDA receptor-mediated mechanisms.

IntroductionGeneralized spike and wave (SW) discharges occurring at 2.5±4 Hz

in patients presenting with primary generalized epilepsies are

commonly associated with brief periods of impaired consciousness

(the absence seizures). Over the last few decades, several investiga-

tors have analysed the mechanisms of SW discharges, most often by

using models in which normal animals were made `epileptic' by

convulsant treatment or by brain electrical stimuli (see for review

Avoli et al., 1990, 2001; Snead, 1995). These studies have suggested

that SW discharges originate from oscillatory thalamocortical inter-

actions similar to those involved in sleep spindles. However, the

modalities by which thalamocortical interactions lead to the slow

rhythm of SW discharge remain elusive.

SW discharges in humans occur spontaneously and have a genetic

origin (Lennox & Lennox, 1960; Metrakos & Metrakos, 1961).

Hence, knowledge of the pathophysiogenesis of generalized SW

activity should gain from naturally occurring genetic animal models

of absence seizures. These models, which include the Genetic

Absence Epilepsy in Rats from Strasbourg (GAERS) (Vergnes et al.,

1990) and the Wistar Albino Glaxo/Rijswijk rat (WAG/Rij) (van

Luijtelaar & Coenen, 1986; Peeters et al., 1990), are characterized by

generalized SW discharges at 6±10 Hz that respond to antiepileptic

drugs in a way similar to what has been reported in patients with

primary generalized epilepsy. These genetically determined absence

seizures have been analysed with microphysiological (including

intracellular) recordings that were obtained in vivo from cerebral

cortex and from different thalamic nuclei (Pinault et al., 1998, 2001;

Seidenbecher et al., 1998; Charpier et al., 1999; Seidenbecher &

Pape, 2001; Staak & Pape, 2001; Meeren et al., 2002; Slaght et al.,

2002). These studies, recently reviewed by Crunelli & Leresche

2002), have (i) shown the occurrence of speci®c patterns of cortical

Correspondence: Dr M. Avoli, 1Montreal Neurological Institute, as above.E-mail: [email protected]

Received 7 October 2002, accepted 14 October 2002

doi:10.1046/j.1460-9568.2002.02411.x

European Journal of Neuroscience, Vol. 16, pp. 2383±2393, 2002 ã Federation of European Neuroscience Societies

and thalamic neuron activity during SW discharges, (ii) characterized

the GABA receptor subtypes involved in the periods of neuronal

silence that are recorded in thalamic cells, and (iii) identi®ed a

primary role of the cortex in the initiation of SW discharges.

However, the relative contributions provided by thalamic and cortical

networks to the occurrence of SW discharges in these genetic models

of absence seizures remain to date unclear.

We have recently adapted the thalamocortical slice preparation

originally described in mice by Agmon & Connors (1991) to obtain

rat slices in which reciprocal thalamocortical connectivity is suf®-

ciently preserved to express rhythmic oscillations resembling sleep

spindles (Tancredi et al., 2000; Biagini et al., 2001). Here, we have

used this in vitro approach to study the thalamocortical interactions

occurring in epileptic (> 160 days old) WAG/Rij and in age-matched,

nonepileptic control (NEC) rats. To this end, we used simultaneous

®eld potential recordings in thalamocortical slices that were bathed in

medium containing zero [Mg2+], high [K+] (8.25 mM), or low

concentrations of 4-aminopyridine (4AP; 0.5±1 mM). Only 4AP

application proved to be capable of triggering oscillatory patterns

that were different in WAG/Rij and NEC animals. Therefore, we

focused on this in vitro model of thalamocortical synchronization to

identify the contributions of cerebral cortex and thalamus to these

rhythms and to characterize their sensitivity to excitatory amino acid

receptor antagonists.

Materials and methods

Epileptic WAG/Rij (> 160 day-old) and age-matched NEC rats (Crl/

Wi/br/Wistar obtained from Charles River) were used according to

the procedures established by the European Union and Canadian

Councils of Animal Care. By analysing the behaviour of the WAG/

Rij rats housed before the day of the in vitro experiment, we could

document that these animals displayed frequent episodes (duration 4±

10 s) of behavioural arrest accompanied by myoclonic twitches.

Previous studies performed in this genetic strain have demonstrated

that these clinical events correspond to bilaterally generalized SW

discharges at 6±10 Hz (van Luijtelaar & Coenen, 1986; Meeren et al.,

2002).

Both epileptic WAG/Rij and age-matched NEC rats were decapi-

tated under halothane anaesthesia, and their brains were quickly

removed and cooled in cold, oxygenated arti®cial cerebral spinal ¯uid

(ACSF; see below) in which NaCl was isoosmotically replaced with

sucrose to increase slice viability (Aghajanian & Rasmussen, 1989;

Zhang et al., 1996). Thalamocortical slices (550±650 mm) were

FIG. 1. Synchronous ®eld potential activity recorded in the cortex (Cx) and ventrobasal complex (VB) of NEC and epileptic WAG/Rij rat slices duringapplication of ACSF containing (A) zero [Mg2+], or (B) high [K+]. Note that the patterns of synchronous activity induced by these experimental proceduresare similar in the two different rat strains. The insert in A (NEC panel) shows an enlargement of the onset of the discharge recorded in the deep layers of thecortex (» 800 mm from the pia). Note that the steep negative shift is associated with occurrence of small amplitude population spikes at 20±30 Hz.

2384 G. D'Arcangelo et al.

ã 2002 Federation of European Neuroscience Societies, European Journal of Neuroscience, 16, 2383±2393

obtained using procedures previously described by us (Tancredi et al.,

2000; Biagini et al., 2001). Slices were transferred to a tissue

chamber where they lay in an interface between oxygenated ACSF

and humidi®ed gas (95%O2 : 5% CO2) at 32±35 °C (pH 7.4). ACSF

composition, unless otherwise speci®ed, was (in mM) NaCl, 124;

KCl, 2; KH2PO4, 1.25; MgSO4, 1; CaCl2, 2; NaHCO3, 26; and

glucose, 10. In some experiments MgSO4 was omitted to obtain a

nominally Mg2+- free medium (hereafter termed zero [Mg2+]

medium) while in other experiments KCl was elevated to 7 mM to

reach a total K+ concentration of 8.25 mM (high [K+] medium).

Chemicals were obtained from Sigma (St Louis, USA) with the

exception of the NMDA receptor antagonist 3,3-(2-carboxypiper-

azine-4-yl)-propyl-1-phosphonate (CPP), which was purchased from

Tocris Cookson (Langford, UK).

Field potentials were recorded with ACSF-®lled electrodes

(resistance 2±8 MW) which were positioned under visual control in

the cerebral cortex, the ventrobasal complex (VB) and/or the nucleus

reticularis of the thalamus (nRT). The cortical recording electrode

was positioned in most of the experiments at a depth presumably

corresponding to layer IV. Signals were fed to high-impedance

ampli®ers and were processed through second-stage ampli®ers with

®ltering capability. The high-pass and low-pass ®lters were often set

at 0.2 and 100 Hz, respectively. Field potential recordings were

displayed on a Gould recorder and at times digitized to obtain average

power spectra of selected epochs (overall length > 1 s). In some

experiments a bipolar stainless steel electrode was used to deliver

extracellular stimuli (50±150 ms; < 200 mA) to the cerebral cortex,

the thalamus or the internal capsule.

Pathway transections were made under direct visual guidance with

micromanipulator-mounted blades. In some experiments kynurenic

acid (5 mM, Sigma; diluted in ACSF) or CPP (0.2 mM, diluted in

ACSF) were pressure-applied to restricted areas of the slice by

employing a superfusion system similar to that used by Biagini et al.

(2001). In brief, a broken-tip theta glass pipette (diameter 1.5 mm)

was connected through two Te¯on tubes to a peristaltic pump with

¯ow rate set at » 0.5 mL/min and used as a push±pull cannula. This

pipette was mounted on a micromanipulator and positioned over the

cortex or the thalamus. The area of the slice covered by the drug

applied through the push±pull cannula was < 1 mm2. To avoid

diffusion of the drug from the superfused area to distant regions,

slices were placed in such a way that the treated area was downstream

with respect to the ACSF ¯ow. The spread of the applied solution was

visualized by including phenol red in the superfused medium.

Our work is based on data from 42 and 19 thalamocortical slices

that were obtained from epileptic WAG/Rij and NEC rat brains,

respectively. Measurements are expressed as mean 6 SD and n

indicates the number of experiments. Statistical analysis of the data

was performed with Student's t-test. Data were considered signi®-

cantly different if P < 0.05.

Results

Synchronous activity in NEC and WAG/Rij rat thalamocorticalslices

Thalamocortical slices obtained from either NEC or epileptic WAG/

Rij rats did not generate spontaneous ®eld potential activity when

bathed in normal ACSF (cf. Zhang et al., 1996; Tancredi et al., 2000;

Biagini et al., 2001). Therefore, we used experimental procedures

known to increase neuronal excitability in vitro to obtain synchronous

events similar to those found to occur spontaneously in vivo. In a ®rst

series of experiments we analysed the effects induced by super-

perfusing slices with zero [Mg2+] medium. As reported by Zhang

et al. (1996), Mg2+ wash-out induced sequences of ®eld potential

oscillations (FPOs) that had intraoscillatory frequencies ranging from

6.5 to 15 Hz, lasted 1.3±4 s and were recorded in synchronicity in

thalamus and cortex (Fig. 1A). The onset of these FPOs was often

characterized in cortical DC recordings by a steep negative shift

associated with a brief (< 200 ms) discharge of small-amplitude

population spikes at 20±30 Hz (Fig. 1A, expanded trace in the NEC

panel). Moreover, there was a tendency for the FPOs to decrease their

intraoscillatory frequency through the sequence. As summarized in

Table 1, FPOs induced by zero [Mg2+] medium had similar

characteristics (i.e. duration, rate of occurrence, and intraoscillatory

frequency) in thalamocortical slices obtained from epileptic WAG/

Rij (n = 5 slices) and NEC (n = 6) rats. During prolonged application

of zero [Mg2+] medium (particularly after 4±5 h), slices obtained

from either rat strain could produce synchronous epileptiform

discharges which closely resembled electrographic seizures (not

illustrated, but see Zhang et al., 1996).

Similarly to what was observed in slices superfused with zero

[Mg2+] medium, application of ACSF containing high [K+] induced

synchronous activity that was similar in the two types of tissue. In

both NEC (n = 3) and epileptic WAG/Rij (n = 4) thalamocortical

slices it consisted of brief (< 1 s) FPOs at 12±17 Hz which occurred

in both cortex and VB (Fig. 1B). However, the rate of occurrence of

these synchronous discharges was consistently higher in NEC slices,

even though this difference was not signi®cant (P < 0.08). The

quantitative characteristics of the spontaneous events induced by

superfusing NEC and epileptic WAG/Rij slices with high [K+]

medium are also summarized in Table 1.

Unlike the experiments performed with zero [Mg2+] or high [K+]

medium, bath application of low concentrations of 4AP (0.5±1 mM)

induced spontaneous FPOs which had strain-dependent features. In

NEC slices (n = 7), 4AP caused only one pattern of FPO, which

occurred in thalamus and/or cortex at 9.2±16.3 Hz (12.8 6 1.9 Hz,

n = 5) (Fig. 2A, NEC panel) (cf. Tancredi et al., 2000; Biagini et al.,

2001). In contrast, in epileptic WAG/Rij thalamocortical slices

(n = 23), 4AP induced two coexisting patterns of FPOs: one type was

characterized by frequencies of 9.5±16.1 Hz (13.1 6 2.1 Hz, n = 19)

while the other had frequencies ranging from 5.0 to 8.9 Hz

(7.7 6 1.1 Hz, n = 19) (Fig. 2A, WAG/Rij panel). These two types

of rhythms will be hereafter termed fast and slower FPOs, respect-

ively. It should be emphasized that in both NEC and epileptic WAG/

Rij thalamocortical slices the ranges of FPO frequencies were

narrower in any given experiment.

Power spectra obtained from segments of fast and slower FPOs

recorded in the cortex and thalamus of an epileptic WAG/Rij slice are

shown in Fig. 2B. By plotting the peak values of the power spectra

TABLE 1. Characteristics of the synchronous activity recorded in NEC and

epileptic WAG/Rij rat thalamocortical slices

Slices(n)

Intraoscillatoryfrequency (Hz)

Duration(s)

Occurrence(events/min)

Zero [Mg2+]NEC 6 7.8 6 2.5 1.9 6 0.7 4.7 6 0.6WAG/Rij 5 8.2 6 2.4 1.7 6 0.5 4.8 6 0.7

High [K+]NEC 3 14.3 6 0.6 0.23 6 0.15 27.6 6 2.9WAG/Rij 4 13.8 6 2.2 0.20 6 0.09 25.1 6 12.2

Slices were bathed in medium containing zero [Mg2+] or high [K+].Measurements were obtained from the cortical recording.

Thalamocortical oscillations in vitro 2385


obtained during segments of fast and slower FPOs recorded in the

cortex and VB of each of 19 WAG/Rij slices, we found that the

frequency of the two rhythms was highly correlated (r2 = 0.84) and

displayed a slower : fast frequency ratio of » 0.61 (Fig. 2C). In

addition, slower FPOs were characterized by amplitudes that were

larger than those seen in the same experiment during the fast FPOs

(Figs 2A and 3). As illustrated in Fig. 2A (WAG/Rij panel), the

slower FPOs were often identi®ed ®rst in the cortical recording,

becoming apparent in the thalamic area after 1±3 cycles. However,

this ®nding depended on the cortical and thalamic sites from which

recordings were obtained. Further quantitative details of the FPOs

induced by 4AP in the two types of thalamocortical slices are

provided in Table 2.

Fast FPOs generated by epileptic WAG/Rij or NEC slices could

often occur independently in cortex and thalamus, and appeared

simultaneously in these two structures only when the recording

electrodes were placed within speci®c areas of the cortical or

thalamic sectors (Figs 2 and 3A). In contrast, the slower FPOs

recorded in epileptic WAG/Rij slices were often synchronous in both

structures. This characteristic is further shown in Fig. 3A, which

illustrates fast and slower FPOs successively recorded from different

cortical and thalamic areas in the same slice. As illustrated in Fig. 3B,

FIG. 2. An application of 4AP discloses different patterns of ®eld potential oscillations in the somatosensory cortex (Cx) and ventrobasal complex (VB) ofNEC and epileptic WAG/Rij thalamocortical slices. (A) Only fast FPOs (9.3±10 Hz) are recorded from the NEC slice; in addition, these oscillations occur(a) independently in Cx and VB unless (b) recording sites with intact functional connectivity are identi®ed. In contrast, both fast (10±14 Hz) and slower (6.9±7.8 Hz) FPOs are recorded in an epileptic WAG/Rij thalamocortical slice; note that in this experiment as well, fast FPOs occur independently in Cx and VB,while slower FPOs occur synchronously in cortex and thalamus at these recording sites. Note also that the sequence of slower FPOs in this speci®c sampleappears to initiate in the cortical recording. (B) Power spectra of the rhythmic oscillatory activity recorded in the epileptic WAG/Rij rat slice shown in A andlabelled 1±4. The peak values of the power spectra are shown in each panel. (C) Plot of the power spectrum peaks of fast and slower FPOs recorded in thecerebral cortex (Cx) and the ventrobasal complex (VB) in each of 19 experiments. Note that there is a close relationship (r2 = 0.84) between the peaks of thefast and slower FPOs, with a ratio of » 0.6.



both fast and slower FPOs generated by epileptic WAG/Rij

thalamocortical slices were also recorded in nRT (n = 5), where

they appeared to be synchronized with those seen in VB. In these

experiments as well, the slower FPOs recorded in nRT displayed

amplitudes that were larger than those seen during fast FPOs.

Moreover, fast FPOs occurred synchronously in nRT and VB even

when they were not re¯ected in the cortical recordings (Fig. 3B,

asterisk). In these experiments we also found that single-shock

electrical stimuli delivered into the white matter could readily elicit

slower FPOs that were similar to those occurring spontaneously

(Fig. 3B, triangle).

Contribution of thalamic and cortical networks to 4AP-inducedoscillations in epileptic WAG/Rij slices

Having established that 4AP application can disclose different

patterns of thalamocortical activity in NEC and epileptic WAG/Rij

slices, we analysed the role played by cortical and thalamic networks

in elaborating the FPOs recorded in epileptic WAG/Rij rat slices.

First, we studied the changes caused by surgical isolation of the

cerebral cortex and thalamus (n = 5) on both spontaneous and

stimulus-induced slower FPOs. As shown in Fig. 4, isolated cortical

networks did not generate either fast or slower FPOs. In contrast, after

cutting the connections between the cerebral cortex and thalamus we

FIG. 3. (A) Different degrees of synchronization characterize fast (column a) and slower (column b) FPOs induced by 4AP application in an epileptic WAG/Rij thalamocortical slice. Position of the recording electrodes in the frontoparietal cortex (Cx) and in VB are numbered in the insert. Note that fast SLOs aresynchronized in cortex and thalamus only in panel a2, while synchronization is always present for the slower FPOs. (B) Slow speed chart recording of thespontaneous ®eld potential activity recorded in the cerebral cortex (Cx), ventrobasal complex (VB) and nucleus reticularis thalami (nRT) of an epilepticWAG/Rij rat thalamocortical slice during 4AP application. Note that synchronous, fast FPOs are recorded in both thalamic locations, but not in the cortex;also, slower FPOs occur in all recordings both spontaneously and in response to single-shock electrical stimulation of the white matter (m).

TABLE 2. Characteristics of the synchronous activity induced by 4APin NEC and epileptic WAG/Rij rat thalamocortical slices

Slices(n)

Intraoscillatoryfrequency (Hz)

Duration(s)

Occurrence(events/min)

Fast Slower Fast Slower Fast Slower

NEC 7 12.8 6 1.9 ± 2.1 6 0.54 ± 5.7 6 1.3 ±WAG/Rij 19 13.1 6 2.1 7.7 6 1.1 1.9 6 0.6 3.2 6 0.9 2.6 6 1.0 3.4 6 0.8

Measurements were obtained from the cortical recording.



could still record sequences of fast FPOs in VB and nRT, both

spontaneously and following single-shock electrical stimuli delivered

in the thalamus (Fig. 4).

Next, we used local application of the glutamatergic antagonist

kynurenic acid to cortical or thalamic areas of the slice to selectively

decrease the excitability of either neuronal network, and thus to

establish their contribution to the FPOs recorded in epileptic WAG/

Rij slices. As illustrated in Fig. 5A, applying kynurenic acid to the

cortex (n = 4) abolished both fast and slower FPOs in the cerebral

cortex while only fast FPOs continued to occur in the thalamic areas.

FIG. 4. Effects induced by cutting the connections between the cerebral cortex and thalamus on the slow ®eld potentials oscillations induced by 4AP in anepileptic WAG/Rij rat thalamocortical slice. (A) Drawings depict the thalamocortical slice under control conditions (left panel) and after complete separationof the thalamus from the cortex with two cuts performed with a micromanipulator-mounted blade (right panel). Recordings were obtained simultaneouslyfrom the cortex (Cx), the ventrobasal complex (VB) and the nucleus reticularis (nRT). FPOs shown in B occurred spontaneously before and after the cut,while those in C were induced by single electrical stimuli delivered into the internal capsule (m) or into the white matter underlying the cortex (d). Notethat, after surgical separation, both spontaneous and stimulus-induced FPOs are absent from the cortex, while they are still observed in VB and nRT, thoughwith higher frequencies than under control conditions.



Hence, depressing cortical excitability transformed thalamic slower

FPOs into fast FPOs. In contrast, application of this glutamatergic

antagonist to the thalamus made both fast and slower FPOs disappear

in thalamic and cortical areas (n = 3) (Fig. 5B). Under these

conditions focal electrical stimuli in the internal capsule induced

®eld potential responses in the cerebral cortex and VB that lasted

< 180 ms and were characterized by an isolated negative or positive±

negative transient (Fig. 5B, triangle in `Kynurenic acid in VB + nRT'

panel). The effects induced by either cortical or thalamic application

of kynurenic acid were reversible following removal of the pipette

and 20±30 min of washout of the glutamatergic receptor antagonist

from the site of application (not shown). The graphs in Fig. 5C

summarize the results obtained in these experiments.

NMDA receptor-dependent characteristics of the 4AP-inducedoscillations

Sequences of slower FPOs induced by 4AP in WAG/Rij thalamo-

cortical slices (n = 6) became less frequent and eventually disap-

peared when increasing concentrations of the NMDA receptor

antagonist CPP were added to the bathing medium. The blockade

of these slower FPOs was seen concomitantly in cortex and VB, and

it was paralleled by an increase in the recurrence of fast FPOs; such

an effect was more striking with CPP concentrations that completely

abolished the slower oscillatory activity (Fig. 6). In contrast, no

measurable changes were observed when similar concentrations of

CPP were tested in NEC slices on the 4AP-induced fast FPOs (n = 4)

(Fig. 6).

In order to identify the site of action of CPP within the WAG/Rij

thalamocortical slice, we administered this NMDA receptor antag-

onist locally in the cerebral cortex or VB + nRT. In six experiments,

application of CPP to the cerebral cortex markedly decreased and

eventually abolished slower FPOs, while the occurrence of fast FPOs

displayed a tendency to increase (Fig. 7A and C). In contrast, local

application of CPP to the thalamic areas (n = 7) did not modify the

rate of occurrence of either fast or slower FPOs (Fig. 7B and C).

Discussion

We have found that rat thalamocortical slices maintained in vitro in

the presence of minimal concentrations of 4AP generated patterns of

thalamocortical oscillations with electrographic features which

depend on the animal strain. Speci®cally, fast FPOs could occur in

both types of tissue, while slower FPOs were only recorded in

epileptic WAG/Rij rat slices. In addition, slower FPOs were of larger

amplitude than the fast FPOs in both cortex and thalamus, suggesting

that larger neuronal populations were presumably involved in the

slower thalamocortical rhythm. In keeping with this view, cortical

and thalamic slower FPOs occurred in a synchronous fashion over

much of the cortical and thalamic areas investigated with ®eld

FIG. 5. Effects induced by local application of the glutamatergic antagonist kynurenic acid to the cortex or thalamus on FPOs recorded in two slices obtainedfrom epileptic WAG/Rij rats. (A) Kynurenic acid application to the cortex abolishes slower FPOs while fast FPOs continue to occur in VB. (B) In contrast,kynurenic acid application to the thalamus (VB + nRT) makes both fast and slower FPOs disappear in thalamic and cortical areas; note that under theseconditions focal electrical stimuli in the internal capsule (m) induces an isolated ®eld potential response lasting > 200 ms. (C) Summary of the effects inducedby kynurenic acid application to the cortex (n = 4 slices) and thalamus (n = 3) on the fast and slower FPOs recorded in cortex (upper panel) and VB (lowerpanel).



potential recordings, but fast FPOs were synchronized only when

recording from speci®c locations (cf. Tancredi et al., 2000; Biagini

et al., 2001).

Epileptic WAG/Rij rats present with ictal behavioural and EEG

manifestations that are similar to those seen in patients with absence

seizures (van Luijtelar & Coenen, 1986; Peeters et al., 1990), even

though the generalized SW activity in this and in another well-

characterized genetic rat model (i.e. the GAERS) is characterized by

intraburst frequencies that are higher than in humans (i.e. 6±10 Hz vs.

2.5±4 Hz, respectively). Indeed, Destexhe et al. (1999) have proposed

by using mathematical modelling of SW discharges that this may be

caused by differences in low-threshold Ca2+ current and/or GABAB

receptor function. In vivo studies have shown that generalized SW

discharges in WAG/Rij rats re¯ect highly synchronized oscillations in

thalamocortical networks (Staak & Pape, 2001; Meeren et al., 2002).

Similar ®ndings have been obtained in GAERS rats (Seidenbecher

et al., 1998) and in the feline generalized penicillin (FGPE) model

(Avoli et al., 1983), in which transformation of sleep spindles into

generalized SW discharges is accompanied by a progressive increase

in ®ring correlation between cortical and thalamic cells (McLachlan

et al., 1984). Therefore, even though generalized SW discharges

could not (for obvious reasons) be reproduced in the slice preparation,

we are inclined to propose that the strain-dependent ability to

generate slower SLOs, along with their high degree of thalamocor-

tical synchronization, may represents an in vitro hallmark of the

epileptogenicity that characterizes WAG/Rij rats.

The oscillatory patterns recorded in epileptic WAG/Rij rat

thalamocortical slices, regardless of their frequency and degree of

synchronicity, require the function of thalamic networks, whose

output activity must reach cortical targets. Accordingly, no rhythmic

oscillatory activity was generated by cortical neurons when the cortex

was surgically separated from the thalamus or when the broad

spectrum glutamatergic antagonist kynurenic acid was locally applied

to VB-nRT. Similar ®ndings have been reported by us while studying

fast thalamocortical oscillations induced by 4AP in slices obtained

from young control rats (Tancredi et al., 2000). Moreover, early work

in the FGPE model has demonstrated that both EEG spindles and

generalized SW discharges disappear during transient depression of

thalamic function by local microinjection of KCl (Avoli & Gloor,

1981). It is, indeed, well established that spindle activity originates in

the thalamus and relies on the function of nRT networks (Steriade

et al., 1985). Interestingly, nRT cells are also intimately involved in

the generalized SW discharges recorded in GAERS rats (Slaght et al.,

2002). It should, however, be emphasized that, after intracortical

injection of bicuculline, SW activity at 2±3 Hz is recorded in the

cortex of cats which had undergone total ipsilateral thalamectomy

with abolition of sleep spindles (Steriade & Contreras, 1998) Hence,

these data demonstrate that in this experimental model cortical

networks may sustain SW discharges even in the absence of

thalamocortical inputs.

An important issue in the pathophysiogenesis of generalized SW

discharges in patients with absence epilepsy is whether the

synchronous thalamocortical oscillations that accompany the electro-

graphic pattern originate in cortex or thalamus. We have reported

here that only fast FPOs were recorded from VB or nRT after surgical

separation from the cortex and that slower FPOs disappeared in all

areas during cortical application of a glutamatergic receptor antag-

onist. Therefore, our data suggest that the ability of epileptic WAG/

Rij thalamocortical slices to generate slower FPOs depends on the

involvement of cortical networks in this type of rhythm and

speci®cally on their ability to drive both relay (i.e. VB) and nRT

cells via corticothalamic inputs (Deschenes & Hu, 1990; Deschenes

et al., 1998). This view is in keeping with the ability of cortical

spreading depression to change thalamic SW discharges into spindles

in cats injected with large doses of penicillin (Avoli & Gloor, 1982a).

Moreover, penicillin-induced SW discharges in cats are absent in the

thalamus following removal of the cortex (Avoli & Gloor, 1982b).

FIG. 6. Summary of the effects induced by bath application of twoconcentrations of the NMDA receptor antagonist CPP on the rate ofoccurrence of fast and slower FPOs in epileptic WAG/Rij thalamocorticalslices (n = 4) and on the fast FPOs recorded in NEC tissue (n = 3). Notethat CPP reduced and eventually abolished the slower FPOs in epilepticWAG/Rij thalamocortical slices, whereas fast FPOs increased their rate ofoccurrence. In contrast, CPP did not modify the pattern of fast FPOsrecorded in NEC slices. *P < 0.05.



Recent evidence obtained in slices of the ferret visual thalamus has

demonstrated that the frequency of thalamic network oscillations is

changed by varying the strength of corticothalamic tract stimuli that

were triggerd by the spontaneous activity of thalamic relay cells in

the geniculate nucleus (Bal et al., 2000; Blumenfeld & McCormick,

2000). In particular, it has been shown that weak feedback activation

leads to oscillations at 6±10 Hz, while strong corticothalamic

feedback stimuli cause a more synchronized, slower (2±4 Hz)

oscillatory process. Intracellular recordings obtained in vivo from

GAERS have shown that during SW discharges nRT cells generate

robust discharges of action potentials (presumably in response to

corticothalamic inputs) that cause a tonic hyperpolarization in the

majority of thalamocortical neurons (Pinault et al., 1998; Slaght et al.,

2002). These data are in keeping with an earlier intracellular study of

the activity of thalamocortical neurons in anaesthetized cats that

spontaneously developed SW activity at 2±4 Hz (Steriade &

Contreras, 1995). It was proposed in this study that the hyperpolar-

ization of thalamocortical cells and the concomitant ®ring silence

may represent the cellular correlate of the unresponsiveness seen

during absence seizures.

A leading role of cortical networks in generalized SW discharges

of epileptic WAG/Rij rats has also been proposed by Meeren et al.

(2002), who have employed non-linear association analysis of the

EEG signals recorded in vivo from multiple thalamic and cortical

sites during SW discharges. Interestingly, these experiments have

identi®ed a `consistent cortical focus' of SW activity within the peri-

oral region of the somatosensory cortex. Indeed, this cortical area was

presumably included in most of the thalamocortical slices used by us

in the present study. It must be emphasized that both the cortical

origin of SW discharges and the ability of cortical networks to lead

the thalamus during this rhythm have been identi®ed in several

studies performed in cats by Steriade and collaborators (Steriade &

Amzica, 1994; Steriade & Contreras, 1995; Neckelmann et al.,

1998).We have also established here that the generation of slower

FPOs in epileptic WAG/Rij thalamocortical slices depends on the

function of NMDA receptor-mediated mechanisms. In line with this

view, slower FPOs were no longer recorded during bath application

of the competitive NMDA receptor antagonist CPP, while the

occurrence of fast FPOs increased. Interestingly, CPP application to

NEC thalamocortical slices treated with 4AP did not cause any

change in the rate of occurrence of the fast FPOs, thus suggesting that

the increase in fast FPO occurrence in epileptic WAG/Rij rats during

CPP application may re¯ect the block of slower FPOs. NMDA

receptors have been proposed as contributing to SW discharge

generation in epileptic WAG/Rij (Peeters et al., 1989, 1990) and

GAERS (Pumain et al., 1992; Koerner et al., 1996) rats. However, it

was unclear from these in vivo studies whether NMDA dysfunction

resided in the cortex (Pumain et al., 1992), the thalamus (Koerner

et al., 1996) or, perhaps, in both structures. By using local application

of CPP to cortex or thalamus we have demonstrated that slower FPOs

induced by 4AP in epileptic WAG/Rij thalamocortical slice disappear

only when CPP is cortically applied. Therefore, these data demon-

strate that NMDA receptor-mediated mechanisms responsible for the

occurrence of slower FPOs should reside in the cortex. In addition,

this pharmacological evidence further supports the role of cortical

networks in generating slower FPOs, and suggests that NMDA

receptors do not play any major role in the genesis of fast FPOs.

It should be emphasized that the speci®c role played by NMDA

receptor-mediated mechanisms in the genesis of slow FPOs in WAG/

Rij rat slices is supported by the inability of the other two procedures

tested in this study to elicit different, strain-dependent patterns of

thalamocortical oscillations. It is well established that both zero

[Mg2+] and, to a lesser extent, medium containing high [K+], lead to

activation of NMDA receptor-mediated conductances. Hence, both

FIG. 7. (A and B) Effects induced by local application of CPP to the cerebral cortex and thalamus (VB + nRT) of an epileptic WAG/Rij thalamocortical slicethat was continuously superfused with 4AP-containing medium. Note that during application of CPP to the cortex, slower FPOs disappeared in both cortexand thalamus while the fast FPOs continued to occur in both areas. (C) Summary histograms of the rate of occurrence of 4AP-induced fast and slower FPOsrecorded in cerebral cortex (upper panel) and VB (lower panel) of epileptic WAG/Rij thalamocortical slices under control conditions and during localapplication of CPP to the cortex (n = 4) or to VB + nRT (n = 5).



manipulations could mask the subtle differences in NMDA receptor

function that are present in the cortex of WAG/Rij rat as compared

with NECs.

In conclusion, we have demonstrated here that epileptic WAG/Rij,

but not NEC, rat thalamocortical slices can produce sequences of

slower oscillatory rhythms during bath application of 4AP. The

occurrence of these events requires reciprocally connected and

functional thalamic and cortical networks. In particular, our data

indicate that thalamic networks are necessary to provide rhythmo-

genesis that, however, is not expressed at a slower intraoscillatory

frequency unless corticothalamic inputs are operant. Therefore, an

important determinant for the production of generalized SW

discharges in the WAG/Rij rat resides in the cortex which is

overexcitable (Luhmann et al., 1995) and endowed with the ability to

promote a fast intracortical spread of the epileptiform activity

(Meeren et al., 2002). Overall, as originally proposed by Steriade &

Contreras (1998), our data point at the cortex as a target for

antiepileptic drug action in this genetic rodent model of generalized

SW discharges and presumably in human absence epilepsy.

Acknowledgements

This study was supported by the Canadian Institutes of Health Research (GrantMOP-43964), the National Research Council of Italy and the SavoyFoundation. M.D. and R.A.W. were recipients of a Fellowship from theFragile X Research Foundation of Canada and of a Scholarship from the Fondsde la Recherche en SanteÂ du QueÂbec, respectively. We thank Dr Florin Amzicafor reading an early draft of this paper and Toula Papadopoulos for secretarialassistance.

Abbreviations

4AP, 4-aminopyridine; ACSF, arti®cial cerebrospinal ¯uid; CPP, 3,3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonate; FPOs, ®eld potential oscilla-tions; GAERS, Genetic Absence Epilepsy in Rats from Strasbourg; NEC,nonepileptic control; nRT, nucleus reticularis of the thalamus; SW, spike andwave; VB, ventrobasal complex; WAG/Rij, Wistar Albino Glaxo/Rijswijk.

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Thalamocortical oscillations in a genetic model of absence seizures

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