INTRODUCTION

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There are several reports indicating that cholinergic neurotransmission has a complex role in the induction of epileptic activity in both human patients and animal models of epilepsy. In human patients presenting a temporal lobe epilepsy, a reduction of muscarinic neurotransmission has been observed (Kish et al. 1988; Müller-Gärtner et al. 1993; Pennell et al. 1999). Recently, familial frontal lobe epilepsy has been linked to a mutation altering the functional properties of nicotinic cholinergic receptors (Lena and Changeux 1997; Steinlein et al. 1995, 1997). In animals, convulsive manifestations are induced by systemic injection or local application of various cholinergic agonists such as carbachol (Brudzynski et al. 1995; Mraovitch and Calando 1995; Snead 1983), pilocarpine (Turski et al. 1989), or nicotine (Westerlain and Fairchild 1985). However, some contradictory results were reported since cholinergic antagonists may also provoke seizures (Segal 1991).

We have developed a model of focal cortical epilepsy in baboons and rats by continuously infusing GABA into the motor cortex (Brailowsky et al. 1987, 1988, 1990). On cessation of the GABA infusion, continuous paroxysmal electroencephalographic discharges appear in the infusion area associated with myoclonic twitches of the contralateral corresponding body territory. This epilepsy named "GABA withdrawal syndrome" (GWS) is a model of local status epilepticus (Brailowsky et al. 1988). In neocortical slices obtained from rats presenting GWS, a great number of pyramidal neurons present intrinsic bursts induced by intracellular depolarizing current injection and/or paroxysmal depolarization shifts (PDSs) induced by white matter stimulation (Silva-Barrat et al. 1989, 1992).

An immunocytochemical study revealed the presence in the GABA-injected cortical area of significantly more numerous choline acetyltransferase immunopositive neurons than those seen in the contralateral area or in control rats (having received an intracortical infusion of saline), suggesting the appearance in epileptic rats of neurons neosynthetizing choline acetyltransferase and possibly acetylcholine (Araneda et al. 1994). It is known that the cholinergic innervation of the cerebral cortex in normal rats mainly comes from the basal forebrain (Rye et al. 1984). Given these data, we investigated possible changes in the effects of cholinergic agonists that might have resulted from the GWS. We tested the effects on bursting cells of acetylcholine and carbachol, acting on both muscarinic and nicotinic receptors. These effects were compared with those observed on regular spiking cells from both control (nonepileptic) and GWS (epileptic) rats. The respective participation of muscarinic and nicotinic receptors was assessed using the selective agonists methyl-acetylcholine and nicotine. Results demonstrate that acetylcholine and carbachol through muscarinic receptor activation might have a protective role against the synaptic spread of the epileptic activity in this model of focal epilepsy.

	Methods

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The methods were similar to those described in related papers (Silva-Barrat et al. 1989, 1992). In brief, rats (Wistar male, 150-180 g) were anesthetized by ip injection of a ketamine-xylazine mixture and implanted with an intracortical cannula in the left fronto-parietal cortex (2 mm posterior to bregma, 2 mm lateral and 1.0 mm depth), according to the rat brain atlas by Paxinos and Watson (1982). The implanted zone corresponds to the hindlimb motor area. An ALZET osmotic minipump, filled with GABA (100 µg/µl, delivery rate 1 µl/h), was placed under the skin of the animal's back and connected with a subcutaneous catheter to the intracortical cannula. Five days later, GABA infusion was stopped and the appearance of GWS was verified by the appearance of myoclonic twitches of the right hindlimb. A group of control rats was either naive (receiving no infusion) or infused with saline. In these two situations, obtained data showed no differences, as had already been observed in the past (Silva-Barrat et al. 1989, 1992, 1994).

After decapitation of the animals, the cortex was removed and transverse slices (450 µm) were cut with a Campden vibratom at the level of the infusion site. Slices were then transferred to the recording chamber and superfused with warm (32 ± 0.1°C), oxygenated (95% O₂-5% CO₂) Ringer-Krebs solution composed of (in mM) 124 NaCl, 3.2 KCl, 1.2 NaH₂PO₄, 1.2 MgSO₄, 2.4 CaCl₂, 26 NaHCO3, and 10 glucose, pH 7.4. The cholinergic agonists were added to the perfusate from freshly prepared stock solutions to reach the indicated final concentration: acetylcholine chloride (Ach, 200-1000 µM); carbamylcholine chloride (carbachol or Cch, 10-50 µM), a nonhydrolizable agonist of the Ach receptor; acetyl--methylcholine chloride (mAch, 100-400 µM); atropine sulfate (1-50 µM), a selective antagonist of the muscarinic Ach receptor; and nicotine hydrogen tartrate (50-100 µM). Drug applications lasted 5 to 10 min. To avoid degradation of the agonists or desensitization of the cells to the agonists, Ach and nicotine at high concentrations were applied by drops to the surface of the slice near the recording electrode. In this case, given the relative volumes of the recording chamber and perfusion bolus, and given the perfusion speed, the concentration in the bath medium at the end of application was estimated at a tenth of the initial value. Application by drops was effective to activate cholinergic receptors in a comparable slice preparation (Sorenson and Chiappinelli 1990). Tetrodotoxine (TTX, 0.5-1 µM) was added directly to the perfusate. All drugs were obtained from Sigma Chemical (St. Louis, MO).

Intracellular recordings were made with 4 M potassium acetate-filled micropipettes (60-120 M) in the vicinity of the GABA infusion site. Membrane potential recordings and intracellular current injections were performed with an Axoclamp-2A amplifier in a current clamp, bridge mode. Pulses of current of 0.1-1 nA for 250-400 ms were delivered through the intracellular electrode to monitor input resistance and action potentials (APs). Single stimuli delivered every 5 s (0.05 ms duration and 0.5-1.0 mA intensity) were applied to white matter (WM) by a concentric bipolar electrode. Voltage transients and injected currents were monitored with an oscilloscope, displayed on a thermal arraycorder (Ankersmit), and stored on a digital magnetic tape (Biologic). The analysis consisted of an A/D conversion through a Cambridge Electronic Device (CED 1401) and the computer treatment of digital data by an averaging program (SIGAVG), or through an Axon Instruments device (Digidata 1200B acquisition system) and the treatment by Axoscope software. Data were plotted and analyzed using ORIGIN, Microcal software (Northampton, MA). The statistical significance of data were evaluated by analysis of variance (ANOVA).

The recorded bursting cells were situated in the upper part of layer V (150-250 µm below the cortex surface) and selected on the basis of stable membrane potential and AP amplitude over 3- to 8-h periods. These cells presented intrinsic bursts induced by intracellular depolarizing current injection and/or PDSs induced by WM stimulation similar to those previously described (Silva-Barrat et al. 1989, 1992, 1994). The intrinsic bursts consisted of two to five APs riding on a slow wave. The synaptically induced bursts consisted of three to six APs riding on a large depolarizing wave or PDS. They were evoked at resting membrane potential by suprathreshold WM stimulation with an intensity 1.5-2 times higher than required for evoking an EPSP.

PDSs observed during GWS are Ca²⁺- and NMDA-related processes superimposed on classical, mostly -amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA), excitatory postsynaptic potential (EPSPs) (Silva-Barrat et al. 1992). These PDSs were not observed in intrinsic bursting cells of control rats. We investigated the synaptic generation of PDSs by measuring synaptic responses through the following parameters (Figs. 1 and 2):

View larger version (50K): [in this window] [in a new window]   Fig. 1. Amplitude of synaptic responses and membrane potentials of recorded cells. Columns represent mean values and standard deviations of excitatory postsynaptic potential (EPSP) amplitudes and membrane potentials measured on regular spiking cells of control rats (A, C), and of paroxysmal depolarization shifts (PDSs) and membrane potentials measured on bursting cells of GABA withdrawal syndrome (GWS) rats (B, D), before (predrug) and during cholinergic drug applications. Measurements were made as follows: amplitude from the resting level to the top of the wave of EPSPs or PDSs and resting membrane potential from the amplitude of the abrupt deflection obtained on withdrawal of the recording electrode from the cell. Statistical differences between predrug and drug applications are indicated by stars representing the standardized level of significance [analysis of variance (ANOVA) test]. Amplitude of synaptic responses of bursting cells before the drug application is significantly higher (P < 0.001) than that for the regular spiking cells; otherwise, no significant difference appears in the membrane potential values.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Effects of drug applications on PDSs. Columns represent mean values and standard deviations of the amplitude of EPSPs when PDS can be triggered (A), the PDS amplitude from the onset to the top of the PDS immediately after the end of the burst firing (B), and the number of APs associated with the PDS (C), before (predrug) and during cholinergic drug applications. Stars indicate the standardized level of significance of differences between predrug and drug applications (ANOVA test).

1) EPSP amplitude from the resting level to the top of the EPSP.

2) EPSP amplitude when PDS can be triggered.

3) PDS amplitude from the resting level to the top of the PDS, immediately after the end of burst firing.

4) Latency between the stimulus and the onset of the PDS.

5) Failure of PDS generation indicated by the percentage of absence of PDS within the 500-ms period of time following the stimulus, i.e., approximately 10 times the delay observed in predrug condition.

6) Changes in input resistance (Ri) with respect to predrug value; this parameter was taken as an index to evaluate the possible impact of Ri on potentials.

7) Changes in membrane potential (Em) similar to those induced by agonist applications. These changes were produced by steady current injection to evaluate the modifications of EPSP amplitude by the drug-induced depolarization.

	RESULTS

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Of the 72 recorded cells in slices obtained from 65 rats presenting GWS, 30 were bursting cells presenting synaptic and/or intrinsic bursts of APs and the remaining 42 were nonbursting cells presenting regular spiking. Twenty-four regular spiking cells and three bursting cells were recorded in slices obtained from 27 control rats.

In all cell types of GWS and control rats, Cch and Ach caused a slow depolarization of the membrane potential (Figs. 1, C and D, and 3A) that lasted 4 to 15 min after the end of application. This depolarization was associated with an increase of action potential firing induced by depolarizing current injection and, in most cells, with an increase of input resistance from 27-33 to 36-47 M. These effects were concentration-dependent with threshold response in the low micromolar range. The ability of Cch to provoke a depolarization was preserved in the presence of TTX (0.5 µM, n = 3, not shown) indicating that the depolarization was not mediated by an action potential-dependent release of neurotransmitters.

View larger version (32K): [in this window] [in a new window]   Fig. 3. Carbachol (Cch) preserves GWS-related intrinsic burst generation despite membrane depolarization. A: chart paper record showing the Cch-induced slow depolarization and the associated increase of action potential firing in a bursting cell of a GWS rat. B: neuronal activity recorded in the same cell, in the absence of Cch, and during the depolarization by steady current injection at the same level as during Cch effects. C1: before Cch application, intrinsic bursts are induced by injection of a depolarizing current pulse (0.3 nA, 200 ms) (Em 64 mV). C2: during the Cch-induced depolarization, bursting activity appears spontaneously. C3: expanded example during steady current injection as in B, no bursting activity occurs even though AP firing is increased.

None of the three bursting cells recorded in slices obtained from control rats presented PDSs. Regular spiking cells from control and from GWS rats showed no significant differences (ANOVA test) in passive properties (Em and Ri) and EPSP amplitudes. However, in four out of nine regular spiking cells recorded in GWS slices, Cch provoked some special modifications described hereafter.

Cch and Ach promote intrinsic bursting and depress PDSs

In bursting cells of GWS rats, in addition to the slow depolarization and increase of AP firing (Fig. 3A) and input resistance, Cch (n = 6) and Ach (n = 12) provoked the appearance of spontaneous bursting (Fig. 3C2). Such spontaneous bursting is due to cholinergic agonists and not to membrane depolarization since it could not be reproduced when the membrane potential was depolarized by steady current injection at the same level as during cholinergic effects (Fig. 3, B and C3). Moreover, Cch and Ach increased the number of bursts induced by depolarizing current injection (Fig. 4A2). In contrast, these cholinergic agonists were unable to induce intrinsic bursting activities in regular spiking cells. In four out of nine regular spiking cells recorded in GWS slices, Cch at the same dose provoked oscillatory variations of the membrane potential associated with an important rhythmic activation of AP firing (not shown) but completely different from epileptic-like activity. This activity was not seen in control rats (n = 6), suggesting that regular spiking cells of GWS rats could have some properties different from those of nonepileptic rats. Therefore muscarinic agonists had an epileptogenic action on the intrinsic membrane properties of GWS neurons, but not of regular spiking cells.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Cch exerts opposite effects on GWS-related intrinsic bursts and PDSs. Intrinsic bursts (A1, A2, A3) and PDSs (B1, B2, B3) of a bursting cell of a GWS rat recorded before Cch application (predrug, Em 58 mV), during Cch application (Cch 50 µM, Em 53 mV), and during combined application of Cch (50 µM) and atropine (50 µM) (Cch + atropine, Em 60 mV).

The effects of muscarinic agonists on synaptically induced PDSs were completely different: Cch and Ach decreased significantly the amplitude of PDSs as well as the number of APs associated with PDSs (Figs. 2, B and C, and 4B2). The PDS latency (65.7 ± 19.2 ms) was not significantly modified.

We tested whether or not the depression of PDSs induced by Cch is caused by the Cch-induced depolarization. First, a bursting cell in the absence of Cch (Em 64 mV, Fig. 5C) was depolarized by steady current injection (Em 58 mV, Fig. 5A) at the level reached during Cch action (Fig. 5B). This procedure was unable to induce the PDS depression. Second, the cell depolarized by Cch was brought back to resting level without any effect on the PDS depression (Fig. 5D). As for Cch, these effects were not reproduced when the cell was depolarized by a steady current injection at the same potential as during Ach action. The failure of PDS generation (evaluated as described in METHODS) considered during the period of Cch- or Ach-induced depolarization was 45-50%. Ach was less efficient than Cch (effects less reproducible), probably because Cch is a nonhydrolizable agonist of the Ach receptor (Haj-Dahmane and Andrade 1996).

View larger version (11K): [in this window] [in a new window]   Fig. 5. Cch blocks generation of PDSs. A: PDS recorded in a bursting cell of a GWS rat in the absence of Cch, during a depolarization at the same level as during Cch (Em 58 mV) by steady current injection (predrug). B: the same cell during the Cch-induced depolarization. C: PDS recorded at resting membrane potential (Em 64 mV) in the absence of Cch. D: PDS remained blocked during the Cch application when the membrane potential was brought back to the resting membrane potential.

In regular spiking cells of both control and GWS rats, Cch (n = 15) and Ach (n = 7) induced a slow depolarization associated with the appearance of spontaneous spikes and with an increase of the number of APs induced by the depolarizing pulse, and a decrease of EPSP amplitude that was not as significant as for PDSs (Fig. 1A). In two intrinsic bursting cells recorded from control rats, Ach provoked a depolarization (~5 mV) and a shift in the firing pattern from burst to regular spiking activity; this latter effect was reproduced when depolarizing the neurons by steady current injection (not shown).

Muscarinic receptors are involved in the increase of intrinsic bursting and depression of PDSs

To identify pharmacological effects of Cch, we tested the effects of atropine that antagonize muscarinic action. Atropine alone was unable to generate changes in PDSs or intrinsic bursts, thus exhibiting neither proepileptogenic nor antiepileptogenic action in GWS slices. When atropine was applied after or in combination with Cch (n = 2), a reversal of both Cch effects, i.e., PDS depression and intrinsic burst facilitation, was observed (Fig. 4, A3 and B3) with no significant effect on membrane potential. This atropine effect was dose-dependent, being more efficient with atropine at 50 µM than at 1 µM.

To determine the specific effects of muscarinic receptor activation, we also tested the effects of mAch, a selective muscarinic agonist on both control (n = 10) and GWS rats (n = 11). Similarly to Cch and Ach, mAch provoked in bursting cells from GWS rats a slow depolarization (Fig. 1D) associated with a significant decrease or blockade of PDS (Fig. 2, B and C), and an increase in the number of bursts induced by depolarizing current injection. In three bursting cells of GWS rats, atropine (50 µM) applied in combination with methyl-acetylcholine (mAch) prevented the development of these effects. These results suggest that the above-described effects of Ach and Cch are mediated by muscarinic receptors. In regular spiking cells mAch also induced a slow depolarization (Fig. 1C) associated with an activation of APs during depolarizing current pulses. EPSPs were reduced (Fig. 1A), but this effect was significantly smaller (6.4 ± 4.6%) than the decrease of PDSs in bursting cells (12.6 ± 4.0%) and was reversed when atropine was applied in combination with mAch.

Nicotine potentiates synaptically induced PDSs

In bursting cells of GWS rats (n = 4), nicotine provoked a slow depolarization (Fig. 1) and an increase of input resistance stronger than that observed with the other tested agonists (18-22 M). During nicotine application, the intrinsic bursts induced by depolarizing current injection were replaced by isolated spikes, while in the same cells, nicotine potentiated PDSs (Fig. 6) by increasing PDS duration and delaying the process of PDS termination. Contrarily to Ach, Cch, and mAch, which suppressed the synaptically induced bursts and the underlying PDS, nicotine increased the number of APs in the bursts (Fig. 2C) without a significant modification in the rising time of the underlying PDS (n = 4, Fig. 6).

View larger version (18K): [in this window] [in a new window]   Fig. 6. Opposite control of GWS-related PDS amplitude by nicotine and mAch. Top: superimposition of bursts before (predrug) and during nicotine application (nicotine). In the predrug situation the burst was recorded at the same membrane potential (Em 56 mV) as during nicotine-induced depolarization. Bottom: superimposition of burst and EPSP before (predrug) and during mAch application (mAch). Both responses were recorded at the same membrane potential (Em 64 mV).

In regular spiking cells of control (n = 2) and GWS rats (n = 5), the slow depolarization and AP discharges induced by nicotine were stronger than that observed with the other tested agonists (Fig. 1C), but synaptic responses were not transformed into bursts of APs even though the afterdepolarization following the spike became larger (not shown). This effect of nicotine contrasts with those previously described for noradrenaline which increased the amplitude of afterdepolarization and induced bursts (Silva-Barrat et al. 1994). From these data, we can conclude that GWS leads to a differential effect of nicotine and muscarinic agonists. In addition, these nicotinic effects were not seen under Cch or Ach application, providing evidence for the prevalence of muscarinic effects with agonists acting on both muscarinic and nicotinic receptors.

	DISCUSSION

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The present study provides evidence for the first time showing that during epileptogenic GWS cholinergic agonists exert opposite effects on intrinsic (membranar) and extrinsic (synaptic) epileptic events in cortical neurons. During a previous analysis on GWS slices, blockers of K⁺ and Ca²⁺ currents as well as antagonists of glutamatergic and catecholaminergic receptors were found to exert similar effects on both intrinsic bursts and PDSs (Silva-Barrat et al. 1992, 1994). In contrast, we show that the activation of muscarinic receptors (by Ach, Cch, or mAch) favors intrinsic bursting, while it significantly depresses synaptically induced PDSs. Because nicotinic effects were not induced by Cch or Ach in the presence of atropine, we considered that the effects of muscarinic agonists were almost exclusively mediated by muscarinic receptors in our experimental conditions. These data provide a basis for a complex modulation of cortical epileptic activities by cholinergic agonists acting through muscarinic receptors during GWS.

Muscarinic increase of burst generation in GWS

We have observed that in bursting cells of GWS rats, the slow depolarization and the increase of input resistance induced by cholinergic agonists (Ach or Cch) are associated with an activation of intrinsic bursting activity. These effects are atropine-sensitive, i.e., mediated by muscarinic receptors. In cortical neurons from nonepileptic animals, Krnjevic et al. (1971), McCormick and Prince (1986), and McCormick et al. (1993) have described the induction by cholinergic agonists of a slow depolarization and an increase of input resistance which have been attributed to the activation of cholinergic receptors by inhibiting K⁺ channels or by activating voltage-dependent nonselective cationic current (Haj-Dahmane and Andrade 1996). Moreover, McCormick et al. (1993) and Wang and McCormick (1993) have observed that the slow depolarization is associated with a shift in the firing pattern from bursting to single spike activity. In cortical neurons during GWS, we have observed the same effect during norepinephrine application (Silva-Barrat et al. 1994). In contrast, the present study shows that bursting activity of GWS but not of control rats persists at higher frequency during the slow depolarization induced by cholinergic agonists or intracellular current injection. Altogether, these observations indicate that cholinergic activation of intrinsic bursts observed in GWS does not result from the slow depolarization that develops at the same time.

Bursting neurons situated in the epileptic focus area of GWS rats are tolerant to GABA, as a result of an excessive influx of Ca²⁺ (Silva-Barrat et al. 1989); the decrease in the GABA efficacy leads to a disinhibition that could favor the reactivity of neurons to cholinergic agonists. In support of this hypothesis, Bianchi and Wong (1994) and Psarropoulou and Dallaire (1998) demonstrated that bursting activities could be induced by Cch in the hippocampus (CA3) slices of the guinea pig in the absence of GABAergic transmission. In this region, the appearance of rhythmic bursts and sustained depolarization in the presence of Cch were attributed to a direct action on muscarinic receptors involving a reduction in K⁺ conductances. It is therefore possible that in the neocortex during GWS, intrinsic bursting is increased by the combined effects of tolerance to GABA and the postsynaptic activation of muscarinic receptors on epileptic-like neurons by cholinergic agonists.

Muscarinic decrease of synaptically induced PDSs

In contrast to the muscarinic exaggeration of intrinsic bursting, a blockade of synaptically induced bursts and a reduction of the PDS amplitude are induced by Ach, Cch, and mAch in bursting neurons of GWS rats. Intrinsically induced bursts are increased by a muscarinic facilitatory action at the postsynaptic level. Synaptic depression, therefore, does not result from these muscarinic effects on postsynaptic electroresponsiveness and the intrinsic bursting activity of neurons. We rather suggest that muscarinic agonists exert a potent presynaptic inhibitory action on the release of other neurotransmitters. This presynaptic action is probably mediated by a muscarinic reduction in Ca²⁺ currents through voltage-dependent channels, as demonstrated in several cell types (Gahwiler and Brown 1987; Toselli and Lux 1989) through pertussis toxin-sensitive and -insensitive G-protein-gated receptors (Howe and Surmeier 1995; Wanke et al. 1994). Therefore the opposite muscarinic effects on PDSs and intrinsic bursts may result from an action at different presynaptic and postsynaptic cellular levels.

We have observed that in regular spiking neurons of both control and GWS rats, Ach and Cch provoke a nonsignificant modification of EPSPs which were slightly depressed by mAch, indicating that this effect was much weaker than that observed on PDSs. Given that the muscarinic depression of EPSPs was much less important than for PDSs, we suggest that GWS may exaggerate the function of presynaptic muscarinic receptors. Previous studies of voltage-dependent currents have shown that GWS causes an excessive influx of Ca²⁺ in neurons (Silva-Barrat et al. 1989). Muscarinic agonists may reduce this Ca²⁺ influx presynaptically and consequently strongly depress synaptically induced PDSs.

Possible functional consequences of muscarinic action

Bursting neocortical neurons recorded in GWS rats are pyramidal neurons located in the cortical layer V (Silva-Barrat et al. 1992). They have an axonal arborization that largely remains within layer V and may therefore mediate synchronization of intralaminar activity especially when intracortical inhibitory mechanisms are depressed by prolonged GABA infusion. These neurons receive a high cholinergic terminal density from the basal forebrain (Eckenstein et al. 1988) and it is known that the endogenous Ach is able to modify the rhythmic bursting activity (Metherate et al. 1992). In GWS rats, cholinergic synaptic transmission is probably modified in the cortex, thereby explaining some of the effects observed in the present study during the exogenous application of Ach. In GWS rats we observed overexpression of choline acetyltransferase (ChAT) RNA messengers and immunoreactive cell bodies in the cortical focus area but not in the homotopic controlateral region or in saline-infused rats (Araneda et al. 1994; Menini et al. 1992), suggesting the appearance of neurons neosynthetizing ChAT and possibly Ach. Because atropine totally lacks antiepileptogenic effects on bursting neurons, it is unlikely that in the slice preparation the cholinergic function of these neurons is active. The observed modifications in the intrinsic bursting behavior due to cholinergic drugs which are described in the present paper do not contribute to the epileptogenesis in GWS in the slice preparation. However, it is possible that, in vivo, the endogenous cholinergic system contributes to enhance the epileptogenesis in the focus area and to diminish the spread of epileptic activity to other structures. In vivo, the cholinergic neurons in the focus area may be activated by external afferents resulting in complex effects (Rye et al. 1984). Moreover, Menini et al. (1992) and Araneda et al. (1994) reported an overexpression of tyrosine hydroxylase RNA messengers and immunoreactive cell bodies in the cortical focus area, suggesting that noradrenaline could also be neosynthetized in nonnoradrenergic cell bodies. Nevertheless, the muscarinic action contrasts with the action of noradrenaline which has similar effects on the PDSs and on the intrinsic bursts: in nonbursting cells of GWS rats noradrenaline provoked the appearance of intrinsic bursts and PDSs in the focus area. Whether or not the noradrenergic and cholinergic neurons appearing in the GWS focus are active to regulate GWS remains to be investigated in vivo. Thus we now can conclude that muscarinic depression of PDSs seems to be a potent mechanism that could prevent the synchronization of intralaminar activity and the spread and generalization of the epileptic focus. The functions of muscarinic and catecholaminergic neuromodulations appear therefore complementary rather than redundant in controlling the generation and propagation of epilepsy during GWS.