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The Journal of Physiology logoLink to The Journal of Physiology
. 2008 Jan 31;586(Pt 7):1867–1883. doi: 10.1113/jphysiol.2007.146159

Network hyperexcitability within the deep layers of the pilocarpine-treated rat entorhinal cortex

Philip de Guzman 1, Yuji Inaba 1, Enrica Baldelli 2, Marco de Curtis 3, Giuseppe Biagini 2, Massimo Avoli 1,4
PMCID: PMC2375734  PMID: 18238812

Abstract

In this study we report that in the presence of normal buffer, epileptiform discharges occur spontaneously (duration = 2.60 ± 0.49 s) or can be induced by electrical stimuli (duration = 2.50 ± 0.62 s) in the entorhinal cortex (EC) of brain slices obtained from pilocarpine-treated rats but not in those from age-matched, nonepileptic control (NEC) animals. These network-driven epileptiform events consist of field oscillatory sequences at frequencies greater than 200 Hz that most often initiate in the lateral EC and propagate to the medial EC with 4–63 ms delays. The NMDA receptor antagonist CPP depresses the rate of occurrence (P < 0.01) of these spontaneous epileptiform discharges but fails in blocking them. Paradoxically, stimulus-induced epileptiform responses are enhanced in duration during CPP application. However, concomitant application of NMDA and non-NMDA glutamatergic antagonists abolishes spontaneous and stimulus-induced epileptiform events. Intracellular recordings from lateral EC layer V cells indicate a lower frequency of spontaneous hyperpolarizing postsynaptic potentials in pilocarpine-treated tissue than in NEC (P < 0.002) both under control conditions and with glutamatergic receptor blockade; the reversal potential of pharmacologically isolated GABAA receptor-mediated inhibitory postsynaptic potentials has similar values in the two types of tissue. Finally, immunohistochemical analysis shows that parvalbumin-positive interneurons are selectively reduced in number in EC deep layers. Collectively, these results indicate that reduced inhibition within the pilocarpine-treated EC layer V may promote network epileptic hyperexcitability.

The entorhinal cortex (EC) is a limbic structure that is heavily involved in processing information from cortical and hippocampal regions. Activation of the EC can originate from the subicular complex whose efferent fibres terminate in layer V (Kohler, 1985; Tamamaki & Nojyo, 1995), while neocortical efferents enter the EC through layers II/III (Deacon et al. 1983; Dolorfo & Amaral, 1998b). In addition, the parallel information flow from EC layer V can reach the neocortex (Swanson & Kohler, 1986; Insausti et al. 1997), or extend to EC layers II/III; in turn, the latter route provides hippocampal re-entry to the dentate gyrus or hippocampal CA1/subiculum via the perforant and temporoammonic pathways, respectively (Ruth et al. 1988; Dolorfo & Amaral, 1998a; Baks-Te Bulte et al. 2005). Due to its information processing, the EC is anatomically and functionally subdivided into medial EC and lateral EC (Hamam et al. 2000, 2002). The medial EC receives inputs from visual associational, posterior parietal and cingulate cortices; in contrast, the lateral EC receives anatomical inputs from the piriform, insular and perirhinal cortices (Burwell & Amaral, 1998; Kerr et al. 2007). These network connections contribute to the EC physiological function and to its role in spatial memory and learning (Hafting et al. 2005). In pathophysiological conditions such as human temporal lobe epilepsy (TLE) the EC exhibits dysfunctional neurotransmission (Jamali et al. 2006), neuronal death (Du et al. 1993) and volumetric reduction (Bernasconi et al. 1999).

In vitro electrophysiological studies of the EC have demonstrated that bath application of convulsants promotes robust epileptiform activity. This experimental approach has allowed us to further understand the EC network machinery as it may exploit synaptic or intrinsic properties unique to this structure (Avoli et al. 1996; Dickson et al. 2000; de Guzman et al. 2004; Uva et al. 2005). However, these experiments focused on seizure-like events involving tissue that did not exhibit the network changes that are associated with chronic conditions such as TLE. Furthermore, pharmacological manipulations (e.g. the application of convulsant drugs) alter EC excitability, making it difficult to identify subtle functional alterations that may be present in the epileptic tissue.

Investigating chronic models of TLE can address these limitations. Thus, studies in kainic acid- or pilocarpine-treated animals have shown patterns of neuronal death similar to those observed in human TLE along with alterations in network function (Ben-Ari, 1985; Du et al. 1995; Covolan & Mello, 2000; van Vliet et al. 2004; Biagini et al. 2005; Tolner et al. 2005). Most of these investigations have addressed the superficial layers of the medial EC and have identified both enhanced network interactions and altered intrinsic neuronal properties (Kobayashi et al. 2003; Shah et al. 2004; Tolner et al. 2005; Wozny et al. 2005; Kumar & Buckmaster, 2006). In addition, layer V of the medial EC of pilocarpine-treated tissue has been reported to exhibit changes in excitatory presynaptic activity (Yang et al. 2006). These experiments indicate that network changes within the medial EC can lead to hyperexcitability thus contributing to epileptiform synchronization and limbic seizures. However, the contribution of the lateral EC to TLE development remains under-investigated. Therefore, by employing field potential and intracellular recordings along with immunohistochemistry, we assessed here the network interactions of layer V networks of the lateral EC in slices obtained from non-epileptic control (NEC) and pilocarpine-treated rats.

Methods

Preparation of pilocarpine-treated rats

Adult, male Sprague–Dawley rats (150–200 g) were subjected to intraperitoneal injections with the cholinergic agonist pilocarpine (380 mg kg−1, i.p.) (cf. de Guzman et al. 2006) according to procedures approved by the Canadian Council of Animal Care. All efforts were made to minimize the number of animals used and their suffering. To prevent discomfort induced by peripheral muscarinic receptor stimulation, rats were treated with i.p. injection of scopolamine methylnitrate (1 mg kg−1) 30 min prior to pilocarpine injection. Animal behaviour was monitored for 6 h after pilocarpine injection and scored according to the classification of Racine (1972). Pilocarpine treated rats that experienced status epilepticus (stages 3–5) for 30 min or more (duration = 46 ± 5 min, n = 52) were defined as the experimental group and studied within 4 months (17 ± 1 week; n = 52) subsequent to pilocarpine injection. Previous studies have established that adult rats experiencing pilocarpine-induced status epilepticus will develop chronic seizures (Cavalheiro et al. 1991; Priel et al. 1996). As such, pilocarpine-treated animals that were video-monitored showed spontaneous behavioural seizures (n = 26). Age-matched NEC rats – injected i.p. with saline – did not develop status epilepticus or any other form of epileptic behaviour.

Electrophysiology procedures

Brain slices from NEC and pilocarpine-treated epileptic rats were obtained according to the procedures established by the Canadian Council of Animal Care. Animals were decapitated under halothane anaesthesia, and the brain was extracted and placed in cold (1–3°C) oxygenated artificial cerebrospinal fluid (ACSF). Brain slices (450 μm thick) including the EC, subiculum and hippocampus proper were cut with a vibrating microtome along a horizontal plane of the brain that was tilted by approx. 10 deg along a posterosuperior–anteroinferior plane passing between the lateral olfactory tract and the brainstem base (Avoli et al. 1996). Slices were transferred to an interface tissue chamber and superfused with oxygenated (95% O2, 5% CO2) ACSF at 32–34°C. ACSF composition was (mm): NaCl 124, KCl 2, KH2PO4 1.25, MgSO4 2, CaCl2 2, NaHCO3 26, and glucose 10. Ifenprodil (10 μm), 3,3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonate (CPP, 10 μm), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μm) and picrotoxin (50 μm) were bath applied. Chemicals were acquired from Sigma-Aldrich Canada Ltd (Oakville, Ontario, Canada) and Tocris Cookson Inc. (Ellisville, MO, USA).

Field potential recordings were performed with ACSF-filled glass electrodes (tip diameter: < 8 μm; resistance: 2–10 MΩ) that were connected to a Cyberamp 380 amplifier (Axon Instruments, Union City, CA, USA). Lateral EC neurons were recorded intracellularly with sharp electrodes that were filled with 3 m potassium acetate (tip resistance = 90–120 mΩ) and coupled to an Axoclamp 2A amplifier (Axon Instruments) with an internal bridge circuit for intracellular current injection. Resistance compensation was monitored throughout the experiment and adjusted as required. The fundamental electrophysiological parameters of lateral EC cells were measured as follows: (i) resting membrane potential (RMP) after cell withdrawal, (ii) apparent input resistance (Ri) from the maximum voltage change in response to hyperpolarizing current pulses (< –0.5 nA), (iii) action potential amplitude (APA), and (iv) action potential duration (APD). Neuronal network activation was made via a concentric bipolar electrode (Frederick Haer and Co., Bowdoinham, ME, USA) positioned in lateral EC layer V and the minimum stimulus intensity (duration = 100 μs) eliciting a reliable response was selected. Field potential and intracellular signals were fed to a computer interface (Digidata 1322A, Axon Instruments) and were acquired and stored using the pCLAMP 8.0 software (Axon Instruments). Subsequent data analysis was performed with the Clampfit 9 software (Axon Instruments).

For each field potential trace obtained from the lateral or medial EC layer V, the onset of epileptiform activity was determined relative to the earliest deflection from the baseline recording. Consequently, time lag histograms (Fig. 3) were constructed according to the time difference between the initial deflection point in either the lateral or medial EC and the subsequent deflecting waveform in the respective EC subdivision. To preclude any biasing effect, we used three randomly selected in vitro epileptiform events. The reversal potential of stimulus-induced, pharmacologically isolated inhibitory postsynaptic potentials (IPSPs) was determined by linear regression from the plot of their amplitude versus membrane potential.

Figure 3. Spontaneous network activity propagates between both structures within the pilocarpine-treated EC.

Figure 3

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Dual field potential electrodes were positioned within layer V of the lateral EC (LEC) and medial EC (MEC) combined with a bipolar electrode stationed in LEC layer V (slice schematic). A, single shock stimulation (triangle) in NEC tissue elicits a negative deflection followed by a biphasic waveform in the LEC and MEC, respectively. Expanded inset: network activity from the LEC propagates towards the MEC following stimulation. B, in two different slices, spontaneous epileptiform activity originates from the LEC (Ba: expanded inset) or MEC (Bb: expanded inset) and subsequently propagates to the MEC or LEC, respectively. Electrical stimulation in LEC (triangle) elicits network discharge that spreads to the MEC (expanded inset: Ba and Bb). C, graphical distribution of the delay times (ms) of spontaneous activity from the LEC→MEC and MEC→LEC in slices from 9 pilocarpine-treated rats. D, a more confined time lag distribution (i.e. LEC→MEC) emerges when electrical stimuli are delivered in LEC.

Immunohistochemical procedures

Pilocarpine-treated and NEC animals were anaesthetized (chloral-hydrate 450 mg kg−1i.p.) and perfused via the ascending aorta with 100 ml saline followed by Zamboni fixative (pH 6.9). Brains were postfixed for an additional 4 h in the same fixative at 4°C and after cryoprotection by immersion in 15 and 30% sucrose–phosphate buffer solutions, they were frozen and cut horizontally from the ventral side with a freezing microtome (Biagini et al. 2005). Changes in parvalbumin-positive cells were assessed with a mouse monoclonal antibody (Swant, Bellinzona, Switzerland) used at 1: 2000 on 50 μm thick horizontal sections obtained, respectively, at levels −7.10 to 6.60 from bregma (Paxinos & Watson, 2007). Of every two sections, one was stained with toluidine blue to identify the EC anatomical subdivisions. Immunohistochemistry was performed with the avidin–biotin complex technique and diaminobenzidine as chromogen (cf. Biagini et al. 2005). Endogenous peroxidase was blocked by 0.1% phenylhydrazine in phosphate-buffered saline (PBS) for 20 min, followed by several washes in PBS preceding incubation with primary antibodies. Secondary antibodies and the avidin–peroxidase complex were purchased from Amersham Italia (Milan, Italy) and diluted 1: 200 and 1: 300, respectively. Stained sections were analysed using the image analysis software KS300 (Zeiss Kontron, Munich, Germany) (cf. de Guzman et al. 2006). Four sections for each animal were investigated and averaged for statistical analysis.

Statistical methods

Measurements in the text are expressed as means ±s.e.m. and n indicates the number of samples studied under each specific protocol. Results were compared with Student's t test, the Mann–Whitney test or the chi-square test and were considered statistically significant if P < 0.05.

Results

Epileptiform activity is a hallmark of the pilocarpine-treated EC

As illustrated in Fig. 1A, spontaneous hypersynchronous activity was not recorded from the EC in brain slices obtained from NEC animals (n = 35 slices from 24 rats). In these experiments extracellular focal stimuli delivered in the EC deep layers elicited transient monophasic or biphasic field potentials (duration = 0.14 ± 0.05 s, n = 16; Fig. 1Aa). In contrast, spontaneous field discharges occurred in 36 of 49 pilocarpine-treated EC slices obtained from 31 animals (Fig. 1B). These epileptiform discharges (duration = 2.60 ± 0.49 s; interval of occurrence = 35.2 ± 4.3 s; n = 36) consisted in the lateral EC layer V of a series of negative deflections arising from a slow negative shift coinciding with an initial positive waveform in layers II and III (Fig. 1Ba). Similar field events (duration = 2.50 ± 0.62 s, n = 49) were elicited in all pilocarpine-treated slices by electrical stimuli (Fig. 1Bb).

Figure 1. Spontaneous network discharges can be recorded in the pilocarpine-treated lateral EC.

Figure 1

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In the NEC and pilocarpine-treated tissues, three simultaneous field potential electrodes were positioned in lateral EC layers II, III and V combined with a bipolar stimulator in lateral EC layer V (see slice schematic). A, no spontaneous field potential activity occurs in the NEC tissue while single-shock stimulation produces a negative–positive deflecting transient waveform (expanded inset in Aa). B, in contrast the pilocarpine-treated lateral EC demonstrates spontaneous and stimulus-induced (triangle, Bb) network discharges that consist of multiple population spikes (expanded insets: Ba and Bb) and appear to propagate throughout layers II, III and V.

As illustrated in Fig. 2, the onset of both spontaneous and stimulus-induced epileptiform events recorded from the lateral EC of pilocarpine-treated slices consisted of transient repetitive runs of fast oscillatory activity (duration = 52.8 ± 5.7 ms, n = 22). Power spectral analysis (following a band pass filtering from 100 Hz to 1000 Hz) of the initial component of the field discharges recorded from the lateral EC layer V demonstrated fast oscillatory ripple activity exceeding 200 Hz (Fig. 2Ab and c and Bb and c). In contrast, quantification of the frequency of the late component of these epileptiform events identified the occurrence of population spikes at 20 Hz (not shown) each containing oscillatory activity at frequencies > 190 Hz (Fig. 2Ab and d and Bb and d).

Figure 2. Fast oscillatory ripple activity occurs in the pilocarpine-treated lateral EC layer V.

Figure 2

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A and B, spontaneous and stimulus-induced (triangle) network bursting consists of a negative deflecting waveform on which multiple population spikes occur. Band pass filtering (100–1000 Hz: Ab and Bb) reveals a transient discharge of fast oscillatory activity (expanded insets). Power spectral analyses of spontaneous (Ac) and stimulus induced (Bc) transient discharges demonstrate network oscillations greater than 200 Hz, while the individual spontaneous (Ad) and stimulus induced (Bd) network bursts within the late phase consisted of frequencies at approximately 190 Hz.

Network interactions within the pilocarpine-treated EC

Next, we assessed the initiation and propagation of epileptiform discharges in the deep layers of both lateral and medial EC subdivisions. The slice schematic in the top part of Fig. 3 illustrates the position of the field potential recording electrodes and of the stimulating electrode that was located in the lateral EC. Electrical stimuli delivered in NEC slices – which did not generate any spontaneous field activity – elicited a monophasic negative deflection in the lateral EC that was followed by a biphasic positive–negative waveform in the medial EC (n = 6; Fig. 3A). In contrast, slices from pilocarpine-treated rats generated spontaneous epileptiform events originating from the lateral (n = 6, Fig. 3Ba) or medial EC (n = 3, Fig. 3Bb) and subsequently propagated to the medial and lateral EC, respectively; a trend (P = 0.08, chi-square test) toward a more frequent occurrence of the spontaneous epileptiform events was observed in the lateral EC. Propagation delays between the two areas produced a variable travel time between 4 and 63 ms (n = 9). In the distribution histogram shown in Fig. 3C, we arbitrarily defined territorial lag times from the lateral EC towards the medial EC as negative whereas the reverse direction (medial EC→lateral EC) was classified as positive. This variability in bidirectional propagation could be restricted to a unidirectional movement from lateral to medial EC along with a more confined time lag distribution during stimulation of lateral EC layer V (4–24 ms; n = 9) (Fig. 3D).

Firing properties of layer V lateral EC neurons in both NEC and pilocarpine-treated tissue

We further investigated whether the firing patterns and the intrinsic properties of lateral EC layer V neurons were altered in the epileptic tissue compared to NEC. Intracellular injection of depolarizing current pulses (duration = 1 s) induced two patterns of firing in lateral EC neurons analysed in both types of tissue. The first consisted of regular, repetitive firing only (Fig. 4Aa and Ba), while the second was characterized by an initial burst of action potentials followed by regular firing (Fig. 4Ab and Bb). Quantification of these two firing patterns in NEC (regular firing, n = 25; intrinsic bursting, n = 6) and pilocarpine-treated slices (regular firing, n = 47; intrinsic bursting, n = 8) demonstrated in both cases a higher incidence of regular firing neurons compared to intrinsic bursters. In addition, lateral EC neurons recorded in the two types of tissue displayed similar fundamental intrinsic properties (Table 1).

Figure 4. Intrinsic firing patterns of lateral EC layer V neurons in NEC and pilocarpine-treated tissue.

Figure 4

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Two types of firing patterns are recorded from layer V lateral EC neurons in NEC (A) and pilocarpine-treated (B) slices during injection of depolarizing current: (i) regular repetitive action potential firing (Aa and Ba) or (ii) initial bursts (expanded insets) followed by regular action potential spiking (Ab and Bb).

Table 1.

Intrinsic neuronal properties of NEC and pilocarpine-treated lateral EC layer V neurons

Firing Pattern RMP (mv) Ri (MΩ) APA (mV) APD (ms)
NEC lateral entorhinal cortex
 Regular firing (n = 25) −70.1 ± 1.0 46.1 ± 2.5 90.2 ± 1.1 1.3 ± 0.1
 Intrinsic bursting (n = 6) −66.7 ± 2.9 46.7 ± 7.5 82.6 ± 2.7 1.3 ± 0.1
Pilocarpine-treated lateral entorhinal cortex
 Regular firing (n = 47) −69.2 ± 0.8 49.3 ± 2.2 87.1 ± 1.1 1.2 ± 0.1
 Intrinsic bursting (n = 8) −69.8 ± 1.9 49.9 ± 4.4 96.6 ± 1.9 1.1 ± 0.1
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These properties included resting membrane potential (RMP), input resistance (Ri), action potential amplitude (APA) and action potential duration (APD).

Intracellular characteristics of spontaneous and stimulus-induced events in NEC and pilocarpine-treated lateral EC neurons

Single-shock stimulation produced an initial depolarizing response that was followed by biphasic hyperpolarizing components in lateral EC layer V neurons recorded at RMP in NEC slices (Fig. 5A, −67 mV trace). Cell membrane hyperpolarization to values more negative than −80 mV with injection of steady negative current increased the amplitude of the stimulus-induced depolarizing component and markedly reduced the subsequent hyperpolarizations (Fig. 5A, −80 mV trace). In contrast, depolarization of these cells disclosed a robust hyperpolarizing response (Fig. 5A, −51 mV trace) that was at times preceded by a single action potential (not shown). Hence, all layer V neurons (n = 26) recorded from the NEC lateral EC generated sequences of depolarizing and hyperpolarizing postsynaptic sequences in response to single-shock electrical stimuli.

Figure 5. Single-shock stimulation and spontaneous network activity are synaptically driven in pilocarpine-treated lateral EC layer V neurons.

Figure 5

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A, single-shock stimulation (triangle) elicits a depolarizing hyperpolarizing postsynaptic response at −67 mV (RMP) in a NEC lateral EC layer V neuron. Electrical stimuli at hyperpolarized (−80 mV) and depolarized (−51 mV) membrane potentials elicit depolarizing and hyperpolarizing PSPs, respectively. B, stimulus-induced (triangle) and spontaneous epileptiform activity recorded at RMP (−69 mV and −71 mV) in pilocarpine-treated tissue is characterized by action potential bursting. The amplitude of the depolarizing envelope increases at −82 mV, whereas a reduction occurs at −50 mV and −52 mV. The selected RMPs in NEC and pilocarpine-treated tissue were used as comparisons to demonstrate the absence of stimulus induced PSPs in the pilocarpine-treated EC.

In contrast, stimulus-induced (n = 56/75 neurons from 72 slices) and spontaneous (n = 50/75 neurons from 72 slices) action potential bursting was recorded intracellularly from layer V neurons of the lateral EC in pilocarpine-treated slices. At RMP both stimulus-induced and spontaneous discharges were characterized by a depolarizing envelope that was overridden by action potential bursting (Fig. 5B, −69 mV and −71 mV traces). Membrane hyperpolarization to values more negative than RMP increased the amplitude of the stimulus-induced and spontaneous depolarizing envelopes (Fig. 5B, −82 mV and −82 mV traces, respectively). Conversely, steady depolarization of these neurons to membrane values around −50 mV reduced the amplitude of these depolarizations (Fig. 5B, −50 mV and −52 mV traces, respectively). Within the range of membrane potentials tested in these experiments (i.e. from −85 to −50 mV), stimulus-induced and spontaneous depolarizations triggered similar amounts of action potential discharge. In addition, as expected from a network-driven synaptic event, changing the neuron membrane potential did not modify the rate of occurrence of the spontaneous epileptiform depolarizations.

Epileptiform activity in pilocarpine-treated EC persists during NMDA receptor antagonism but is abolished by a non-NMDA glutamatergic antagonist

We further characterized the dependence of spontaneous network-driven epileptiform activity (n = 5) on ionotropic glutamatergic mechanisms (Fig. 6A). Bath application of the NR2B receptor antagonist ifenprodil (10 μm, n = 5) did not modify the duration or the rate of occurrence of these spontaneous events. Synchronous discharges also persisted following application of CPP (10 μm, n = 5) although this NMDA receptor antagonist increased significantly (P < 0.01) their interval of occurrence (Fig. 6A). Subsequent application of CNQX blocked spontaneous network bursting (Fig. 6A; n = 5). The effects of these ionotropic glutamatergic antagonists on the duration and rate of occurrence of spontaneous epileptiform events are summarized in Fig. 6B and C.

Figure 6. Spontaneous epileptiform activity and NMDA receptor blockade in pilocarpine-treated slices.

Figure 6

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A, simultaneous intracellular and field potential recordings demonstrate spontaneous network bursting within the pilocarpine-treated lateral EC layer V. Note that spontaneous bursting persists in the presence of ifenprodil (10 μm) and CPP (10 μm), but is blocked subsequent to CNQX (10 μm) application; under this pharmacological procedure, single-shock stimulation induces a postsynaptic response. B and C, quantitative summary of the effects induced by the glutamatergic receptor antagonists on the duration and interval of occurrence of the spontaneous network discharges; note that these parameters are not significantly altered by ifenprodil while CPP significantly increases the interval of occurrence (P < 0.01).

In these experiments we also tested the effects of NMDA and non-NMDA glutamatergic antagonism on the paroxysmal depolarizations elicited by single-shock stimuli delivered in lateral EC layer V of pilocarpine-treated slices. Bath application of ifenprodil did not influence these stimulus-induced epileptiform discharges (n = 5), while subsequent application of CPP (n = 6) caused an enhanced network response characterized by a single action potential followed by sustained discharge (Fig. 7A). This apparent augmented response was associated with a significant increase in the latency of the epileptiform event following the stimulus. Finally, subsequent addition of CNQX (10 μm, n = 6) abolished the stimulus-induced epileptiform discharges and could reveal a monosynaptic postsynaptic potential (PSP) (not shown). The effects induced by these ionotropic glutamatergic antagonists on the duration and latency of the stimulus-induced epileptiform responses are quantified in the plots shown in Fig. 7B and C.

Figure 7. NMDA receptor blockade paradoxically enhances the duration of single-shock-induced epileptiform discharges.

Figure 7

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A, single-shock stimulation (triangle, duration = 100 μs) in lateral EC layer V in the pilocarpine elicits a network epileptiform response as indicated by simultaneous intracellular and field potential recordings. Bath application of ifenprodil does not change this epileptiform response while CPP (10 μm) enhances its duration and increases its latency. Note also that the stimulus-induced epileptiform response is blocked following application of CNQX (10 μm). B and C, quantitative summary of the duration and latency of the stimulus-induced epileptiform discharges measured from field potential recordings. Note that a prolongation and an increased latency of these responses (P < 0.00001 in both cases) occur following NMDA receptor blockade with CPP.

Reduced GABAergic inhibition in the pilocarpine-treated EC

Spontaneous hyperpolarizing postsynaptic potentials (PSPs) were recorded from NEC EC layer V neurons analysed at RMP under control conditions (Fig. 8A, −73 mV trace). These events occurred at intervals ranging from 7.4 to 34.9 s (12.5 ± 1.1 s, n = 7 neurons); this value differed from what was observed in pilocarpine-treated layer V lateral EC (Fig. 8A, −68 mV trace) as similar PSPs occurred at significantly longer intervals in these cells (Figs 8B and 28.5 ± 4.9 s, n = 12, P < 0.002 independent t test).

Figure 8. Reduced frequency of IPSP activity in layer V of the pilocarpine-treated lateral EC.

Figure 8

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A, intracellular recordings at RMP from lateral EC layer V neurons in NEC and pilocarpine-treated tissue demonstrate spontaneous hyperpolarizing PSPs under control conditions. B, graphic representation of the intervals of occurrence of hyperpolarizing PSPs recorded under control conditions from lateral EC layer V neurons in NEC and pilocarpine-treated tissue; note that the interval of occurrence is significantly longer in pilocarpine-treated neurons than in NEC (P < 0.002). C, intracellular recordings at RMP from layer V lateral EC neurons in NEC and pilocarpine-treated tissue reveal spontaneous hyperpolarizing PSPs during blockade of NMDA (CPP 10 μm) and AMPA/kainate receptors (CNQX 10 μm). D and E, quantitative summary of the interval of occurrence and amplitude of spontaneous hyperpolarizing PSPs recorded from NEC and pilocarpine-treated neurons under glutamatergic receptor blockade; note that significant differences (P < 0.00001 and P < 0.0002, respectively) occur for both parameters. Data shown in B, D and E were calculated using 20–30 min long epochs. F, stimulus-induced PSP recorded in the presence of CPP (10 μm) + CNQX (10 μm) at different membrane potentials from NEC and pilocarpine-treated EC neurons. G, graphical display of the stimulus-induced PSP reversal points in NEC and pilocarpine-treated EC cells; note that these values are not significantly different (P > 0.05).

Spontaneous hyperpolarizing PSPs, recorded at RMP during concomitant application of CPP (10 μm) and CNQX (10 μm), were also more frequent in NEC (interval of occurrence = 14.4 ± 1.6 s, n = 6) as compared to pilocarpine-treated neurons (interval of occurrence = 73.3 ± 18.9 s, n = 7; P < 0.00001) (Fig. 8C and D). As shown in Fig. 8C and E, the amplitude of these PSPs was smaller in pilocarpine-treated cells (2.15 ± 0.17 mV, n = 6) than in NEC (5.14 ± 1.14 mV, n = 6; P < 0.0002). Finally, during blockade of glutamatergic receptors, NEC and pilocarpine-treated EC neurons responded to local electrical stimuli with intracellular potentials that were characterized by similar reversal values (Fig. 8F and G; −75.4 ± 1.1 mV, n = 6 for NEC and −73.2 ± 1.6 mV, n = 6, for pilocarpine-treated EC cells; P > 0.05, independent t test). Subsequent bath application of the GABAA receptor antagonist picrotoxin (50 μm, n = 6) blocked both spontaneous and stimulus-induced IPSP activity in both types of tissue (not illustrated).

Reduced parvalbumin-positive interneurons in the pilocarpine-treated EC

Finally we investigated whether the reduced occurrence of spontaneous hyperpolarizing PSPs corresponded to changes in parvalbumin-positive interneurons. These cells were well-stained throughout all EC layers (Fig. 9A) but were mainly localized in superficial layers (Wouterlood et al. 1995) of NEC rats (n = 5). No changes were apparent in the superficial EC layers (cf. de Guzman et al. 2006) of pilocarpine-treated rats (n = 8, Fig. 9B) while immunoreactivity was faint in the deep regions (Fig. 9C and D). Accordingly, counts of parvalbumin-positive cells revealed a decrease (approx. −40%) in neuronal cell densities in both V and VI EC layers, but this change was significant (P < 0.01, Mann–Whitney test) in layer V only (Fig. 9E). Similar parvalbumin cell counts were identified in layers II–IV of NEC and pilocarpine-treated EC (Fig. 9E).

Figure 9. Reduced parvalbumin-positive cells in layer V of the pilocarpine-treated lateral EC.

Figure 9

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A, parvalbumin-positive interneurons are identified throughout the EC of a NEC rat by means of a monoclonal antiparvalbumin antibody. Note that the staining is prevalent in superficial layers (I–IV), whose boundaries are identified by asterisks. The boundary with the perirhinal cortex (Prh) is also indicated. In B, the parvalbumin staining is well preserved in the superficial layers of pilocarpine-treated rats, while it is faint in deep layers. Magnification of deep layers in NEC (C) and pilocarpine-treated (D) EC reveals a difference in the number of positively stained cell bodies; note that the arrows identify easily recognizable interneurons stained, respectively, in A and B. Quantification of parvalbumin cell densities in E demonstrates a decrease in interneurons of deep layers in pilocarpine-treated rats. **P < 0.01, Mann–Whitney test. Scale bars, 200 μm.

Discussion

We have found that the EC in slices obtained from pilocarpine-treated animals can generate spontaneous epileptiform events that do not occur in NEC tissue. These discharges initiated preferentially within the lateral EC, and entrained the medial EC. We have also demonstrated that this type of epileptiform activity is not NMDA receptor-dependent, as spontaneous and stimulus-induced synchronous bursting persisted following NMDA receptor antagonism. Finally, we have established that spontaneous, presumably GABAA receptor-mediated, PSPs in epileptic lateral EC neurons were reduced in amplitude and in frequency, thus suggesting that network hyperexcitability in the pilocarpine-treated lateral EC may result from impaired inhibition possibly due to loss of interneurons in deep layers.

Network hyperexcitability within the pilocarpine-treated EC

Experiments performed in kindled rats have provided evidence for EC hyperexcitability, although no spontaneous network activity was reported (Fountain et al. 1998). Employing simultaneous field potential recordings, we have found that spontaneous network discharges in the absence of acute application of epileptogenic agents occur within the lateral and medial portions of the EC in pilocarpine-treated animals. In addition, we could identify a trend toward a more frequent initiation of these epileptiform events in the lateral EC along with a rather wide range of propagation delays between the two areas suggesting the presence of variable sites of initiation within the EC. Convulsant treatments in control slices have demonstrated that synchronous epileptiform discharges are generated in the medial EC as well as that this epileptiform activity originates in the deep layers (Jones & Heinemann, 1988; Dickson & Alonso, 1997; Lopantsev & Avoli, 1998a,b).

The spontaneous network discharges recorded from EC slices of pilocarpine-treated animals exhibit bidirectional routes of propagation between the medial and lateral component of this limbic structure. Similar characteristics have been reported for the epileptiform activities recorded in the EC of control tissue slices in the presence of convulsants (Klueva et al. 2003; de Guzman et al. 2004; Uva et al. 2005). These studies collectively demonstrate that enhanced excitation can promote network reverberation within the EC as reported in in vitro whole brain studies (Biella et al. 2002a,b; Gnatkovsky & de Curtis, 2006). It should also be emphasized that the paroxysmal network synchronization identified in pilocarpine-treated lateral EC is supported by the occurrence of brief bursts of transient, high frequency oscillations in layer V at the onset of spontaneous and stimulus-induced epileptiform events. High frequency oscillations exceeding 200 Hz, deemed pathological, may serve as a surrogate marker of epileptogenicity (Bragin et al. 2004) as suggested by intracranial recordings performed in the EC of TLE patients (Bragin et al. 2002b; Jirsch et al. 2006) and by in vivo recordings in the temporal cortex of kainic acid-treated rodents (Bragin et al. 2002a; Tolner et al. 2005). The hyperexcitabilty of pilocarpine-treated layer V lateral EC cells also emerged following electrical stimulation. Single-shock stimuli in NEC slices elicited PSPs consisting of an initial depolarizing response (presumably an EPSP that could eventually trigger a single action potential) followed by biphasic hyperpolarizing components (Williams et al. 1993; Behr et al. 1998). In contrast, EC neurons recorded in slices from pilocarpine-treated animals responded to similar electrical stimuli by generating paroxysmal epileptiform activity. A decreased threshold of network responses to stimuli has been reported to occur in EC superficial layers (Kobayashi et al. 2003; Kumar & Buckmaster, 2006) and subiculum (Cohen et al. 2002; de Guzman et al. 2006) of epileptic tissue.

We also addressed whether network hyperexcitability within the pilocarpine-treated EC could be attributed to changes in intrinsic neuronal properties. RMP, Ri, and APA were not different in layer V neurons recorded in NEC and pilocarpine-treated animals. In both conditions, an overwhelming number of regular firing cells was observed compared to intrinsic bursters, which is in line with studies performed in layer V of the lateral (Hamam et al. 2002; Rosenkranz & Johnston, 2006) and medial (Hamam et al. 2000) EC in control tissue. As such, we are inclined to conclude that factors involving recurrent excitatory connectivity and attenuated inhibition contribute to the hyperexcitablity of the deep layer EC (Dhillon & Jones, 2000; Woodhall et al. 2005). However, the absence of different intrinsic properties that emerges from our sharp-electrode intracellular analysis should be taken with caution as changes in voltage-gated currents have been recently identified in neurons recorded from epileptic animals with whole-cell, patch-clamp techniques (Shah et al. 2004).

Reduced network inhibition in the pilocarpine-treated EC

Previous studies in control tissue have shown a significantly reduced network inhibition in EC layer V compared to layer II (Woodhall et al. 2005). In our pilocarpine-treated tissue, lateral EC layer V neurons revealed a lower frequency of spontaneous postsynaptic events. These results, which suggest decreased network inhibition within the pilocarpine-treated lateral EC layer V, were further reinforced by data obtained in the presence of glutamatergic antagonists. We have found that lateral EC layer V neurons in pilocarpine-treated slices produced a lower frequency of IPSP activity along with significantly reduced amplitudes than NEC cells. Thus, these findings indicate a diminished GABAergic inhibition of layer V pyramidal cells when compared to the NEC. In parallel with these results, EC layer II is subject to reduced network inhibition in the pilocarpine-treated EC (Kobayashi et al. 2003). However, the attenuated inhibition identified in EC layer V neurons was not associated with a change in GABAA receptor-mediated IPSP reversal potential, unlike previous studies performed in the subiculum of human epileptic patients (Cohen et al. 2002) and pilocarpine-treated animals (de Guzman et al. 2006).

A recent study of the epileptic EC layer V has indicated that functional alterations in presynaptic GABAB receptors may promote increased and reduced frequencies of excitatory postsynaptic current and inhibitory postsynaptic current, respectively (Thompson et al. 2007). In addition, we have described here a 40% reduction in parvalbumin inhibitory interneurons in EC deep layers that could potentially explain the attenuated inhibition of principal cells in pilocarpine-treated rats. Parvalbumin-positive cells in the lateral EC represent about 50% of GABAergic interneurons (Miettinen et al. 1996). This finding is at variance with the preservation of parvalbumin interneurons in superficial layers, as also described in our previous experiments (de Guzman et al. 2006). Overall, our data suggest that disinhibition within the EC could result from reduced inhibitory input rather than alterations in Cl− extrusion mechanisms. Attenuated network inhibition resulting from a reduced excitatory drive on inhibitory interneurons has been reported in the hippocampus and EC layers II/III (Bekenstein & Lothman, 1993; Williams et al. 1993; Sloviter et al. 2003; Kumar & Buckmaster, 2006).

Glutamatergic mechanisms in the pilocarpine-treated EC

The presence of spontaneous epileptiform activity in pilocarpine-treated EC involves reduced network inhibition leading to a presumed over-expression of glutamatergic mechanisms. We have, however, found that EC network hyperexcitability is not dependent upon NMDA receptors. Previous investigations in control tissue have revealed that NMDA receptor blockade abolishes ictal-like activity (Avoli et al. 1996; de Guzman et al. 2004). In contrast, both spontaneous and stimulus-induced epileptiform discharges in pilocarpine-treated EC appeared to be resistant to NMDA receptor antagonism.

A recent investigation of EC layer V neurons in pilocarpine-treated slices has revealed an alteration in NR2B receptor function thus suggesting a developmental regression of this NMDA receptor subtype (Yang et al. 2006). In contrast, we have found in our experiments that spontaneous or stimulus-induced epileptiform discharges were not influenced by NR2B receptor antagonism. Since the presence of the NR2B receptor is reported to be age dependent, these different results may reflect the younger age of the animals used in the study of Yang et al. (2006). However, we have also found that spontaneous epileptiform discharges in pilocarpine-treated EC persisted in the presence of full NMDA receptor blockade even though it occurred at longer intervals. Interestingly, single-shock stimulation during CPP application produced a delayed response consisting of a prolonged bursting sequence, thereby suggesting an apparent augmentation of network discharge. The delayed occurrence, combined with the gradual rising and sustained action potential firing, following single-shock stimuli during NMDA receptor blockade may be caused by reduced NMDA-dependent depolarization and synchronization. Thus, under these experimental conditions, blockade of NMDA receptors could delay the development of a bursting sequence that is sustained by AMPA/kainate receptors coupled with the subsequent recruitment of cholinergic synapses to promote network reverberation (Cobb & Davies, 2005).

Kindling studies in the dentate gyrus indicate that NMDA receptor blockade does not preclude seizure development (Brandt et al. 2003) nor contribute to the maintenance of seizure activity (Sayin et al. 1999). Therefore, factors such as reduced inhibitory inputs (Kumar & Buckmaster, 2006) as well as increased synaptic sprouting (de Guzman et al. 2006) leading to over-function of AMPA/kainate receptor-mediated mechanisms should be taken into account for the hyperexcitability demonstrated in the pilocarpine-treated rats. In keeping with this view, spontaneous and stimulus-induced epileptiform discharges were abolished upon AMPA/kainate receptor antagonism.

Conclusions

Our study indicates that the pilocarpine-treated EC is hyperexcitable. The epileptic EC has been demonstrated to interact with the subiculum and CA1; this limbic circuit has been suggested to enhance network reverberation and to increase excitation under epileptogenic conditions (D'Antuono et al. 2002; Biagini et al. 2005; Wozny et al. 2005). As such, the pilocarpine-treated EC exhibits hyperexcitable network properties that contribute to the generation and propagation of seizure activity across limbic structures. Most investigations have focused on the medial EC while the lateral EC has received scarce attention. Nonetheless, in chronic models of seizure activity, the lateral EC layer III enhances subicular activity (de Guzman et al. 2006), displays altered intrinsic neuronal properties (Shah et al. 2004) and expresses high levels of FosB-related antigens (Biagini et al. 2005). Furthermore, lateral EC ablation attenuates limbic seizures thus underscoring the epileptic role of this area (Kopniczky et al. 2005). Overall, our findings suggest that further investigation of the lateral EC is required as this area may represent an important therapeutic target for controlling seizures in TLE.

Acknowledgments

This study was supported by grants from the Canadian Institutes of Health Research (CIHR; grant 8109), the Savoy Foundation and the Pierfranco and Maria Luisa Mariani Foundation (R-06–50). Enrica Baldelli was recipient of a Mariani Foundation–University of Modena and Reggio Emilia Fellowship (Borsa di Studio per la Ricerca e Formazione Avanzata, 2006/07). We thank Dr Daniela Longo for participating in the parvalbumin experiments.

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