Abstract

Seizure patterns in temporal lobe epilepsies have been described both in humans and in animal models. The involvement of specific hippocampal–parahippocampal subregions in the initiation and progression of temporal lobe seizures is not defined yet. We analyzed limbic network dynamics during seizures induced by 3-min arterial perfusion of 50 µM bicuculline in the in vitro isolated guinea pig brain preparation. As for human and animal temporal lobe epilepsies, 2 seizure types characterized at onset by either fast activity (FA) or hypersynchronous activity (HSA) were observed in our acute model. Simultaneous extracellular recordings were performed from ventral hippocampal–parahippocampal subregions with multichannel electrodes, and laminar analysis and propagation directions were computed to define reciprocal interactions during seizures. FA seizures started with fast oscillations generated in CA1-subiculum and entorhinal cortex, followed by irregular spikes and progressively regular bursts well defined in all subfields, with the exception of pre- and parasubiculum that do not participate in seizure activity. Dentate gyrus was not involved at FA seizure onset and became prominent during the transition to bursting in both FA and HSA patterns. HSA seizures were similar to FA events, but lacked initial FA. During seizures, reliable and steady propagation within the intra-hippocampal re-entrant loop was observed.

Introduction

The anatomical circuits that connect the hippocampus and the parahippocampal region (PHR) form parallel and local networks that can be altered in pathological conditions, thus facilitating synchronization and generation of epileptiform discharges. Seizures recorded from the PHR–hippocampal region of patients suffering from temporal lobe epilepsy (TLE) and in animal TLE models organize in peculiar discharge patterns (Lothman et al. 1991; Marks et al. 1995; Bragin, Engel, Wilson, Fried et al. 1999; Bragin, Engel, Wilson, Vizentin et al. 1999; Velasco et al. 2000; Bartolomei et al. 2001; Avoli et al. 2002; Wendling et al. 2003; Ogren et al. 2009). Experimental studies suggest that subfields of the hippocampal–parahippocampal area contribute differently to ictogenesis. Dentate gyrus (DG) works as a gate that controls propagations of synchronized activity from the entorhinal cortex (EC; Heinemann et al. 1992; Lothman et al. 1992; Sloviter 1994; Coulter and Carlson 2007); this gate can be disabled in condition of acute (Paré et al. 1992) or chronic hyperexcitability (Behr and Heinemann 1996; Cohen et al. 2003; Bonislawski et al. 2007). Enhanced synaptic excitation was shown in area CA1 in different models of epileptiform activity (Cossart et al. 2001; Denslow et al. 2001; Wu and Leung 2003; Wozny, Gabriel et al. 2005). In combined hippocampal–EC rat slices, CA3-driven interictal spikes were found to propagate to CA1, EC, and DG after acute proconvulsant applications (Walther et al. 1986; Avoli et al. 1996; Barbarosie and Avoli 1997). In the 4-aminopyridine model, ictal discharges prevailed in EC only after disconnection from the hippocampus and the duration of such seizure-like events depended on the integrity of subicular–EC connections (Barbarosie and Avoli 1997; Barbarosie et al. 2000; Panuccio et al. 2010). Behr and Heinemann (1996) showed for the first time spontaneous epileptiform activity in subicular rat slices; other studies later confirmed the contribution of this structure to acute and chronic epileptic conditions (Wellmer et al. 2002; Benini and Avoli 2005; Knopp et al. 2005). The capability of the subiculum to generate epileptiform discharges was confirmed in post-surgical tissue from operated TLE patients (Cohen et al. 2002; Wozny, Knopp et al. 2005; Huberfeld et al. 2011), suggesting a role for this structure in ictogenesis. The involvement of para- and presubiculum to ictogenesis has not been investigated yet.

We previously demonstrated in the guinea pig brain maintained in vitro the early involvement of CA1 and EC in the generation of epileptiform discharges induced by arterial administration of bicuculline (Uva et al. 2005), pilocarpine (Uva et al. 2008), or 4-aminopyridine (Carriero et al. 2010). Here we further study the involvement of hippocampal region–PHR structures, such as subiculum, parasubiculum, and presubiculum, in seizure discharges induced by transient and incomplete dis-inhibition by brief arterial perfusion of the GABAA receptor antagonist, bicuculline. Moreover, we will verify if the 2 distinct ictal patterns (low-voltage fast and hypersynchronous ictal onset patterns) described in TLE patients and rats are observed also in our experimental model. The findings of this study have been previously reported in abstract form (Boido et al. 2011).

Materials and Methods

Hartley guinea pig brains (150–200 g, Charles River, Italy) were isolated and maintained in vitro according to the standard procedure described elsewhere (de Curtis et al. 1991, 1998; Muhlethaler et al. 1993). Briefly, animals anesthetized with sodium thiopental (125 mg/kg i.p., Farmotal, Pharmacia, Italy) were trans-cardially perfused with a cold (10 °C), carboxygenated (95% O2, 5% CO2) solution containing: 126 mM NaCl, 3 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 2.4 mM CaCl2, 26 mM NaHCO3, 15 mM glucose, 2.1 mM HEPES, and 3% dextran M.W. 70 000, at pH 7.1. After decapitation, brains were isolated and transferred into a recording chamber (de Curtis et al., 1998). A cannula was inserted in the basilar artery to restore brain perfusion with the solution mentioned above (7 mL/min, pH 7.3, 15 °C) via a peristaltic pump (Minipulse 3, Gilson, France). Before starting the electrophysiological experiment, the temperature of both the perfusate and the chamber was slowly raised (steps of 0.2 °C/min) to 32 °C using a temperature controller (PTC 10, NPI, Germany).

Experiments were performed on 23 isolated guinea pig brains. The number of animals utilized in the present study was minimized according to the international guidelines on ethical use of animals (European Communities Council Directive of 24 November 1986 – 86/109/EEC). The experimental protocol was reviewed and approved by Committee on Animal Care and Use and by the Ethics Committee of the Fondazione Istituto Neurologico.

To test the viability of the preparation, at the beginning of the experiments we recorded in DG, CA1, subiculum, pre- and parasubiculum, and medial EC (mEC) of the ventral hippocampus region–PHR the activity evoked by stimulation of the lateral olfactory tract (LOT) through an isolation unit driven by a pulse generator (Telefactor S88, Grass, RI, USA; Biella and de Curtis 2000). The stimulating electrode was a twisted Teflon-insulated silver wire. Field potentials signals were amplified with a multichannel differential amplifier (Biomedical Engineering, NY, USA).

Recording electrodes (0.9 M NaCl-filled glass pipettes or tungsten wires; 5–10 MΩ resistance) were positioned under visual control with a stereoscopic microscope; single-shaft 16-channel silicon probes (50 or 100 µm inter-site spacing; Neuronexus, Ann Arbor, MI, USA) were placed in different structures at variable depths, depending on the subregion to be inspected. Electrode insertion was guided by the LOT-stimulation-evoked responses and was later anatomically verified. To confirm electrode positions, at the end of the experiments electrolytic lesions were performed by passing a constant DC current (150 µA for 10 s) through tungsten probes and reference. Silicon probe tracks and lesions were reconstructed on histological sections.

Electrophysiological data were digitized via AT-MIO-64E3 National A/D Board (National Instruments, Italy), stored, and analyzed with a custom-made software (ELPHO©) developed in Labview by Dr. Vadym Gnatkovsky.

One-dimensional current source density (CSD) analysis (Mitzdorf 1985; de Curtis et al. 1994; Biella and de Curtis 1995) was performed on 16-channel laminar profiles as previously described by using a custom-made Matlab script (The MathWorks Inc., Natick, MA, USA). Pre-processing consisted in 1 s-window offset removal. The CSD trace corresponding to each contact of the 16-channel probe was computed considering 2 contacts above and 2 contacts below along the probe shaft.

Brains were fixed with 4% paraformaldehyde (pH 7.2), subsequently sliced by vibratome (Leica VT1000S Leica Microsystems GmbH, Wetzlar, Germany) in 100 µm coronal sections mounted with 20% chromalin solution and stained with thionine.

Epileptiform activities were induced by 3-min arterial applications of the GABAA receptor antagonist bicuculline methiodide (BMI; Sigma Aldrich, Italy; 50 µM).

In all the experiments, standard electrode configuration consisted of tungsten electrodes or glass pipettes and two 16-channel silicon probes. Across different experiments, all the regions mentioned in Results were investigated with the multichannel probes.

Propagation patterns were analyzed by considering activity during early and late bursting phases (b-early and b-late) and by measuring time intervals between couples of consecutive spikes recorded in different regions. Propagation times <5 ms were not considered in the analysis (considering 7–10 ms as the mean value for a monosynaptic propagation). For each propagation direction, the average of propagation intervals was calculated separately for the 2 considered phases and further averaged over 14 seizures. Significant differences among phases were evaluated by Student's t-test; a confidence level of 95% was considered sufficient to reject the null hypothesis; whenever verified, higher levels of confidence were also reported. We collected all measured time intervals for each propagation direction to construct distribution histogram for non-parametric analysis (Kolmogorov–Smirnov test, 95% confidence level) and to verify possible multi-modal distributions.

The analysis of the modulation of propagations inside each burst was done by means of ad hoc algorithm which provides a parameter M as shown below 

formula
where pi is the preceding and pj the subsequent time propagation (ms), the difference of consecutive time propagation of spikes between 2 structures inside a burst was computed and averaged. Positive M values mean that the propagation times globally increased, while a negative M value indicates a progressive increase in the propagation velocity inside a burst. Note that averaging compensates large differences whenever their sign is opposite, being in that way insensible to large variations without any clear trend toward increase or decrease in propagation interval. Then the ratio between averaged propagation differences and averaged time interval propagation between 2 structures in a burst was computed, to avoid influence by the duration of the specific propagation direction. Finally, M values belonging to each burst of every seizure were averaged, dividing them on the basis of the phase (b-early and b-late; see Supplementary Fig. 1).

Results

Two Distinct Seizure-Like Patterns

Arterial perfusion of BMI (50 µM; 3 min) in the isolated guinea pig brain maintained in vitro induced seizure-like activities (hereafter termed seizures) that could be recorded in the ventral hippocampal region–PHR (n = 72; Fig. 1). As previously reported (Gnatkovsky et al. 2008), the short BMI perfusion induced a transient and incomplete (ca. 40%) reduction in inhibition. In 39 of 72 recorded seizures, we observed an electrophysiological pattern defined as fast activity (FA) at onset (Fig. 1A) that showed quite a standard progression with different recognizable phases. The epileptiform activity started with few large (pre-ictal) spikes, followed by fast-activity (fa; highlighted in the lower insert in Fig. 1A) at 20–30 Hz, irregular spiking phase (irrs) and bursting activity (b). At the transition between irrs and b phases, short bursts occurred at irregular frequency and with a variable duration (b-early phase). The transition to more regular rhythmic bursts was characterized by a progressive decrease in frequency and increase in duration of bursts (b-late phase). Electrode positions for the illustrated seizure recordings are shown in Figure 1B,C.

Figure 1.

(A) Example of seizure-like event with FA at onset induced by arterial perfusion of BMI (50 µM; 3 min). Field potentials were simultaneously recorded in presubiculum (presub), subiculum (sub), CA1, dentate gyrus (DG) of the ventral hippocampus and in mEC. An enlargement of fa recorded in mEC is reported at the bottom of the panel. Different phases were identified and reported above the traces (see text). fa = fast activity phase; irrs = irregular spiking phase; b-early and b-late = early and late bursting phases. (B) Drawing of the arrangement of the recording electrodes in the isolated guinea pig brain is shown. (C) Histological reconstructions on coronal thionine-stained sections of the position of the electrodes utilized to record activities in A. In the upper panels, the tracks of two 16-channel silicon probes inserted in CA1 (left) and in DG (right) are shown. In the lower panels, electrolytic lesions of the tips of tungsten electrodes in the presubiculum (left) and subiculum (right) are illustrated. Calibration bar = 1 mm. (D) Ictal event with hyper-synchronous activity at onset recorded in presub, CA1, DG, and mEC. An enlargement of the ictal onset recorded in mEC is reported at the bottom of the panel. irrs = irregular spiking phase; b-early and b-late = early and late bursting phases.

Figure 1.

(A) Example of seizure-like event with FA at onset induced by arterial perfusion of BMI (50 µM; 3 min). Field potentials were simultaneously recorded in presubiculum (presub), subiculum (sub), CA1, dentate gyrus (DG) of the ventral hippocampus and in mEC. An enlargement of fa recorded in mEC is reported at the bottom of the panel. Different phases were identified and reported above the traces (see text). fa = fast activity phase; irrs = irregular spiking phase; b-early and b-late = early and late bursting phases. (B) Drawing of the arrangement of the recording electrodes in the isolated guinea pig brain is shown. (C) Histological reconstructions on coronal thionine-stained sections of the position of the electrodes utilized to record activities in A. In the upper panels, the tracks of two 16-channel silicon probes inserted in CA1 (left) and in DG (right) are shown. In the lower panels, electrolytic lesions of the tips of tungsten electrodes in the presubiculum (left) and subiculum (right) are illustrated. Calibration bar = 1 mm. (D) Ictal event with hyper-synchronous activity at onset recorded in presub, CA1, DG, and mEC. An enlargement of the ictal onset recorded in mEC is reported at the bottom of the panel. irrs = irregular spiking phase; b-early and b-late = early and late bursting phases.

As observed in chronic mesial TLE models (Bragin, Engel, Wilson, Vizentin et al. 1999), another seizure pattern was found in our in vitro recordings (n = 33), characterized by hypersynchronous activity (HSA) at onset (Fig. 1D). During the pre-ictal phase bursts were recorded; we identify as seizure onset an abrupt increase of burst frequency (b-early; highlighted in the lower insert in Fig. 1D). Neither fa nor irrs phases were observed in HSA seizures. As for FA seizures, bursts occurring later in HSA seizures progressively become larger in amplitude and slower in rate (b-late).

In 11 out of 23 experiments, BMI perfusion induced more than one seizure. A second BMI perfusion was performed in 16 out of 23 experiments after a 2-h washout of the preceding BMI application. All BMI perfusions (first and second) consistently induced seizures. The FA pattern was predominant in first seizures recorded immediately after BMI perfusion (Fig. 2A,B, n first seizures = 39), while the HSA pattern was typically observed in seizures that occurred after the first one (Fig. 2A,B, n other seizures = 33; P < 0.01 χ2 test with Yates' correction).

Figure 2.

(A) Raster plot of seizures occurrence and their duration after arterial perfusions of BMI (50 µM; 3 min). The first arterial perfusion of BMI performed in each experiment (numbered on the left) induces recurrent ictal events illustrated in the left column; the right column shows seizures induced after a second perfusion of BMI (same concentration and duration) performed in 16 out of 23 experiments no less than 120 min after the first one. FA-onset and HSA-onset seizures are illustrated by black and grey bars, respectively. Time line is shown in the upper part of the panels. (B) FA ictal pattern was predominant in the first ictal event after BMI perfusion, while HSA seizures were more frequent for subsequent ictal events (χ2 test with Yates correction, **P < 0.01). (C) Proportion of FA and HSA patterns for the first seizures after the first and the second BMI perfusions. (D) Duration of FA and HSA ictal events induced by both first and second BMI perfusions. (E) Duration of the first seizures induced by the first and second BMI perfusion.

Figure 2.

(A) Raster plot of seizures occurrence and their duration after arterial perfusions of BMI (50 µM; 3 min). The first arterial perfusion of BMI performed in each experiment (numbered on the left) induces recurrent ictal events illustrated in the left column; the right column shows seizures induced after a second perfusion of BMI (same concentration and duration) performed in 16 out of 23 experiments no less than 120 min after the first one. FA-onset and HSA-onset seizures are illustrated by black and grey bars, respectively. Time line is shown in the upper part of the panels. (B) FA ictal pattern was predominant in the first ictal event after BMI perfusion, while HSA seizures were more frequent for subsequent ictal events (χ2 test with Yates correction, **P < 0.01). (C) Proportion of FA and HSA patterns for the first seizures after the first and the second BMI perfusions. (D) Duration of FA and HSA ictal events induced by both first and second BMI perfusions. (E) Duration of the first seizures induced by the first and second BMI perfusion.

The second perfusion with BMI always induced a series of seizures, but in the present study only the first seizures were considered in the analyses and were reported in the raster plot in Figure 2.

HSA onset seizures showed a not significant shorter duration compared with FA ictal events (Fig. 2C). We found no differences in both pattern (Fig. 2D) and duration (Fig. 2E) between the first seizures elicited by the first and the second BMI perfusion (n = 23 and n = 16, respectively), suggesting that brain excitability completely recovered before the second BMI perfusions. Complete recovery of brain excitability was also verified by evaluating amplitude and shape of LOT-evoked responses (not shown). Therefore, we pooled together the results obtained by the 2 tests for the following analyses.

Involvement of Different Hippocampal Region–PHR Structures During Ictal Patterns

To evaluate the reliability of activity patterns recorded in different regions during the progression of the first seizures (induced by either the first or the second BMI application) and, whenever present, the subsequent seizures recorded after the first BMI administration, we recorded simultaneously from different regions of the temporal lobe. CSD analysis was performed on laminar profiles recorded with 16-channel silicon probes to localize activities in different structures, as illustrated in the representative experiment in Figure 3. The fa phase in FA seizures was principally generated in CA1/subiculum and mEC (see Figs 4 and 5). The subsequent irrs phase (Fig. 3B) was characterized by irregular spiking of several structures, including CA1, mEC, and subiculum. DG-located events followed by sinks in CA1/subiculum occurred during the transition to phase b-early (Fig. 3C) and during the late bursts phase (b-late), which terminates the ictal events (Fig. 3D).

Figure 3.

(A) On the left, microphotograph of the electrode tracks of the 16-channel silicon probe (50 µm spacing between recording sites) utilized to record the activity in DG and CA1, as illustrated in the other panels. The coronal section of the hippocampus is stained with thionine. The position of the recording leads is represented by dotted lines. Calibration bar: 1 mm. In the middle, superimposed laminar profile of the activity evoked by LOT stimulation (marked by arrow-heads), recorded with the silicon probe positioned in CA1 and DG. The corresponding CSD contour plots computed from DG and CA1 laminar profiles are shown on the right. Sinks and sources are represented by black and gray contour lines, respectively. CSD iso-current lines = 100 mV/mm2. (B, C and D) Field potentials (upper panels) and relative CSDs (lower panels) recorded in CA1 and DG with two 16-channel silicon probes. During early irregular spiking phase (irrs) sinks localized in CA1 were observed. During b-early phase (C) spikes tended to organize in irregular duration/occurrence bursts with sinks emerging also in the DG. In the b-late phase (D) bursts were more reliable in duration and frequency and the characteristic patterns of DG > CA1 propagation became evident.

Figure 3.

(A) On the left, microphotograph of the electrode tracks of the 16-channel silicon probe (50 µm spacing between recording sites) utilized to record the activity in DG and CA1, as illustrated in the other panels. The coronal section of the hippocampus is stained with thionine. The position of the recording leads is represented by dotted lines. Calibration bar: 1 mm. In the middle, superimposed laminar profile of the activity evoked by LOT stimulation (marked by arrow-heads), recorded with the silicon probe positioned in CA1 and DG. The corresponding CSD contour plots computed from DG and CA1 laminar profiles are shown on the right. Sinks and sources are represented by black and gray contour lines, respectively. CSD iso-current lines = 100 mV/mm2. (B, C and D) Field potentials (upper panels) and relative CSDs (lower panels) recorded in CA1 and DG with two 16-channel silicon probes. During early irregular spiking phase (irrs) sinks localized in CA1 were observed. During b-early phase (C) spikes tended to organize in irregular duration/occurrence bursts with sinks emerging also in the DG. In the b-late phase (D) bursts were more reliable in duration and frequency and the characteristic patterns of DG > CA1 propagation became evident.

Figure 4.

Evaluation of the contribution (% presence) of local activity in the inspected regions during subsequent phases of FA (A) and HSA (B) ictal events. During HSA seizures fa and irrs phases are absent. As shown in the legend in the upper part of (A), different structures are symbolized by different black-to-white shading.

Figure 4.

Evaluation of the contribution (% presence) of local activity in the inspected regions during subsequent phases of FA (A) and HSA (B) ictal events. During HSA seizures fa and irrs phases are absent. As shown in the legend in the upper part of (A), different structures are symbolized by different black-to-white shading.

Figure 5.

(A) Large-amplitude local spikes recorded in DG started several seconds after the beginning of FA ictal event at the transition into b-early phase. (B) In the middle panel, multichannel electrode recording showed spikes in DG, confirmed by the lesion produced by the electrode probe in the thionine stained coronal slice on the left (calibration bar: 0.5 mm). On the right, the sharp, large-amplitude DG spike outlined by the box is illustrated at expanded time scale and the polarity inversion of the spike is appreciable. (C) Delay of occurrence of the first large-amplitude DG spikes in FA (black column) and HSA (grey column) seizures from the ictal onset.

Figure 5.

(A) Large-amplitude local spikes recorded in DG started several seconds after the beginning of FA ictal event at the transition into b-early phase. (B) In the middle panel, multichannel electrode recording showed spikes in DG, confirmed by the lesion produced by the electrode probe in the thionine stained coronal slice on the left (calibration bar: 0.5 mm). On the right, the sharp, large-amplitude DG spike outlined by the box is illustrated at expanded time scale and the polarity inversion of the spike is appreciable. (C) Delay of occurrence of the first large-amplitude DG spikes in FA (black column) and HSA (grey column) seizures from the ictal onset.

In Figure 4, the involvement of investigated regions to each phase of FA and HSA seizures is summarized. The numbers of seizures analyzed with CSD are the following: DG = 29, CA1 = 20, presub = 17, sub = 14, mEC = 29, parasub = 5 for FA seizures; DG = 15, CA1 = 13, presub = 6, sub = 6, mEC = 15, parasub = 6 for HSA seizures (Fig. 4A,B). The majority of regions explored in our experiments actively participated to the ictal event, with the exception of the parasubiculum and the presubiculum (Fig. 4). Parasubiculum never produced epileptic activity (n = 11). As parasubiculum, presubiculum was almost inactive because CSD analysis demonstrated that events recorded in this region were not locally generated, apart from activations recorded in a rather small number of FA seizures (4 of 17; Fig. 4A). In the large majority of experiments, pre-ictal spikes were generated in CA1, mEC, subiculum, and DG (Fig. 4). DG was poorly involved in the fa phase of FA seizures (Fig. 4A,C) and was maximally recruited only between irrs and b phases, possibly boosting bursting activity of both FA and HSA ictal events (Fig. 4). Typical sharp (2–5 ms in duration) and high-amplitude spikes appeared in DG at the transition between irrs and b-early phases in almost all ictal events (95%, n = 44; Fig. 5A,B) and continued until the end of seizures. Large DG spikes were observed during the b-early phase of both FA and HSA ictal events; as shown in Figure 5C, robust DG activation started later during the FA pattern, because of the lack of fa phase in HSA seizures (n = 23 for FA, n = 15 for HSA).

Spike Propagation During the Ictal Events

To further study dynamic changes in neuronal network during seizures, we verified the reliability of the activity propagation among recorded structures during the epileptic discharges. To accomplish this task we measured the time delays between 2 consecutive locally generated spike (as verified by CSD analysis) in the inspected regions (see Materials and Methods) during b-early and b-late phases. As a rule, activity was simultaneously recorded from no less than 3 regions, using 2 silicon probes and a single electrode (see example in Fig. 6A,B). Spike propagation between recorded structures become more stereotyped and consistent during b-early and b-late phases. As illustrated in Figure 6A,B, a reliable propagation along the DG > CA1/subiculum > mEC > DG putative re-entrant hippocampal loop was observed. The time delays measured for mEC > DG propagation (11.7 ± 1 ms; n = 7) and for CA1 > mEC propagation (13.7 ± 0.8 ms; n = 6) were compatible with monosynaptic activations, in accordance with the anatomical data. Longer and possibly polysynaptic delays were measured for DG > CA1 (19.7 ± 1.3 ms; n = 10). As mentioned above, no local activity was observed in pre- and parasubiculum. No differences were found between FA- and HSA-onset seizures with respect to propagation delays.

Figure 6.

(A) Burst recorded with a 16-channel silicon probe in DG-CA1 and with a single pipette in mEC (lower trace). (B) An example of sequential propagation of spikes generated in the 3 regions. (C) Quantification of propagation intervals for the main pathways computed averaging all ictal events across experiments during the subsequent bursting phases of the seizure: DG > CA1 propagation was significantly faster during the last phase (b-late), while no difference was found for the other main pathways. (D) CV is shown for each propagation reported above. Significant decrease in the CV interestingly parallels the significance found for DG > CA1 propagation, confirming a minor spread in the time intervals measured in the b-late phase.

Figure 6.

(A) Burst recorded with a 16-channel silicon probe in DG-CA1 and with a single pipette in mEC (lower trace). (B) An example of sequential propagation of spikes generated in the 3 regions. (C) Quantification of propagation intervals for the main pathways computed averaging all ictal events across experiments during the subsequent bursting phases of the seizure: DG > CA1 propagation was significantly faster during the last phase (b-late), while no difference was found for the other main pathways. (D) CV is shown for each propagation reported above. Significant decrease in the CV interestingly parallels the significance found for DG > CA1 propagation, confirming a minor spread in the time intervals measured in the b-late phase.

An indirect confirmation of the synaptic nature of spike propagation is given by the time delay distributions for the main propagation directions. Both parametric (Fig. 6C) and non-parametric analyses of inter-structures delays (Fig. 7) indicated a significant decrease only in the time lag occurring in DG > CA1 propagation during the b-late phase with respect to b-early phase (n = 10 in b-early, n = 11 in b-late). To better analyze the presence of a real shift in the distribution of delays during the b-late phase, we computed the coefficient of variation (CV) for the experiments considered in the parametric test. A significant decrease in the DG > CA1 CV correlated with the decrease of the mean delay; this is possibly due to the synchronization of activities along the synaptic pathway, causing a diminished scattering of delay distribution in the DG > CA1 pathway (Fig. 6D; same n as above). No significant changes were observed for propagation directions CA1 > mEC (n = 6 in b-early, n = 8 in b-late) and mEC > DG (n = 7 in b-early, n = 9 in b-late).

Figure 7.

Histograms of the distributions of propagation intervals for DG > CA1 (upper row), CA1 > mEC (middle row), and mEC > DG directions (lower row), respectively, during the last 2 phases of ictal events. On the right column, the cumulative curves corresponding to the histograms shown in the 2 left panels are shown. The decrease in the propagation time from DG to CA1 in b-late phase was also confirmed by non-parametric statistics (**P > 0.01 K-S test).

Figure 7.

Histograms of the distributions of propagation intervals for DG > CA1 (upper row), CA1 > mEC (middle row), and mEC > DG directions (lower row), respectively, during the last 2 phases of ictal events. On the right column, the cumulative curves corresponding to the histograms shown in the 2 left panels are shown. The decrease in the propagation time from DG to CA1 in b-late phase was also confirmed by non-parametric statistics (**P > 0.01 K-S test).

The measured propagation directions were taken from the central–final part of the bursts; it is possible that the pathways and, maybe, even the mechanisms of the propagations at burst onset or in the other phases of the seizures differ from those discussed here. Finally, we verified whether there were changes in time propagation of series of spikes recorded within the same burst between the 2 analyzed structures. The measurement of intra-burst percentage delay change of spikes propagation in phases b-early and b-late (see Materials and Methods) did not show any significant difference in time intervals recorded both during FA and HSA seizures (Fig. 8; t-test, P < 0.05).

Figure 8.

(A) An example of a burst in which the algorithm adopted to measure the subsequent propagation was applied. The 3 main identified propagation directions are indicated by arrows. (B) The mean percentage variation of propagation intervals (M) within analyzed bursts (DG > CA1, CA1 > mEC and mEC > DG, respectively) during b-early and b-late phases. Positive (and negative) values correspond to increased (and decreased) time delays inside bursts. Not significant variations were found among the subsequent phases.

Figure 8.

(A) An example of a burst in which the algorithm adopted to measure the subsequent propagation was applied. The 3 main identified propagation directions are indicated by arrows. (B) The mean percentage variation of propagation intervals (M) within analyzed bursts (DG > CA1, CA1 > mEC and mEC > DG, respectively) during b-early and b-late phases. Positive (and negative) values correspond to increased (and decreased) time delays inside bursts. Not significant variations were found among the subsequent phases.

Discussion

As reported in previous studies, hippocampal region and PHR are key actors for the generation of in vitro epileptic seizure-like events induced by BMI, 4-aminopirydine, and high K+ application (Avoli et al. 2002; Dzhala and Staley 2003; Uva et al. 2005; Gnatkovsky et al. 2008; Carriero et al. 2010). Our previous work demonstrated that seizure-like events induced by BMI in the isolated brain preparation are generated in EC–hippocampus (Uva et al. 2005). The present report is the extension of this study and shows that, as for in vivo TLE models and human TLE, limbic areas in our model generate 2 different patterns of seizures: FA and HSA. We outline for the first time the role of hippocampal and parahippocampal subregions in the generation of these 2 patterns. We demonstrate for the first time that presubiculum and parasubiculum are not involved in seizure generation, whereas subiculum is largely recruited in most phases. We confirmed the crucial role in seizure progression of DG, which is increasingly involved during the transition from irregular spiking to bursting pattern. Finally, we observed that there are no major changes in the main pathways of activity propagation throughout bursting phases of seizures. A schematic summary of the involvement of different regions during the phases of seizure-like events is illustrated in Figure 9. In this figure, no distinction between HSA and FA seizures was made, based on the assumption (see below) that network interactions during bursting phase in HSA are similar to those observed in FA seizures.

Figure 9.

Scheme showing the involvement and the propagation of seizure-like activity in the investigated regions. No difference between HSA and FA seizures is reported, based on the demonstration that network interactions during HSA seizures are similar to those observed in the bursting phase of FA seizures. Light shading indicates the structures not involved in the epileptiform discharge. Pre-ictal phase is characterized by the involvement of CA1/sub and mEC that can be followed by DG activation. fa can arise from either CA1 or mEC without the recruitment of DG which, in turn, is more and more engaged during the irrs and b-phases. The repetitive input from EC to DG during fa phase possibly promotes the DG as generator of the ictal activity in irrs phase. In b-phase, the propagation pathways become more reliable and it is virtually impossible to identify the region that initiates the discharge. Para and presubiculum are never involved in ictal activities.

Figure 9.

Scheme showing the involvement and the propagation of seizure-like activity in the investigated regions. No difference between HSA and FA seizures is reported, based on the demonstration that network interactions during HSA seizures are similar to those observed in the bursting phase of FA seizures. Light shading indicates the structures not involved in the epileptiform discharge. Pre-ictal phase is characterized by the involvement of CA1/sub and mEC that can be followed by DG activation. fa can arise from either CA1 or mEC without the recruitment of DG which, in turn, is more and more engaged during the irrs and b-phases. The repetitive input from EC to DG during fa phase possibly promotes the DG as generator of the ictal activity in irrs phase. In b-phase, the propagation pathways become more reliable and it is virtually impossible to identify the region that initiates the discharge. Para and presubiculum are never involved in ictal activities.

We previously proposed that a brief (3-min) arterial perfusion of 50 µM BMI induced a 40% reduction of the paired-pulse inhibition of disynaptic responses in the piriform–EC (Gnatkovsky et al. 2008). Seizures usually occur several minutes after the end of BMI perfusion, suggesting that we reach a threshold brain concentration when seizure initiate. The 3-min arterial BMI perfusion likely produces a low intra-parenchymal concentration of the drug, possibly lower than 5 µM, a concentration that typically induces seizure activity in cortical slices.

Epileptic seizures recorded with intracranial EEG techniques in patients with mesial TLE recognize 2 main patterns observed at onset: HSA and low-voltage FA (Bragin, Engel, Wilson, Fried et al. 1999; 2005; Velasco et al. 2000; Bartolomei et al. 2001; Wendling et al. 2003; Ogren et al. 2009). Bragin, Engel, Wilson, Fried et al. (1999; Bragin, Engel, Wilson, Vizentin et al. 1999) made a bridge between the human EEG patterns and the seizure onset recorded in vivo in rats, in which a chronic epileptic condition mimicking TLE was induced by unilateral intrahippocampal injection of kainic acid. Here we introduce the same classification for seizure-like events acutely induced by arterial perfusion of BMI in our in vitro model of isolated whole brain of guinea pig. Unlike humans and the kainic acid model, FA- and HSA-onset seizures are induced in a naïve brain by acute manipulations with a pro-convulsive agent in a non-chronic epileptic condition.

We propose a segmentation of seizures in phases, based on the activity pattern. According to this partitioning, we were able to discriminate FA to HSA seizures, defining the latter as lacking fa and irrs phases and being composed only by burst activity. We noticed that the majority of seizures induced immediately after the BMI perfusion is of the FA type, while HSA seizures are generated in brains already exposed to a preceding FA-onset seizure. In humans, HSA seizures have been proposed to be focally generated in the hippocampus (Velasco et al. 2000), whereas FA seizures may involve extrahippocampal areas (Bragin, Engel, Wilson, Vizentin et al. 1999). We demonstrate that mEC, CA1, and subiculum are involved in all the phases in both FA- and HSA-onset seizures, suggesting that both patterns are locally generated within the limbic area. Previous experiments demonstrated that extra limbic regions, such as the temporal neocortex and perirhinal region, are not involved in seizures, unless recurrent activation of seizure discharges occurred (Uva et al. 2005). Since HSA seizures show network patterns similar to the late phases of FA seizures, the possibility that HSA seizures are initiated in limbic regions that are not monitored with electrodes should be considered.

The DG has an important role in the maintenance and development of the seizure, even though it is almost always inactive at the onset of FA seizures (Fig. 9; dotted arrow in fa panel). During the transition from irrs to the bursting phase, large amplitude spikes initiate in DG and this structure becomes more involved in seizure progression (see irrs and b panels in Fig. 9). Under normal conditions, the DG works as a low-excitability filter that hinders propagation of excessive activity to other subregions of the hippocampus. An impairment of GABAergic inhibition in the DG is followed by an increase in the excitability in the whole hippocampus (Lothman et al. 1991, 1992; Sloviter 1991, 1994; Heinemann et al. 1992; Paré et al. 1992, Doherty and Dingledine 2001). Stringer and Lothman (1989) showed that the activation of the DG can be obtained by stimulation of the angular bundle at a specific rate (15–30 Hz) that, interestingly, corresponds to the frequency of FA recorded at the onset of FA seizure in our experiments. Therefore, FA in the beta range could be interpreted as a pattern that promotes the transition toward DG disinhibition, thus liberating the recurrence of large amplitude potentials. Disinhibited DG sustains highly synchronous periodic bursting along the polysynaptic hippocampal connectivity, possibly promoting the re-entrant activation of the hippocampal loop (Paré et al. 1992). Interestingly, a study performed on post-surgical in vitro slices of hippocampi from patients with drug-resistant mesial TLE demonstrated that DG can produce seizure-like events (Gabriel et al. 2004) and intracranial recordings with microelectrodes during pre-surgical evaluation in mesial TLE patients demonstrated the presence of pathologic high-frequency oscillation within the DG (Bragin et al. 2011).

Surprisingly, pre- and parasubiculum provided minimal, if any, participation to seizure activity despite the documented anatomical connections with the EC and the hippocampal formation (Fig. 9; van Groen and Wyss 1990; van Strien et al. 2009) and the evidence of epileptiform components in the evoked responses described both in chronic epileptic rats (Tolner et al. 2005) and in slices exposed to picrotoxin (Funahashi et al. 1999). Indeed, in the present work, field potentials could be recorded from presubiculum, but in the large majority of the experiments CSD analysis supported a passive volume-conducted nature, since no local sinks/sources were associated with such an activity. However, the involvement of presubiculum and parasubiculum in TLE cannot be excluded, since cell loss in both structures was documented in rats exposed to pilocarpine and electrically induced status epilepticus and intense electrical stimulation (Cardoso et al. 2011; van Vliet et al. 2004).

In the present study, we simultaneously explored different subregions of the hippocampus, with the exception of CA3. Even if silicon probes were inserted in the ventral hippocampus to aim at this region, local CA3 responses could never be recorded both after LOT stimulation and during seizure activity (data not shown). This could be possibly due to the distribution of the mossy fibres in both strata radiatum and oriens of CA3 (Swanson et al. 1978) that may prevent the organization of laminar dipoles compatible with the generation of large amplitude field potentials. This aspect was previously discussed in a study focused on the distribution patterns of olfactory inputs to the hippocampus (Uva and de Curtis 2005). However, the analysis of the spike propagation times, in particular from DG to CA1, indirectly suggests the involvement of an intermediate CA3 station between the 2 structures. The time lag between DG and CA1/subiculum of the spikes recorded during phases b-early and b-late was, indeed, 20–25 ms and was then compatible with a di/trisynaptic connection mediated by the interposed CA3 activation. Interestingly, CA3 has been rarely reported as seizures initiator in in vitro models of epilepsy (Luhmann et al. 2000). CA3 relevance in ictogenesis has been emphasized for inter-ictal glutamatergic spikes generation in acute hippocampal region–PHR slices treated with 4-aminopyridine (Barbarosie and Avoli 1997; Barbarosie et al. 2000). In particular, CA3 inter-ictal spikes have been demonstrated to exert an anti-epileptic function restraining ictal discharge generation in the EC. On the other hand, seizure-like events persist in spite of the presence of CA3 interictal activity in hippocampal–EC slices bathed with either low magnesium (Walther et al. 1986) or BMI (Borck and Jefferys 1999).

The spike propagation intervals between hippocampal region and PHR subfields revealed to be very reliable during bursting phases of seizures. Only the DG > CA1 pathway showed a significant acceleration in propagation with a corresponding significant lower CV of the intervals during successive bursts during the last seizure phase (b-late). A possible mechanism underlying the higher efficiency in DG > CA1 synaptic connection could be an increase in non-synaptic interaction among the ordered CA1 pyramidal neurons, favored by a mild swelling due to the intense neuronal activity (Jefferys 1995). Another possibility resides in an increase in the neurotransmitter release probability during an activity regime that mimics the stimulation patterns utilized to promote long-term potentiation (for a review, see Malenka and Nicoll 1999). These data suggest that DG-CA1 network wiring becomes more efficient and reliable during seizure-mediated reactivation of the hippocampal–EC network and exclude the possibility that acute repetitive bursting may support a functional rewiring of intrahippocampal connectivity, as suggested for the pilocarpine-induced chronic model of TLE (Avoli et al. 2002). This is probably due to the absence of cell damage in CA3 and CA1 regions in our acute model, in comparison to a chronic TLE model.

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/.

Funding

The study was supported by a grant of the Italian Health Ministry for Young Investigators (Grant Giovani Ricercatori RF 114-2008) and by an ERANET-NEURON grant (2p-imaging 2010-13).

Notes

We want to thank Dr. Barbara Cipelletti for her valuable technical help. Conflict of Interest: None declared.

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