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Keywords:

  • Bicuculline;
  • Cortical dysplasia;
  • Electrographic seizure episode;
  • Epileptic focus;
  • Hippocampus;
  • Interictal spikes;
  • Neocortex

Summary

  1. Top of page
  2. Summary
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

Purpose:  Periventricular nodular heterotopia (PNH) is, in humans, often associated with difficult-to-control epilepsy. However, there is considerable controversy about the role of the PNH in seizure generation and spread. To study this issue, we have used a rat model in which injection of methylazoxymethanol (MAM) into pregnant rat dams produces offspring with nodular heterotopia-like brain abnormalities.

Methods:  Electrophysiologic methods were used to examine the activity of the MAM-induced PNH relative to activity in the neighboring hippocampus and overlying neocortex. Recordings were obtained simultaneously from these three structures in slice preparations from MAM-exposed rats and in intact animals. Bath application or systemic injection of bicuculline was used to induce epileptiform activity.

Key Findings:  In the in vitro slice, epileptiform discharge was generally initiated in hippocampus. In some cases, independent PNH discharge occurred, but the PNH never “led” discharges in hippocampus or neocortex. Intracellular recordings from PNH neurons confirmed that these cells received synaptic drive from both hippocampus and neocortex, and sent axonal projections to these structures—consistent with anatomic observations of biocytin-injected PNH cells. In intact animal preparations, bicuculline injection resulted in epileptiform discharge in all experiments, with a period of ictal-like electrographic activity typically initiated within 2–3 min after drug injection. In almost all animals, the onset of ictus was seen synchronously across PNH, hippocampal, and neocortical electrodes; in a few cases, the PNH electrode (histologically confirmed) did not participate, but in no case was activity initiated in the PNH electrode. Interictal discharge was also synchronized across all three electrodes; again, the PNH never “led” the other two electrodes, and typically followed (onset several milliseconds after hippocampal/neocortical discharge onset).

Significance:  These results do not support the hypothesis that the PNH lesion is the primary epileptogenic site, since it does not initiate or lead epileptiform activity that subsequently propagates to other brain regions.

Medically intractable forms of epilepsy are often associated with abnormal brain development (Schwartzkroin & Walsh, 2000; Porter et al., 2002; Schwartzkroin et al., 2004). Abnormalities in neuronal migration, where newly developed neurons fail to reach their proper destinations, result in aberrant brain structure, often termed cortical dysplasia (CD). Dysplastic cell clusters and/or surrounding structures (e.g., in neocortex or hippocampus) are often identified as the sites in which epileptic activities are generated, and invasive surgical resection of these “lesions” has been used to provide seizure control (Hirabayashi et al., 1993; Francione et al., 2003; Russo et al., 2003; Aghakhani et al., 2005; Stefan et al., 2007). The condition of periventricular nodular heterotopia (PNH), in which a collection of neurons protrude into the ventricular space, has been described as a genetic disorder associated with intractable seizure activity (Guerrini, 2005; Sheen et al., 2005; Cardoso et al., 2009). Although the PNH lesion is relatively rare, it serves as a “model” of other epilepsy-associated migration disorders, and provides a useful context in which to explore the relative epileptogenicity of the aberrant structure (the nodule) versus the relatively “normal” surrounding tissue (Battaglia et al., 2006).

Direct recording from human PNH is invasive, and electrographic data from human patients provide only indirect evidence of the involvement of these aberrant structures in the generation of seizure activity (Dubeau & Tyvaert, 2010). Functional imaging approaches have been useful for gaining additional insight into the relationship between the nodular lesions and surrounding tissue (Archer et al., 2010). But given the difficulty of drawing “cause–effect” conclusions from these studies, animal models of PNH may provide valuable insights into the role of the nodular lesion in seizure generation.

Several animal models have been developed to study neuronal migration disorders. Chemical treatments [e.g., using the DNA-alkylating agent, methylazoxymethanol acetate (MAM)] (e.g., Baraban & Schwartzkroin, 1995, 1996; Germano & Sperber, 1997; Chevassus-au-Louis et al., 1999a,b; Castro et al., 2001), in utero irradiation (Roper et al., 1997), genetic manipulations of important cell development genes (Lee et al., 1997), and cortical freeze-lesions (Jacobs et al., 1996, 1999; Scantlebury et al., 2004; Takase et al., 2009) have provided valuable experimental tools to study different aspects of the basis of epileptic activities in neuronal migration disorders.

In utero exposure of rats to MAM results in reproducible brain abnormalities, including microcephaly, heterotopic cell clusters in hippocampus and cortex, and abnormally located cell clusters in the periventricular region (Singh, 1977; Baraban & Schwartzkroin, 1996; Chevassus-au-Louis et al., 1998; Sancini et al., 1998; Tschuluun et al., 2005). Previous MAM studies have focused primarily on heterotopia within the hippocampus and cortex, using coronal rat slices. Tract-tracing methods were used to demonstrate abnormal reciprocal connectivity between heterotopia and ipsilateral and contralateral cortical and hippocampal regions (Colacitti et al., 1998), and abnormal connectivity has been described in the malformed tissue (e.g., tangential cortico-cortical axon bundles, partial deafferentation of neocortical heterotopia, and abnormal ectopic mossy fiber projections within the hippocampus) (Chevassus-au-Louis et al., 1999a).

The periventricular localization of dysplastic cell clusters resembles PNH as seen in patients with intractable epilepsy (Aghakhani et al., 2005; Stefan et al., 2007; Giannopoulos et al., 2008). Clinical studies suggest that the PNH may serve as the initiator of epileptic activities in these patients—or at least contribute to a more widespread “epileptic” network (Aghakhani et al., 2005; Tassi et al., 2005; Stefan et al., 2007). Data from clinical imaging studies (Aghakhani et al., 2005; Tassi et al., 2005; Tyvaert et al., 2008; Archer et al., 2010) as well as results from experimental animal studies (Chevassus-au-Louis et al., 1998, 1999b; Colacitti et al., 1998) suggest that PNH-like lesions are functionally integrated into the surrounding cortical and hippocampal tissue. Based on our previous studies (Tschuluun et al., 2005), we have suggested that the PNH structure provides aberrant connectivity between normally unconnected brain regions, and thus creates a novel pathway by which epileptiform activity can more easily spread/generalize to relatively normal brain regions.

In the current experiments, we have used the MAM rat model of PNH to address the following questions: (1) Does the PNH initiate epileptiform discharges in the brain of MAM rats? (2) Does the PNH integrate structurally into neighboring tissue so as to facilitate the propagation of the epileptiform discharges? We used both in vitro (slice) and in vivo (intact rat) electrophysiologic recording approaches to examine these issues. In acute brain slice preparations, the PNH did not initiate (i.e., lead) epileptiform discharges (either ictal or interictal electrographic activities), although the nodule appeared capable of generating independent electrographic “bursts.” We also found that PNH neurons are reciprocally integrated into the neighboring cortical and hippocampal networks, and thus can serve as functional “shortcuts” for epileptiform discharges initiated in the hippocampus to propagate into the cortex. Recordings from the intact rat brain containing PNH-like lesions confirmed our general results from slice experiments: The PNH never initiated epileptiform discharges when the rat was injected with bicuculline, but usually participated (i.e., generated synchronized discharge) in events initiated in ipsilateral hippocampus or neocortex.

Material and Methods

  1. Top of page
  2. Summary
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

All procedures were carried out in accordance with NIH animal use guidelines, and were approved by the UC Davis Institutional Animal Care and Use Committee. Experimental procedures were designed to minimize the number of animals used and to minimize animal suffering.

MAM administration

As described previously (Tschuluun et al., 2005), pregnant Sprague-Dawley rats (Harlan, Indianapolis, IN, U.S.A.) were injected with either 25 mg/kg MAM (MRI, Kansas City, MO, U.S.A.) [25 mg dissolved in 1 ml 0.9% sodium chloride (NaCl)] or vehicle (NaCl) alone. A single intraperitoneal (i.p.) injection of MAM was administered at gestational day 15.

In vitro slice preparation and electrophysiology

Details of slice preparation, intracellular recording, and cell staining have been published (Tschuluun et al., 2005) and are provided in Supporting Information. In pilot studies, we used morphologic methods to study connectivity of MAM-induced PNH in both transverse and sagittal slices as well as intact brain, and found that the general orientation for single and/or multiple PNH follows an axis from rostromedial to laterocaudal, corresponding to the anatomic borders of the lateral ventricles between cortex (corpus callosum) and caudatoputamen/septum/hippocampus. We adopted this orientation for the current study, since it largely preserves anatomic connections (i.e., axons) between the PNH and cortex/hippocampus, both in slices and in intact brain, as seen with biotinylated dextran amine tracer experiments and intracellular biocytin staining of individual PNH neurons.

Simultaneous field potential recordings were obtained from the PNH, the hippocampus, and the neocortex (Fig. 1A) to study the spread of the spontaneous epileptiform discharges in the brain. Low-resistance electrodes (approximately 1 megaohm resistance) were pulled from 1-mm outside diameter (OD) borosilicate glass and filled with 3 m NaCl. Electrodes were placed in the PNH, CA1 or CA3 region of the hippocampus, and in the deeper layers (V–VI) of the neocortex, and voltage changes were recorded. In order to elicit spontaneous epileptiform discharges, slices were perfused with 50 μm bicuculline methiodide (BMI; Sigma Aldrich, St. Louis, MO, U.S.A.) and 5 mm KCl in the artificial cerebrospinal fluid (ACSF). After robust episodes of epileptiform discharges were established (recorded for at least 10 min), a cut was made between the PNH and the hippocampus using a razor blade fragment. The completeness of the cut was visually verified under the microscope. Field potential changes were recorded for another 10 min, and another cut—between the PNH and neocortex—was made. Discharge activity was recorded for an additional 10 min. In slices in which hippocampal and cortical discharges were still time-locked after the second cut, a third cut was made to separate the subiculum from the overlying neocortex. In some slices, after completing this series of recordings, we attempted to remove the PNH from the slice tissue (gentle suction) and the PNH recording electrode was placed in the “empty” space to measure field potential spread from the surrounding tissue.

image

Figure 1.  (A) Sagittal slice through the periventricular nodule (PNH), including hippocampus (Hip) and neocortex (Ctx). Recording electrodes were placed in Hip, Ctx, and PNH in order to monitor simultaneously epileptiform activity when the slice was bathed in bicuculline. EC, entorhinal cortex; cc, corpus callosum. (B) Surgical exposure showing electrodes entering the rat brain through holes in the overlying skull. A single probe, with two attached recording wires, was used to monitor activity in neocortex and hippocampus (Ctx-Hip). A second probe, with attached tungsten wire, was stereotaxically targeted to the PNH.

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In vivo recording from control and MAM-treated rats

Two-month-old to 3-month-old male and female rats, both control and MAM-treated, were anesthetized with 4% isoflurane and intubated. Animals were placed in a stereotaxic head-holder and 2–2.5% isoflurane was delivered via the intubation tube. The body temperature was held at constant 37°C via a heating pad. An incision was made to expose the skull, and bregma coordinates were determined. A hole for introducing the PNH recording electrode was marked at: 0.4 mm posterior and 1.5 mm lateral from bregma. For ipsilateral hippocampal and neocortical electrodes, a hole was marked at the following coordinates: 2 mm posterior and 1.5 mm lateral from bregma. Holes were drilled using a 0.7 mm drill bit. Two insulated, low-resistance fine tungsten wires (0.4 mm OD) were used to record from the hippocampus and neocortex. These two electrodes were introduced together, with the tip of the cortical recording electrode approximately 1 mm above the tip of the hippocampal electrode. They were mounted on a stereotaxic manipulator and carefully lowered into the brain until the hippocampal electrode reached a depth of 1.7 mm below the dura (Fig. 1B). A sharp insulated tungsten electrode (FHC, Bowdoin, ME, U.S.A.) was used to record from the PNH. It was mounted on a second stereotaxic manipulator, introduced through the skull hole over the PNH, and carefully lowered into the brain. All three electrodes were connected to differential AC amplifiers [Model 1700; A-M Systems, Sequim, WA, U.S.A.] via dedicated head stages. Activity from the PNH electrode (monitored on an oscilloscope) was monitored when the electrode tip reached a target depth of 2 mm from the dura; the electrode was carefully lowered from that point until cellular discharge (reflecting a pocket of neurons) was apparent. After a 10-min baseline recording period, 0.5 mg/kg bicuculline (dissolved in DMSO, dimethylsulfoxide) or 25 mg/kg pentylenetetrazol (PTZ, dissolved in 0.9% NaCl) was administered via a tail vein injection. Recordings continued for another 20 min. Signals were digitized (DigiData 1200; Molecular Devices, Sunnyvale, CA, U.S.A.) using Axoscope 9 (Molecular Devices) and further analyzed using Clampfit 9 (Molecular Devices). In some animals, a 0.25 μA current was passed through the recording electrode (15 s) to mark the tip of the recording electrode; in the remaining animals, electrode location was established by histologic reconstruction of the electrode track.

At the conclusion of the recording procedure, electrodes were removed, the scalp incision was sewn together, and the animal was perfused with 4% paraformaldehyde in order to allow histologic verification of electrode locations.

Tissue preparation for light microscopy/histochemistry

Details have been published previously (Tschuluun et al., 2005) and are provided in Supporting Information.

Results

  1. Top of page
  2. Summary
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

Histologic features of PNH neurons

Single MAM injections into pregnant female Sprague-Dawley rats reliably induced morphologic brain abnormalities in the offspring, including microcephaly, disarrangement of cortical lamination, and neuronal heterotopia in neocortex and hippocampus. The current electrophysiologic study focused on PNH located in the dorsal wall of the lateral ventricles and extending between the corpus callosum and dorsal hippocampus. In about one-fourth of these cases, intrahippocampal heterotopia was also present (see also Dubeau et al., 1995). Interestingly, according to Battaglia et al. (2003), periventricular heterotopia and intrahippocampal heterotopia are formed by different neurogenic patterns during cerebral neurogenesis. The results of our PNH experiments did not differ between the cases with and without intrahippocampal heterotopia, in slices or in intact animals.

Histologic abnormalities, including cellular features of neurons (including interneurons) in hippocampal and/or PNH, have been described in detail previously (Baraban & Schwartzkroin, 1995; Chevassus-au-Louis et al., 1999a; Battaglia et al., 2003; Tschuluun et al., 2005). In the current study, individual electrophysiologically identified neurons located within the PNH were filled with biocytin, and compared with normotopic pyramidal neurons of hippocampus or neocortex. Although many of the biocytin-filled neurons were irregularly oriented and no lamination patterns were observed within the heterotopia (but see Garbelli et al., 2009), many of the neurons exhibited structural features of pyramidal-like cells (Fig. 2A). Most of the neurons exhibited a variably oriented apical main dendrite with many branched and variably oriented lateral dendrites. The number of basal dendrites appeared to be higher than seen in normal hippocampal pyramidal cells. Many of the neurons were similar in their morphologic appearance to those of neurons observed in layer II/III of the neocortex (Tschuluun et al., 2005). All of the neurons showed dendritic spines; spine morphology, spine distribution, and density appeared to be similar to normal pyramidal cells. From a total of 15 completely biocytin-filled neurons located within the PNH (from separate slices from different animals), eight neurons exhibited axonal projections into the adjacent cortical layers (7) and/or the hippocampus (6). Termination of their axonal collaterals was seen in neocortical layers III–IV, as well as in strata oriens and pyramidale of hippocampal CA1 and CA3 subfields. However, many of the axonal collaterals were cut off after passing into the corpus callosum, suggesting that they may have changed their direction/trajectory. As illustrated in Fig. 2B, four of the biocytin-filled neurons exhibited double axonal projections into both neocortex and hippocampus.

image

Figure 2.  (A) Two-dimensional reconstruction of a biocytin-filled neuron in a PNH. The cell shows anatomic similarity to layer II/III neocortical pyramidal neurons, with an apical dendrite (black arrowhead) (random orientation across neurons) and axon collaterals (black arrows) extending throughout the PNH (and beyond). The axon of this neuron projected into the neocortex. Scale bar: 50 μm. (B) Two-dimensional reconstruction of a biocytin-filled neuron in a PNH, with axon collaterals ramifying within the PNH and extending into both cortex (Ctx) and hippocampus (CA3). Micrographs showing axonal segments in Ctx and CA3 (insets). PNH, periventricular nodular heterotopia; V, ventricle. Scale bar: 100 μm.

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In vitro slice electrophysiology

Field recording experiments

Twenty-three experiments were performed on acute brain slices of MAM rats and five experiments on tissue from control rats in order to study the connectivity between the periventricular nodular heterotopia (PNH), the hippocampus (Hip), and the neocortex (Ctx). In 19 MAM experiments, all three structures exhibited epileptiform discharges following bath application of BMI; these experiments provided the data for further analysis. In all 19 MAM-slices (and in the five control rat experiments), the bicuculline-elicited epileptiform discharges always initiated in the hippocampus; in 17 of 19 experiments, time-locked epileptiform bursts occurred subsequently in PNH and then in the cortex (Fig. 3A,C). In two experiments, PNH bursts occurred asynchronously (i.e., not associated with bursts in hippocampus or neocortex), whereas cortical and hippocampal discharges were time-locked with each other.

image

Figure 3.  (A) Field potentials recorded simultaneously from CA3 hippocampus (Hip), neocortex (Ctx) and the periventricular nodular heterotopia (PNH) in a slice from a MAM-exposed rat. Slices were bathed in bicuculline to induce spontaneous epileptiform discharges [left traces at slow time-scale; single event (boxed) expanded to the right]. Diagram at right (see Fig. 1A for description) shows recording arrangement. (B) Same recording arrangement (same slice) following a cut (CUT I) between the PNH and the hippocampus (dashed line). (C, D) Latency time plot showing onset of epileptiform discharges in PNH and Hip relative to Ctx, before (C) and after (D) CUT I. (E, F). Same recording arrangement shows epileptiform events following CUT II between PNH and Ctx (E) and following CUT III between Ctx and subiculum (F). (G, H) Latency time plot showing onset of epileptiform discharges in PNH and Hip relative to Ctx after CUT II and CUT III, respectively. (I) Same recording arrangement following CUT IV, along the corpus callosum.

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We made a series of cuts to study the connectivity between the PNH and surrounding structures. When CUT I (Fig. 3B) was made along the border between the PNH and the hippocampus, the sequence of the epileptiform bursting changed. In eight such experiments, cortical discharges preceded those in the PNH (Fig. 3B,D); in 10 experiments, PNH discharges were no longer synchronized with those of the hippocampus and the cortex; and in one experiment, the PNH no longer exhibited epileptiform discharges (not shown). When CUT II along the border between the PNH and the cortex was added (Fig. 3E), the PNH still generated epileptiform discharges that were independent from hippocampal and cortical discharges (in 10 experiments); in nine experiments, epileptiform discharges no longer occurred in the PNH (Fig. 3E,G); in all 19 experiments, hippocampal and cortical discharges remained time-coupled. A third cut (CUT III) was made in cortex close to the subiculum in order to disrupt the connectivity between the hippocampus and cortex (Fig. 3F). In 13 experiments, epileptiform discharges were then no longer seen in the cortex, but in six experiments the connectivity between the hippocampus and the cortex persisted (Fig. 3H). In those latter slices, a final cut along the corpus callosum (CUT IV) successfully disrupted the connectivity between the hippocampus and the cortex (Fig. 3I). This last cut presumably more completely separated overlying cortex from hippocampus, severing aberrant connections descending from the cortex and PNH, which cross the corpus callosum at different levels (as shown in our preliminary tracer studies).

These results suggest that: (1) of the structures preserved in the slice, hippocampus has the lowest seizure threshold and tends to drive discharge in the remaining tissue; (2) in most cases, CA1 hippocampus projects to neocortex via the subiculum and entorhinal cortex (i.e., there is no “direct” route); and (3) the PNH provides a new, more direct pathway by which activity in the hippocampus may influence neocortical activity.

Single cell recordings

We studied activity of single cells in the PNH in response to stimulation within the PNH itself or in neighboring tissue. In 27 experiments in which a stimulating electrode was placed in the PNH, excitatory postsynaptic potentials (EPSP) were elicited in PNH neurons (Fig. 4A). In 18 of those experiments, we recorded IPSPs to the same stimulus (Fig. 4A), suggesting activation of local inhibitory interneurons. In 11 experiments, we recorded antidromic action potentials (Fig. 4A inset). A stimulating electrode was placed in the deeper layers of the neocortex (adjacent to a PNH) in 12 experiments. In 10 of those experiments, we recorded EPSPs in PNH neurons, in four experiments we registered IPSPs to the cortical stimulations (Fig. 4A), and in five experiments we also detected antidromic action potentials (data not shown). Inhibitory postsynaptic potentials were bicuculline-sensitive (blocked when BMI was applied). In 27 experiments, one of the stimulating electrodes was placed in the CA3 region adjacent to the PNH. In 26 experiments, stimulation at this site elicited excitatory postsynaptic potentials (EPSPs) from PNH neurons; in 12 of these experiments, we recorded inhibitory postsynaptic potentials (IPSPs) (Fig. 4B); and in eight experiments, we recorded antidromic action potentials (Fig. 4B) (indicating a projection of the recorded cell to the hippocampus). Fig. 4C summarizes the findings from the intracellular recording and labeling experiments. Our data show that PNH cells receive excitatory and inhibitory inputs in response to activation of both the hippocampus and the cortex, and in return can project into both neighboring brain regions.

image

Figure 4.  (A) Intracellular recording showing responses of a PNH neuron to an intracellular depolarizing pulse (left), intra-PNH stimulation (single asterisk, middle), and cortical stimulation (double asterisk, right) at different membrane potentials. An expanded time scale (insets above) shows antidromic activation and postsynaptic responses to PNH stimulation, and synaptic drive (EPSP and IPSP) in response to stimulation in cortex. The diagram to the right shows placement of recording and stimulating electrodes. (B) Similar display, showing responses of a PNH neuron to intracellular depolarization (left), intra-PNH stimulation (single asterisk, middle), and hippocampal (Hip) stimulation (triple asterisk, right). (C) Schematic illustration of connectivity between PNH and cortex (Ctx) and hippocampus (Hip), as suggested by these stimulation experiments in slices. Int, inhibitory interneuron.

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In vivo electrophysiologic recordings from control and MAM rats

Twenty-six intact MAM-exposed rats were studied in acute experiments in which bicuculline was injected systemically. In 12 experiments, the PNH electrode was confirmed histologically to be in the PNH; in 12 experiments, the PNH electrode was nearby, but not in the PNH; in two experiments, there was no PNH (although the animals had been exposed to MAM in utero). Five experiments were conducted on age-matched control rats in order to compare hippocampal and cortical activities. Only those preparations in which we have histologic confirmation of electrode placement in the PNH (Fig. 5A) are described below.

image

Figure 5.  (A) Histologic section through bilateral PNH of a rat used for recording, showing electrode track confirmed to PNH recording site. PNH, periventricular nodular heterotopia; Ctx, cortex; V, ventricle (B). Spontaneous epileptiform event in a rat injected with bicuculline. Traces at left—slow recording speed; traces at right—expanded sweep speed of boxed event (at left), to illustrate the relative “simultaneity” of event onset in the cortex, hippocampus, and nodule. (C). A different experiment, illustrating the coincidence of epileptiform events in the three electrodes, but an occasional “independent” event recorded from the PNH (asterisk). (D). Slow recording speed, to show onset of an ictal-like episode (arrow), seen simultaneously in all three electrodes.

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At baseline, low frequency high-amplitude spiking activity was seen in most preparations (probably resulting from the anesthesia treatment). This discharge was synchronized in hippocampus and neocortical electrodes, with the PNH sometimes also participating (Fig. 5B). In rare instances, independent discharge arose from the PNH but was not seen in the other two electrodes (Fig. 5C).

The bicuculline injection resulted in high-amplitude epileptiform discharge in all experiments. Increases in burst frequency and increases in amplitude of discharge, leading to onset of an electrographic seizure event, were observed at all three recording sites. Ictal activity was typically initiated within 2–3 min after drug injection. In almost all animals, the onset of ictus was seen synchronously across all three electrodes (Fig. 5D); that is, our analysis could not detect a discrete onset site. In three cases, the PNH electrode did not participate in the ictal discharge. Electrographic seizure activity in the PNH was never observed to “lead” activity in hippocampus or neocortex. Interictal discharge (before or after the seizure episode) was also synchronized across all three electrodes; again, discharge in the PNH never led discharge in the other regions, and typically followed (onset several milliseconds after hippocampus/neocortex onset). Generally, PNH discharges closely followed discharge in the hippocampus, although in 3 of 13 experiments the PNH discharges were more closely associated with cortical discharges. In 4 of 13 experiments, PNH generated independent discharges, but these events did not “drive” discharge in the other two regions.

Similar results were seen with PTZ injection (37 experiments; electrode tip in the PNH in six animals, near or perforating the PNH in four animals). In no case did PNH discharge lead epileptiform activity in the cortex or hippocampus. Details of those experiments are not reported here since PTZ was not used in any slice experiments, and so we cannot make direct comparisons of in vivo and in vitro data.

Discussion

  1. Top of page
  2. Summary
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

In these studies, we have attempted to address the question of if/how activity in a discrete dysplastic lesion affects epileptogenicity. Using a rat model of PNH, we have found that the aberrant structure does not appear more excitable than the surrounding tissue (hippocampus and neocortex), and does not appear to initiate or lead epileptiform activities. In both in vitro slice and in vivo intact rat preparations, epileptiform activity in the nodule was generally synchronized with discharge in surrounding tissue, but did not serve as a “driver” for either ictal or interictal events. These electrophysiologic results appear within a context of obvious structural connectivity between the nodule and surrounding brain tissue. The slice data further suggest that the aberrant nodule provides a bridge that facilitates communication between brain regions that in normal brain do not communicate directly. It should be emphasized, however, that these data do not address the questions of whether (1) the presence of the nodule affects general brain epileptogenicity, or (2) to what extent the surrounding tissue has become structurally (and functionally) abnormal as a result of the developmental process that also gives rise to the nodule. These latter questions require additional experimental investigation.

PNH constitutes a subclass of cortical dysplasia with well-defined pathologic features (Aghakhani et al., 2005; Tassi et al., 2005). Although seizures are highly correlated with this usually genetically determined brain disorder, clinical studies have not provided clear results regarding the role of the PNH in the initiation and spread of the electric activity during seizures (Tassi et al., 2002, 2005; Aghakhani et al., 2005; Stefan et al., 2007). Common findings from clinical studies are generally consistent with our experimental results; that is: (1) The PNH generates electrical ictal activity closely synchronized with cortical and hippocampal discharges; (2) the PNH is sometimes capable of generating independent interictal-like discharges; and (3) it is anatomically (and functionally) connected to neighboring cortical structures (Aghakhani et al., 2005; Stefan et al., 2007).

One of the best-studied animal models of PNH is the MAM-treated rat (Baraban & Schwartzkroin, 1995, 1996; Germano & Sperber, 1997; Germano et al., 1998; Tschuluun et al., 2005; Marchi et al., 2006; Harrington et al., 2007). MAM rats rarely exhibit spontaneous seizures (but see Harrington et al., 2007), but they show lower threshold for epileptogenic insults (Baraban & Schwartzkroin, 1996; Germano & Sperber, 1997; Germano et al., 1998). Our previous in vitro study on tissue from MAM rats (Tschuluun et al., 2005) suggested that dysplastic neurons in hippocampal heterotopia contribute to the spread of the epileptic discharges in brain slices. Based on these data, we postulated that although the dysplastic lesion may not itself be hyperexcitable, it provides a “low resistance bridge” that allows a more efficient, faster spread of epileptiform discharge, and, therefore, facilitates synchronization of aberrant discharge patterns. The initiator of that abnormal activity, however, remains to be determined.

The aberrant developmental program in MAM-exposed rats (and in human with genetic mutations that give rise to dysplastic structures) not only generates a “lesion” with obvious anatomic abnormality, but also gives rise to surrounding tissue with more subtle abnormalities (Castro et al., 2001; Garbelli et al., 2009). In the current study, we found the nodule was capable of independent discharge but did not appear to drive epileptiform discharge in surrounding tissue, consistent with previous work from our own lab and from others (Aghakhani et al., 2005; Tschuluun et al., 2005; Tyvaert et al., 2008). Further, inhibition is clearly relatively “intact” in the nodule; histology/immunocytochemistry reveals γ-aminobutyric acid (GABA)ergic inhibitory interneurons in the PNH, and stimulation of cortex and hippocampus elicits not only excitatory but also inhibitory synaptic events in PNH neurons. Therefore, it may be that surrounding tissue constitutes the initiator/driver, as has been proposed for the freeze-lesion model of epileptogenicity associated with abnormal structure (Jacobs et al., 1996, 1999; Scantlebury et al., 2004).

There is, of course, the question of if/how well this animal model (or any animal model) mimics the pathophysiology of human PNH. Most etiologic studies of epilepsies associated with PNH abnormalities have focused on genetic causes (e.g., Guerrini, 2005; Parrini et al., 2006; Ferland et al., 2009; Sprour et al., 2011). The MAM model represents, however, an extrinsic insult—perhaps more akin to developmental insults such as in utero stroke. Montenegro et al. (2002) suggested that prenatal injury may play a significant role in the genesis of development abnormalities; in almost half of their study group, prenatal events were identified that might have contributed (along with genetics) to the pathogenesis of cortical malformations. Another issue for interpreting the MAM rat model data arises from the simple geometrical differences between rat and human brain. In the rat, the PNH is physically close to both cortex and hippocampus, and thus connectivity can be “easily” established. In contrast, in the human brain, the PNH may be distant, especially from the hippocampus—and the hippocampus may, therefore, not be so involved in PNH-associated seizure generation (Li et al., 1997). The clinical literature suggests that not only may PNH pathology be heterogeneous (Sprour et al., 2011), but so also is seizure onset; therefore, in some patients, seizures may in fact originate directly from the nodules (Kothare et al., 1998; Scherer et al., 2005). Indeed, in a small number of patients with PNH, selective thermocoagulation of nodules has resulted in reduced epileptogenicity (Catenoix et al., 2008)—certainly suggestive of a causal role for PNH in seizure generation.

Epilepsy associated with periventricular nodular heterotopia often presents as medically intractable (Li et al., 1997; Guerrini, 2005; Poduri et al., 2005; Guerrini & Marini, 2006; Parrini et al., 2006). As has been shown for tubers in tuberous sclerosis complex, surgical removal of the aberrant structure may serve to control seizure activity. However, this empirical result does not “prove” that the lesion is epileptogenic, since such resection not only removes the purported “epileptic focus” but also disrupts a potential “epileptic network” (as is also the case for temporal lobe resection). Furthermore, such clinical interventions invariably also remove surrounding tissue, which may play a major role in epileptogenicity. The question of whether the dysplastic lesion is somehow responsible for brain epileptogenicity, therefore, remains an unresolved. Surgical interventions that specifically target the nodule may provide empirical evidence that guides therapeutic intervention (e.g., see Catenoix et al., 2008). If, however, it is not the features of the nodule per se that underlie epileptic function, but rather the associated abnormalities in surrounding tissue (e.g., aberrant connectivity, altered inhibitory function, etc.—see Jacobs et al., 1999; Schwarz et al., 2000), then such surgical intervention may be fruitless. It is, therefore, important for future research to provide more revealing insight into the relationship between abnormal structure and abnormal function.

Acknowledgment

  1. Top of page
  2. Summary
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

This research was support by NIH grant NS 57209 (PAS). We are grateful to Ms. Laurie Beninsig for assistance with the figures.

Disclosure

  1. Top of page
  2. Summary
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

The authors have no conflicts of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

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  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

Data S1. Methods.

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