Propagation dynamics of epileptiform activity acutely induced by bicuculline in the hippocampal-parahippocampal region of the isolated Guinea pig brain

PURPOSE: Aim of the study is to investigate the involvement of parahippocampal subregions in the generation and in the propagation of focal epileptiform discharges in an acute model of seizure generation in the temporal lobe induced by arterial application of bicuculline in the in vitro isolated guinea pig brain preparation. METHODS: Electrophysiological recordings were simultaneously performed with single electrodes and multichannel silicon probes in the entorhinal, perirhinal, and piriform cortices and in the area CA1 of the hippocampus of the in vitro isolated guinea pig brain. Interictal and ictal epileptiform discharges restricted to the temporal region were induced by a brief (3-5 min) arterial perfusion of the GABA(A) receptor antagonist, bicuculline methiodide (50 microM). Current source density analysis of laminar field profiles performed with the silicon probes was carried out at different sites to establish network interactions responsible for the generation of epileptiform potentials. Nonlinear regression analysis was conducted on extracellular recordings during ictal onset in order to quantify the degree of interaction between fast activities generated at different sites, as well as time delays. RESULTS: Experiments were performed in 31 isolated guinea pig brains. Bicuculline-induced interictal and ictal epileptiform activities that showed variability of spatial propagation and time course in the olfactory-temporal region. The most commonly observed pattern (n = 23) was characterized by the initial appearance of interictal spikes (ISs) in the piriform cortex (PC), which propagated to the lateral entorhinal region. Independent and asynchronous preictal spikes originated in the entorhinal cortex (EC)/hippocampus and progressed into ictal fast discharges (around 25 Hz) restricted to the entorhinal/hippocampal region. The local generation of fast activity was verified and confirmed both by CSD and phase shift analysis performed on laminar profiles. Fast activity was followed by synchronous afterdischarges that propagated to the perirhinal cortex (PRC) (but not to the PC). Within 1-9 min, the ictal discharge ceased and a postictal period of depression occurred, after which periodic ISs in the PC resumed. Unlike preictal ISs, postictal ISs propagated to the PRC. CONCLUSIONS: Several studies proposed that reciprocal connections between the entorhinal and the PRC are under a very efficient inhibitory control (1). We report that ISs determined by acute bicuculline treatment in the isolated guinea pig brain progress from the PC to the hippocampus/EC just before ictal onset. Ictal discharges are characterized by a peculiar pattern of fast activity that originates from the entorhinal/hippocampal region and only secondarily propagates to the PRC. Postictal propagation of ISs to the PRC occurred exclusively when an ictal discharge was generated in the hippocampal/entorhinal region. The results suggest that reiteration of ictal events may promote changes in propagation pattern of epileptiform discharges that could act as trigger elements in the development of temporal lobe epilepsy.     

Ictal spikes: a marker of specific hippocampal cell loss.

Spontaneous seizures recorded from mesial temporal depth electrodes in the human are commonly manifested by one of two onset patterns: a high frequency discharge or a periodic spike discharge morphologically similar but clearly distinguished from ongoing interictal activity. We categorized medial temporal lobe seizure onset for the presence of periodic ictal spikes at a frequency of less than 2 Hz lasting for more than 5 sec to investigate the relationship of this ictal pattern to anatomical changes in the resected temporal lobe tissue. Fifty-one patients had hippocampal depth electrode recordings of spontaneous seizures, subsequent hippocampal resection, and quantitative cell counts of hippocampal subfields. Thirty-two of these patients had ictal spikes lasting at least 5 sec in more than 50% of their seizures. The presence of ictal spikes was significantly correlated with decreased cells in CA1 only (P = 0.015). The correlation of a common ictal pattern with focal cell loss in the hippocampus suggests that electrophysiological manifestations of seizures provide a clue to the underlying pathological substrate. Ictal spikes may be a cause or result of the cell loss. These observations should be correlated with independent investigations in humans and animal models that reflect the CA1 cell loss associated with temporal lobe epilepsy.     

Ictal spikes: a marker of specific hippocampal cell loss

Spontaneous seizures recorded from mesial temporal depth electrodes in the human are commonly manifested by one of two onset patterns: a high frequency discharge or a periodic spike discharge morphologically similar but clearly distinguished from ongoing interictal activity. We categorized medial temporal lobe seizure onset for the presence of periodic ictal spikes at a frequency of less than 2 Hz lasting for more than 5 sec to investigate the relationship of this ictal pattern to anatomical changes in the resected temporal lobe tissue. Fifty-one patients had hippocampal depth electrode recordings of spontaneous seizures, subsequent hippocampal resection, and quantitative cell counts of hippocampal subfields. Thirty-two of these patients had ictal spikes lasting at least 5 sec in more than 50% of their seizures. The presence of ictal spikes was significantly correlated with decreased cells in CA1 only (P=0.015). The correlation of a common ictal pattern with focal cell loss in the hippocampus suggests that electrophysiological manifestations of seizures provide a clue to the underlying pathological substrate. Ictal spikes may be a cause or result of the cell loss. These observations should be correlated with independent investigations in humans and animal models that reflect the CA1 cell loss associated with temporal lobe epilepsy.     

Do Interictal Discharges Promote or Control Seizures? Experimental Evidence from an In Vitro Model of Epileptiform Discharge

Summary: Interictal and ictal discharges are recorded from limbic structures in temporal lobe epilepsy patients. In clinical practice, interictal spikes are used to localize the epileptogenic area, but they also are assumed to promote ictal events. Here I review data obtained from combined slices of mouse hippocampus–entorhinal cortex that indicate an inverse relation between interictal and ictal events. In this preparation, application of 4-aminopyridine or Mg²⁺-free medium induce (a) interictal discharges that originated from CA3 and propagate (via the Schaffer collaterals) to CA1 and entorhinal cortex, to return to the hippocampus through the dentate area; and (b) ictal discharges that initiate in the entorhinal cortex and propagate to the hippocampus via the dentate gyrus. Interictal activity occurs throughout the experiment (up to 6 h), whereas ictal discharges disappear after 1–2 h. Schaffer collateral cut abolishes interictal discharges in CA1, entorhinal cortex, and dentate and reestablishes entorhinal ictal discharges. Moreover, ictal discharge generation in the entorhinal cortex after Schaffer collateral cut is prevented by mimicking CA3 activity with rhythmic electrical stimulation of CA1 outputs. Thus hippocampal interictal activity controls the ability of the entorhinal cortex to generate seizures. It also may be proposed that Schaffer collateral cut may model the epileptic condition in which CA3 damage results in loss of hippocampal control over the entorhinal cortex. In conclusion, these experiments demonstrate that interictal activity controls rather than promotes ictal events, and functional integrity of CA3 constitutes a critical control mechanism in temporal lobe epilepsy.     

CA3 axonal sprouting in kainate-induced chronic epilepsy

Latency between an early neurological insult and development of spontaneous recurrent seizures suggests aberrant chronological reorganization in patients with mesial temporal sclerosis associated epilepsy. Kainate-induced status similarly results in delayed development of spontaneous recurrent seizures. Mossy fiber sprouting by the dentate granule cells is a well-characterized manifestation of such temporal structural reorganization in both patients and animal models. However, alterations in other components of hippocampal circuitry have not been evaluated. We present results from studies using precise anterograde and retrograde tract tracing methodologies to evaluate the reorganization of outflow of the CA3 pyramidal cells. Although septotemporal relationships of the normal CA3 outflow tract through the Schaffer collaterals are well known, their aberrant reorganization following kainate-induced spontaneous recurrent seizures is not known. We provide the first definitive evidence of widespread CA3 structural reorganization in the form of sprouting of CA3 axons to widespread areas throughout the hippocampus and entorhinal cortex. This includes an apparent increase in the density of projection to areas that normally receive CA3 outflow such as CA1 and subiculum as well as novel projections beyond the confines of the hippocampus to the pre and parasubiculum and medial and lateral entorhinal cortex. We provide the first evidence of novel CA3 Schaffer collateral projection to the entorhinal cortex. The sprouting of CA3 outflow to widespread regions of the hippocampus and the entorhinal cortex may provide insight into how the injured hippocampus propagates unconventional impulse excitation to cortical fields which have a critical role in providing excitatory inputs into the hippocampus possibly setting up reverberating excitatory circuits as well as widespread connections throughout the cortical mantle. Sprouting-related mechanisms may also explain the latency associated with development of spontaneous recurrent seizures, the hallmark of temporal lobe epilepsy.     

Anatomic Correlates of Interhippocampal Seizure Propagation Time

The relation between interhippocampal seizure propagation time (IHSPT) and anatomic alterations in the human epileptic hippocampus may provide insight into the pathophysiology of temporal lobe epilepsy (TLE). Using depth electrode recordings, we measured the time required for spontaneous seizures with onset in one hippocampus to become manifest in the contralateral hippocampus in 50 patients who underwent resection of the temporal lobe of seizure origin. Cell densities in individual hippocampal subfields were determined and correlated with mean IHSPT for each patient. Mean IHSPT was significantly and inversely correlated with cell counts in CA4 only (r = -0.38, p less than 0.01, Pearson's product correlation; r = -0.52, p less than 0.001, Spearman's rank order correlation). In 5 patients with bilateral independent hippocampal seizure onset who had temporal lobectomy and a diagnosis of mesial temporal sclerosis, mean IHSPT was consistently longer from the sclerotic temporal lobe than to it. These observations suggest that anatomic changes associated with chronic epilepsy alter propagation patterns. Because CA4 is believed to modulate the output of dentate granule cells and also has commissural connections to the contralateral homotopic area, the association of decreased CA4 cells with prolongation of IHSPT suggests that the observed anatomic alterations may actively (through increased inhibition) or passively (through decreased recruitment) interfere with various routes of seizure propagation.     

Functional connections and epileptic spread between hippocampus, entorhinal cortex and amygdala in a modified horizontal slice preparation of the rat brain

The hippocampus, the entorhinal cortex and the amygdala are interconnected structures of the limbic system that are implicated in memory and emotional behaviour. They demonstrate synaptic plasticity and are susceptible to development of temporal lobe epilepsy, which may lead to emotional and psychological disturbances. Their relative anatomical disposition has limited the study of neurotransmission and epileptic spread between these three regions in previous in vitro preparations. Here we describe a novel, modified-horizontal slice preparation that includes in the same plane the hippocampus, entorhinal cortex and amygdala. We found that, following application of bicuculline, each region in our preparation could generate spontaneous bursts that resembled epileptic interictal spikes. This spontaneous activity initiated in the hippocampal CA3/2 region, from where it propagated and controlled the activity in the entorhinal cortex and the amygdala. We found that this spontaneous bursting activity could spread via two different pathways. The first pathway comprises the well-known subiculum-entorhinal cortex-perirhinal cortex-amygdala route. The second pathway consists of a direct connection between the CA1 region and perirhinal cortex, through which the hippocampal bursting activity can spread to the amygdala while bypassing the entorhinal cortex. Thus, our experiments provide a new in vitro model of initiation and spread of epileptic-like activity in the ventral part of the limbic system, which includes a novel, fast and functional connection between the CA1 region and perirhinal cortex.     

Interictal-ictal interactions and limbic seizure generation.

Interictal discharges are used in clinical practice to localize the epileptogenic focus in patients with partial epilepsy. However, the interaction between interictal and ictal discharges remains debatable. For instance, interictal events may lead to seizure onset in some models of epileptiform discharge. By contrast, in other models, disappearance of interictal activity (for example by activation of GABAB receptors) induces or potentiates ictal events. We have recently obtained new evidence for a control exerted by interictal discharges on ictal activity in rodent combined slices of hippocampus-entorhinal cortex. In this preparation continuous application of 4-aminopyridine induces: (i) interictal activity which initiates in CA3 and propagates via CA1 and subiculum to the entorhinal cortex, and return to the hippocampus through the dentate gyrus; and (ii) ictal discharges, which originate in the entorhinal cortex and propagate via the dentate gyrus to the hippocampus. Ictal discharges disappear over time, while synchronous interictal discharges continue to occur. Lesioning the Schaffer collaterals abolishes interictal discharges in CA1, entorhinal cortex and dentate gyrus and discloses entorhinal ictal discharges that propagate, via the dentate gyrus, to the CA3 subfield. Interictal activity of CA3 origin also prevents the occurrence of ictal events recorded in the entorhinal cortex in Mg(2+)-free medium. Moreover, in both models, ictal discharge generation in the entorhinal cortex after Schaffer collateral cut is prevented by mimicking CA3 activity through rhythmic electrical stimulation of CA1 hippocampal outputs. Hence, our data demonstrate that hippocampus interictal discharges control the expression of electrographic seizures in entorhinal cortex. Sectioning the Schaffer collaterals may model the epileptic condition in which cell damage in the CA3 subfield results in loss of CA3 control over the entorhinal cortex. Hence, the functional integrity of hippocampal CA3 neurons may represent a critical control point in temporal lobe epilepsy.     

Differentiability of simulated MEG hippocampal, medial temporal and neocortical temporal epileptic spike activity.

Previous studies have shown that magnetoencephalography (MEG) can measure hippocampal activity, despite the cylindrical shape and deep location in the brain. The current study extended this work by examining the ability to differentiate the hippocampal subfields, parahippocampal cortex, and neocortical temporal sources using simulated interictal epileptic activity. A model of the hippocampus was generated on the MRIs of five subjects. CA1, CA3, and dentate gyrus of the hippocampus were activated as well as entorhinal cortex, presubiculum, and neocortical temporal cortex. In addition, pairs of sources were activated sequentially to emulate various hypotheses of mesial temporal lobe seizure generation. The simulated MEG activity was added to real background brain activity from the five subjects and modeled using a multidipole spatiotemporal modeling technique. The waveforms and source locations/orientations for hippocampal and parahippocampal sources were differentiable from neocortical temporal sources. In addition, hippocampal and parahippocampal sources were differentiated to varying degrees depending on source. The sequential activation of hippocampal and parahippocampal sources was adequately modeled by a single source; however, these sources were not resolvable when they overlapped in time. These results suggest that MEG has the sensitivity to distinguish parahippocampal and hippocampal spike generators in mesial temporal lobe epilepsy.     

Anticonvulsant effects of N6-cyclohexyladenosine microinjected into the CA1 region of the hippocampus on entorhinal cortex-kindled seizures in rats.

In this study, the role of adenosine A1 receptors of the hippocampal CA1 region in entorhinal cortex-kindled seizures was investigated in rats. Animals were kindled by daily electrical stimulation of the entorhinal cortex. In fully kindled rats, N(6)-cyclohexyladenosine (CHA; a selective A1 receptor agonist) and 1, 3-dimethyl-8-cyclopenthylxanthine (CPT; a selective A1 receptor antagonist) were microinfused bilaterally into the hippocampal CA1 region. Rats were stimulated and seizure parameters were measured. Results obtained showed that CHA (10 and 50 micro moles) decreased the afterdischarge duration (ADD) in the hippocampal CA1 region and entorhinal cortex, stage 5 seizure duration (S5D) and seizure duration (SD) only at the dose of 50 micro moles, and significantly increased the latency to stage 4 (S4L). Intrahippocampal CPT increased ADD and S5D, and significantly reduced the latency to stage 4 (S4L) at the dose of 10 micromoles. Pretreatment of rats with CPT (5 micro moles) before CHA (50 micro moles), significantly reduced the effect of CHA on seizure parameters. The results suggest that the CA1 region of the hippocampus plays an important role in spreading seizure spikes from the entorhinal cortex to other brain regions and activation of adenosine A1 receptors in this region participates in the anticonvulsant effects of adenosine agonists.