Ictogenesis

Abstract

*Michel Le Van Quyen, †Pascale Quilichini, †Yehezkel Ben‐Ari, †Christophe Bernard, and †Henri Gozlan ( *Neurodynamics Group, LENA‐CNRS UPR640, Hôpital de la Salpêtrière, Paris , and †INMED‐INSERM U 29 Marseille, France ).Network oscillations in the gamma‐frequency band (40–100 Hz) have a central role in the transition to seizure. Here we analyzed spontaneous ictal‐like events (ILE) at distinct postnatal periods P1–P8 in intact rat corticohippocampal formations (CHF) exposed to low Mg2+ artificial cerebrospinal fluid (Quilichini et al., 2002). Quantitative time‐frequency analyses of field recordings showed that gamma oscillations can be observed very early at postnatal day (P) 3. Between P3–P5, spontaneous gamma oscillations were seen as brief bursts (<200 ms) at 30–60 Hz frequencies, superposed on slow epileptiform activities at the beginning of the ILE. After P6, the oscillations gained amplitude and extended also to higher frequencies (>100 Hz). The study of simultaneous recordings of various regions (CA1, CA3, dentate gyrus, neocortex, two interconnected CHF) showed that gamma oscillations primarily emerge in the hippocampal circuitries, but propagate to all other brain structures. Gamma oscillations persist after blockade of GABAA receptors by bicuculline or after blockade of glutamate receptors by CNQX. Furthermore, gamma oscillations are not sensitive to the gap junction blocker, carbonexolone. This suggests that an interactive network of interneurons may play a pivotal role in the gamma oscillations of early life seizures. Surprisingly, their generation seems not to be exclusively associated with GABAergic synaptic interactions or with electrotonic coupling. We conclude that intact and immature brain structures provide a unique opportunity to study in vitro the implication of gamma oscillations in ictogenesis, as well as their involvement in the production of chronic epileptic conditions.*James W.Y. Chen and *Arthur W. Toga ( *Department of Neurology, The David Geffen School of Medicine at UCLA, Los Angeles, CA, U.S.A. ). Purpose: It was noted in our study that optical intrinsic signal (OIS) imaging represents seizure activity with a high degree of correlation (Neurology 2000;55:312–5). Our group also reported triphasic OIS changes during cortical spreading depression (CSD) (NeuroReport 2000;11:2121–5). Our recent findings in OIS imaging of acute seizures are summarized. Methods: Please refer to the listed publications for methods. Results: (a) In 14 rats for which optical CSD was induced by seizures, the CSD showed characteristic triphasic responses, which spread symmetrically in all directions, at a rate of ∼3.5 mm/min. The optical CSD showed an intricate back‐and‐forth reciprocal interaction, with the seizure activity at the interface of CSD and seizures. (b) In eight rats, before epileptiform discharges were detectable on EEG, the region near penicillin application increased light reflectance (∼10% changes from the baseline). The surrounding region showed decreased light reflectance (∼5% changes from the baseline). During seizure induction, the central region gradually decreased, and the reversal of the light reflectance changes coincided with the onset of spikes. (c) In 10 rats, OIS imaging at 610 nm, which detected deoxyhemoglobin concentration changes, was sensitive to the early detection of seizures. Imaging at 610 nm does not follow spike‐to‐spike fluctuation well, but instead shows a tonic response correlating with the overall seizure intensity. This response dissociates from seizure intensity when seizures intensify. Imaging at 850 nm, known to correlate with neuronal activation, shows “optical spikes” that correlate well with single spikes on EEG. Conclusions: (a) Seizure‐induced CSD showed reciprocal inhibitory effects on seizure activity. (b) OIS imaging at 610 nm detected optical changes in the preictal phase during seizure induction, and reversal of light reflectance could define seizure onset. (c) Imaging at 610 nm represented overall seizure intensity and, at 850 nm, correlated well with single spikes. (Supported by NINDS grant K08 NS42708.)*Massimo Avoli, *Giuseppe Biagini, *Giovanna D'Arcangelo, *Margherita D'Antuono, and *Virginia Tancredi ( *MNI/McGill University, Montreal, Quebec, Canada ).Mesial temporal lobe epilepsy (MTLE) patients present with seizures involving the limbic system and with a pattern of brain damage characterized by neuronal loss in CA1/CA3 areas, dentate hilus, and entorhinal cortex (EC), layer III (Houser CR. Adv Neurol 1999;79:743–61). Similar findings are seen in laboratory animals following pilocarpine injection (Turski WA, et al. Behav Brain Res 1983;9:315–35). This procedure induces an initial convulsive response, which is followed within 2–3 weeks by recurrent seizures. Limbic network hyperexcitability in MTLE and in animal models results from seizure‐induced brain damage leading to (a) synaptic reorganization (Cavazos JE, et al. J Neurosci 1991;11:2795–803; Houser CR. Adv Neurol 1999;79:743–61) and (b) changes in GABA receptor–mediated inhibition (Buhl EH, et al. Science 1996;271:369–7; Doherty J, Dingledine R. J Neurosci 2001;21:2048–57. However, it is unclear how these changes lead to a chronic epileptic condition.CA3‐driven interictal activity induced in normal brain tissue by epileptogenic stimuli inhibits the EC from generating ictal discharges (Barbarosie M, Avoli M. J Neurosci 1997;17:9308–14), suggesting that CA3 damage causes a decrease of hippocampal output activity that would release EC ictogenesis and establish a chronic epileptic condition. Accordingly, slices obtained from pilocarpine‐treated epileptic mice respond to 4‐aminopyridine (4AP) application by generating (a) CA3‐driven interictal activity that is less frequent than in nonepileptic control (NEC) tissue, and (b) ictal discharges that do not disappear over time and propagate to the CA1‐subiculum via the temporoammonic path (D'Antuono M, et al. J Neurophysiol 2002;87:634–9). From these findings, we predicted that limbic seizures result from EC–subiculum interactions. Using brain slices obtained from pilocarpine‐treated, epileptic rats, we found that decreased CA3 output function, along with reverberation between EC and subiculum networks, lead to in vitro epileptogenesis. First, intense activation of EC and subiculum was identified with intrinsic optical signal (IOS) recordings in pilocarpine‐treated, but not in NEC slices. Second, using field potential recordings during 4AP application, we established that CA3‐driven interictal activity occurs at lower frequency in pilocarpine‐treated slices and that disconnection of the EC from the subiculum attenuates 4AP‐induced ictal discharges in pilocarpine‐treated, but not in NEC slices. Third, the distribution of FosB/FosB‐related proteins in epileptic tissue demonstrated distinct patterns overlapping those seen with IOS recordings, with the highest intensity in layer III of the lateral EC.In conclusion, our data show that hippocampal damage in epileptic rats, and perhaps in MTLE patients, hampers the ability of CA3 output activity to control ictogenesis in the EC. Such a process is reinforced by interactions between subiculum and EC networks.*Ricardo Mario Arida, *Fulvio Alexandre Scorza, *Reinaldo de Amorin Carvalho, and *Esper Abrão Cavalheiro ( *Neurologia Experimental‐Escola Paulista de Medicina, São Paulo, Brazil ).The potential interest of Proechimys guyannensis (PG), a spiny rat living in the Amazonian region, as an animal model of anticonvulsant mechanisms prompted the investigation of the susceptibility of PG to different epileptogenic paradigms. The findings pointed out a remarkable resistance of these animals to different models of experimental epilepsy. (1) Amygdala kindling development. Proechimys animals demonstrated a striking resistance to reaching stage 5 of kindling. Of 43 Proechimys rats submitted to the kindling process, only three animals reached stage 5. Of 40 animals that did not reach the kindled state, 16 did not get beyond stage 1, 15 did not get beyond stage 2, seven did not get beyond stage 3, and three did not get beyond stage 4. Amygdala electrical stimulations were followed by very long afterdischarges, mainly in stages 1–4. (2) Intrahippocampal kainic acid (KA). A remarkable sensibility to intrahippocampal KA was noticed in PG. One‐tenth of the KA dose usually used in Wistar rats elicited self‐sustained electrographic status epileptus (SE) in PG animals, which lasted for >48 h with increased mortality rate. On the other hand, none of the surviving animals presented spontaneous seizures in the long‐term observation period (up to 120 days). Neuropathological examinations of the hippocampus of Proechimys animals after KA injection showed a complete neuronal destruction at the injected hippocampal formation, more pronounced in CA1/CA3 areas, and with less marked changes in the contralateral hippocampus. (3) Pilocarpine. Pilocarpine (350–380 mg/kg, doses regularly used in Wistar rats), when administered to PG, induced severe tonic seizures followed by death of all animals. A dose slightly lower (300 mg/kg) than those previously mentioned was able to induce repetitive electrographic and behavioral seizures that culminated in SE 20–30 min following pilocarpine. However, pilocarpine‐induced SE in PG had a shorter duration, rarely exceeding 2 h, clearly in contrast to the 8‐ to 12‐h long SE in the Wistar rat. Of 60 animals injected with pilocarpine, 48 presented with SE and only two with presented some spontaneous seizures (∼1/week for 8 weeks) after silent periods of 60 and 66 days. The histological analysis of the brain of these two animals revealed neuronal loss in the CA3 area, in the hilus of the dentate gyrus (DG) and mossy fibers sprouting in the supragranular layer of DG, and in the stratum radiatum of CA3. Altogether these data indicate that PG, although extremely sensitive to chemical convulsants (i.e., KA and pilocarpine) and presenting longer afterdischarge following electrical stimulation of the amygadala, is unable to establish a circuitry appropriated to the elaboration of spontaneous seizures, i.e., epilepsy. In other words, it seems probable that limbic circuitries, such as those found in the amygdala or the hippocampus, are highly affected by excitatory stimulation. However, some “external factors,” such as inputs originating in extralimbic areas, seem to prevent or inhibit the formation of a true epileptic focus. Some behavioral aspects observed in PG during the three experimental situations described previously give hints that this hypothesis may be justified. During amygdala kindling stimulation, and also during the seizures observed after systemic pilocarpine or intrahippocampal KA, a cataleptic behavior was clearly observed in PG animals characterized by opistotonus and S tail, suggesting the participation of the opiate system in this process. These findings indicate that PG rats may have natural endogenous antiepiletic mechanisms and further investigations (anatomical, biochemical, etc.) need to be carried out to clarify this phenomenon.*Stiliyan Kalitzin, *Piotr Suffczynski, †Demetrios Velis, †Jaime Parra, and ‡Fernando Lopes Da Silva ( *Medical Physics Department , †Department of Clinical Neurophysiology , and ‡Workgroup Advanced Diagnostics, SEIN ).We previously have shown that a measure of enhanced temporal phase consistency, called relative phase clustering index (rPCI), measured in EEG/MEG signals during intermittent photic stimulation of patients with photosensitive epilepsy, can be related to the probability of eliciting photoparoxysmal responses. We apply the same paradigm in temporal lobe epilepsy patients with implanted electrodes. The analysis of periictal SEEG data collected during electric stimulation with periodic pulse trains shows that high rPCI values are indicative of the seizure onset site. In addition, a further increase of rPCI extensive to neighboring recording sites suggests increased probability for seizure occurrence. In this contribution, neural network simulations yielded the following major results: (a) A realistic corticothalamic model has a pair of simultaneously existing dynamic phase space attractors. A steady‐state attractor corresponds to a normal (interictal) state and a limit cycle type of attractor represents ictal activity. (b) The introduction of additional fast inhibitory synapses in the population of cortical interneurons leads to gamma‐frequency resonance‐generating rPCI. (c) Increasing the slope of the response function of the cortical interneurons or the average intracortical excitation leads to both increasing the levels of rPCI and to facilitation of the ictal transition. (d) Similar hippocampal neural network model confirms the presence of high PCI in the gamma band (>40 Hz) with no significant amplitude increase.REFERENCES1. Kalitzin S, Parra J, Velis D, Lopes da Silva F. IEEE-TBME 2002;49: 1279–86. 2. Parra J, Kalitzin S, Iriarte J, Blanes W, Velis D, Lopes da Silva F. Brain 2003;126: 1164–72. 3. Velis DN, Kalitzin SN, Van Engelen FAM, Lopes da Silva FH. Epilepsia 2002;43():51. 4. Kalitzin S, Velis D, Parra J, Blanes W, Van Engelen F, Suffczynski P, Lopes da Silva F. Physica Medica 2003;XIX: 67. *Claire Leroy, †Catherine Roch, ‡Pierrick Poisbeau, †Izzie Jacques Namer, and *Astrid Nehlig ( *Psychopathologie et Pharmacologie de la Cognition INSERM U405 , †Institut de Physique Biologique UMR 7004 ULP/CNRS , and ‡Neurophysiologie Cellulaire et Intégrée ULP/CNRS 7519, Strasbourg, France ). Purpose: Epileptic seizures originating in the temporal lobe are one of the main intractable epilepsies in humans. Hippocampal sclerosis and parahippocampal dysfunctions are frequently observed in patients with temporal lobe epilepsy (TLE). However, the respective roles of the different structures in epileptogenesis remain to be clarified, and also whether hippocampal sclerosis is the cause or the consequence of epilepsy. We used the lithium‐pilocarpine (Li‐pilo) model of epilepsy in 21‐day‐old (P21) and adult rats, which reproduce most clinical, neuropathological, and developmental features of TLE. The consequences of Li‐pilo‐induced status epilepticus (SE) are age dependent, and while all adult rats become epileptic, only a subset of P21 rats will develop epilepsy. Methods: In adult and P21 rats, we studied neuropathologic, vascular, and metabolic changes at different times to identify the critical structures in epileptogenesis and maintenance of spontaneous seizures. (a) T2‐weighted sequences of MRI (4,7 T) and neuronal counting (thionine staining) were performed to determine morphological changes and neuronal damage. (b) Autoradiography with 14C‐a‐aminoisobutyric acid (AIB) was performed 90 min after the onset of SE to determine the role of the permeability of the blood–brain barrier (BBB) in the establishment of early neuronal damage. (c) 14C‐2‐deoxyglucose autoradiography was used to assess changes in local cerebral glucose use that occur during epileptogenesis and epilepsy. (d) Finally, in adult rats, whole‐cell patch clamp recording was performed in the granule cells of the dentate gyrus to study changes in synaptic GABAA receptors during epileptogenesis and epilepsy. Results: There was no clear correlation between the anatomical distribution of BBB opening during SE and occurrence of neuronal damage, which appeared to rather depend on excitotoxic mechanisms due to major neuronal hyperactivity. MRI and histological data showed that entorhinal and piriform cortices were the initial structures damaged (as soon as 6 h after onset of SE). The lack of signal in piriform and entorhinal cortices in P21 rats that did not become epileptic confirmed the critical role of these cortices in the early establishment of epileptic networks. On the other hand, the development of hippocampal sclerosis was delayed (36/48 h after SE onset) and progressively worsened; this delayed damage could be the consequence of the primary cortical injury or reflect different properties of hippocampal neurons. The late involvement of hippocampus in epileptogenesis appears as a relative hypermetabolism compared to the number of neurons surviving in the hilus of the dentate gyrus in adult and P21 epileptic rats during the silent and chronic phase of this epilepsy. Finally, the electrophysiological properties of granule cells of adult rats subjected to Li‐pilo SE, which are the first input of the perforant path to the hippocampus, showed alterations of the pharmacological properties of GABAA receptors as soon as 24 h after SE onset that progressed after the appearance of epilepsy. Conclusions: The results detailed above illustrate that the use of a variety of techniques in rats at different ages allowed characterization of the nature of the structures involved in the process of epileptogenesis in the Li‐pilo model of TLE.* Diogo Lösch De Oliveira, * Ingrid Schweigert, * Fernando Scheibel, * Diogo O. Souza, †Suzana Wofchuk, and * Luiz Santos Perry ( *Dep. Bioquímica–Instituto de Ciências Básicas da Saúde & Departamento de Ciências da Saúde–UNIJUI , and †Faculdade de Farmácia, Pontificia Universidade Católica do Rio Grande do Sul Marcos ).The population groups most vulnerable to undernutrition are women and young children. Undernutrition in the form of protein‐energy malnutrition remains high in parts of some countries and affects mainly preschool children. Undernutrition can be seen in the prevalence of low‐birthweight babies born to underweight mothers. This has an impact on morbidity and mortality from infectious diseases and on the full mental and physical development of children. Nevertheless, there are few works correlating undernutrition and seizures. In this work, we investigate the effect of gestational and postnatal undernutrition on the picrotoxin‐induced seizures and on GABA uptake on cerebral cortex slices. Young Wistar rats (P25) of both sexes were obtained from a local breeding colony. For picrotoxin‐induced seizures, animals were divided in two groups: (a) rats that received a diet with 25% of casein (G25) and (b) undernourished rats that received a diet with 7% of casein (G7). Picrotoxin was administered i.p. at 1 mL/kg. Animals were observed for 60 min for latency and occurence of seizures. The seizures were classified as: (a) minimal seizures characterized by clonuses of head muscles and forelimbs, with righting ability preserved, and (b) major seizures usually beginning with running followed by a loss of posture, followed by the tonic phase and, after some seconds, occurrence of long‐lasting clonic seizures of all limbs (status epilepticus). Only major seizures were considered. For GABA uptake assay, the animals were divided in four groups: (a) G25, (b) G7, (c) G25, which received an i.p. picrotoxin injection, and (d) G7, which received an i.p. picrotoxin injection. The doses of picrotoxin used for GABA uptake experiments were doses that caused seizures in all animals. Lower doses of picrotoxin, 3.2 and 4.0 mg/kg, induced major seizures in 11% and 60% of undernourished animals, respectively. However, G25 group showed no major seizures with 3.2 mg/kg and 20% with 4.0 mg/kg. At the 4.8 mg/kg dose, picrotoxin induced seizures in 100% and 60% of G25 and G7 groups, respectively. Higher doses of picrotoxin (5.6 and 6.4 mg/kg) induced seizures in all animals from both groups. Animals submitted to a protein‐restricted diet showed a decreased latency compared with the G25 animals. The undernutrition increased the GABA uptake (0.099 nmol/mg protein/min; p < 0.05) compared to the G25 group (0.065 nmol/mg protein/min). The picrotoxin had no effect on the GABA uptake in any group. Therefore, protein malnutrition decreased resistance to picrotoxin‐induced seizures but increased GABA uptake in young rats. While very little is known about the relationships between protein malnutrition and GABAergic signaling, the regulatory mechanisms involving the picrotoxin susceptibility and GABA uptake are under investigation in our lab.