NEURONAL MECHANISMS OF EPILEPTOGENESIS Topic Editor Roberto Di Maio CELLULAR NEUROSCIENCE Frontiers in Cellular Neuroscience December 2014 | Neuronal mechanisms of epileptogenesis | 1 ABOUT FRONTIERS Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals. FRONTIERS JOURNAL SERIES The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing. 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Cover image provided by Ibbl sarl, Lausanne CH ISSN 1664-8714 ISBN 978-2-88919-382-0 DOI 10.3389/978-2-88919-382-0 Frontiers in Cellular Neuroscience December 2014 | Neuronal mechanisms of epileptogenesis | 2 Topic Editor: Roberto Di Maio, University of Pittsburgh, USA; Ri.MED Foundation, Palermo, Italy NEURONAL MECHANISMS OF EPILEPTOGENESIS Frontiers in Cellular Neuroscience December 2014 | Neuronal mechanisms of epileptogenesis | 3 Table of Contents 05 Neuronal Mechanisms of Epileptogenesis Roberto Di Maio 07 Perirhinal Cortex and Temporal Lobe Epilepsy Giuseppe Biagini, Margherita D'Antuono, Ruba Benini, Philip de Guzman, Daniela Longo and Massimo Avoli 17 Homeostatic Control of Brain Function – New Approaches to Understand Epileptogenesis Detlev Boison, Ursula S. Sandau, David N. Ruskin, Masahito Kawamura Jr. and Susan A. Masino 29 The Role of Dopamine Signaling in Epileptogenesis Yuri Bozzi and Emiliana Borrelli 41 Changes in the Sensitivity of GABA A Current Rundown to Drug Treatments in a Model of Temporal Lobe Epilepsy Pierangelo Cifelli, Eleonora Palma, Cristina Roseti, Gianluca Verlengia and Michele Simonato 48 Resilience to Audiogenic Seizures is Associated with p-ERK1/2 phosphorylation in the Subiculum of Fmr1 Knockout Mice Giulia Curia, Fabio Gualtieri, Regina Bartolomeo, Riccardo Vezzali and Giuseppe Biagini 61 K + Channelepsy: Progress in the Neurobiology of Potassium Channels and Epilepsy Maria Cristina D'Adamo, Luigi Catacuzzeno, Giuseppe Di Giovanni, Fabio Franciolini and Mauro Pessia 82 The Possible Role of GABA A Receptors and Gephyrin in Epileptogenesis Marco I. Gonzalez 89 Contribution of Apoptosis-Associated Signaling Pathways to Epileptogenesis: Lessons From Bcl-2 Family Knockouts David C.Henshall and Tobias Engel 100 Molecular Mechanism of Circadian Rhythmicity of Seizures in Temporal Lobe Epilepsy Chang-Hoon Cho 109 Neural Circuit Mechanisms of Post–Traumatic Epilepsy Robert F. Hunt, Jeffery A. Boychuk and Bret N. Smith Frontiers in Cellular Neuroscience December 2014 | Neuronal mechanisms of epileptogenesis | 4 123 Expressional Analysis of the Astrocytic Kir4.1 Channel in a Pilocarpine–Induced Temporal Lobe Epilepsy Model Yuki Nagao, Yuya Harada, Takahiro Mukai, Saki Shimizu, Aoi Okuda, Megumi Fujimoto, Asuka Ono, Yoshihisa Sakagami and Yukihiro Ohno 133 Role of Hormones and Neurosteroids in Epileptogenesis Doodipala Samba Reddy 153 Age Dependency of Trauma-Induced Neocortical Epileptogenesis Igor Timofeev, Terrence J. Sejnowski, Maxim Bazhenov, Sylvain Chauvette and Laszlo B. Grand 167 Dentate Gyrus Network Dysfunctions Precede the Symptomatic Phase in a Genetic Mouse Model of Seizures Oana Toader, Nicola Forte, Marta Orlando, Enrico Ferrea, Andrea Raimondi, Pietro Baldelli, Fabio Benfenati and Lucian Medrihan 182 Are Vesicular Neurotransmitter Transporters Potential Treatment Targets for Temporal Lobe Epilepsy? Joeri Van Liefferinge, Ann Massie, Jeanelle Portelli,Giuseppe Di Giovanni and Ilse Smolders 206 Seizure-Like Activity in Hyaluronidase-Treated Dissociated Hippocampal Cultures Maria Vedunova, Tatiana Sakharnova, Elena Mitroshina, Maya Perminova, Alexey Pimashkin, Yuri Zakharov, Alexander Dityatev and Irina Mukhina 216 Immune Mechanisms in Epileptogenesis Dan Xu, Stephen D. Miller and Sookyong Koh EDITORIAL published: 21 February 2014 doi: 10.3389/fncel.2014.00029 Neuronal mechanisms of epileptogenesis Roberto Di Maio* Department of Neurology, Pittsburgh Institute for Neurodegenerative Disease, Ri.MED Foundation, University of Pittsburgh, Pittsburgh, PA, USA *Correspondence: rdimaio@hs.pitt.edu Edited and reviewed by: Egidio D’Angelo, University of Pavia, Italy Keywords: epileptogenesis, neuronal epileptic damage, hippocampal damage, epilepsy, Temporal Lobe Epilepsy (TLE), TLE prevention The primary purpose of this topic is to collect scientific contri- butions providing novel insights in the cellular and molecular mechanisms of epileptogenesis as potential targets for innovative therapeutic approaches aimed at preventing the chronic epileptic disorder. Prevention of chronic epileptic disorder with an appropri- ate intervention might represent the most ambitious goal in the clinical treatment of this epileptic disorder, but has been largely unsuccessful to this point. Clinical trials aimed at prevention of chronic epilepsy have often produced negative, disappointing results. However, in most cases, these studies ultimately evaluated the downstream clinical manifestations, failing to monitor early, specific molecular epileptogenic events. Therefore, elucidation of the underlying mechanisms of epileptogenesis, are essential. Several types of brain injuries are causes of acquired epilepsy, including brain trauma, one of the most common causes of idiopathic epilepsy (Hunt et al., 2013; Timofeev et al., 2013). Genetic mutations enhancing structural and functional alter- ations of key proteins including pre-synaptic complexes (Toader et al., 2013) and potassium channels (D’Adamo et al., 2013) are also related to the occurrence of epileptic disorders. Consistently with these findings obtained in genetic animal models of epilepsy, studies conducted in animal models of acquired epilepsy addressed the critical role of vesicular neurotransmitters trans- porters (VNTs) (Van Liefferinge et al., 2013) and non-neuronal potassium channel (Kir4.1) (Nagao et al., 2013) expression during epileptogenesis. Temporal Lobe Epilepsy (TLE) is the most common form of refractory epileptic disorder often related to childhood seizures. The symptomatic manifestations of TLE appear only after a widespread irreversible damage of entorhinal cortex (Bartolomei et al., 2005), hippocampus (Mathern et al., 2002) and perirhi- nal cortex, which has a major role in the spread of limbic seizures (Biagini et al., 2013), These pathological features of TLE reduce the possibility of successful therapeutic approaches, often rendering the disease refractory. The difficult clinical manage- ment of chronic TLE and the limited success rate of surgical approaches, increase the incapacitating nature of this specific epileptic disorder. Despite its complex etiology, a common feature of the epileptic disorders is a paroxysmal excitatory activity, which is able to pro- duce the same pathological features that are ultimately recognized clinically as epileptic disease. Only recently the role of oxidative stress in epilepsies has begun to be recognized. Neuronal hyper-excitability is associated with a calcium-dependent activation of intracellular oxidant systems, including NOX2, which is the major NMDAR-regulated source of superoxide (Di Maio et al., 2011). This early phenomenon occurring during the epileptic onset might be responsible for the long-term neuronal dysfunction leading to the chronic epileptic disorder (Di Maio et al., 2012). Excitatory/inhibitory unbalance and oxidative-related events might be determinant in the epileptic pathogenesis of neu- ronal networks mediating a complex disruption of self-regulatory homeostatic mechanisms such as the bioenergetics systems (Boison et al., 2013). Epileptic neurons may develop short and long-term adap- tive changes in sensitivity to GABA-ergic neurotransmission by means of GABA A receptor (Cifelli et al., 2013), worsening the excitatory/inhibitory unbalance and reducing the possibil- ity of successful therapeutic approaches with the conventional Antiepileptic Drugs. Interesting insights have been recently pro- vided on this regard. Epileptogenic changes of GABA A recep- tor may be caused by altered expression of scaffolding proteins involved in the trafficking and anchoring of GABA A recep- tors. This phenomenon could directly impact the stability of GABA-ergic synapses and promote impairment of the neuronal response to the inhibitory GABA-ergic input. These findings offer novel potential therapeutic targets to prevent the development of epilepsy. Dopaminergic projections to limbic system play also a crit- ical role in the control of seizures. Dopaminergic activity in limbic structure exerts a complex neuromodulation of neu- ronal excitability mainly through D1 and D2 receptors subtypes. Impairment of the fine tuning mediated by dopamine (DA) receptors activity can contribute to spread of seizures in the lim- bic system. Recent evidences on the identification of intracellular signaling pathways activated by DA receptors activity are leading to promising studies aimed at the identification of novel targets for the treatment of epilepsy (Bozzi and Borrelli, 2013). An increasing number of experimental evidences suggest a major involvement of inflammation in epileptogenesis. Seizure activity elicits release of pro-inflammatory cytokines and acti- vates immune responses. These phenomena have been widely related to an increased brain susceptibility to seizure, synaptic reorganization and neuronal death (Xu et al., 2013). Inflammatory processes in brain can affect the extracellular neuronal matrix (ECM) integrity. ECM plays a critical role in the modulation of AMPA receptor mobility, paired-pulse depres- sion, L-type voltage-dependent Ca 2 + channel activity and LTP processes. Noteworthy, an original study published in this topic, suggests that changes in the expression of Hyaluronic acid, the Frontiers in Cellular Neuroscience www.frontiersin.org February 2014 | Volume 8 | Article 29 | CELLULAR NEUROSCIENCE 5 Di Maio Neuronal mechanisms of epileptogenesis major component of neuronal ECM, can lead to neuronal hyper- excitability and calcium dysregulation (Vedunova et al., 2013). Neuronal cell death has been implicated as a causal factor leading to the development of the epileptic disorder. The find- ings reported in this topic support the idea that repeated seizures mediate neuronal necrosis and apoptosis prevalently associated to the activation of certain distinct anti/pro-apoptotic Bcl-2 family factors. Thus, epileptogenesis elicits apoptotic events by means of a specific pattern of Bcl-2 family proteins, which might represent a possible target of intervention to protect against the epileptic damage (Henshall and Engel, 2013). Hormones play an important role in the epileptic disorders. Corticosteroids, progesterone, estrogens, and neurosteroids have been shown to affect seizure activity in animal models and in human. However, the impact of hormones on epileptogenesis is still underexplored and controversial. Further studies are required in the field to generate evidences on the therapeutic potential of hormonal agents in epileptogenesis (Reddy, 2013). The circadian pattern of seizures is one of the first phenomena described in the epileptic disorders. However, due to the lack of promising hypotheses, has not attracted enough scientific atten- tion. Recent findings provide novel insights in the implication of circadian rhythm in modulating transcription factors governing clock genes expression, and the mTOR signaling pathway, one of the most relevant signaling pathway in epilepsy (Cho, 2012). REFERENCES Bartolomei, F., Khalil, M., Wendling, F., Sontheimer, A., Regis, J., Ranjeva, J. P., et al. (2005). 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Immune mechanisms in epileptogenesis. Front. Cell. Neurosci. 7:195. doi: 10.3389/fncel.2013.00195 Received: 09 December 2013; accepted: 03 February 2014; published online: 21 February 2014. Citation: Di Maio R (2014) Neuronal mechanisms of epileptogenesis. Front. Cell. Neurosci. 8 :29. doi: 10.3389/fncel.2014.00029 This article was submitted to the journal Frontiers in Cellular Neuroscience. Copyright © 2014 Di Maio. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or repro- duction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Cellular Neuroscience www.frontiersin.org February 2014 | Volume 8 | Article 29 | 6 REVIEW ARTICLE published: 29 August 2013 doi: 10.3389/fncel.2013.00130 Perirhinal cortex and temporal lobe epilepsy Giuseppe Biagini 1 , Margherita D’Antuono 2 , Ruba Benini 2 , Philip de Guzman 2 , Daniela Longo 1 and Massimo Avoli 2,3 * 1 Laboratory of Experimental Epileptology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy 2 Montreal Neurological Institute and Departments of Neurology and Neurosurgery and of Physiology, McGill University, Montréal, QC, Canada 3 Faculty of Medicine and Dentistry, Department of Experimental Medicine, Sapienza University of Rome, Roma, Italy Edited by: Roberto Di Maio, University of Pittsburgh, USA Reviewed by: Marco Capogna, Medical Research Council, UK Roberto Di Maio, University of Pittsburgh, USA *Correspondence: Massimo Avoli, Montreal Neurological Institute and Departments of Neurology and Neurosurgery and of Physiology, McGill University, 3801 University Street, Montréal, QC H3A 2B4, Canada e-mail: massimo.avoli@mcgill.ca The perirhinal cortex—which is interconnected with several limbic structures and is intimately involved in learning and memory—plays major roles in pathological processes such as the kindling phenomenon of epileptogenesis and the spread of limbic seizures. Both features may be relevant to the pathophysiology of mesial temporal lobe epilepsy that represents the most refractory adult form of epilepsy with up to 30% of patients not achieving adequate seizure control. Compared to other limbic structures such as the hippocampus or the entorhinal cortex, the perirhinal area remains understudied and, in particular, detailed information on its dysfunctional characteristics remains scarce; this lack of information may be due to the fact that the perirhinal cortex is not grossly damaged in mesial temporal lobe epilepsy and in models mimicking this epileptic disorder. However, we have recently identified in pilocarpine-treated epileptic rats the presence of selective losses of interneuron subtypes along with increased synaptic excitability. In this review we: (i) highlight the fundamental electrophysiological properties of perirhinal cortex neurons; (ii) briefly stress the mechanisms underlying epileptiform synchronization in perirhinal cortex networks following epileptogenic pharmacological manipulations; and (iii) focus on the changes in neuronal excitability and cytoarchitecture of the perirhinal cortex occurring in the pilocarpine model of mesial temporal lobe epilepsy. Overall, these data indicate that perirhinal cortex networks are hyperexcitable in an animal model of temporal lobe epilepsy, and that this condition is associated with a selective cellular damage that is characterized by an age-dependent sensitivity of interneurons to precipitating injuries, such as status epilepticus Keywords: cholecystokinin, hippocampal formation, interneurons, neuropeptide Y, parvalbumin, perirhinal cortex, pilocarpine, temporal lobe epilepsy BACKGROUND The perirhinal cortex is a limbic structure that is closely inter- connected with the lateral entorhinal cortex, the amygdala, and with unimodal and polymodal association cortices (Suzuki and Amaral, 1994; Burwell et al., 1995; Kealy and Commins, 2011). Hippocampal networks exchange information with the neocor- tex through the rhinal cortices (Van Hoesen, 1982; Naber et al., 1999; Kealy and Commins, 2011) ( Figure 1 ), and it has been consistently demonstrated that the perirhinal cortex is intimately involved in learning and memory (Zola-Morgan et al., 1989; 1993; Murray et al., 1993; Suzuki et al., 1993; Suzuki, 1996; Weintrob et al., 2007; Kealy and Commins, 2011). Knowledge on the memory functions of the perirhinal cor- tex has been obtained from patients presenting with temporal lobe epilepsy. Initial observations in patients undergoing epilepsy neurosurgery reported vivid recollection or sensation of familiar- ity known as déjà vu and déjà vécu when the temporal lobe was electrically stimulated (Penfield and Perrot, 1963; Bancaud et al., 1994). In addition, Bartolomei et al. (2004) found that similar experiential phenomena were elicited more frequently by stimu- lating the rhinal cortices than the amygdala or the hippocampus. Specifically, they reported that déjà vu was obtained following stimulation of the entorhinal cortex, whereas reminiscence of memories occurred during perirhinal cortex stimulation. The perirhinal cortex has also been investigated for the poten- tial contribution of this region to ictogenesis in the limbic system (McIntyre and Plant, 1989; Kelly and McIntyre, 1996). Pioneering investigations based on the kindling protocol identified the amyg- dala and the piriform cortex as major epileptogenic areas (Kelly and McIntyre, 1996). For this reason, McIntyre and his collabo- rators proposed an in vitro amygdala-piriform slice preparation to characterize the properties of these limbic areas. Because of the limited spontaneous epileptiform activity observed in the slice preparation, they challenged neuronal networks with a modi- fied bathing medium, devoid of magnesium; this experimental procedure revealed a prominent epileptiform activity that was generated in the perirhinal cortex (McIntyre and Plant, 1989). These findings gave rise to a series of in vivo experiments demon- strating that: (i) the piriform cortex is not crucial in the spread of seizures originated in the hippocampus; (ii) the perirhinal cortex is kindled in a faster manner compared to other limbic regions and, above all, presents with the lowest latency to seizure spread Frontiers in Cellular Neuroscience www.frontiersin.org August 2013 | Volume 7 | Article 130 | CELLULAR NEUROSCIENCE 7 Biagini et al. Perirhinal cortex in epilepsy FIGURE 1 | Scheme of the main afferent/efferent connections of the perirhinal cortex (PC, area 35) under physiological conditions. The drawing corresponds to a section taken at 7 .6 mm from the bregma according to the Paxinos and Watson (2007) atlas. Major afferents projections to the perirhinal cortex (blue arrows) originate from olfactory insular and piriform cortices, lateral amygdala (LA) and entorhinal cortex (EC); conspicuous efferent projections of the perirhinal cortex (red arrows) are directed to these areas as well. Note that subcortical efferents from the perirhinal cortex (green arrows) terminate in several brain regions, including the basal ganglia (cf., Furtak et al., 2007). to frontal cortex motor areas; and (iii) the posterior region of the perirhinal cortex is critical to the propagation of hippocampal seizures (Kelly and McIntyre, 1996). Compared to other limbic areas, the perirhinal cortex remains overlooked, and in particular detailed information on its dysfunc- tional characteristics are scarce. Over the last decade, however, some studies have begun to unveil the fundamental electrophys- iological properties and the morphological features of perirhinal cortex cells (Bilkey and Heinemann, 1999; Faulkner and Brown, 1999; Beggs et al., 2000; D’Antuono et al., 2001; Furtak et al., 2007). In addition, new pathophysiological roles for this limbic structure in epileptogenesis and ictogenesis are emerging. Our paper is aimed at: (i) reviewing the electrophysiological charac- teristics of neurons that are recorded in the perirhinal cortex in an in vitro slice preparation; (ii) summarizing data regarding the ability of perirhinal cortex neuronal networks to generate epilep- tiform discharges when challenged with acute epileptogenic phar- macological procedures; (iii) highlighting the changes in neuronal excitability that occur in the pilocarpine model of temporal lobe epilepsy; and (iv) elucidating the contribution of selective interneuron subtype damage in promoting epileptogenesis. FUNDAMENTAL INTRINSIC AND SYNAPTIC PROPERTIES Intracellular studies performed in the perirhinal cortex have shown that neurons include fast-spiking, burst-spiking and regular-spiking cells (Kelly and McIntyre, 1996; Faulkner and Brown, 1999; Kealy and Commins, 2011). In addition, Beggs et al. (2000) have described late-spiking pyramidal cells that are capa- ble of generating delayed action potential discharges, and pro- posed that these neurons may play a role in encoding “long-time intervals” during associative learning. By employing sharp intra- cellular recordings (D’Antuono et al., 2001; Benini et al., 2011), we found that most of the neurons recorded in the perirhinal cortex correspond morphologically to spiny pyramidal cells and FIGURE 2 | (A) Photomicrograph of a neurobiotin-filled perirhinal cortex cell that corresponds to an upright pyramidal neuron (cf., Furtak et al., 2007). Scale bar, 100 μ m. (B) Regular repetitive firing with adaptation is generated by a pyramidal-like neuron recorded in the perirhinal cortex during injection of a pulse of depolarizing current. (C) Effects induced by bath application of CsCl (3 mM) on the voltage responses to intracellular current pulses; note that under control conditions stepwise hyperpolarization of the membrane leads to the appearance of a sag toward the resting level as well as that addition of CsCl to the bath increases the neuronal input resistance and abolishes the sag. Bottom traces represent current monitor. (D) Power spectra of the intracellular signals recorded in the cell shown in the insets at two different levels of depolarization are illustrated in (a) ; note that the spectrum obtained from the signal recorded at − 58 mV (orange line) is characterized by a peak at ∼ 5 5 Hz. In (b) , histogram of the peak frequencies of membrane oscillations recorded in 29 perirhinal cortical neurons. (E) Responses recorded intracellularly from a perirhinal cortex neuron following local single-shock extracellular stimuli of progressively increasing strength (from a to c ). Inserts in panel (b) illustrate the intracellular responses recorded during injection of depolarizing ( − 59 mV trace) or hyperpolarizing ( − 73 mV trace) current. are regularly firing ( Figures 2A,B ). These neurons generate sev- eral types of sub-threshold responses during injection of intracel- lular current pulses including: (i) tetrodotoxin-sensitive inward rectification in the depolarizing direction (not illustrated) and (ii) Cs + -sensitive inward rectification during injection of hyper- polarizing current pulses ( Figure 2C ). In addition, the repetitive firing generated by these neurons is characterized by adaptation and is followed by a slow after-hyperpolarization upon termi- nation of the depolarizing current pulse ( Figure 2B ). We have also found that both phenomena are greatly reduced by appli- cation of Ca 2 + channel blockers, indicating that Ca 2 + -activated K + conductances play an important role in controlling the intrin- sic excitability of pyramidal cells in the perirhinal cortex. These intrinsic properties are indeed similar to those demonstrated in Frontiers in Cellular Neuroscience www.frontiersin.org August 2013 | Volume 7 | Article 130 | 8 Biagini et al. Perirhinal cortex in epilepsy principal cells recorded intracellularly in several cortical struc- tures (Constanti and Galvan, 1983; Stafstrom et al., 1985; Spain et al., 1987; Mattia et al., 1997). Pyramidal neurons in the perirhinal cortex are also capable of generating voltage-gated, subthreshold membrane oscillations at 5–12 Hz during steady injection of depolarizing current (Bilkey and Heinemann, 1999). As illustrated in Figure 2Da (inserts), when neurons were recorded at resting membrane potential (more negative than − 70 mV), no significant oscillatory activ- ity was observed; however, when they were depolarized with injection of steady intracellular current, sinusoidal-like oscilla- tions became evident along with “clustered” or “tonic” action potential firing. This phenomenon is further identifiable in the power spectrum of the intracellular signals recorded at − 70 and − 58 mV ( Figure 2Da ), while the plot histogram in Figure 2Db summarizes the peak frequencies of the subthreshold mem- brane oscillations recorded from several perirhinal cortical cells. It should be emphasized that as reported in entorhinal cor- tex or subicular cells (Alonso and Llinas, 1989; Mattia et al., 1997), this voltage-dependent oscillatory activity persisted during blockade of glutamatergic and γ -aminobutyric acid (GABA)ergic transmission with specific receptor antagonists as well as during application of Ca 2 + channel blockers. However, it disappeared during application of tetrodotoxin suggesting that voltage-gated Na + electrogenesis contributes to this oscillatory phenomenon. As shown in Figure 2E , perirhinal principal cells generate synaptic potentials with polarity and amplitude that depend on the intensity of the extracellular stimulus; thus, stimuli at threshold strength ( Figure 2Ea ) often induced a hyperpolariz- ing inhibitory postsynaptic potential (IPSP) while, at progres- sively higher intensities, an excitatory postsynaptic potential (EPSP)-IPSP sequence ( Figure 2Eb ) and eventually an EPSP- single action potential ( Figure 2Ec ) occurred. Moreover, these responses changed in amplitude during injection of depolarizing or hyperpolarizing current ( Figure 2Eb ) and the early hyperpo- larizing component of the IPSP was characterized by reversal potential values at approximately − 80 mV (not illustrated). Overall these findings indicate that the intrinsic properties of principal cells in the perirhinal cortex reproduce those reported for cortical pyramidal cells in several areas of the brain. The pres- ence of fast-spiking cells (Faulkner and Brown, 1999) that are known to release GABA is mirrored by the ability of principal neurons in the perirhinal cortex to generate robust inhibitory responses both spontaneously and following electrical stimuli (Benini et al., 2011). EPILEPTIFORM SYNCHRONIZATION in vitro Experiments performed in vitro in extended brain slices com- prising the hippocampus along with the entorhinal and perirhi- nal cortices have shown that interictal and ictal discharges are generated during bath application of the convulsant drug 4- aminopyridine or Mg 2 + -free medium (de Guzman et al., 2004). These epileptiform patterns were only identified after severing the connections between these parahippocampal areas and the hip- pocampus; such a procedure abolished the propagation of CA3- driven fast interictal discharges that controlled the propensity of parahippocampal neuronal networks to generate “slow” interictal events along with prolonged ictal discharges (see for review, Avoli and de Curtis, 2011). As illustrated in Figure 3A , the epileptiform events recorded under control conditions from the entorhinal and perirhinal cortices occurred synchronously in these two areas, could initiate from any of them, and propagated to the neighbor- ing structure with delays ranging from 8 to 66 ms. However, cut- ting the connections between entorhinal and perirhinal cortices generated independent epileptiform activity in both structures ( Figure 3A , EC/PC cut); interestingly, these procedures short- ened ictal discharge duration in the entorhinal but not in the perirhinal cortex. These experiments have also demonstrated that network synchronization underlying ictogenesis in the perirhi- nal cortex is N-Methyl-D-aspartate (NMDA) receptor-dependent (de Guzman et al., 2004). We have recently reported that 4-aminopyridine-induced ictal discharges in the rat entorhinal cortex are preceded by an isolated “slow” interictal discharge or suddenly initiate from a pattern of frequent polyspike interictal discharges; only rarely ictal dis- charge onset was characterized by an acceleration of interictal event rates (Avoli et al., 2013). These findings contrast with what has been observed in the perirhinal cortex since retrospective analysis of the experiments published by de Guzman et al. (2004) indicates that in this area approximately half of the slices treated with 4-aminopyridine presented with ictal discharge onset char- acterized by acceleration of interictal events ( Figure 3Ba ) while in the remaining experiments ictal discharges are preceded by a “slow” interictal discharge ( Figure 3Bb ). These electrographic FIGURE 3 | (A) Ictal discharges occur synchronously in entorhinal (EC) and perirhinal (PC) cortices in an in vitro brain slice treated with 4-aminopyridine (Control panel). Note that cutting the connections between entorhinal and perirhinal areas causes the occurrence of independent ictal activity in these two structures (EC/PC cut panel). (B) Two types of ictal discharge initiation can be recorded from the perirhinal cortex during bath application of 4-aminopyridine. Note that the ictal discharge shown in (a) is characterized by acceleration of the interictal events preceding its onset while that in (b) is characterized by a “slow” interictal event. Frontiers in Cellular Neuroscience www.frontiersin.org August 2013 | Volume 7 | Article 130 | 9 Biagini et al. Perirhinal cortex in epilepsy characteristics are reminiscent of the hypersynchronous onset and of the low-voltage, fast activity onset patterns, respectively, that have been reported to occur in vivo in both epileptic patients (Velasco et al., 2000; Ogren et al., 2009) and animal models (Bragin et al., 1999, 2005; Lévesque et al., 2012, 2013). Overall, these in vitro data indicate that the perirhinal cor- tex may be more prone to generate ictal discharges as compared with the entorhinal cortex. In line with this view, in vivo studies have shown that kindling within the perirhinal cortex promotes seizure activity more rapidly than stimulation of the piriform cor- tex, amygdala or dorsal hippocampus (McIntyre et al., 1993, 1999; Sato et al., 1998). Moreover, lesioning the perirhinal cortex (Kelly and McIntyre, 1996; Fukumoto et al., 2002) or applying gluta- matergic receptor antagonists (Tortorella et al., 1997) or adeno- sine A1 receptor agonists (Mirnajafi-Zadeh et al., 1999) to the perirhinal cortex attenuated and even prevented the appearance of seizure activity following amygdala kindling. CHANGES IN EXCITABILITY IN PILOCARPINE-TREATED EPILEPTIC RATS By using in vitro electrophysiological recordings we have recently reported that brain slices obtained from pilocarpine-treated epileptic rats present with remarkable changes in synaptic excitability when compared to age-matched, non-epileptic con- trols (Benini et al., 2011). The pilocarpine model of temporal lobe epilepsy—which consists of an initial status epilepticus induced by i.p. injection of this cholinergic agonist that is followed 1–4 weeks later by a chronic condition of recurrent limbic seizures— is presumably the most commonly used model for studying this epileptic disorder (Curia et al., 2008). It provides the opportu- nity of controlling epilepsy severity and associated brain damage by pharmacologically regulating the duration of the initial sta- tus epilepticus . Moreover, in contrast to other chronic epilepsy models, spontaneous seizures recur frequently and consistently in virtually all pilocarpine-treated rats. Neurons recorded intracellularly from the deep layers of the perirhinal cortex of non-epileptic control and pilocarpine-treated animals had similar intrinsic and firing properties (Benini et al., 2011). Moreover, they generated spontaneous depolarizing and hyperpolarizing postsynaptic potentials with comparable dura- tion and amplitude. However, spontaneous and stimulus-induced epileptiform discharges could be recorded with fiel