In this study, we used in vitro electrophysiology along with immunohistochemistry and molecular techniques to study the subiculum--a limbic structure that gates the information flow from and to the hippocampus--in pilocarpine-treated epileptic rats. Comparative data were obtained from age-matched nonepileptic controls (NEC). Subicular neurons in hippocampal-entorhinal cortex (EC) slices of epileptic rats were: (i) hyperexcitable when activated by CA1 or EC inputs; and (ii) generated spontaneous postsynaptic potentials at higher frequencies than NEC cells. Analysis of pharmacologically isolated, GABA(A) receptor-mediated inhibitory postsynaptic potentials revealed more positive reversal potentials in epileptic tissue (-67.8 +/- 6.3 mV, n = 16 vs. -74.8 +/- 3.6 mV in NEC, n = 13; P < 0.001) combined with a reduction in peak conductance (17.6 +/- 11.3 nS vs. 41.1 +/- 26.7 nS in NEC; P < 0.003). These electrophysiological data correlated in the epileptic subiculum with (i) reduced levels of mRNA expression and immunoreactivity of the neuron-specific potassium-chloride cotransporter 2; (ii) decreased number of parvalbumin-positive cells; and (iii) increased synaptophysin (a putative marker of sprouting) immunoreactivity. These findings identify an increase in network excitability within the subiculum of pilocarpine-treated, epileptic rats and point at a reduction in inhibition as an underlying mechanism.

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Temporal lobe epilepsy (TLE) is a chronic epileptic disorder involving the hippocampal formation. Details on the interactions between the hippocampus proper and parahippocampal networks during ictogenesis remain, however, unclear. In addition, recent findings have shown that epileptic limbic networks maintained in vitro are paradoxically less responsive than non-epileptic control (NEC) tissue to application of the convulsant drug 4-aminopyridine (4AP). Field potential recordings allowed us to establish here the effects of 4AP in brain slices obtained from NEC and pilocarpinetreated epileptic rats; these slices included the hippocampus and parahippocampal areas such as entorhinal and perirhinal cortices and the amygdala. First, we found that both types of tissue generate epileptiform discharges with similar electrographic characteristics. Further investigation showed that generation of robust ictal-like discharges in the epileptic rat tissue is (i) favored by decreased hippocampal output (ii) reinforced by EC-subiculum interactions and (iii) predominantly driven by amygdala networks. We propose that a functional switch to alternative synaptic routes may promote network hyperexcitability in the epileptic limbic system.

In this study we report that in the presence of normal buffer, epileptiform discharges occur spontaneously (duration = 2.60 ± 0.49 s) or can be induced by electrical stimuli (duration = 2.50 ± 0.62 s) in the entorhinal cortex (EC) of brain slices obtained from pilocarpine-treated rats but not in those from age-matched, nonepileptic control (NEC) animals. These network-driven epileptiform events consist of field oscillatory sequences at frequencies greater than 200 Hz that most often initiate in the lateral EC and propagate to the medial EC with 4-63 ms delays. The NMDA receptor antagonist CPP depresses the rate of occurrence (P < 0.01) of these spontaneous epileptiform discharges but fails in blocking them.

Fast oscillations at approximately 200 Hz, termed ripples, occur in the hippocampus and cortex of several species, including humans, and are thought to play a role in physiological (e.g., sensory information processing or memory consolidation) and pathological (e.g., seizures) processes. Blocking gamma-aminobutyric acid type A (GABA(A)) receptor-mediated inhibition represents one of the most often used models of epileptiform discharge. Here we found that bath application of the GABA(A) receptor antagonist picrotoxin (50 microM) to mouse hippocampus-entorhinal cortex slices induced spontaneous epileptiform activity (duration 536.6 +/- 146.1 msec, mean +/- SD; interval of occurrence 14.8 +/- 3.3 sec, n = 12) with two distinct phases of discharge; the first was characterized, in the dentate gyrus only, by high-frequency, field oscillations (ripples) at 206.3 +/- 23.4 Hz (n = 12), whereas the second component corresponded to afterdischarges in the theta range frequency. Ripples, which were also recorded in "minislices" only of the dentate gyrus, were unaffected by application of the mu-opioid receptor agonist (D-Ala2-N-Me-Phe,Gly-ol)enkephalin (10 microM; n = 6) or the N-methyl-D-aspartate (NMDA) receptor antagonist 3-(2-carboxy-piperazine-4-yl)-propyl-l-phosphonate (10 microM; n = 5). In contrast, the non-NMDA glutamatergic receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (10 microM; n = 5) completely blocked all picrotoxin-induced activities. In addition, application of the GABA(B) receptor agonist baclofen (0.01-0.5 microM; n = 6) dose dependently and reversibly abolished all picrotoxin-induced activities. We also found that application of the gap-junction decouplers carbenoxolone (0.2-0.5 mM; n = 6) or octanol (0.2-0.5 mM; n = 3) blocked the second phase while leaving ripples unchanged. These findings demonstrate that the disinhibited dentate gyrus can generate ripple activity at approximately 200 Hz that is contributed by ionotropic glutamatergic mechanisms and is not dependent on either GABA(A) receptor-mediated or gap-junction mechanisms.

Abstract4-Aminopyridine (4AP, 50 μM) induces interictal-and ictal-like discharges in brain slices including parahippocampal areas such as the entorhinal cortex (EC) but the relation between these two types of epileptiform activity remains undifined. Here, by employing field potential recordings in rat EC slices during 4AP application, we found that: (i) interictal events have a wide range of duration (0.4-3.3 s) and interval of occurrence (1.4-84 s); (ii) ictal discharges are either preceded by an isolated "slow" interictal discharge (ISID; duration=1.5±0.1 s, interval of occurrence=33.8±1.8 s) or suddenly initiate from a pattern of frequent polispike interictal discharge (FPID; duration=0.8±0.1 s; interval of occurrence=2.7±0.2 s); and (iii) ISID-triggered ictal events have longer duration (116±7.3 s) and interval of occurrence (425.8±42.3 s) than those initiating suddenly during FPID (58.3±7.8 s and 202.1±21.8 s, respectively). Glutamatergic receptor antagonists abolished ictal discharges in all experiments, markedly reduced FPIDs but did not influence ISIDs. We also discovered that high-frequency oscillations (HFOs, 80-500 Hz) occur more frequently during ISIDs as compared to FPIDs, and mainly coincide with the onset of ISID-triggered ictal discharges. These findings indicate that interictal events may define ictal onset features resembling those seen in vivo in low-voltage fast activity onset seizures. We propose a similar condition to occur in vivo in temporal lobe epileptic patients and animal models.

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.

We obtained field, K(+) selective and "sharp" intracellular recordings from the rat entorhinal (EC) and perirhinal (PC) cortices in an in vitro brain slice preparation to identify the events occurring at interictal-to-ictal transition during 4-aminopyridine application. Field recordings revealed interictal- (duration: 1.1 to 2.2s) and ictal-like (duration: 31 to 103s) activity occurring synchronously in EC and PC; in addition, interictal spiking in PC increased in frequency shortly before the onset of ictal oscillatory activity thus resembling the hypersynchronous seizure onset seen in epileptic patients and in in vivo animal models. Intracellular recordings with K-acetate+QX314-filled pipettes in PC principal cells showed that spikes at ictal onset had post-burst hyperpolarizations (presumably mediated by postsynaptic GABAA receptors), which gradually decreased in amplitude. This trend was associated with a progressive positive shift of the post-burst hyperpolarization reversal potential. Finally, the transient elevations in [K(+)]o (up to 4.4mM from a base line of 3.2mM) - which occurred with the interictal events in PC - progressively increased (up to 7.3mM) with the spike immediately preceding ictal onset. Our findings indicate that hypersynchronous seizure onset in rat PC is caused by dynamic weakening of GABAA receptor signaling presumably resulting from [K(+)]o accumulation.