Limbic network interactions leading to hyperexcitability in a model of temporal lobe epilepsy

In mouse brain slices that contain reciprocally connected hippocampus and entorhinal cortex (EC) networks, CA3 outputs control the EC propensity to generate experimentally induced ictal-like discharges resembling electrographic seizures. Neuronal damage in limbic areas, such as CA3 and dentate hilus, occurs in patients with temporal lobe epilepsy and in animal models (e.g., pilocarpine- or kainate-treated rodents) mimicking this epileptic disorder. Hence, hippocampal damage in epileptic mice may lead to decreased CA3 output function that in turn would allow EC networks to generate ictal-like events. Here we tested this hypothesis and found that CA3-driven interictal discharges induced by 4-aminopyridine (4AP, 50 microM) in hippocampus-EC slices from mice injected with pilocarpine 13-22 days earlier have a lower frequency than in age-matched control slices. Moreover, EC-driven ictal-like discharges in pilocarpine-treated slices occur throughout the experiment (< or = 6 h) and spread to the CA1/subicular area via the temporoammonic path; in contrast, they disappear in control slices within 2 h of 4AP application and propagate via the trisynaptic hippocampal circuit. Thus, different network interactions within the hippocampus-EC loop characterize control and pilocarpine-treated slices maintained in vitro. We propose that these functional changes, which are presumably caused by seizure-induced cell damage, lead to seizures in vivo. This process is facilitated by a decreased control of EC excitability by hippocampal outputs and possibly sustained by the reverberant activity between EC and CA1/subiculum networks that are excited via the temporoammonic path.     

An analysis of penicillin-induced generalized spike and wave discharges using simultaneous recordings of cortical and thalamic single neurons

To study the relationship between cortical and thalamic single-neuron activity during spike and wave (SW) discharge of feline generalized penicillin epilepsy (FGPE), extracellular single-unit and local electroencephalogram (EEG) activity were recorded simultaneously from pairs of neurons, one located in the cortex of the middle suprasylvian gyrus (MSS), the other in the dorsal thalamic nuclei (n. lateralis posterior or pulvinar). These two areas are anatomically and functionally closely interrelated. Computer-generated EEG averages and histograms of single-unit activity triggered by either peaks of cortical or thalamic EEG transients or by cortical or thalamic action potentials (aps) showed that cortical neurons in the MSS fired at the time of the spike of the SW complex, while at the time of the wave they became silent. Two populations of thalamic neurons also fired maximally during the spike of SW discharge, but they differed in the precise timing of their firing in relation to that of the simultaneously recorded cortical neuron. The first group of thalamic neurons tended to fire 5-45 ms before the cortical neuron. Of these 28 neurons, 9 were antidromically and 2 orthodromically activated by cortical stimulation. The neurons of the second group tended to fire 0-45 ms after the cortical neuron. Cortical stimulation activated 15 of these 19 neurons orthodromically and 2 antidromically. A third and smaller population of thalamic neurons (n = 8) increased its firing probability during the wave of the SW complex and decreased it during the spike. In 74% of the pairs of neurons the cyclic alternation of excitation and "inhibition" associated with SW activity appeared in the cortex by 1-3 cycles earlier than in the thalamus. This was most common when the thalamic neuron of the pair reached its peak firing probability before the simultaneously recorded cortical neuron. In 11 pairs of neurons the same rhythmic alternation of excitation and "inhibition" of neuronal firing was seen in both the cortex and thalamus during SW discharges evoked by single-shock stimulation of nucleus centralis medialis. These data demonstrate that both cortical and thalamic neurons participate in the SW firing pattern of FGPE by undergoing periods of mutually phase-locked cyclic alternations of excitation and "inhibition" at the frequency of the EEG SW rhythm. Although the initial steps leading to generalized SW discharge in FGPE take place in the cortex, the thalamus soon becomes entrained in the SW rhythm.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bicuculline-induced epileptogenesis in the human neocortex maintained in vitro

Summary Intracellular and extracellular recordings were made from human neocortical slices of the temporal lobe maintained in vitro. The slices were treated with bicuculline methiodide to reduce synaptic inhibition mediated by tha gamma-aminobutyric acid A (GABA_A) receptor. Spontaneously occurring epileptiform activity was never observed in over 60 slices examined. All epileptiform discharges were elicited by single-shock stimuli delivered in the underlying white matter or within the cortical layers. Intracellularly, the stimulus-induced epileptiform discharge resembled the paroxysmal depolarization shift (PDS). This potential was observed in neurons located between 200 and 2200 m from the pia. It was characterized by a 100–1800 ms long depolarization which triggered burst firing of action potentials, and was at times followed by an afterdischarge. Simultaneous intracellular and extracellular recordings showed that each PDS was reflected by the synchronous discharge of a neuronal aggregate. The voltage behaviour of the PDS and its preceding EPSP was analyzed in cells that were injected with the lidocaine derivative QX-314. The amplitudes of the PDS depolarizing envelope measured at its peak and during its falling phase both behaved as a monotonic function of the membrane potential by increasing in amplitude during hyperpolarization. In addition, the PDS peak amplitude showed a much greater rate of increase than the early EPSP peak amplitude, thus suggesting that the synaptic conductance underlying the PDS was much greater. Perfusion of the neocortical slices with the N-Methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-phosphonovaleric acid (APV) reduced both the duration and the amplitude of the paroxysmal field discharge in a dose related fashion. The effects of APV were reflected intracellularly by an attenuation of the PDS's late phase and a blockade of the afterdischarge. Similar findings were also obtained by using the NMDA receptor antagonist 3-((±)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid. These data indicate that reduction or blockade of the GABA_A receptor is sufficient to elicit epileptiform discharges in the human neocortex maintained in vitro. Mechanisms dependent upon the NMDA receptor contribute to this type of epileptiform response mainly by prolonging the stimulus-induced depolarizing potential and the associated burst of firing.     

Excitatory synaptic transmission mediated by NMDA and non-NMDA receptors in the superficial/middle layers of the epileptogenic human neocortex maintained in vitro.

Conventional intracellular recordings were made from regular-spiking cells located in layers II-IV to examine the involvement of excitatory amino acid receptors in synaptic transmission in epileptogenic human neocortical slices maintained in vitro. Extracellular stimuli that were below the threshold for generating action potentials evoked an excitatory postsynaptic potential (EPSP) with short latency to onset (0.8-4 ms). When suprathreshold stimuli were delivered, 95% of the neurons fired a single action potential. In 5% of the population, however, an all-or-none bursting discharge was observed. The EPSP and the bursting discharge were tested with the N-methyl-D-aspartate (NMDA) antagonist 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP, 5 microM) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 4 microM). In the presence of CNQX the peak amplitude of the EPSP was reduced by 85% and the bursting discharge was abolished completely. By contrast, CPP reduced the peak amplitude of the EPSP by 52%, attenuated the late phase of the bursting discharge and increased its threshold. These results indicate that excitatory amino acids function as excitatory transmitters in the human brain. While the involvement of non-NMDA receptors in the EPSP is in line with data from normal neocortical slices of other mammals, the participation of NMDA-mediated conductances to the EPSP appears peculiar to the epileptogenic human neocortex. This evidence, together with the contribution of NMDA and non-NMDA receptors to the all-or-none bursting discharge suggests that excitatory amino acid-mediated transmission might be modified in the epileptogenic human neocortex.     

Synaptic and non-synaptic mechanisms underlying low calcium bursts in the in vitro hippocampal slice

Summary 1. The epileptiform activity generated by lowering extracellular [Ca⁺⁺] was studied in the CA1 subfield of rat hippocampal slices maintained in vitro at 32° C. Extracellular and intracellular recordings were performed with NaCl and KCl filled microelectrodes. 2. Synaptic potentials evoked by stimulation of the stratum radiatum and alveus were blocked upon perfusion with artificial cerebrospinal fluid (ACSF) containing 0.2 mM Ca⁺⁺, 4 mM Mg⁺⁺. Blockade of synaptic potentials was accompanied by the appearance of synchronous field bursts which either occurred spontaneously or could be induced by stimulation of the alveus. 3. Both spontaneous and stimulus-induced low Ca⁺⁺ bursts recorded extracellularly in stratum pyramidale consisted of a negative potential shift with superimposed population spikes. This extracellular event was closely associated with intracellularly recorded action potentials rising from a prolonged depolarization shift. Steady hyperpolarization of the cell membrane potential decreased the amplitude of the depolarizing shift suggesting that synaptic conductance were not involved in the genesis of the low Ca⁺⁺ burst. 4. Spontaneous depolarizing inhibitory potentials recorded in normal ACSF with KCl filled microelectrodes were reduced in size in low Ca⁺⁺ ACSF. However, small amplitude potentials could still be observed at a time when low Ca⁺⁺ bursts were generated by hippocampal CA1 pyramidal neurons. 5. Bicuculline methiodide, an antagonist of -aminobutyric acid (GABA), was capable of modifying the frequency of occurrence and the shape of synchronous field bursts. The effects evoked by bicuculline methiodide were, however, not observed when 81–100% of NaCl was replaced with Na-Methylsulphate. Hence, it was concluded that in low Ca⁺⁺ ACSF even though large release of transmitter such as those following electrical activation of stratum radiatum or alveus cannot be observed, small spontaneous release of the inhibitory transmitter GABA seems to persist. 6. Substitution of NaCl with Na-Methylsulphate also caused changes in the synchronous field bursts which were different from those observed following application of bicuculline methiodide. These findings suggest that in low Ca⁺⁺ ACSF, in addition to residual GABAergic Cl^- mechanisms, non-synaptic Cl^- conductances might play a role in controlling the excitability of hippocampal neurons.Supported by grants from the MRC of Canada (MA-8109) and Sick Children Foundation to MA     

Extracellular K+ accumulations and synchronous GABA-mediated potentials evoked by 4-aminopyridine in the adult rat hippocampus

Transient changes in extracellular potassium concentration ([K⁺]₀) and field potentials were evoked by 4-aminopyridine (4-AP; 50–100 M) and recorded with ion-selective microelectrodes in CA1b, CA3b and dentate sectors of adult rat hippocampal slices. Long-lasting field potentials recurred at a frequency of 1/60 s (0.016±0.003 Hz) in association with increases in [K⁺]₀ which were largest and most sustained in the dendritic regions where afferent fibers terminate (dentate>CAl>CA3) and in the hilus. In stratum radiatum of CA1 or stratum moleculare of the dentate these fields had a peak amplitude of 1.4±0.29 mV, duration 8.3±1.6 s, and were accompanied by increases in [K⁺]₀ of 1.8±0.22 mM that lasted 32±5.5 s (n = 17 slices). Interictal epileptiform potentials, which were brief (<0.2 s) and more frequent at 1/3 s (0.30±0.02 Hz) were also present in CA1, CA3 and the hilus and associated with small increases in [K⁺]₀ (0.5 mM, duration 2 s). Interictal activity was blocked by 6-cyano-7-nitroquinoxalone-2,3-dione (CNQX; 5–20 M); the slow, less frequent potentials were resistant to both CNQX and dl-2amino-5-phosphonovaleric acid (APV; 50 M) and reversibly blocked (or attenuated by 80%) by bicuculline methiodide (BMI) (25–100 M). The BMI-sensitive potentials were also abolished by baclofen (100 M), an effect which was reversed by 2-OH-saclofen (100 M). Focal application of KCl or GABA in the absence of 4-AP evoked long-lasting field and [K⁺]₀ potentials which were similar to those evoked by 4-AP but more sustained. The proportional relationship between the amplitudes of field and K⁺ potentials with GABA closely resembled that observed for 4-AP; in contrast the slope of KCl-evoked responses was lower. Our results demonstrate that in the adult rat hippocampus 4-AP induces in many different regions accumulations of [K⁺]₀ in synchrony with the long-lasting field potentials, which are known to correspond to an intracellular long-lasting depolarization of the pyramidal cells. These changes are smaller than those which occur in the immature rat hippocampus — which may be related to differences in Na-K-ATPase and susceptibility to seizures. These events involve the activation of GABA_A receptors, are under the modulatory control of GABA_B receptors, and likely arise from the activity of GABAergic interneurons and/or afferent terminals. The long-lasting field potentials appear to reflect mainly the direct depolarizing actions of GABA and to a much more limited extent the associated accumulation of [K⁺]₀.     

Topiramate depresses carbachol-induced plateau potentials in subicular bursting cells

Intracellular recordings were made in an in vitro slice preparation to establish whether the antiepileptic drug topiramate reduces the depolarizing plateau potentials (PPs) induced in the rat subiculum by intracellular pulses of depolarizing current, in the presence of the cholinergic agonist carbachol (CCh, 70-100 microM). PPs lasted up to about 2 s, and disappeared during application of the muscarinic receptor antagonist atropine. Topiramate (10-100 microM, n = 22 neurons) decreased and eventually abolished in a dose-dependent manner these PPs, even when the function of excitatory amino acid and GABAA receptors was blocked. Hence, topiramate depresses muscarinic receptor-dependent PPs in the rat subiculum, thus suggesting that this form of excitation may represent a target for the mechanism of action of this antiepileptic compound.     

A GABAergic depolarizing potential in the hippocampus disclosed by the convulsant 4-aminopyridine

Intracellular recordings from hippocampal pyramidal cells in the CA1 subfield of the 'in vitro' slice in the presence of 4-aminopyridine (4-AP, 5-50 microM) revealed a long-lasting (up to 1.5 s) depolarizing potential which occurred either spontaneously or following orthodromic stimulation. This potential was: capable of blocking both direct and synaptic activation of the cell; sensitive to bath application of low concentrations of bicuculline methiodide; and associated to an extracellular current sink in the dendrites as suggested by the extracellular field potentials recorded at different levels along an axis perpendicular to the stratum pyramidale. It is concluded that the long-lasting depolarizing potential evoked by 4-AP is caused by the activation of GABA receptors localized in the dendritic region of the CA1 subfield.     

Effects of post-ictal depression on experimental spike and wave discharges

The effects of post-ictal depression on spike and wave (SW) discharges of feline generalized penicillin epilepsy (FGPE) were studied. After tonic-clonic seizures which are not uncommon in FGPE spindle bursts appeared during the post-ictal period. Upon recovery spontaneous and thalamically evoked SW discharges reappeared. Spindles before penicillin and during post-ictal depression showed a similar intraburst frequency (twice that of SW discharges) in the same animal. These findings add further evidence to the notion that any depression of cortical excitability in FGPE leads to replacement of SW by spindles and thus supports the hypothesis that SW discharges occur in hyperexcitable cortex in response to normally spindle-inducing thalamocortical volleys.