Docosahexaenoic acid inhibits synaptic transmission and epileptiform activity in the rat hippocampus

Docosahexaenoic acid (DHA) has been suggested to be required for neuronal development and synaptic plasticity. However, in view of the fact that DHA facilitates NMDA responses and blocks K(+) channels, it might predispose the neurons to epileptiform bursting. By using extracellular recording of population spikes in the CA1 region of rat hippocampal slices, we tested this possibility by examining the effect of DHA on the epileptiform activity induced by bicuculline or in Mg(2+)-free medium. When stimuli were delivered to the Schaffer collateral/commissural pathway every 20 or 30 sec, DHA had no significant effect on the epileptiform activity. However, when the frequency of stimulation was increased to 0.2 Hz, DHA attenuated the amplitude of the bursting activity induced by bicuculline to 57.5+/- 10.8% and those induced by Mg(2+)-free ACSF to 65.8+/-13.9% of control. DHA reduced the slope of field excitatory postsynaptic potential (fEPSP) to 77.1+/-7.4% of baseline, without significant effect on the ratio of paired-pulse facilitation (PPF). By intracellular recording of neurons in the stratum pyramidale of rat hippocampal slices, we found that DHA markedly inhibited the repetitive firing of action potentials elicited by depolarizing current pulses but did not affect the initial action potential. Thus, DHA may attenuate epileptic activity mainly through the frequency-dependent blockade of Na(+) channels.     

Epileptiform activity induced by low extracellular magnesium in the human cortex maintained in vitro.

Extracellular field potentials and [K+]o were recorded in slices of human epileptogenic neocortex maintained in vitro during perfusion with Mg(2+)-free artificial cerebrospinal fluid (ACSF). The human neocortex was obtained during neurosurgical procedures for the relief of seizures that were resistant to medical treatment. Spontaneous epileptiform activity and episodes of spreading depression appeared within 1.5 to 2 hours of perfusion with Mg(2+)-free ACSF. The epileptiform discharges consisted of negative field potential shifts (amplitude, 0.8-10 mV) that lasted 2.5 to 80 seconds and recurred at intervals ranging between 4 and 160 seconds. Both duration and frequency of occurrence of epileptiform events were not significantly different when measured in slices obtained from spiking tissue compared with those gathered from nonspiking neocortical areas. Transient increases in [K+]o of up to 10.5 mM were associated with each epileptiform discharge; these changes were maximal and fastest in the middle neocortical layers. Spreading depression episodes were characterized by 20 to 30-mV negative shifts that lasted up to 200 seconds and were accompanied by increases in [K+]o of approximately 100 mM. Epileptiform discharges and spreading depressions did not occur during perfusion with Mg(2+)-free ACSF that contained either competitive or noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptor subtype. In contrast, pharmacological blockade of non-NMDA receptors did not influence the epileptiform activity observed in Mg(2+)-free ACSF. These findings demonstrate that decreasing [Mg2+]o leads to the appearance of both spontaneous epileptiform discharges and spreading depression in the human epileptogenic neocortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low magnesium epileptogenesis in the rat hippocampal slice: electrophysiological and pharmacological features

Extra- and intracellular recording techniques were used to study the epileptiform activity generated by rat hippocampal slices perfused with Mg2(+)-free artificial cerebrospinal fluid (ACSF). This procedure induced in both CA1 and CA3 subfields the appearance of synchronous, spontaneously occurring epileptiform discharges which consisted of extracellularly recorded 100-800 ms long, positive shifts with superimposed negative going population spikes. Simultaneous, extracellular recordings from CA1 and CA3 subfields revealed that the epileptiform discharges in CA3 preceded those occurring in CA1 by 5-25 ms. Surgical separation of the two areas led to the disappearance of spontaneous events in the CA1 but not in the CA3 subfield. In this type of experiment CA1 pyramidal cells still generated epileptiform discharges following orthodromic stimuli. The intracellular counterpart of both spontaneous and stimulus-induced epileptiform discharges in CA1 and CA3 pyramidal cells was a large amplitude depolarization with high frequency discharge of action potentials which closely resembled the paroxysmal depolarizing shift recorded in the experimental epileptogenic focus. A hyperpolarizing potential triggered by alvear stimuli was recorded in CA1 cells perfused with Mg2(+)-free ACSF. This hyperpolarization was blocked by bicuculline methiodide (BMI) indicating that it represented a GABAergic inhibitory postsynaptic potential (IPSP). BMI also caused a prolongation of both spontaneous and stimulus-induced Mg(+)-free epileptiform discharges. Perfusion of the slices with the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphono-valerate (APV) reduced and eventually abolished the Mg(+)-free epileptiform discharges. These effects were more pronounced in the CA1 than in the CA3 subfield. APV also reduced the amplitude and the duration of the alveus-induced IPSP. These data demonstrate that Mg(+)-free epileptiform activity is present in the hippocampal slice at a time when inhibitory GABAergic potentials are operant as well as that in the CA1 subfield this type of epileptiform activity is dependent upon NMDA-activated conductances. Our experiments also indicate that NMDA receptors might be involved in the neuronal circuit responsible for the hyperpolarizing IPSP generated by CA1 pyramidal neurons.     

Ethanol inhibits epileptiform activity and NMDA receptor-mediated Synaptic transmission in rat amygdaloid slices

The effect of ethanol on the epileptiform activity induced by Mg(++)-free solution was studied in rat amygdalar slices using intracellular recording techniques. The spontaneous and evoked epileptiform discharges consisting of an initial burst followed by afterdischarges were observed 20-30 min after switching to Mg(++)-free medium. Superfusion with ethanol (20-100 mM) reversibly reduced the duration of spontaneous and evoked bursting discharges in a concentration-dependent manner. Synaptic response mediated by N-methyl-D-aspartate (NMDA) receptor activation was isolated by application of a solution containing the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and either in Mg(++)-free solution or in the presence of 50 microM bicuculline. Application of ethanol reversibly suppressed the duration of NMDA receptor-mediated synaptic response. These results suggest that intoxicating concentrations of ethanol possess anticonvulsant activity through blocking the NMDA receptor-mediated synaptic excitation. In addition, the observed effect of ethanol on NMDA receptor-mediated synaptic response could be relevant to the cognitive and behavioral manifestations seen in some alcoholics.     

Spatiotemporal organization and thalamic modulation of seizures in the mouse medial thalamic-anterior cingulate slice

Purpose: Seizure‐like activities generated in anterior cingulate cortex (ACC) are usually classified as simple partial and are associated with changes in autonomic function, motivation, and thought. Previous studies have shown that thalamic inputs can modulate ACC seizure, but the exact mechanisms have not been studied thoroughly. Therefore, we investigated the role of thalamic inputs in modulating ACC seizure‐like activities. In addition, seizure onset and propagation are difficult to determine in vivo in ACC. We studied the spatiotemporal changes in epileptiform activity in this cortex in a thalamic–ACC slice to clearly determine seizure onset. Methods: We used multielectrode array (MEA) recording and calcium imaging to investigate the modulatory effect of thalamic inputs in a thalamic–ACC slice preparation. Key Findings: Seizure‐like activities induced with 4‐aminopyridine (4‐AP; 250 μm ) and bicuculline (5–50 μm ) in ACC were attenuated by glutamate receptor antagonists, and the degree of disinhibition varied with the dose of bicuculline. Seizure‐like activities were decreased with 1 Hz thalamic stimulation, whereas corpus callosum stimulation could increase ictal discharges. Amplitude and duration of cingulate seizure‐like activities were augmented after removing thalamic inputs, and this effect was not observed with those induced with elevated bicuculline (50 μm ). Seizure‐like activities were initiated in layers II/III and, after thalamic lesions, they occurred mainly in layers V/VI. Two‐dimensional current‐source density analyses revealed sink signals more frequently in layers V/VI after thalamic lesions, indicating that these layers produce larger excitatory synchronization. Calcium transients were synchronized after thalamic lesions suggesting that ACC seizure‐like activities are subjected to desynchronizing modulation by thalamic inputs. Therefore, ACC seizure‐like activities are subject to desynchronizing modulation from medial thalamic inputs to deep layer pyramidal neurons. Significance: Cingulate seizure‐like activities were modulated significantly by thalamic inputs. Repeated stimulation of the thalamus efficiently inhibited epileptiform activity, demonstrating that the desynchronization was pathway‐specific. The clinical implications of deep thalamic stimulation in the modulation of cingulate epileptic activity require further investigation.     

Effect of levetiracetam on epileptiform discharges in human neocortical slices

PURPOSE: The anticonvulsant effects of the novel antiepileptic drug (AED) levetiracetam (LEV) were tested in neocortical slice preparations from 23 patients who underwent surgery for the treatment of refractory epilepsy. METHODS: Slices were used to evaluate the effects of LEV on two different models of epilepsy: low-Mg2+-induced untriggered and bicuculline-evoked stimulus-triggered epileptiform burst discharges and spontaneously appearing rhythmic sharp waves. RESULTS: LEV (0.1-1 mM) did not influence spontaneously appearing rhythmic sharp waves or Mg2+-free aCSF-induced epileptiform field potentials. LEV affected neither the amplitudes or duration nor the repetition rates of burst discharges in these epilepsy models. However, LEV (100-500 microM) significantly suppressed the ictal-like discharges elicited by the gamma-aminobutyric acid subtype A (GABAA)-receptor antagonist bicuculline. A marked reduction of the amplitude and duration of bicuculline-evoked field response in the presence of LEV was observed. CONCLUSIONS: The results indicate the potential for LEV to inhibit epileptiform burst discharges in human neocortical tissue, which is consistent with its effects in animal models of epilepsy. These results also support the seizure reduction observed in clinical trials and support that this may, in part, be related to the ability of LEV to inhibit epileptiform discharges.     

Effects of retigabine on rhythmic synchronous activity of human neocortical slices

The antiepileptic effects of the novel antiepileptic drug retigabine (D-23129) [N-(2-amino-4-(4-flurobenzylamino)phenyl) carbamid acid ethyl ester] were tested in neocortical slice preparations (n=23) from 17 patients (age, 3-42 years) who underwent surgery for the treatment of intractable epilepsy. Epileptiform events consisted of spontaneously occurring rhythmic sharp waves, as well as of epileptiform field potentials (EFP) elicited by superfusion with Mg(2+)-free solution without or with addition of 10 micromol/l bicuculline. (1) Spontaneous rhythmic sharp waves (n=6), with retigabine application, the repetition rate was decreased down to 12-47% of initial value (10 micromol/l, n=3) after 180 min or suppressed completely within 12 min (50 micromol/l, n=3). (2) Low Mg(2+) EFP (n=9), with retigabine application, the repetition rate was decreased down to 50 and 65% of initial value (10 micromol/l; n=2) after 180 min or suppressed completely after 9-55 min (10, 50 and 100 micromol/l; n=2 in each case). In one slice only a transient reduction of the repetition rate was seen with 10 micromol/l retigabine. (3) Low Mg(2+) EFP with addition of bicuculline (n=8), with retigabine application, the repetition rate was decreased down to 12-55% of initial value (10 micromol/l; n=4) after 180 min or suppressed completely after 6-30 min (50 and 100 micromol/l; n=2 in each case). The depressive effect of retigabine was reversible in all but one slice. The results show a clear antiepileptic effect of retigabine in human neocortical slices on spontaneously occurring rhythmic sharp waves and different types of induced seizure activity.     

Effect of recurrent epileptiform discharges induced by magnesium-free treatment on developing cortical neurons in vitro

As seizures in infants and children often originate from the neocortex, neocortical epilepsy models may be appropriate for studying epileptiform activity and seizure-induced injury in the developing nervous system. However, the characterization of epileptiform activity or seizure-induced injury in cultured developing cortical neurons has seldom been reported. Therefore, We attempted to establish a cultured developing cortical neuronal epilepsy model, and to study the subsequent effect on neurons. Cultures were exposed to Mg(2+)-free media for 3 h, and then returned to regular media. Using whole-cell patch-clamp intracellular recording techniques, we found that spontaneously recurrent epileptiform discharges for at least 72 h could be induced after transient Mg(2+)-free treatment. Neuron morphology following Mg(2+)-free treatment demonstrated no prominent alterations. At different time points (6, 24 and 72 h) after Mg(2+)-free treatment, neuronal viability, identified by trypan blue staining and LDH activity, and apoptosis, measured by flow cytometry, showed modest but non-significant (P>0.05) changes compared with the age-matched control group after various culture periods (6 and 17 days) in vitro. Mitochondrial metabolic activity, measured by MTT assay, significantly decreased by 15% at 6 h after Mg(2+)-free treatment (P<0.05) in neurons cultured for 6 days, and at 24 h showed a 29% decrease in neurons cultured for 17 days (P<0.05). In conclusion, brief Mg(2+)-free treatment constitutes a cultured developing cortical neuron 'seizure' model, and can induce transient mitochondrial dysfunction without cell loss.     

Pattern of intracortical potential distribution during focal interictal epileptiform discharges (FIED) and its relation to spinal field potentials in the rat

The pattern of intracortical potential distribution during focal interictal epileptiform discharges (FIED) was analysed with respect to the occurrence of descending neuronal activity to the spinal cord recorded as spinal field potentials (SFPs). The experiments were performed in rats. Epileptiform activity was elicited by application of penicillin to the motor cortex. The spread of active penicillin was limited by penicillinase in part of the experiments. (1) When penicillinase was applied 10--20 sec before penicillin to the cortical surface typical FIED appeared in the epicortical lead. During well-established focal activity they were accompanied by negative field potentials at a depth of 300 micrometers and 600 micrometers and by positive field potentials in deeper records. This pattern of intracortical potential distribution was not associated with characteristic SFPs. (2) When penicillinase was applied simultaneously with penicillin, the fully developed epicortical FIED were accompanied by negative intracortical field potentials which in this case reached a depth of 900 micrometers. In the layers below predominantly positive potential fluctuations occurred. This pattern of intracortical potential distribution was associated with characteristic SFPs. (3) Intracortical application of penicillin at a depth of 800--900 micrometers led to negative field potentials of large amplitude in all intracortical records, with the concomitant epicortical potentials being positive in polarity. In this case SFPs occurred throughout the interictal activity. Since seizure activity can be restricted to only a few cortical laminae, descending neuronal activity to the spinal cord need not be correlated with definite epicortical potentials. A prerequisite for cortical output is intracortical activity reflected negative potentials at a depth of approx. 900 micrometers.     

Prenatal morphine exposure enhances seizure susceptibility but suppresses long-term potentiation in the limbic system of adult male rats

The present study examined the effects of prenatal morphine exposure on NMDA-dependent seizure susceptibility in the entorhinal cortex (EC), and on activity-dependent synaptic plasticity at Schaffer collateral and perforant path synapses in the hippocampus. During perfusion with Mg(2+)-free ACSF, an enhancement of epileptiform discharges was found in the EC of slices from prenatally morphine-exposed male rats. A submaximal tetanic stimulation (2x50 Hz/1 s) in control slices elicited LTP at the Schaffer collateral-CA1 synapses, but neither LTP nor LTD was evoked at the perforant path-DG synapses. In slices from prenatally morphine-exposed adult male rats, long-term potentiation of synaptic transmission was not observed at Schaffer collateral-CA1 synapses, while the submaximal tetanus now elicited frank LTD of synaptic EPSPs at perforant path synapses. These data suggest that prenatal morphine exposure enhances the susceptibility of entorhinal cortex to the induction of epileptiform activity, but shifts long-term plasticity of hippocampal synapses in favor of LTD.