Search

The influence of epileptic activity on both the fine structure of neuronal processes and the subcellular distribution of calcium-binding sites was investigated in an epileptic model system, the buccal ganglion of Helix pomatia. Pentylenetetrazole was used to induce epileptic activity. Calcium-binding sites were visualized as electron-dense precipitates. Epileptic and control activity was intracellularly recorded from neuron B3 labeled with neurobiotin. After epileptic treatment, many processes contained vacuolated or electron-lucent areas next to morphologically intact areas. Most of these areas were enveloped by layers of endoplasmic reticulum. Lamellar formations of membranes occurred frequently. Calcium cytochemistry revealed a high content of dense precipitates within these formations of the endoplasmic reticulum. Local accumulations of diffuse precipitates were more frequent after epileptic activity than in controls. In contrast, structures such as lamellar bodies, cytosomes, and synapse-like formations, all of which contained many electron-dense precipitates, were apparently unchanged after epileptic activity. This study demonstrates that epileptic activity can lead to local degeneration of neuronal fibers and an associated increase in calcium-binding sites. It is suggested that calcium sequestration is locally increased within neuronal processes during epileptic activity.

The specific L-type calcium channel blocker verapamil exerts an antiepileptic effect on neurons. This effect is assumed to depend on the blockade of transmembraneous calcium flux during epileptic discharges. In order to test this hypothesis, fura-dextran loaded snail neurons were rendered epileptic by pentylenetetrazole (40 mmol/l). The effect of verapamil (20 or 40 mumol/l) on free intracellular calcium ([Ca2+]i) transients was investigated by means of fluorescence ratio-imaging and simultaneous intracellular membrane potential recording. During epileptic depolarization [Ca2+]i increased especially in the outermost submembraneous areas of the neuron. [Ca2+]i reached peak values 6-22 s after the onset of epileptic depolarizations. Application of verapamil progressively shortened the epileptic depolarizations. This shortening of epileptic depolarizations developed along with a diminution of the submembraneous calcium signals down to noise level. The effect was found to be reversible. It is concluded that the antiepileptic effect of verapamil depends largely on its ability to block transmembraneous calcium flux.

The influence of the glutamate subreceptor agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) on cortical field potentials and on changes in extracellular free calcium concentration ([Ca2+]o) was tested on human neocortical slices (eleven from nine different patients). The tissue used was a small portion of that which is normally removed for the treatment of a brain tumor. [Ca2+]o and field potentials were measured by Ca(2+)-selective microelectrodes. Local pressure-microejection of NMDA (100 mumol/l)- and AMPA (1 mmol/l)-induced negative field potentials with maximal amplitudes of 0.9 +/- 0.1 mV (11 slices, mean +/- S.E.M.) and 1.0 +/- 0.1 mV (nine slices), respectively. The negative field potentials induced by NMDA were accompanied by monophasic decreases of [Ca2+]o (0.8 +/- 0.1 mmol/l, nine slices). AMPA elicited no (three slices) or only minor decreases of [Ca2+]o (0.2 +/- 0.1 mmol/l, five slices). The responses to the glutamate subreceptor agonists NMDA and AMPA were reversibly depressed by adding their specific antagonists DL-2-amino-5-phosphonovalerate (APV, 100 mumol/l, six slices) and 6-cyano-7-nitroquinoxalin-2,3-dion (CNQX, 5 mumol/l, four slices), respectively. The results correspond to findings in animal experiments and are consistent with the interpretation that in the human neocortex the Ca2+ permeability of channels gated by NMDA is higher than those gated by AMPA.

With epileptic activity, neurons show paroxysmal depolarization shifts (PDSs) corresponding to epileptic field potentials (EFPs) generated by the neuronal population. Experimental results indicate that calcium and calcium-dependent currents participate in the generation of these events. Consequently neuronal PDS/EFP were depressed by organic calcium channel blockers. This justifies the hope that calcium channel blockers might be useful in the treatment of human epilepsies.

Changes of free calcium concentration at the outer neuronal surface during paroxysmal depolarization shifts elicited by pentylenetetrazol were measured. Investigations were performed on the identified neuron B3 of the buccal ganglion of Helix pomatia. Extracellular calcium concentration was recorded by calcium-selective microelectrodes. The extracellular calcium concentration steeply decreased with the commencement of paroxysmal depolarization and started to reincrease when the paroxysmal depolarization had reached its plateau level. It is concluded that an influx of calcium ions takes place during paroxysmal depolarization shifts.

Epileptic activity was elicited in the rat's motor cortex by local application of penicillin. At the neuronal level it consisted of typical paroxysmal depolarization shifts. The calcium agonist BAY K 8644 was injected into neurons showing such a discharge pattern. The application of this drug increased amplitude and after depolarization of paroxysmal neuronal depolarizations.

Interference of the epileptogenic drug pentylenetetrazol (PTZ) on the Ca2+-selective microelectrode based on the neutral carrier ETH 1001 is described. It is suggested that tetraphenylborate, a component of the Ca2+-selective membrane, is responsible for this interference. Though the sensitivity of the Ca2+-selective microelectrode to PTZ is low, the error in measurements of the free Ca2+ concentration in biological preparations treated with PTZ has to be considered, since pathological changes in the extracellular Ca2+ concentration also produce relatively small changes in the relative electrode potential.

Intracellular Ca2+ is known to activate an early unspecific inward and a potassium outward current in various vertebrate and invertebrate preparations. In the identified neurons B1, B2 and B3 of the buccal ganglia of Helix pomatia an intracellular application of Ca2+ induced a late long-lasting inward current besides the currents mentioned. Ca2+ was injected by ionophoresis or pressure pulses. The long-lasting inward current was reversed at membrane potentials of between -30 and -40 mV, whereas the equilibrium potential of the unspecific inward and potassium outward current was in the range of -20 to +10 mV and -50 to -70 mV, respectively. The late inward current was accompanied by an increase of the membrane conductance and suppressed by the Cl- channel blocker 9-anthracencarbonic acid. The equilibrium potential shifted with changes in the extracellular Cl- concentration. The long-lasting inward current is suggested to be activated by intracellular Ca2+ and to be carried by Cl-.

Focal epileptiform activity was induced by local application of penicillin to the surface of the rat motor cortex. Neurons located within the epileptic focus displayed typical paroxysmal depolarization shifts (PDS). The participation of membrane calcium currents in the generation of PDS was examined by injecting the quaternized calcium entry blocker D890 into single neurons by iontophoresis or by pressure pulses. After intracellular injections of D890, PDS were depressed in amplitude by up to 55%. In a few cases the depression of PDS following intracellular application of D890 was preceded by a transient increase. Similar increases of PDS amplitude were obtained by injections of the calcium chelator EGTA. Control experiments in preparations without epileptic activity revealed that excitatory potentials elicited by thalamic stimulation and Cl(-)-dependent inhibitory postsynaptic potentials evoked by epicortical stimulation were not affected by intracellular D890. In these experiments successful intracellular drug application was verified by monitoring the transient shift of the Cl(-)-equilibrium potential induced by injection of KCl together with D890. It is concluded that in the penicillin-induced epileptic focus of the motor cortex Ca2+ inward currents participate in the generation of neuronal PDS.

The actions of intracellularly applied D890 on membrane currents of the identified neurons B1, B2 and B3 of Helix pomatia were investigated. The TTX-resistant component of the inward current, the inward currents in Na+-free sucrose solution and in Ca2+-free Ba2+ solution were reduced. In Ca2+-free Co2+ solution the inward current was not affected. The late outward currents were strongly reduced. In solutions containing 20 mmol/l NiCl2 the remaining parts of these currents were blocked only to a lesser extent. The early outward current remained unchanged. It is concluded that intracellularly applied D890 mainly exerts its effects on the calcium current.

The ionic composition of the currents underlying the acetylcholine (ACh) depolarizations in the identified neurons B1 and B3 of the buccal ganglia of Helix pomatia was analysed. The equilibrium potential of the ACh responses was -2.8 +/- 0.6 mV (N = 49) and -4.0 +/- 0.7 mV (N = 79; mean +/- SEM) in the neurons B1 and B3, respectively. Replacement of NaCl in the bath solution by sucrose shifted the ACh equilibrium potential into the negative direction. A similar but less pronounced shift occurred when Ca2+ was substituted for Na+. Substitution of Cl- in the bath solution by propionate or an increase of the intracellular Cl- concentration did not affect the ACh equilibrium potential. Changes of K+ concentration in the bath between 1 and 50 mmol/l left the ACh equilibrium potential nearly unaffected when the Na+ concentration was at the control level. With a simultaneous reduction of extracellular Na+ an increase of K+ concentration shifted the ACh equilibrium potential towards more positive potentials. The findings are compatible with calculated K+ permeabilities if a K+ redistribution across the cell membrane is considered. In the neurons B1 and B3, channels operated by ACh are permeable for K+, Na+ and Ca2+, with the relative permeabilities 1.6:1.0:0.1.

In animal experiments, focal epileptic activity and tonic-clonic seizures were induced by local application of penicillin and repeated intraperitoneal injections of pentetrazol (PTZ), respectively. With epileptic activity neocortical and hippocampal neurons showed typical paroxysmal depolarization shifts. Experimental findings indicate that a calcium inward current and calcium dependent membrane currents participate in the generation of these events. Paroxysmal depolarizations were depressed by intra- and extracellular applications of the organic calcium channel blockers N,N-dimethyl-N-(4-cyan-4-(3,4,5-trimethoxyphenyl)-5-methyl-hexyl)- N-(beta- 3,4-dimethoxyphenylethyl)-ammonium-chloride-monohydrate (D 890) and verapamil. The same depressive effect was exerted by flunarizine in hippocampal but not in neocortical neurons. Paroxysmal depolarizations were enhanced by intracellular injections of the calcium agonist methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5- carboxylate (Bay K 8644) and of the calcium chelator ethyleneglycol-bis(aminoethylether)N,N,N',N'-tetraacetate (EGTA). Focal seizure activity of the neocortex was reduced and often abolished by intracerebroventricular perfusion of verapamil, with the frequency of occurrence of epileptic discharges being decreased. Generalized tonic-clonic seizures were depressed to a great extent and often abolished during an intracerebroventricular verapamil perfusion. Simultaneously, the negative shift of the DC potential evoked by the PTZ injections turned over to a positive displacement. In non-epileptic preparations, organic calcium antagonists had no depressive effects on neuronal bioelectrical activity.

Potassium- and calcium conductances regulate neuronal excitability and epileptiform activity. In this study, the effects of different extracellular potassium concentrations ([K+]o) were investigated on the modulatory effect of the L-type transmembranous calcium currents on epileptiform discharges. The in vitro brain slice technique was used to examine the effects of calcium channel blockers, verapamil and nifedipine, on the repetition rate, amplitude, and duration of epileptiform field potentials (EFP) in the presence of low, physiological, and high background [K+]o in guinea pig hippocampal slices. Epileptiform activity was induced by omission of Mg2+ from artificial cerebrospinal fluid contained 2, 4, and 8 mM [K+]o. Both verapamil and nifedipine suppressed EFP after a transient increase in repetition rate. The extent of EFP frequency rate acceleration significantly increased with reduction of [K+]o. The increase in EFP frequency rate induced by application of verapamil and nifedipine was accompanied by a reduction in the EFP amplitude and a reversible increase in the burst discharge duration. The extent of burst discharge prolongation was also significantly higher with decreasing [K+]o. Further application of verapamil and nifedipine suppressed the epileptiform burst activity in the presence of different [K+]o. The latency of EFP depression was significantly diminished both with increased and decreased background potassium concentrations. The data indicate the importance of the effect of the L-type transmembranous calcium currents on the regulatory effect of background [K+]o on epileptiform burst discharge frequency and duration. [Copyright &y& Elsevier]