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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.

Locally applied epileptogenic drugs directly affect the local cells which project their epileptic activity into the surrounding tissue. This paper describes an experimental set-up which allows differentiation of direct effects of an epileptogenic drug from indirect ones, which are synaptically projected. The set-up consisted of an experimental chamber which enabled the isolated superfusion of a part of a nervous system with a drug. Isolated superfusion was obtained by means of a divider between two compartments of the experimental chamber and of an outlet for the bath solutions below the divider. Quality of isolation was tested with unilateral application of the epileptogenic drug pentylenetetrazole (PTZ) and of the dye methylene blue. Distribution of extraganglionic and intraganglionic PTZ was measured by PTZ-selective microelectrodes. It was found that the drug could not be detected at the non-drug side. Correspondingly, unilateral dye application resulted in a sharp border between the treated and untreated side. The experimental set-up was used to study projected events evoked by sustained epileptic activity of defined parts of a nervous system. It was found that the non-treated cells became progressively coupled to the epileptic neurons in the course of the experiments.

The action of the epileptogenic agent pentylenetetrazol (PTZ) on a cloned potassium channel of the rat brain was studied. The Kv1.1 channel was expressed in oocytes of Xenopus laevis and potassium currents were investigated in outside-out and inside-out membrane patches. The results show that PTZ increased the multi-channel potassium currents at strongly negative potentials and decreased them at potentials positive to -35 mV both in outside-out and inside-out membrane patches. The extent and manner of PTZ action, the concentration dependence as well as the onset and time course of the PTZ effect were the same both in outside-out and inside-out membrane patches. The single-channel potassium currents showed an increase in open probability and frequency of opening and a decrease in close time at -50 mV and vice versa at 0 mV with application of PTZ. The amplitude of single-channel current, the open time and the latency to the first channel opening remained almost unchanged under PTZ. The results indicate that PTZ acts via the cell membrane and influences the membrane-associated part of the potassium channel. Thereby, PTZ accelerates the transition from the inactivated to the open state of the channel at strongly negative potentials and reduces it at slightly negative and positive potentials. This mechanism may be the basis for a gate function which is in favour of the development of epileptic discharges.

For further investigation of the epileptogenic properties of pentylenetetrazol (PTZ), membrane currents elicited by PTZ were analysed in native Xenopus oocytes. PTZ elicited a sequence of membrane currents. Two inward currents have been described to be due to a decrease in potassium permeability and an increase in chloride permeability. Experiments performed up to 3 days after preparation of the oocytes showed that PTZ is also able to activate an outward current. This current is: (1) reversed near the potassium equilibrium potential, (2) associated with a decrease in membrane resistance, (3) reduced by tetraethylammonium and caesium, (4) abolished by defolliculation, and (5) blocked by glibenclamide. Thus, the current can be interpreted to be due to an activation of ATP-sensitive potassium (KATP) channels located in the follicle cells. An activation of KATP channels by PTZ may contribute to termination and re-initiation of seizure activity.

The convulsive agent pentylenetetrazol (PTZ) antagonized gamma-aminobutyric acid (GABA)-induced membrane currents on RNA-injected Xenopus oocytes with both extra- and intracellular applications. With extracellular administration PTZ enters the cell within a few minutes and reaches concentrations in the millimolar range. The permeability of the plasma membrane makes it possible for systemically applied PTZ to elicit its effect on the GABA-induced currents via extra- as well as intracellular sites of action.

The effects of the epileptogenic agent pentylenetetrazol (PTZ) on current responses to glutamate (Glu) and to the Glu receptor agonist N-methyl-D-aspartate (NMDA), kainate (KA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and quisqualate (QA) were studied on RNA-injected Xenopus oocytes. PTZ antagonized the reactions to Glu as well as to NMDA, KA, AMPA and QA. With increasing concentration of AMPA, however, the depressive effect of PTZ turned to an augmentation. The complete change from a decreasing to an increasing effect of PTZ with elevated agonist concentration was unique for current responses by AMPA.

The effect of the epileptogenic agent pentylenetetrazol (PTZ) on the cloned rat brain potassium channel Kv1.1 (labelled also RCK1) was investigated in the Xenopus laevis oocyte expression system. The Kv1.1 channel was affected by PTZ in a voltage-dependent manner. PTZ increased the potassium currents at more negative potentials and decreased them at more positive potentials. At a potential of -50 mV the potassium currents were increased by 0.97 and at -20 mV decreased by 0.21 of control value with 100 mmol/l PTZ. The potential at which the inversion from increase to decrease occurred was -33 mV. The inactivation characteristic of the current was shifted to more negative potentials by PTZ. The PTZ effect was obtained at a threshold concentration of 1 mmol/l and increased with rising PTZ concentrations. After removal of the tissues covering the oocyte membrane, the PTZ effect was augmented; with a concentration of 10 mmol/l PTZ the potassium currents at 0 mV were decreased by 0.04 in oocytes with covering tissues and by 0.27 of control value in oocytes without covering tissues. Under current-clamp conditions, PTZ decreased small depolarizations and increased larger depolarizations. This effect of PTZ represents a 'discriminatory function' that may contribute to epileptogenesis in nervous tissues.

The effects of the epileptogenic substance pentylenetetrazol (PTZ) on ligand operated membrane channels were studied. For this purpose serotonin (5-HT) sensitive channels were expressed in RNA injected oocytes of Xenopus laevis. With simultaneous application of both substances, the response to 5-HT was reduced and eventually abolished by PTZ with increasing concentrations (5-100 mM). A reduction of the 5-HT response also appeared when PTZ was applied in various intervals (15 and 240 s) before 5-HT. It may be assumed that PTZ produces a component of its epileptogenic effect by acting on ligand operated membrane channels.

The effects of the epileptogenic agent pentylenetetrazole (PTZ) on membrane currents of native oocytes of Xenopus laevis were studied. PTZ elicits a response that consists of two inward currents. The first one is interpreted to be due to a decrease of potassium permeability since: (1) the input resistance is increased; (2) the equilibrium potential is near that of potassium; (3) the current is decreased during administration of potassium channel blocking agents; and (4) the PTZ response can be mimicked by blocking potassium channels without PTZ application. The second one is interpreted to be due to an increase of chloride permeability since: (1) the input resistance is decreased; (2) the equilibrium potential is near that of chloride; and (3) the response is decreased during administration of chloride blocking agents. These findings correspond to some extent with those made in neurons.

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.

The effect of the epileptogenic drug pentylenetetrazol (PTZ) on the shape of dendrites of identified snail neurons was investigated. Comparison between control and test preparations revealed that changes in the shape of dendrites appeared after PTZ treatment: (i) the number of dendrites, especially filopodia-like structures, increased; (ii) separations and involutions of dendrites occurred. Both of these changes of dendrites were found either separately or in combination.

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.

A double-barrelled microelectrode is described, which permits the continuous measurement of the concentration of the epileptogenic agent pentylenetetrazol (PTZ). The electrode is based on the liquid potassium exchanger (Corning No. 477 317) and enables measurements of PTZ concentration in physiological salines down to 1 mM. The electromotive behaviour of the liquid membrane against PTZ cannot directly be described by the Nicolsky-Eisenman formalism. It is suggested that specific interactions of the PTZ molecule with the Corning ligand are involved in the potential generating mechanisms.

The actions of the convulsant drug pentylenetetrazol (PTZ) were studied on CA3 neurons of hippocampal slices (300-400 microns thick). In 9 out of 109 neurons, epileptic reactions were elicited during a single application of PTZ. After repeated applications of PTZ, 53 neurons showed periodic paroxysmal activity. They developed according to the following sequence: (i) Paroxysmal hyperpolarizations, (ii) burst activity, and (iii) typical paroxysmal depolarization shifts (PDS). The rate of occurrence was about 8/min. Paroxysmal hyper- and depolarizations appeared synchronously in pairs of neurons. The developmental sequence occurred in reverse during washing. After the onset of paroxysmal activity, bursts and PDS persisted if PTZ concentration in tissue ranged between about 2 and 10 mmol/l. When this range was exceeded in either direction, epileptic activity was abolished at a calcium concentration of 2.75 mmol/l. Decreasing and increasing the calcium concentration shifted the epileptogenic concentration range to lower and higher levels, respectively. It is concluded that repetition of PTZ application alters membrane properties of neurons and thus leads to paroxysmal events triggered by synaptic processes.

After systemic application of pentylenetetrazol (PTZ), mammalian as well as molluscan neurons generate epileptic paroxysmal depolarization shifts. For a further analysis of these potential oscillations the membrane currents induced by local application of PTZ onto identified neurons of Helix pomatia were investigated. Different types of responses were obtained at membrane potentials negative and positive to ca. -30 mV. At holding potentials more negative than -30 mV, PTZ as a rule evoked an inward current, sometimes preceded by a brief outward current. In a few experiments only a solitary outward current was found. The amplitudes of the inward and outward currents increased towards more negative potentials. The inward current was associated with a decrease and the outward current with an increase in membrane resistance. Besides these findings pharmacological and ion substitution experiments indicate that the inward current represents an unspecific current. At holding potentials more positive than -30 mV, PTZ evoked a sequence of currents which was the same in all neurons. This stereotyped current sequence consisted of (i) an early inward current, (ii) an intermediate outward current, and (iii) a late long-lasting inward current. The amplitudes of all these components increased towards more positive potentials with the outward current being particularly enhanced. The early inward current and the following outward current were associated with a decrease and the late inward current with an increase of the membrane resistance. Besides these pharmacological and ion substitution experiments suggest that the early inward current represents a mixed sodium and calcium current, the intermediate outward current a calcium activated potassium current. The late inward current is assumed to be due to a decreased potassium conductance. On the basis of the present results, it may be concluded that the unspecific inward current in the negative potential range is involved in the initiation and the calcium dependent potassium current in the termination of spontaneously occurring paroxysmal depolarization shifts.

Antiepileptic actions of the organic calcium antagonists flunarizine (cinnarizine derivate) and verapamil (papaverin derivat) on pentylenetetrazol-induced epileptic bioelectric activity were tested in CA3 neurones of hippocampal slices. In all experiments both calcium antagonists reduced the amplitudes and/or durations of paroxysmal depolarizations as well as their rate of occurrence, when the bath concentrations of flunarizine or verapamil exceeded 20 mumol/l. When they were added to the bath solution before pentylenetetrazol application, recordings of the resting membrane potential, of the membrane resistance, of action potentials and of spontaneous as well as of evoked excitatory and inhibitory postsynaptic potentials gave no indication that the antiepileptic effects of these drugs are due to unspecific depressive actions on neuronal excitability or spread of excitation.