Pacemaker potentials are the physiologic basis of epileptiform activity in the buccal ganglia of Helix pomatia.

Mechanisms of epileptic activity in nervous systems were studied using the identified neurons B1 through B4 in the buccal ganglia of the snail Helix pomatia as a model system. Activities were recorded with intracellular microelectrodes. Epileptiform activity was induced by bath application of an epileptogenic drug (pentylenetetrazol: 1 mM to 40 mM, or etomidate: 0.1 mM to 1.0 mM). Epileptiform potentials recorded from the somata of neurons consisted of paroxysmal depolarization shifts (PDSs). With increasing concentration of an epileptogenic drug, pacemaker potentials in neuron B3 developed into PDS. Simultaneously several types of chemical post-synaptic potentials were suppressed in amplitude. Since on the one hand epileptic seizures only appear when PDS are synchronized in many neurons and since on the other hand synaptic potentials were found to be suppressed during epileptic conditions, mechanisms underlying neuronal synchronization were studied. Evidence was found that, under epileptogenic conditions only, neurons were synchronized by an non-synaptic release of substances. Strong depolarizations accompanied by an increase in intracellular calcium concentration are known to induce an unspecific exocytosis. Thus, an unspecific exocytosis from the dendrites of PDS-generating neurons probably appears under epileptic conditions and synchronizes neighbouring neurons.

Continuous increase of epileptogenic effects following application of proteolytic enzymes (buccal ganglia of Helix pomatia).

Epileptic activity of neurons consists of paroxysmal depolarization shifts (PDS) which can be induced presumably in any nervous system by application of an epileptogenic drug. The spontaneous appearance of epileptic activity, however, is based on a largely unknown process which increases susceptibility to epileptic activity (seizure susceptibility in man). It is presently shown that the treatment of ganglia with proteolytic enzymes (Pronase) decreases the effective concentration of epileptogenic drugs, i.e. increases seizure susceptibility. Since proteolytic enzymes are known to primarily affect glial cells a contribution of glia to seizure susceptibility is discussed.

Epileptogenicity and epileptic activity: mechanisms in an invertebrate model nervous system.

Epileptic seizures are based on paroxysmal depolarization shifts (PDS) which are synchronized in many neurons. Mechanisms underlying PDS and seizures are still not understood. The present review is based on studies using the buccal ganglia of the snail Helix pomatia as a model nervous system. Essential mechanisms of epileptic activity in nervous systems are thought to be identical in whatever part of the human or animal nervous system epileptic activity appears. From studies using the buccal ganglia of Helix pomatia, epileptic activity is essentially non-synaptic. PDS are "giant pacemaker potentials", which are generated non-synaptically by the single neurons. It is, however, not yet clear which processes transform pacemaker potentials into PDS. Synchronization of PDS follows generation of PDS and results mainly from a non-synaptic, unspecific release of intracellular substances from the dendrites of a PDS-generating neuron to the dendrites of neighbouring neurons. This explains the existence of small epileptic foci. From the above observations epileptogenicity is introduced or intensified when the proteins underlying pacemaker potentials are expressed. The first chapter of the present review presents the model system. The second chapter describes epileptiform activity in the model system to correspond in all aspects to epileptiform activity recorded in vertebrate nervous systems including man. Subsequently, antiepileptic and epileptogenic properties of drugs are described using the buccal ganglia. Two following chapters concern neuronal structures and neuronal functions affected by epileptiform activity, and in the final chapter the mechanisms underlying epileptiform activities are described.

Effects of valproate derivatives I. Antiepileptic efficacy of amides, structural analogs and esters.

Derivatives of the antiepileptic drug valproate (VPA, 2-propylpentanoic acid) have been synthesized and tested in order to improve the intracellular availability of VPA. The buccal ganglia of Helix pomatia were used as a test nervous system and antiepileptic efficacies were reconfirmed using rat cortex in vivo. Epileptiform activities consisted of typical paroxysmal depolarization shifts (PDS) which appeared in the identified neuron B3 with application of pentylenetetrazol. Epileptiform activities were found to be accelerated, unaffected or blocked. (i) The Amide-derivatives 2-propylpentanamide and N,N-dipropyl-2-propylpentanamide, and short chain ester derivatives 1-O-(2-propylpentanoyl)-2,3-propandiol, 2,2-di(hydroxymethyl)-1-O-(2-propylpentanoyl)-1,3-propanediol and 2,2-di(hydroxymethyl)-1,3-di-O-(2-propylpentanoyl)-1,3-propanediol accelerated epileptiform activities. Membrane potential often shifted to a permanent depolarization which corresponded to the PDS-inactivation level. (ii) The structural analogs 1-cycloheptene-1-carboxylic acid and cyclooctanecarboxylic acid accelerated epileptiform activities only slightly or were without effects. (iii) The small VPA-ester, 2-propylpentanoic acid ethyl ester, decreased the epileptiform activities in a way that is comparable to the effects of VPA well known from previous studies. It thus could be thought as a VPA-pro-drug. (iv) The mannitol-esters 1-O-(2-propylpentanoyl)-D-mannitol and 3,4;5,6-Di-O-isopropylidene-1-O-(2-propylpentanoyl)-D-mannitol blocked the PDS in a way which is different from the known effects of VPA. These substances are interpreted not to exert their effects after being metabolized to VPA and thus they are thought to be new antiepileptic substances.

Effects of valproate derivatives II. Antiepileptic efficacy in relation to chemical structures of valproate sugar esters.

The structure effect relationships of derivatives of the antiepileptically active ester of valproate (VPA) 3,4:5,6-Di-O-isopropylidene-1-O-(2-propylpentanoyl)-D-mannitol (1) have been studied using intracellular recording to record the membrane potential of single neurons (buccal ganglia, Helix pomatia). Epileptiform activity was induced by the epileptogenic drug pentylenetetrazol. The effects of several derivatives on epileptiform activity were compared with those of the relay compound 1. Most of the synthesized agents decreased the duration of paroxysmal depolarization shifts (PDS) and increased their repetition rate. It was considered that a decreased the duration of PDS is antiepileptic and an increased repetition rate is pro-epileptic. Compared with the effects of compound 1, the following relationships were found: (1) Derivatives containing glucitol or galactitol were of similar antiepileptic potency. (2) Introduction of pyranoses or furanoses rendered the substances inactive or even pro-epileptic. (3) VPA in position 1 and 6 at the sugar acted as an antiepileptic whereas in position 3 and 4 it proved to be ineffective. (4) Replacement of VPA by ethylhexanoyl reduced the antiepileptic potency slightly and pivaloyl strongly. (5) Replacement of isopropylidene bridges by penta-O-acetyl or cyclohexylidene residues led to largely inactive substances. (6) Compounds having isopropylidene bridges in position 2,4;3,5 proved to be antiepileptic whereas bridges especially in positions 2,3:4,5 slightly enhanced epileptic activities.

Heptanol exerts epileptiform effects in identified neurons of the buccal ganglia of Helix pomatia.

1-Heptanol (0.2-5.0 mM) known to block electrical contacts was tested under epileptic and non-epileptic conditions in the buccal ganglia of Helix pomatia. Synchronicity of epileptiform activity was not affected. In concentrations below 1 mM, heptanol accelerated epileptiform activity induced by pentylenetetrazol. In concentrations above 1 mM, it evoked epileptiform activity without admixture of an epileptogenic drug. Coupling coefficient was increased and decreased in low and high concentration ranges of heptanol, respectively. The measured decrease of coupling is interpreted as a result of the activation of 'epileptiform' membrane conductances accompanied by decreased length constants of neuronal fibers.

Epileptic neurons induce augmenting synaptic depolarizations in non-epileptic neurons (buccal ganglia, Helix pomatia).

Spread of epileptic activity was studied by inducing epileptiform activity (pentylenetetrazol, PTZ) in one part of a nervous system and by analyzing responses of neurons in a non-PTZ-treated part (identified neurons, paired buccal ganglia, Helix pomatia). Paroxysmal depolarization shifts (PDS) induced time-locked depolarizations in non-epileptic neurons (latency ca. 5 s, duration ca. 1 min, amplitude < or =20 mV). Amplitudes were augmenting during several hours of epileptic activity. Depolarizations were accompanied by an increase in membrane resistance and they were blocked in 'high Mg-low Ca' solutions. It is assumed that the potentials represent a typical widespread response of non-epileptic neurons to PDS of other neurons. This response may be induced via non-specific releases of substances of the epileptically active neurons thereby activating neighboring neurons which in turn activate neurons in control ganglion.

Prediction of neurotoxic potency of hazardous substances with a modular in vitro test battery.

Neurotoxic action was investigated on different model nervous systems linked to a modular in vitro test battery. Voltage operated potassium channels and glutamate operated ion channels expressed in oocytes of the clawed frog Xenopus laevis by injection of cRNA (cloned RNA) or mRNA, respectively, as well as isolated neurons and isolated neuronal networks from the buccal ganglia of the snail Helix pomatia, were used as consecutive modules of different complexity. Lead (Pb2+) was chosen as a known neurotoxic model substance to evaluate the suitability of the test battery to predict the neurotoxic potency of hazardous substances, to establish dose-response relationships, and to investigate the basic mechanisms involved in neurotoxicity. All modules delivered consistent results: potassium currents were reduced by lead with a threshold concentration of 0.1 mumol/l. Membrane currents elicited by the glutamate receptor agonists kainate were decreased by lead with a threshold concentration below 0.1 mumol/l, while currents elicited by the agonist AMPA were not affected. Action potentials generated by the isolated B4 snail neuron showed a decrease of potential amplitude and a prolongation of potential duration after application of lead. The neuronal network controlling the feeding activities of the snail reacted with a decrease of the frequency of the spontaneously generated feeding depolarisations, thus showing the direct neurotoxic effect of lead on body functions and behaviour.

A method to study the effects of an epileptic focus on non-epileptic nervous tissue.

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.

Alterations of neuronal fibers after epileptic activity induced by pentylenetetrazole: fine structure investigated by calcium cytochemistry and neurobiotin labeling (buccal ganglia, Helix pomatia).

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.

Simultaneous blockade of intracellular calcium increases and of neuronal epileptiform depolarizations by verapamil.

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.

Epileptic discharges induced by pentylenetetrazol: ultrastructural alterations in identified neurons and glial cells (Helix pomatia).

The effects of sustained epileptic activity induced by pentylenetetrazol on morphology of buccal ganglia of Helix pomatia were investigated. Neuronal somata and processes as well as glial cells were evaluated after 5 hours of epileptic activity and after 5 hours under control conditions. After epileptic activity neurons showed signs of degeneration consisting of condensation of nuclear chromatin, decreased activity of Golgi apparatus, increased numbers of lamellar bodies and multivesicular bodies, clusters of vesicles and vacuoles, loss of microtubuli, and scattered lamellar bodies. Neuronal somata and large neuronal processes appeared less affected than the smaller processes. Glial cells showed signs of phagocytotic activity as increased cell size, numerous degenerating neuronal processes within the cytoplasm as well as lysosome like bodies and vacuoles. The changes developing along with epileptic activity were interpreted to indicate degeneration and subsequent phagocytotic activity of neuronal processes in synaptic regions of the ganglia. Thus, evidence is presented for synaptically induced degenerative processes in an intact nervous tissue that is not affected by seizure-induced alterations of respiration or systemic circulation.

Attenuation of a voltage-dependent sodium current by GABA (identified neurons, buccal ganglia, Helix pomatia).

The action of systemically applied GABA on voltage-dependent currents of the identified neurons B1-B4 in the buccal ganglia of Helix pomatia were investigated by conventional voltage clamp techniques. In the B4 neuron, superfusion with sodium-free solution or addition of tetrodotoxin to the bath medium abolished the voltage dependent inward current. This voltage dependent sodium current was reduced with GABA application. Muscimol exerted the same effect as GABA whereas administration of baclofen had no effect. Voltage-dependent sodium and calcium currents of the neurons B1-B3 remained unchanged with GABA application. It is concluded that GABA is capable of reducing voltage-dependent sodium currents of distinct neuronal individuals via a GABAA receptor-like structure.

Simultaneous and continuous measurement of free concentration of valproate in blood and extracellular space of rat cerebral cortex.

Free concentration of valproate (VPA) was measured simultaneously and continuously in blood and in the extracellular space of cerebral cortex of rats by VPA-selective microelectrodes. Constant amounts of VPA were injected into the femoral vein with differing duration of injection. Immediately after drug application, the concentration of free VPA in blood and brain increased to a peak value, the degree of which increased with the speed of injection. Ten to 15 min after VPA injection, a plateau value was reached. This plateau value was equal in the extracellular space of cortex and in blood. The data indicate that VPA can "freely" cross the blood-brain barrier (BBB).

Identified neuronal individuals in the buccal ganglia of Helix pomatia.

The buccal ganglia of Helix pomatia are used as model nervous structures in neurophysiological and epileptological studies. Many basic problems concerning membrane physics and the functioning of single neurons and neuronal networks can be easily studied using these ganglia. The model character derives mainly from the relative simplicity of this nervous system and the fact that it contains large, visually identifiable neurons. As in other invertebrate nervous systems, the large neurons have proved to be individuals showing the same functional and structural properties from one animal to another.

Diffusion analysis of valproate and trans-2-en-valproate in agar and in cerebral cortex of the rat.

The diffusion of valproate (VPA) and trans-2-en-valproate were studied in agar gel and in the cerebral cortex of the rat using pressure microejection and VPA-selective microelectrodes. From the agar measurements a free diffusion coefficient for VPA of 6.52 x 10(-6) cm2.s-1 and for trans-2-en-VPA of 5.25 x 10(-6) cm2.s-1 for 37 degrees C was determined. The tortuosity value in the cortex was 1.92 for VPA and 1.67 for trans-2-en-VPA. The tortuosity values suggest that VPA and trans-2-en-VPA diffuse mainly in the extracellular space of the brain.

Effects of valproate on early and late potassium currents of single neurons.

The effects of valproate sodium (VPA) on potassium currents were tested in identified neurons of the snail Helix pomatia. VPA was extracellularly and intracellularly applied. VPA (i) had no effects on the current-voltage relation of the early potassium outward current (IA), (ii) shifted the steady state inactivation function of IA to more positive potentials, (iii) increased the amplitude of the late potassium outward currents. It is suggested that the extrasynaptic effects on potassium currents markedly contribute to the antiepileptic and antimanic effects of VPA.

Identified neuronal individuals in the buccal ganglia of Helix pomatia.

The buccal ganglia of Helix pomatia are used as model nervous structures in neurophysiological and in epileptological studies. Many basic problems concerning membrane physics, functioning of the single neurons and of neuronal networks can be studied easily using these ganglia. The model character mainly comes from the relative simplicity of this nervous system and that it contains large visually identifiable neurons. As in other invertebrate nervous systems, the large neurons have proved to be individuals showing the same functional and structural properties from one animal to the next.

Effects of valproate in a model nervous system (buccal ganglia of Helix pomatia): I. Antiepileptic actions.

Cellular actions of valproate (VPA) were studied using intracellular recordings of identified neuronal individuals in the buccal ganglia of Helix pomatia. Under nonepileptic conditions, VPA induced (a) a hyperpolarization, (b) slight changes in action potentials (AP), and (c) an increase in membrane resistance. Under epileptic conditions (i.e., during application of an epileptogenic drug), extracellular application of VPA decreased frequency of occurrence of epileptic depolarizations (early effect) and led to a decay in paroxysmal depolarizations (late effect). Intracellular injection of VPA could block epileptic activity in the treated neuron immediately. A metabolite of VPA (trans-2-en VPA) mainly lacked the late effect (decay in epileptic depolarizations) obtained with VPA. Results suggest that the early antiepileptic effect is exerted from the extracellular side of the neuronal membrane and that the late effect results from intracellular actions of VPA being delayed by slow access to an intracellular site.

Effects of valproate in a model nervous system (buccal ganglia of Helix pomatia): II. Epileptogenic actions.

High concentrations of valproate (VPA; greater than 20 mM) depolarized identified neuronal individuals in the buccal ganglia of Helix pomatia and transiently induced paroxysmal depolarization shifts (PDS). Threshold concentration of VPA for the induction of PDS was decreased (a) by increased seizure susceptibility, (b) by increased concentrations of derivatives of VPA, and (c) by increased H+ concentrations. Intrasomatic injection of VPA did not induce PDS. The epileptogenic action of VPA is believed to be exerted from the extracellular side of the cell membrane.