Ulrich Altrup

Inputs and outputs of giant neurons B1 and B2 in the buccal ganglia ofHelix pomatia: an electrophysiological and morphological study

The identified giant neurons B1 and B2 in the buccal ganglia of Helix pomatia were studied with electrophysiological and morphological techniques in order to establish a baseline for the study of neuronal communication in a relatively simple nervous system. Different synaptic inputs to neurons B1 and B2 were found to come from neurons of the buccal ganglia and to come via buccal nerves. In the epithelium of the pharynx, fibers of bipolar peripheral neurons could be stained, the central fibers of which probably contribute to the synaptic inputs of neurons B1 and B2. The dendrites of neurons B1 and B2 are mainly situated in anterior and lateral parts of the neuropil of the buccal ganglia, respectively. The axon of neuron B1 was traced to the esophagus/stomach. Intracellular stimulation of the neuron induces both a contraction of the longitudinally oriented fibers and an increase in the amplitude of spontaneously occurring peristaltic contractions of the esophagus. The axons of neuron B2 run to both salivary glands. Thin axon collaterals showing multiple swellings follow the bases of the epithelial cells of the salivary gland. The functioning of neurons B1 and B2, which are homologous to giant neurons in the buccal ganglia of other molluscs, is discussed.

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.

Block of spontaneous termination of paroxysmal depolarizations by forskolin (buccal ganglia, Helix pomatia)

Effects of cAMP-activated protein kinases (PKA) on epileptic activity are at present studied in a model nervous system. Identified neurons in the buccal ganglia of the snail Helix pomatia were recorded with intracellular microelectrodes in a continuously perfused experimental chamber. Epileptiform activity appeared regularly in neuron B3 when the saline contained pentylenetetrazol (20-40 mM). Epileptiform activity consisted of a series of paroxysmal depolarization shifts (PDS). Epileptiform activity was quantified by calculating the percentage of PDS-duration of PDS-periods. High percentage of PDS-duration was regularly found 15-30 min after the start of treatment with pentylenetetrazol. Subsequently, percentage of PDS decreased spontaneously. Adding forskolin (50 microM) to the pentylenetetrazol-containing solution increased percentage of PDS-duration. The increase during forskolin corresponded to the amount of decrease which had taken place spontaneously before. During application of forskolin for up to 4 h, spontaneous PDS decrease was absent, i.e., epileptiform activity corresponded to status epilepticus. Forskolin was not able to induce epileptiform activity when applied without pentylenetetrazol. 1,6-Dideoxy-forskolin (50 microM) did not accelerate epileptiform activity. When pentylenetetrazol was applied twice (1 h each) separated by 2.5 h of control conditions, PDS decrease obtained during the first application was found to be largely preserved during control conditions. When forskolin was applied for 30 min in between both applications of pentylenetetrazol, the second response to pentylenetetrazol did not show a preserved PDS decrease. Results suggest that forskolin blocks an endogenous antiepileptic process and that activation of PKA can maintain epileptic activity and induce status epilepticus.

Endogenous pacemaker potentials develop into paroxysmal depolarization shifts (PDSs) with application of an epileptogenic drug

Well-known invertebrate ganglia (buccal ganglia of Helix pomatia, abdominal ganglia of Aplysia californica) were used to study the contribution of synaptic potentials, central pattern generators, and endogenously generated neuronal potentials to the development of epileptiform activity. Epileptiform activity which was induced with application of pentylenetetrazol (1 to 100 mM) or etomidate (0.12 to 1.0 mM) consisted of paroxysmal depolarization shifts (PDSs) recorded simultaneously from several identified neurons with sharp microelectrodes. With application of an epileptogenic drug, endogenous pacemaker potentials develop into PDSs. With increasing concentration of the drug, (i) amplitude of pacemaker-depolarizations and (ii) delay of pacemaker-repolarization increased progressively finally resulting in PDSs. Additionally, the activation characterists of currents shifted from between -50 and -40 mV (pacemaker potentials, control conditions) to between -100 and -40 mV (PDS, epileptic conditions). Only neurons which generated pacemaker potentials under control conditions could generate PDSs under epileptic conditions. Chemical synaptic inputs triggered or blocked pacemaker potentials as well as PDSs. Activities induced from central pattern generators were identified with simultaneous recordings from several identified neurons. The central pattern generators could trigger or block pacemaker potentials as well as PDSs. Results demonstrate that, in the used model nervous systems, pacemaker potentials which are generated by the single neurons are the physiologic basis of epileptic activity.

Decrease of free calcium concentration at the outer surface of identified snail neurons during paroxysmal depolarization shifts

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.

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

Abstract.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.

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.

Intrasomatically recorded action potentials in snail neurons (Helix pomatia): different shapes with different sites of origin in the neuronal arborization. A combined morphological and electrophysiological study

Fibres of the identified neurons B1 to B3 in the buccal ganglia of Helix pomatia can be divided into three types according to their diameters. Electrical stimulation of nerves containing the different fibres induces typical fast depolarizations in the somata of neurons B1 to B3. The appearance of these depolarizations is strictly correlated to the fibre types. The depolarizations are interpreted as axonal action potentials.

Differentiation between anti- and orthodromic responses to nerve stimulation in neurons with axo-axonal synapses (Helix pomatia).

In identified neurons of Helix pomatia nerve stimulation evoked depolarizations of short latencies. The mechanisms underlying these potentials were studied by conventional electrophysiological and intracellular staining techniques. Most of the depolarizations behaved like chemically mediated postsynaptic potentials. The remaining responses can be regarded either as antidromic axonal action potentials (APs) or as electrotonic junction potentials. The differentiation between the latter alternatives and hence the identification of the axonal pathways of the impaled neurons proved to be difficult using conventional methods.

Epileptogenic drugs in a model nervous system : Electrophysiological effects and incorporation into a phospholipid layer

Mechanisms of epileptiform activity in a model nervous system (buccal ganglia of Helix pomatia) are presented. The ganglia contain the identified giant neurons B1 through B4. For epileptiform activity, pentylenetetrazol (1 mmol/L to 40 mmol/L) or etomidate (12.5 micromol/L to 500 micromol/L) were applied. Membrane pressure was measured using a Wilhelmy film balance. In electrophysiological experiments, both drugs induced several effects in all studied neurons: membrane resistance increased, down-stroke of action potentials declined, and all types of chemical synaptic potentials decreased (the latter concerns pentylenetetrazol only). The threshold was 1 mmol/L of pentylenetetrazol and 12.5 micromol/L of etomidate. Epileptiform potentials developed in neurons that had expressed the membrane mechanisms underlying pacemaker potentials. The threshold of this development was again 1 mmol/L of pentylenetetrazol and 12.5 micromol/L of etomidate. Epileptiform depolarizations appeared with 40 mmol/L of pentylenetetrazol and 500 micromol/L of etomidate. In biochemical experiments, both drugs incorporated into an artificial phospholipids membrane and increased pressure in the membrane. The threshold of pressure increase was 1 mmol/L of pentylenetetrazol and 12.5 micromol/L of etomidate. Pressure increased dose-dependently and was 69% and 63% above starting pressure of 10 mN/m with epileptogenic concentrations of pentylenetetrazol (40 mmol/L) and of etomidate (500 micromol/L), respectively. It is postulated that amphiphilic substances incorporate into cell membranes and increase intramembranous pressure, and that this disturbs several membrane processes mechanically and leads to epileptic depolarizations in pacemaker neurons.