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To elucidate the intracellular mechanism of the bursting activity which is characteristic of seizure discharge, the behavior of the intracellular cyclic nucleotide and the intracellular calcium during pentylenetetrazole (PTZ)-induced bursting activity in snail neurons was investigated. Cyclic AMP was increased about 3-fold by the incubation of ganglia with PTZ. The effect of PTZ on phosphodiesterase activity measured using either cyclic AMP or cyclic GMP as substrate showed a slight increase in cyclic on phosphodiesterase activity. The release of calcium from the lysosome fraction was increased by the incubation of ganglia with dibutyryl cyclic AMP. Protein kinase activity was stimulated by the incubation of ganglia with PTZ. Adenylate cyclase activity was stimulated by the incubation of ganglia with PTZ. These findings suggest that PTZ-induced bursting activity in snail neurons is initiated by an intracellular increase of cyclic AMP, which promotes calcium release from lysosomes and induces protein kinase activation.

In order to clarify its role in the provocation of seizure activity, the effects of cyclic AMP were examined on the intracellular calcium concentration of pentylenetetrazol (PTZ)-sensitive neurons as well as of PTZ-non-sensitive neurons of the Japanese land snail, Euhadra peliomphala . Extracellular application of isobutylmethylxanthine (IBMX) and dibutyryl cyclic AMP showed bursting activity-like firing in the PTZ-sensitive neurons. When 5′-guanylylimidodiphosphate (GNP-PNP) was injected into PTZ-sensitive neurons in the extracellular presence of IBMX and dibutyryl cyclic AMP, bursting activity followed by long-lasting hyperpolarization occurred. Intracellular injection of cyclic AMP into PTZ-sensitive neurons caused hyperpolarization coincident with an increase in intracellular calcium concentration. This increase in intracellular calcium concentration was the same under conditions in which the calcium influx was inhibited by the substitution of extracellular calcium chloride by cobalt chloride. In PTZ-non-sensitive neurons, cyclic AMP-induced bursting activity was not observed. These results suggest that an increase in cyclic AMP provoked bursting activity via an increase in intracellular calcium concentration.

To elucidate the behavior of the single ionic channels of cerebral cortical neurons during bursting activity, the effects of pentylenetetrazole (PTZ) on a single potassium channel of primary cultured cerebral cortical neurons from mice were examined. All of the examined 10-day-old primary cultured neurons of the cerebral cortex showed clear bursting activity after extracellular application of PTZ using whole-cell patch-clamp recording. Less than half of the examined single potassium channels, both outward and inward, of the 2- and 3-day-old as well as 6-10-day-old primary cultured cerebral cortical neurons showed the bursting-type open-close state by application of PTZ. The PTZ-sensitive single potassium channels were found in the voltage-dependent as well as calcium-activated channels.

The effect of Pentylenetetrazol (PTZ, Metrazol) upon the identified neurons of Japanese land snail was studied.1) In the pleuro-parieto-visceral ganglion of Japanese land snail, Euhadra peliomphala, three types of cells can be differentiated morphologically and functionally as those; depolarized by acetylcholine (D-cell), hyperpolarized by acetylcholine (H-cell) and indifferent to acetylcholine (I-cell).2) PTZ produces a phasic depolarization followed by a sustained depolarization in the D- and H-cells.3) PTZ has no specific effect upon the I-cell.4) By application of d-tubocurarine, atropine and hexamethonium, the typical phasic activity evoked by PTZ is not affected in either the D- or H-cells, suggesting that the effect of PTZ is not of synaptic origin but of endogeneous origin.5) Neither cholinergic nor noncholinergic inhibitory input to the Dand H-cells are affected even at the peak of PTZ activity.

1. In two patiens suffering from urinary retention of various causes, appearance of arrhythmias were seen at the time of overdistension of the urinary bladder, which promptly disappeared by emptying of the bladder by catheterisation.2. Experimental overdistension of the urinary bladder in animals resulted in transitory fall of blood pressure, pictures of coronary insufficiency in EKG and/or appearance of arrhythmias. These changes disappeared by relieving the bladder of overdistension.3. In guinea pigs it was shown that the EKG changes produced by over-distension of the urinary bladder were erased by transection of the spinal cord between the lumbar and sacral segment.4. Using electrophysiological technic, it was shown that the nervous transmission from the bladder wall to the heart was related to vago-vagal reflex.This research was reported at the 11th Congress of the Japanese branch of the international neurovegetative research, on the 8th July 1962.

1. Using a coaxial microelectrode, intra- and extra-cellular potentials were recorded from “idle” cells in the cerebral cortex of the cat. Records were made during repetitive stimulation of the cortical surface and during Metrazol induced paroxysmal activity.2. The intra- and extra-cellular potentials induced by either stimulation of cortical surface or Metrazol injection were of opposite polarity, revealing that the intracellularly recorded potential was a change in membrane potential of the impaled cell and not a field potential generated by other elements.3. The extracellular potential elicited during stimulation of the cortical surface was an approximate mirror image of the intracellular one, and thus accounted for the slow depolarization of the “idle” cell.4. Following Metrazol injection, the extracellular potential, although opposite in polarity to the intracellular one, showed oscillation which had no counterpart in the intracellular record. Similar recordings made from cortical neurons showed intracellularly recorded oscillations which corresponded to extracellular ones. Therefore, the extracellular potential recorded immediately outside an “idle” cell following Metrazol is viewed as reflecting a composite of both glial and neuronal membrane changes.5. Iontophoretic staining of “idle” cells suggests that they are probably glia.

1) The effect of so-called psychostimulant drugs (cytidine diphosphate choline, 5'-cytidine monophosphate choline-2Na, Centrophenoxine and Pyri-thioxine) on the DCR was tested.2) These drugs except 5'-cytidine monophosphate choline-2Na have the al-most identical effect on the DCR: the augmentation of the slow negativity, the increase of the primary negative potential and the exaggeration of the negative DC shift with repetitive stimulation.3) The mechanism of action of these psychostimulant was discussed.The authors wish to express their sincere thanks to Dr. Tatsuji Nomura, Chairman of Central Laboratory for Experimental Animals, for giving them the facilities for this investigation.

A thickness of about 100 angstroms of the cell membrane of an isolated single freeze-dried neuron of the snail can be etched off with an ion shower milling machine. The calcium content of the cell membrane area was more than one-fifth of the whole cell. The calcium content of the cell membrane area increased during pentylenetetrazole-induced bursting activity.

The role of divalent cations, particularly of calcium, has gained increasing interest as a charge carrier and in processes such as regulation of enzymatic activities, in secretions of humoral transmitters and initiation of muscle contraction. The role of calcium has become very important in manifesting the bursting activity of neurons by various electrophysiological techniques, especially by voltage clamping. The distribution of calcium compared with the ultrastructure of nerve cells, however, has not been widely investigated. Analysis by the electron probe X-ray microanalyser(EPXMA) makes this type of research easier. To determine the relationship between calcium localization within the neuron and cellular function, we tried to make an intracellular calcium distribution map of the normal state and that during bursting activity by the computer controlled EPXMA. We also detected the chemical shift between the different states of cellular function caused by the intracellular calcium binding state.