Kagemasa Kajiwara

Cloning and expression of SEZ-6, a brain-specific and seizure-related cDNA

To clarify the molecular mechanism of neuronal bursting activity of seizures, we have constructed a cDNA library from mouse cerebrum cortex-derived cells treated with pentylentetrazole (PTZ), one of the convulsant drugs. Using a differential screening technique, several cDNA clones whose expressions change with PTZ-treatment were obtained. Among these clones, SEZ-6 was characterized by increased expression with PTZ. Detailed northern analysis showed that expression of SEZ-6 was limited to the brain and increased by the administration of PTZ not only in in vitro cultured cells but also in vivo. Analysis of SEZ-6 cDNA revealed multiple motifs, including typical signal sequence, threonine-rich domain, five copies of short consensus repeats (SCRs) or sushi domain (complement C3b/C4b binding site), two repeated sequences which were partially similar to the CUB domain or complement C1r/s-like repeat, one transmembrane domain and a short cytoplasmic segment in the C-terminal region. Although many proteins with multiple SCRs or CUB domains other than complement-related proteins have been found, this is the first report about a brain-specific cDNA which encodes membrane protein with both SCRs and CUB domain-like segments. Based on these findings, it is evident that SEZ-6 encodes a novel type of protein which may be related to seizure.

Intracellular calcium concentration during pentylenetetrazol-induced bursting activity in snail neurons

To clarify the role of intracellular free calcium in the provocation of bursting activity, the intracellular calcium concentration was measured using calcium-sensitive microelectrodes during pentylenetetrazol (PTZ)-induced bursting activity in snail neurons. In the PTZ-sensitive neurons of the snail, Euhadra peliomphala, the intracellular calcium concentration was higher than that in the normal state during PTZ-induced bursting activity, but lower than that which induced the calcium-activated potassium conductance elevation. By application of 50 microM calcium ionophore, A23187, a slight increase in intracellular calcium concentration with a slight depolarization occurred. Then a greater increase of intracellular calcium concentration with bursting activity was observed. By application of 100 microM A23187, a rapid and intense increase of intracellular calcium concentration with hyperpolarization was observed. These findings suggest that, for the provocation of bursting activity, sustained elevation of the intracellular calcium concentration, higher than that in the normal state but lower than that which evokes calcium-activated potassium conductance elevation, is required.

Characteristics of primary cultured neurons from embryonic mutant El mouse cerebral cortex

To elucidate the differences between neurons of epileptogenic animals and those of normal animals, cellular characteristics of neurons of mutant strain El mice which are highly susceptible to seizures were investigated using immunocytochemical techniques. In neurons of 3-day primary cultures, the control ddY mouse neurons showed dividing stages in about 0.2% of neurofilament (NF)-positive neurons, whereas no dividing neurons were observed among the NF-positive El mouse neurons. In 7-day cultures, localization of GD3 ganglioside in the proliferating control ddY mouse neurons was observed, but there was no GD3 ganglioside in the mutant El mouse neuron. The content of GD3 ganglioside detected by high-performance thin-layer chromatography of El mouse cultured cells was ca. 1/4 of that of ddy mice. These findings suggest that neurons of the El mouse are differentiated earlier than those of the control ddY mouse.

Cloning and characterization of pentylenetetrazol-related cDNA, PTZ-17

cDNAs related to pentylenetetrazol-induced bursting activity in neurons were screened by a differential hybridization method using normal and pentylenetetrazol-treated primary cultured neurons from the cerebral cortex of mice. Twenty clones of candidate cDNA with expression increased or decreased by treatment with pentylenetetrazol were obtained. One of them, PTZ-17, was sequenced. Injection of PTZ-17 derived RNA into Xenopus oocytes showed a large calcium inward current with extracellular application of pentylenetetrazol.

Pentylenetetrazole-induced changes of the single potassium channel in primary cultured cerebral cortical neurons

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.

Direct current potential changes of the vagal nuclear area induced by trigeminal stimulation

To elucidate the mechanism of the Reilly syndrome (irritation syndrome), direct current (DC) potential changes in the medulla oblongata were investigated. Ipsilateral and contralateral trigeminal stimulation induced a DC potential shift above the vagal nuclear area in the medulla. Maxillary and mandibular nerve stimulation showed a summation effect. Continuous stimulation of the tooth pulp cavity by croton oil induced a progressively developed DC shift. DC potential change by trigeminal stimulation was accompanied by the same process of elevation of extracellular potassium concentration. These findings suggest that trigeminal stimulation increases the excitability of vagal nuclear neurons and consequently provokes the Reilly syndrome.

Microneedle pH Sensor: Direct, Label-Free, Real-Time Detection of Cerebrospinal Fluid and Bladder pH

Acid-base homeostasis (body pH) inside the body is precisely controlled by the kidneys and lungs and buffer systems, such that even a minor pH change could severely affect many organs. Blood and urine pH tests are common in day-to-day clinical trials and require little effort for diagnosis. There is always a great demand for in vivo testing to understand more about body metabolism and to provide effective diagnosis and therapy. In this article, we report the simple fabrication of microneedle-based direct, label-free, and real-time pH sensors. The reference and working electrodes were Ag/AgCl thick films and ZnO thin films on tungsten (W) microneedles, respectively. The morphological and structural characteristics of microneedles were carefully investigated through various analytical methods. The developed sensor exhibited a Nernstian response of -46 mV/pH. Different conditions were used to test the sensor to confirm their accuracy and stability, such as various buffer solutions, with respect to time, and we compared the reading with commercial pH electrodes. Besides that, the fabricated microneedle sensor ability is proven by in vivo testing in mouse cerebrospinal fluid (CSF) and bladders. The pH sensor procedure reported here is totally reversible, and results were reproducible after several rounds of testing.

Mechanism of Phenytoin Action at the Cellular Level

The mechanism of phenytoin (PHT) action has been studied extensively by various investigators. Most of their conclusions can be summarized by 1) inhibition of the sodium channel in the excitable membrane, 2) inhibition of the calcium action related to neuronal excitation and 3) inhibition of cellular phosphorylation. They are all based on the inhibitory action on excitability of the normally functioning neurons.I* The mechanism of action of PHT has seldom been studied with respect to intracellular events. We described our experiments at the cellular level, and discuss the mechanism of PHT action.

Action of cyclic AMP on intracellular calcium concentration and bursting activity

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.