Kazuo Kawasaki

Effects of morphine, L-DOPA and tetrabenazine on the lamina V cells of spinal dorsal horn.

Experiments were performed to clarify the adrenergic mechanism of morphine action in the spinal cord. Unit activity of lamina V cell of spinal dorsal horn (L6−L7) of the rabbit was recorded using microelectrode. An administration of bradykinin into the femoral artery markedly increased in frequency of the unit activity. Morphine or L-Dopa inhibited the bradykinin-induced response of lumina V cell. In contrast, tetrabenazine facilitated the bradykinin-induced response. Electrical stimuli of the bulbar reticular formation, particularly the nucleus reticularis gigantocellularis markedly inhibited the dorsal horn response induced by bradykinin injection. Effect of stimulation of the bulbar reticular formation was blocked by tetrabenazine and reversed by L-Dopa. These results support the hypothesis that the bulbospinal catecholaminergic neuron inhibits the pain transmission at the dorsal horn of the spinal cord and analgesic action of morphine is mainly mediated by activation of this neuron.

Sprouting of CA3 pyramidal neurons to the dentate gyrus in rat hippocampal organotypic cultures

The understanding of the mechanisms of a functional synaptic plasticity in the hippocampus has been expanded greatly by the use of in vitro slice preparations. The question addressed in the present study was whether morphological plasticity observed in vivo can also be reproduced in hippocampal slices. In vivo, hippocampal commissural and association fibers are known to sprout and occupy synaptic sites vacated by deafferentation of the dentate gyrus (DG). In hippocampal slice preparations, the major input to the DG is eliminated, so that the DG is deafferented. Might intrinsic neurons sprout to the DG if the slice preparation is maintained for weeks? In this study hippocampal slices obtained from 6-day-old rats were cultured. Stimulation of the dentate stratum moleculare produced antidromic field potentials in the CA3 of the slices cultivated for more than 1 week. The antidromic response was not observed in CA1 pyramidal neurons. The CA3 to DG projection response was also observed in a CA3 mini-slice placed near a co-cultured whole hippocampal slice, when the DG in the latter was stimulated. Moreover, stimulation of the CA3 mini-slice induced synaptic responses in the DG of the whole-slice. The conclusion drawn is that deafferentation could induce axonal sprouting in a neuron-specific manner in hippocampal organotypic culture. This preparation would be potentially useful for the screening of chemical factors that influence sprouting of central neurons.

Correlation between anti-ubiquitin immunoreactivity and region-specific neuronal death in N-methyl-d-aspartate-treated rat hippocampal organotypic cultures

Neuronal degeneration appears to be associated with changes in anti-ubiquitin immunoreactivity (UIR). To elucidate the relationship between the two events, we examined the time course of changes in UIR in pyramidal neurons of hippocampal organotypic cultures following exposure to an excitotoxin, N-methyl-D-aspartate (NMDA). In nontreated cultures, weak UIR was confined to the nucleus. Exposure to 100 microM NMDA for 15 min induced degeneration of pyramidal neurons, within 24 h, in the CA1 and CA3c regions. In these neurons, the nuclear UIR was reduced, and instead, UIR developed in the cytoplasm. In response to the same procedure, CA3a,b pyramidal neurons showed slight shrinkage but otherwise virtually normal morphological features. Little perikaryal (cytoplasmic) UIR developed in CA3a,b neurons. Both degeneration and perikaryal UIR were observed in CA3a,b neurons, however, when the culture was exposed to 300 microM NMDA. Immunoblot analysis showed that changes in the amount of a ubiquitin protein conjugate (24 kDa), presumably ubiquitinated histone, are similar to those of nuclear UIR in the same time course. We propose that the changes in the expression of nuclear and perikaryal ubiquitinated proteins represent some process closely related to neuronal death.

Caerulein, a cholecystokinin-related peptide, depresses somatic function via the vagal afferent system

Intravenous doses of 0.5-8 micrograms/kg (0.3-4.9 nmol/kg) of caerulein, a cholecystokinin-related peptide, depressed the crossed extensor reflex response of chloralose-anesthetized rats in a dose-dependent manner. Intraventricularly administered caerulein was effective only at high doses (1-2 micrograms/animal, i.e., 0.6-1.2 nmol/animal). Cervical vagotomy completely abolished the inhibitory effect of peripherally injected caerulein. Electrical stimulation of vagal afferent nerves could simulate the caerulein effect to some extent. These results suggest that the primary site of action of peripherally administered caerulein is located in the gastrointestinal tract and thus the generated afferent vagal impulses mediate the reflex depression via the nucleus tractus solitarius and other as yet unclarified brain stem structures.

An imidazodiazepine derivative, Ro 15-1788, behaves as a weak partial agonist in the crossed extensor reflex

The antagonistic and intrinsic activity of an imidazodiazepine derivative, Ro 15-1788, was manifested on the crossed extensor reflex. Ro 15-1788 antagonized the depressant effect of diazepam and shifted the dose-response curve of diazepam to the right in a parallel manner but could not reverse the depression produced by either phenobarbital or chlorpromazine. Ro 15-1788 also antagonized the enhancement produced by ethyl-beta-carboline-3-carboxylate. When administered alone, Ro 15-1788 showed a weak depressant effect. Thus, Ro 15-1788 seems to be a partial agonist on the benzodiazepine receptors with weak intrinsic activity and pharmacological properties differing from those of ethyl-beta-carboline-3-carboxylate.

Sensitive depressant effect of benzodiazepines on the crossed extensor reflex in chloralose-anesthetized rats

Abstract Benzodiazepine (BZ) derivatives dose-dependently depressed the crossed extensor reflex (CER) in the hind limb of chloralose-anesthetized rats when administered in very small doses that did not cause any behavioral and electrophysiological changes in other reported tests. The intravenous minimum effective doses of diazepam, estazolam, nitrazepam, medazepam and chlordiazepoxide were 0.003, 0.006, 0.0125, 0.125 and 0.25 mg/kg, respectively. Only medazepam showed a characteristic biphasic inhibition, a transient and weak inhibition followed by a long-lasting inhibition. The effects of BZs were clearly antagonized by a subconvulsive dose of bicuculline, but not by strychnine. The ED 50 values of the five BZs in inhibiting the CER very closely correlated with their documented potencies in displacing 3 H-diazepam binding to rat brain benzodiazepine receptors. These results suggest that the CER inhibition by BZ is mediated by GABAergic transmission and the CER in chloralose-anesthetized rats can be used as a favorable indicator to detect the action of BZ via interactions with benzodiazepine receptors in vivo.

GABA-ergic influence on the crossed extensor reflex

Intraventricular administration of muscimol (25-100 ng) and intravenously applied aminooxyacetic acid (2.5-10 mg/kg) depressed the crossed extensor reflex response in a dose-dependent manner. The inhibitory effects of both drugs were clearly antagonized by a subconvulsive dose of bicuculline. A very small dose of bicuculline (10-40 microgram/kg, i.v.) produced a dose-related enhancement of the crossed extensor reflex response without any sign of convulsion. These results suggest that the crossed extensor reflex response is very sensitive to GABAergic drugs and central GABAergic mechanisms play a role in the modulation of the crossed extensor reflex response.

Proliferating cell nuclear antigen in neurones: induction by u.v. irradiation.

Proliferating cell nuclear antigen (PCNA) plays a pleiotropic role in DNA replication, excision repair of injured DNA and apoptosis. We found that PCNA protein is expressed predominantly in CA3 pyramidal neurones in hippocampal slices cultured from 1-week-old rats. Immunoblotting analysis revealed that this PCNA expression is enhanced by u.v. irradiation with no effect on neuronal viability. These results suggest that expression of PCNA in non-proliferating central neurones is associated with repair of injured DNA.

Anti-ubiquitin immunoreactivity associates with pyramidal cell death induced by intraventricular infusion of leupeptin in rat hippocampus

Pathological studies on several neurodegenerative diseases including Alzheimers disease have revealed common deposition of ubiquitin in many inclusion bodies. This implies a possible association of ubiquitin with neurodegeneration. To address this possibility, we examined histochemically the effect of intraventricular infusion of leupeptin, a thiol proteinase inhibitor, which is known to elevate anti-ubiquitin immunoreactivity in rat Purkinje cells. In the leupeptin-infused rat, an intense anti-ubiquitin immunoreactivity in the cytoplasm of neurons occurred not only in cerebellar Purkinje cells but also elsewhere in a wide area of the rat brain. The increase in the immunoreactivity was followed by a gradual depletion of pyramidal neurons in the hippocampal CA1 and CA3 subfields. The immunoreactive neurons disappeared concurrently. The number of anti-ubiquitin immunoreactive neurons was negatively correlated with that of surviving neurons when the duration of leupeptin infusion was varied. These results suggest that increased anti-ubiquitin immunoreactivity associates with neuronal death in leupeptin-treated rat brain.

Ethyl β-carboline-3-carboxylate reverses the diazepam effect on cerebellar cyclic GMP

The administration of ethyl beta-carboline-3-carboxylate (beta-CCE), a ligand for benzodiazepine receptors, did not affect the cerebellar cyclic GMP level in mice, but giving beta-CCE together with diazepam significantly inhibited the diazepam-induced decrease in cyclic GMP. The fact that no antagonism was observed when beta-CCE was given 15 min before the diazepam treatment indicates that beta-CCE is short-acting. These biochemical observations support the conclusions from behavioral and electrophysiological studies which indicated that beta-CCE is a short-acting antagonist of benzodiazepines.