Seitaro Ohkuma

Nitric oxide and peroxynitrite as factors to stimulate neurotransmitter release in the CNS

This review summarizes the stimulatory potentials of NO and peroxynitrite (OONO-) on neurotransmitter release in the central nervous system. Exogenous and endogenous NO stimulates to release neurotransmitter. NO synthesized intracellularly diffuses out through neuronal membrane and acts on the outer side of membrane to depolarize neuronal membrane, which triggers neurotransmitter release. NO-induced release of neurotransmitters is mediated by Ca2+-dependent and -independent processes. The latter process is operated by reverse process of the Na+-dependent carrier-mediated neurotransmitter uptake system or by unknown mechanisms. Ca2+-dependent release of neurotransmitter occurs in part subsequent to increase in Ca2+ influx via VDCCs, although N-type VDCCs may not involve in this action of NO because of suppression of Ca2+ influx through N-type VDCCs by NO. Participation of cGMP formation by NO on neurotransmitter release is controversial. A superoxide scavenger, Ca2+, Zn(2+)-superoxide dismutase, abolishes NO-induced neurotransmitter release and synthesized OONO- induces neurotransmitter release, indicating that OONO- participates in NO-evoked neurotransmitter release.

Development of taurine biosynthesizing system in cerebral cortical neurons in primary culture.

Developmental patterns of taurine biosynthesizing system were investigated using primary cultured neurons prepared from the neopallium of 15‐day‐old fetal mice by a trypsin treatment in comparison with those in cerebral cortices obtained from age‐matched fetal and neonatal mice. The morphological observations by phase contrast and scanning electron micrographies indicated that the cells in primary culture used in the present study possessed typical features of neurons. In addition, the immunohistochemical studies using the antibody to glial fibrillary acidic protein (GFAP), a specific marker for astroglia, revealed that the contamination of astroglias was negligible. The contents of taurine and metabolic intermediates in taurine biosynthesis, cysteine sulfinic acid and cysteic acid, in primary cultured neurons showed decreases during their development, especially during the first week after the inoculation. Similar developmental patterns of these amino acids were observed in cerebral cortices in vivo during perinatal stage which corresponded to the first week of neuronal growth in vitro. On the other hand, the activities of cysteine sulfinic acid decarboxylase and cysteine dioxygenase, both of which are involved in the biosynthesis of taurine, were found to be increased progressively both in primary cultured neurons and in cerebral cortices in vivo during their growth. The immunohistochemical study using antitaurine antibody obtained from rabbit clearly demonstrated that immunoreactive materials were localized in cell bodies and the processes of neurons, and the intensity of the immunoreactivity in primary cultured neurons also showed a reduction with time of culture. These results indicate that primary cultured neurons used in this study possess a similar capacity to synthesize taurine from cysteine as developing brains in vivo. The present results also strongly suggest the well known decrease in cerebral taurine content in vivo during neonatal stages may be predominantly due to the decrease of taurine in neuronal cells.

Simultaneous Monitoring of Conditioned Place Preference and Locomotor Sensitization Following Repeated Administration of Cocaine and Methamphetamine

The paradigm of conditioned place preference has been widely used to demonstrate the rewarding properties of psychomotor stimulants. Such drugs also stimulate locomotor activity. Repeated administration of low doses of psychomotor stimulants causes progressive increases in the locomotor stimulating effect, a phenomenon termed behavioral sensitization. Using a new activity monitor (SCANET MV-10LD) that simultaneously measures the amount of time spent and the distance traveled in each side of a two-compartment chamber, the present study assessed place preference conditioning and locomotor sensitization following repeated administration of cocaine or methamphetamine (MAP) in mice. We examined the effect of environmental factors on these activities using two different types of chamber: one having a single cue, and the other having dual cues for the discrimination of compartments. In both types of chamber, cocaine (5-20 mg/kg) and MAP (1-2 mg/kg) similarly produced conditioned place preference. However, repeated cocaine administration caused the development of locomotor sensitization only in the single-cue chamber. On the other hand, repeated administration of MAP resulted in the development of sensitization in both types of chamber. The findings indicate that environmental factors differentially affect the development of locomotor sensitization, but not place preference conditioning, following repeated administration of cocaine or methamphetamine. The advantages of this new system will be discussed.

Development of γ-aminobutyric acid (GABA)ergic neurons in cerebral cortical neurons in primary culture

The developmental patterns of gamma-aminobutyric acid (GABA)ergic neurons in primary culture obtained from the neopallium of 15-day-old fetus of mouse were investigated in terms of morphological features, GABA metabolism and GABA receptor binding. Morphological investigations revealed that these cells possessed typical features of neurons and the formation of synapses was detected at 10 days after the inoculation. During neuronal growth on polylysine surfaces, GABA contents and activity of GABA transaminase (GABA-T) showed a progressive increase in the time of culture. Similarly, L-glutamic acid decarboxylase (GAD) showed a progressive elevation during neuronal development in vitro, which corresponded well with the change in immunoreactivity to anti-GAD examined immunohistochemically. In addition, the high K+-evoked release of [3H]GABA also showed an enhancement during the growth in vitro. The numbers of binding sites (Bmax) for [3H]muscimol and [3H]flunitrazepam (FLN) also showed increases with the time of incubation, although affinity (Kd) to the labeled ligands did not show any noticeable changes. Moreover, it was observed that [3H]FLN binding was enhanced by GABA even in neurons cultured for 7 days. These results indicate that cerebral cortical neurons in primary culture possess GABA biosynthesizing and degrading systems including a high-affinity uptake mechanism for GABA. The present results also indicate that these cells possess synaptic contacts as well as GABAA receptors coupled with benzodiazepine receptor from a relatively early stage of cellular development.

Nitric oxide-evoked [3H]γ-aminobutyric acid release is mediated by two distinct release mechanisms

Mechanisms underlying the release of [3H] gamma-aminobutyric acid (GABA) evoked by nitric oxide (NO) were investigated by use of primary cultured neurons prepared from the mouse cerebral cortex. NO generators such as sodium nitroprusside (SNP) and S-nitroso-N-a etylpenicillamine (SNAP) increased both [3H]GABA release from the neurons and [45Ca2+] influx into the neurons in a dose-dependent manner, which was significantly diminished by hemoglobin. The removal of Ca2+ significantly reduced the NO-induced [3H]GABA release by about 50%. Nipecotic acid and 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1, 2, 5, 6-tetrahydro-3- pyridinecarboxylic acid (NO-711), GABA uptake inhibitors dose-dependently inhibited the NO-evoked [3H]GABA release in either the presence or absence of Ca2+. The concentration of these GABA uptake inhibitors to suppress the NO-induced release of [3H]GABA was sufficiently lower than that to exhibit the inhibition of [3H]GABA transport into the neurons. In addition, the NO-evoked [3H]GABA release was reduced by approximately 50% when total Na+ in incubation buffer was replaced with equimolar choline, and was also completely abolished by the removal of both Ca2+ and Na+. These results indicate that the release of [3H]GABA evoked by NO is mediated by two release mechanisms, a Ca2+ -dependent release system and the reverse process of the Ca2+ -independent and Na+ -dependent carrier-mediated GABA uptake system.

Muscimol-induced reduction of GABAA receptor α1-subunit mRNA in primary cultured cerebral cortical neurons

The expression of mRNA for GABAA receptor alpha 1-subunit in mouse cerebral cortical neurons in primary culture was examined using RNA blot analysis and ribonuclease protection assay following the treatment of neurons with muscimol, a selective agonist of GABAA receptor. The level of mRNA for GABAA receptor alpha 1-subunit showed a decrease in comparison with that in non-treated cells, whereas no changes in the level of beta-actin mRNA were noted under the same experimental conditions. This muscimol-induced reduction in GABAA receptor alpha 1-subunit mRNA was counteracted by the simultaneous exposure of neurons to both bicuculline, an antagonist of GABAA receptor, and muscimol. The expression of mRNA for GABAA receptor alpha 1-subunit also showed a decline by the treatment of cells with flunitrazepam alone, an agonist of benzodiazepine receptor, and this change was also abolished by the simultaneous exposure of cells to flunitrazepam and Ro15-1788, an antagonist for central benzodiazepine receptor. These results suggest that the continuous stimulation of cerebral GABAA receptor complex may induce the reduced expression of mRNA for the receptor complex.

Peroxynitrite affects Ca2+ influx through voltage-dependent calcium channels

The effect of peroxynitrite (OONO−) on voltage‐dependent Ca2+ channels (VDCCs) was examined by measuring [45Ca2+] influx into mouse cerebral cortical neurones. OONO− time‐ and dose‐dependently increased [45Ca2+] influx and this increase was abolished by manganese (III) tetrakis (4‐benzoic acid) porphyrin, a scavenger for OONO−. Inhibition of cyclic GMP (cGMP) formation did not alter the OONO−‐induced [45Ca2+] influx. OONO−, as well as 30 mm KCl, significantly increased fluorescence intensity of cell‐associated bis‐(1,3‐dibutylbarbituric acid) trimethine oxonol (bis‐oxonol). Tetrodotoxin and membrane stabilizers such as lidocaine dose‐dependently suppressed OONO−‐induced [45Ca2+] influx. Although each of 1 µm nifedipine and 1 µmω‐agatoxin VIA (ω‐ATX) significantly inhibited the OONO−‐induced [45Ca2+] influx and the concomitant presence of these agents completely abolished the influx, 1 µmω‐conotoxin GVIA (ω‐CTX) showed no effect on the influx. On the other hand, OONO− itself reduced 30 mm KCl‐induced [45Ca2+] influx to the level of [45Ca2+] influx induced by OONO− alone, and the magnitude of this reduction was as same as that of KCl‐induced [45Ca2+] influx by ω‐CTX. These results indicate that OONO− increases [45Ca2+] influx into the neurones through opening P/Q‐ and L‐type VDCCs subsequent to depolarization, and inhibits the influx through N‐type VDCCs.

Participation of peroxynitrite in acetylcholine release induced by nitric oxide generators

Peroxynitrite is a product produced by spontaneous reaction of nitric oxide (NO) with superoxide. Functional roles of peroxynitrite in the release of endogenous acetylcholine evoked by NO generators has been examined using primary-cultured cerebral cortical neurons. NO generators, such as sodium nitroprusside and S-nitroso-N-acetylpenicillamine, dose-dependently increased the release of acetylcholine (ACh); such increase of the release was significantly suppressed by hemoglobin which has the capacity to abolish biological effects of NO. Two types of superoxide scavengers, Cu2+, Zn2+ superoxide dismutase and ceruloplasmin, significantly reduced the NO-evoked ACh release. These results indicate that NO requires superoxide to evoke the release of ACh. Synthesized peroxynitrite evoked the release of ACh from cerebral cortical neurons in a dose-dependent manner. Thus, it is indicated that the NO-evoked ACh release is mediated, at least in part, by peroxynitrite produced by the reaction of NO with superoxide.

Psychological stress, but not physical stress, causes increase in diazepam binding inhibitor (DBI) mRNA expression in mouse brains

Effects of conditioned emotional stimuli (CES), which induce psychological stress, on the expression of cerebral diazepam binding inhibitor (DBI) mRNA in mouse were examined using a communication box. Cerebral DBI mRNA expression significantly increased in a time-dependent manner after the application of CES. The maximal enhancement of DBI mRNA expression was observed 2 days after the application of CES, and this increase faded out over 7 days after the treatment. Flunitrazepam (1 mg/kg), an agonist for central benzodiazepine (BZD) receptors, completely abolished the CES-induced elevation of cerebral DBI contents and its mRNA expressions. These results indicate that cerebral DBI is enhanced by psychological stress, which is regulated by central BZD receptors.

Involvement of peroxynitrite in N-methyl-D-aspartate- and sodium nitroprusside-induced release of acetylcholine from mouse cerebral cortical neurons.

Functional roles of peroxynitrite in N-methyl-D-aspartate (NMDA)- and sodium nitroprusside (SNP)-evoked releases of acetylcholine (ACh) from cerebral cortical neurons in primary culture have been investigated. NMDA increased the release of ACh in a dose-dependent manner, which was significantly suppressed by (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cycloheptan-5,10-imine (MK-801), a non-competitive antagonist specific for the NMDA receptor complex, and NO synthase inhibitors. SNP also showed a concentration-dependent increase in ACh release. Hemoglobin significantly abolished the stimulatory effects of both NMDA and SNP on ACh release. In addition, superoxide anion scavengers such as superoxide dismutase and ceruloplasmin significantly reduced the increased ACh release evoked by NMDA and SNP. Synthesized peroxynitrite dose-dependently elevated the release of ACh. These results indicate that the increased release of ACh by NMDA and SNP is mediated through peroxynitrite formed in the reaction of superoxide anion with nitric oxide produced by NMDA receptor activation and liberated from SNP rather than nitric oxide itself.