Takehiko Ito

Effects of cocaine administration on receptor binding and subunits mRNA of GABAA‐benzodiazepine receptor complexes

The effects of intermittent intraperitoneal (i.p.) administration of cocaine (20 mg/kg) on GABAA‐benzodiazepine (BZD) receptors labeled by t‐[35S]butylbicyclophosphorothionate (TBPS), and on several types of mRNA subunits were investigated in rat brain by in vitro quantitative receptor autoradiography and in situ hybridization. Phosphor screen imaging with high sensitivity and a wide linear range of response was utilized for imaging analysis. There was a significant decrease in the level of α1, α6, β2, β3, and γ2 subunits mRNA, with no alteration of [35S]TBPS binding in any regions in the brain of rats at 1 h following a single injection of cocaine. In chronically treated animals, the mean scores of stereotyped behavior were increased with the number of injections. The level of β3 subunit mRNA was decreased in the cortices and caudate putamen, at 24 h after a final injection of chronic administrations for 14 days. In the withdrawal from cocaine, the frontal cortex and hippocampal complexes showed a significant increase in [35S]TBPS binding and α1 and β3 subunit mRNA in the rats 1 week after a cessation of chronic administration of cocaine. These findings suggest that the disruption of GABAA‐BZD receptor formation is closely involved in the development of cocaine‐related behavioral disturbances. Further studies on the physiological functions on GABAA‐BZD receptor complex will be necessary for an explanation of the precise mechanisms underlying the acute effects, development of hypersensitization, and withdrawal state of cocaine. Synapse 38:198–215, 2000.

Oxidative stress alters arginine metabolism in rat brain : Effect of sub-convulsive hyperbaric oxygen exposure

The effect of hyperbaric oxygenation on arginine metabolism was investigated. Rats were exposed to oxygen at 3 atmospheres absolute for 2 h. Under these conditions, lipid peroxidation and activation of the anti-oxidant system were observed. Levels of thiobarbiturate reactive substances and carbon-centered radicals were increased in the cerebral cortex, while superoxide dismutase activity was also increased in the mitochondrial and cytosolic fraction of cerebrocortical homogenates. This suggested that the increase of both Mn and Cu,Zn-superoxide dismutase activities was probably an early compensatory reaction to oxidative stress. Levels of arginine as well as its metabolite, guanidinoacetic acid, were increased in the cerebral cortex. This increase seemed to be, at least in part, explained by a decrease in the arginase activity in the same region. Moreover, arginase activity in the brain showed heterogeneous distribution. Arginine: glycine amidinotransferase activity was decreased in the pons-medulla oblongata. The observed effects of hyperbaric oxygenation seem to favor nitric oxide generation.

A novel quantitative receptor autoradiography and in situ hybridization histochemistry technique using storage phosphor screen imaging

A new technique of image acquisition for quantitative receptor autoradiography and in situ hybridization histochemistry was developed using storage phosphor screen imaging. This method was at least 4-5 times faster than conventional film densitometry. Two of the advantages of the phosphor screen method are high sensitivity and wide linear range of response. Other aspects of this method were compared with those of conventional densitometry. Use of storage phosphor screen imaging will allow greatly increased speed of pharmacological screening procedures that utilize quantitative autoradiography.

Differential expression of GABAA receptor subunit mRNAs and ligand binding sites in rat brain following phencyclidine administration

Recent biochemical observations have suggested the abnormalities in the γ‐amino‐butyric acid (GABA)ergic system in schizophrenic brains. In the present study, we investigated the subunits gene expressions and ligand binding of the GABAA receptor following acute and chronic administration of phencyclidine (PCP), which induces schizophrenia‐like symptoms, in rats using in situ hybridization and in vitro quantitative autoradiography. PCP i.p. administration at a daily dose of 7.5 mg/kg resulted in a significant decrease in expression of α1 subunit mRNA in cerebral cortices (cingulate (‐13%) and temporal cortex (‐6%)) and hippocampal formation (CA1 (‐11%), CA2 (‐10%), CA3 (‐11%) and dentate gyrus (‐12%)) 1 h after a single treatment. In the repeated PCP administrations for 14 days, the expression of β2 mRNA in the cerebellum (‐10%) and of β3 mRNA in the cerebral cortices (cingulate (‐12%), parietal (‐16%) and temporal cortex (‐16%), caudate putamen (‐18%), inferior colliculus (‐18%), and cerebellum (‐15%) were significantly decreased. In addition, [35S]t‐butylbicyclophosphorothionate (TBPS) binding was also reduced in layer IV of the frontoparietal cortex (‐14%), inferior colliculus (‐17%), and cerebellum (‐12%) following chronic PCP treatment, while no changes were observed following acute PCP treatment. These results indicate that single and repeated administrations of PCP independently regulate the expression of GABAA/benzodiazepine (BZD) receptor subunits mRNA and its receptor binding in the brain. Synapse 38:51–60, 2000.

Chronic pentobarbital administration alters γ‐aminobutyric acidA receptor α6‐subunit mRNA levels and diazepam‐insensitive [3H]Ro15‐4513 binding

In order to study the chronic effects of pentobarbital, a positive GABAA receptor modulator, on the inverse agonist binding of the benzodiazepine site, binding of [3H]Ro15‐4513 and levels of GABAA receptor α6‐subunit mRNA were investigated in the brains of pentobarbital‐tolerant/dependent animals, using receptor autoradiography and in situ hybridization histochemistry in consecutive brain sections. Pentobarbital was administered to rats either 60 mg/kg, i.p., once, for acute treatment, or 300 μg/10μl/h i.c.v. continuously for 6 days via osmotic minipumps to render rats tolerant to pentobarbital. Rats assigned to the dependent group were sacrificed 24 h after discontinuance of pentobarbital infusion, while those assigned to the tolerant group were sacrificed at the end of infusion. The α6 subunit mRNA was increased in the tolerant group only. Diazepam‐insensitive [3H]Ro15‐4513 binding was increased in the cerebellar granule layer of pentobarbital‐tolerant and ‐dependent rats. No alterations in these parameters were observed in acutely treated animals. These data suggest that chronic pentobarbital treatment induced expression of α6‐subunit mRNA. This was in contrast to α1‐ and γ2‐subunit mRNA, which in tolerant animals are unchanged, but for which withdrawal triggers a surge in levels. Because the α6‐subunit is a major component of the diazepam‐insensitive [3H]Ro15‐4513 binding site, the increased diazepam‐insensitive [3H]Ro15‐4513 binding implied de novo synthesis of the receptor subunit protein.

Quantitative autoradiographic localization of [3H]3-OH-PCP (1-(1(3-Hydroxyphenyl)cyclohexyl)piperidine) binding sites in rat brain

Binding of a novel radioligand, [3H]3-OH-PCP (1(1(3-hydroxyphenyl) cyclohexyl)piperidine), to N-methyl-D-aspartate (NMDA) receptor-coupled and -uncoupled PCP sites was investigated in the rat brain. The highest densities of [3H]3-OH-PCP binding were observed in the hippocampal formation, notably in the stratum radiatum and oriens of CA1 region, and dentate gyrus. There were relatively high levels of binding in the olfactory system, superficial layer of cortices, the amygdala and the thalamus. In contrast, lower levels of binding were found in the globus pallidus, cerebellum, and brain stem, except for the superior colliculus. These findings demonstrate that [3H]3-OH-PCP binds to discrete regions within the rat brain. Its distribution is consistent with autoradiographic localization of [3H]TCP and [3H]MK-801 binding sites in the rat brain, suggesting that [3H]3-OH-PCP binds to NMDA/PCP ion-channel complexes in preference to sigma sites.

An autoradiographic study of [3H]flunitrazepam binding sites in the brain of rat made tolerant to and dependent on pentobarbital

The effects of continuous administration of pentobarbital on the benzodiazepine receptor labeled by [3H]flunitrazepam were investigated. Animals were made tolerant to pentobarbital by intracerebroventricular (i.c.v.) infusion with pentobarbital (300 micrograms/10 microliters/h) for 6 days through pre-implanted canulae connected to osmotic mini-pumps. The dependent rats were assessed 24 h after cessation of pentobarbital infusion. Changes in [3H]flunitrazepam binding were examined in 37 brain regions at a concentration of [3H]flunitrazepam of 1 nM. In subsequent saturation studies, the binding parameters Bmax and KD were also investigated in 17 brain regions, most of which showed significant changes in [3H]flunitrazepam binding in experiments using a fixed concentration of radioligand. The pentobarbital-tolerant rats showed a significant increase in Bmax with an increase in KD for [3H]flunitrazepam in the ventroposterior nucleus of thalamus. In the dependent rats, a significant increase in Bmax for [3H]flunitrazepam binding, without a change in KD, was observed in all layers of the frontal cortex, the caudate-putamen, olfactory tubercle, and some nuclei in thalamus, compared to those in the control. Increased [3H]flunitrazepam binding in the molecular layer of the olfactory bulb, the ventral pallidum, and the cerebellum of the pentobarbital dependent rats at a fixed concentration of [3H]flunitrazepam was also observed. There was no significant change in [3H]flunitrazepam binding in the hippocampus and several nuclei of the brain stem. These findings suggest that benzodiazepine receptors are closely involved in the development of tolerance to and dependence on pentobarbital. Further studies on changes in gamma-aminobutyric acid (GABA)A receptor subunit mRNA or the effects of pentobarbital on GABAA receptor phosphorylation would be necessary for an explanation of the precise mechanisms underlying the development of tolerance to and dependence on pentobarbital.

Time-course effects of a single administration of cocaine on receptor binding and subunit mRNAs of GABAA receptors

Abstract We investigated the time-course effects of a single administration of cocaine (20 mg/kg) on GABAA receptor binding labeled by t-[35S]butylbicyclophophorothionate (TBPS) and on several types of GABAA receptor subunit mRNAs in the rat brain by in vitro quantitative receptor autoradiography and in situ hybridization. The levels of α1, β2, and β3 subunit mRNAs in several brain regions such as the cortex, cerebellum, and striatum were significantly decreased within 1 h, while β3 subunit mRNA was increased in the dentate gyrus. All of these changes were transient, occurring within 1 h after the injection of cocaine. In the cortex and cerebellum, the reduction in α1 subunit mRNA was followed by a significant decrease in [35S]TBPS receptor binding, which occurred 4 h after cocaine injection. These findings suggest that acute cocaine administration discretely regulates GABAA receptor subunit mRNA levels in several brain regions through a change in transcription or turnover rates of subunit mRNAs, which may be closely related to cocaine-induced behavioral abnormalities.

Changes in [3H]flunitrazepam binding in the brain of rats made tolerant to and dependent upon pentobarbital.

Changes in benzodiazepine binding sites labeled by [3H]flunitrazepan (FNZ) in twenty discrete brain regions of rats made tolerant to and dependent upon pentobarbital were examined. Animals were rendered tolerant by intracerebroventricular (i.c.v.) infusion with pentobarbital (300 micrograms/ 10 microliters/ hr for six days) through pre-implanted cannulae connected to osmotic mini-pumps. The pentobarbital dependence was assessed 24 hr after abrupt withdrawal from pentobarbital. In the tolerant rats, a significant increase in [3H]FNZ binding sites was found in layer IV of frontal cortex and the molecular layer of olfactory bulb. [3H]FNZ binding sites in the pentobarbital dependent rats were significantly increased in layers I-III and V-VI of frontal cortex, caudate-putamen, olfactory tubercle, globus pallidus and ventral pallidum, in addition to those observed in the tolerant group. There was, however, no significant difference in the hippocampus and several regions in the hindbrain in either pentobarbital-treated group. Taken together with characteristics of subtypes of benzodiazepine receptors and changes in GABA-benzodiazepine receptor complexes elucidated in our previous studies, these findings suggest that both types of benzodiazepine receptors are involved in the development of pentobarbital intoxication mediated by GABAA receptors.

Autoradiographic study on the pharmacological characteristics of [3H]3-OH-PCP binding sites in rat brain

The pharmacological characteristics and the regional distribution of [3H]3-OH-PCP (1-[1(3-hydroxyphenyl)-cyclohexyl]piperidine) binding were investigated in rat brain by quantitative autoradiography. Kinetic analysis of [3H]3-OH-PCP binding revealed fast and slow components, in the association and dissociation studies. The regional distribution of binding closely corresponded to those of binding sites labeled by [3H]N-[l-(2-thienyl)-cyclohexyl]3,4-piperidine (TCP) and [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne maleate (MK 801). High densities of [3H]3-OH-PCP binding sites were found in the stratum radiatum and orients of field CA1 in the hippocampus and in the outer layers of cerebral cortices. In contrast, low levels of binding were seen in the brain stem and the granular cell layer of the cerebellum. [3H]3-OH-PCP binding was strongly inhibited by MK 801 and 3-OH-PCP, while the potency of (+)-SKF 10047 in inhibiting [3H]3-OH-PCP binding was less in the cerebral cortex and hippocampus. The antagonists for the glutamate, glycine and polyamine recognition sites at the NMDA/PCP receptor complex displaced [3H]3-OH-PCP binding sites with a potency similar to that of [3H]MK 801. These findings suggest that the [3H]3-OH-PCP binding site is similar or identical to the PCP binding site labeled by [3H]TCP and [3H]MK 801.