Katsuro Shuto

Effect of aging on dopaminergic receptors and uptake sites in the rat brain studied by receptor autoradiography

We studied the age-related alterations of dopaminergic receptors in the brain of Fisher 344 rats with various age (3 weeks and 6, 12, 18 and 24 months) using in vitro receptor autoradiography. [3H]SCH 23390, [3H]spiperone and [3H]nemonapride, and [3H]mazindol were used to label dopamine D1 receptors, dopamine D2 receptors and dopamine uptake sites, respectively. In immature rats (3 weeks old), [3H]SCH 23390 binding showed a significant increase in most brain regions compared to adult animals (6 months old), whereas [3H]spiperone and [3H]nemonapride bindings showed no significant alteration in any brain areas. In contrast, [3H]mazindol binding showed a significant decline in most brain regions. On the other hand, the age-related alterations in [3H]SCH 23390 binding were not observed in any brain regions. [3H]Spiperone and [3H]nemonapride bindings also showed no significant alteration in the brain during aging, except for a transient alteration in [3H]spiperone binding in the nucleus accumbens and hippocampus of 12 months old rats. However, [3H]mazindol binding showed a significant reduction in most brain areas of 12 months old rats. Thereafter, the age-related reduction in [3H]mazindol binding was observed in most brain regions of 18 and 24 months old rats. The results demonstrate that dopamine uptake sites are more susceptible to the aging process than both dopamine D1 and D2 receptors. Furthermore, our results suggest that dopaminergic receptors and dopamine uptake sites may develop with different patterns and speeds after birth. Our studies may provide valuable information concerning the effect of aging on dopaminergic systems.

Autoradiographic mapping of neurotransmitter system receptors in mammalian brain

Regional localization of neurotransmitter system receptors was visualized in the gerbil grain and in the rat brain using receptor autoradiography. [3H]Quinuclidinyl benzilate (QNB), [3H]cyclohexyladenosine (CHA), [3H]muscimol, [3H]MK-801, [3H]SCH 23390, [3H]PN200-110, [3H]spiperone, and [3H]naloxone were label muscarinic receptors, adenosine A1 receptors, GABAA receptors, N-methyl-D-aspartate (NMDA) receptors, dopamine D1 receptors, L-type calcium channels, spirodecanone receptors, and opioid receptors, respectively. Regional localization of [3H]QNB, [3H]muscimol, [3H]MK-801, [3H]SCH 23390, and [3H]PN200-110 binding sites in the gerbil brain was relatively similar to that in the rat brain. In contrast, the autoradiographic distribution of [3H]spiperone and [3H]naloxone binding sites in the gerbil was quite different from that in the rat. This phenomenon was found especially in the hippocampus and the cerebellum. The results suggest that the gerbil differs from the rat with respect to spirodecanone and opioid binding sites in the hippocampus and the cerebellum. This finding may help to further elucidate the species differences and relationships for brain function and behavioral pharmacology.

Alterations in l-NG-nitroarginine binding in brain after transient global or transient focal ischemia in gerbils and rats

We investigated the post-ischemic change in [3H]L-N(G)-nitroarginine binding as a marker of nitric oxide (NO) synthase in the animal brain after transient global ischemia or transient focal ischemia. Transient global ischemia in gerbils was induced for 10 min followed by 1 h to 7 days of recirculation. Transient focal ischemia in rats was induced for 45 min followed by 3 days of recirculation. Following transient global ischemia, [3H]L-N(G)-nitroarginine binding showed a significant increase in the striatum (17-18%) and hippocampal CA1 sector (24%) at 48 and 24 h after recirculation, respectively. The hippocampal CA3 sector also showed a significant elevation (32-40%) in [3H]L-N(G)-nitroarginine binding at 24 and 48 h after global ischemia. Furthermore, the dentate gyrus showed a significant increase (30-32%) in [3H]L-N(G)-nitroarginine binding at 5, 24 and 48 h after global ischemia. Thereafter, a significant reduction in [3H]L-N(G)-nitroarginine binding was observed only in the dentate gyrus 7 days after recirculation. In contrast, [3H]L-N(G)-nitroarginine binding was unchanged in the thalamus throughout the recirculation periods. Histological analysis revealed that transient global ischemia caused severe damage or cellular damage in the striatum and the hippocampal CA1 sector. The hippocampal CA3 sector and thalamus were mildly damaged, whereas the dentate gyrus was morphologically intact. Following transient focal ischemia, a marked elevation (50-52%) in [3H]L-N(G)-nitroarginine binding was found in the regions of the ipsilateral striatum in which severe infarction occurred. Our findings suggest that [3H]L-N(G)-nitroarginine binding increases in the striatum and hippocampus after transient global ischemia or transient focal ischemia. This increase in [3H]L-N(G)-nitroarginine binding may play a pivotal role not only in the pathogenesis of ischemic brain damage, but also in the restoration of injury areas after cerebral ischemia.

Effects of vinconate on age-related alterations in [3H]MK-801, [3H]glycine, sodium-dependent D-[3H]aspartate, [3H]FK-506 and [3H]PN200-110 binding in rats

We investigated the effects of age and (+/-)-methyl-3-ethyl-2,3,3a,4-tetrahydro-1 H-in-dolo[3,2,1-de] [1,5] naphthyridine-6-carboxylate hydrochloride (vinconate), an indolonaphthyridine derivative, on calcium channels, neurotransmitter receptor systems and immunophilin in Fischer rat brain using quantitative receptor autoradiography. [3H]MK-801, [3H]glycine, sodium-dependent D-[3H]aspartate, [3H]FK-506 and [3H]PN200-110 were used to label N-methyl-D-aspartate (NMDA) receptors, glycine receptors, excitatory amino acid transport sites, FK-506 binding proteins (FKBP) and voltage-dependent L-type calcium channels, respectively. [3H]Glycine and sodium-dependent D-[3H]aspartate binding significantly decreased in the frontal cortex, parietal cortex, striatum, nucleus accumbens, hippocampus, thalamus, substantia nigra and cerebellum of 24 month old rats in comparison with 6 month old animals. In contrast, [3H]MK-801, [3H]FK-506 and [3H]PN200-110 binding showed no significant changes in the brain of 24 month old rats. Intraperitoneal chronic treatment with vinconate (10 and 30 mg/kg, once a day for 4 weeks) dose-dependently ameliorated the significant reduction in [3H]glycine and sodium-dependent D-[3H]aspartate binding in the brain of 24 month old rats. These results demonstrate that glycine receptors and excitatory amino acid transport sites are more susceptible to aging processes than NMDA receptors, immunophilin and voltage-dependent L-type calcium channels. Furthermore, our findings suggest that vinconate may have a beneficial effect on age-related changes in glycine receptors and excitatory amino acid transport sites.

Effects of cerebral ischemia on dopamine receptors in the gerbil striatum

Dopamine D1 and D2 receptors and uptake sites were studied in the gerbil striatum and frontal cortex 1 h to 7 days after 10 min of cerebral ischemia caused by occlusion of the bilateral common carotid arteries. [3H]SCH23390 ([N-methyl-3H]R[+]-8-chloro-2, 3,4,5-tetrahydro-3-methyl-5-phenyl-7-ol-benzazepine), [3H]nemonapride and [3H]mazindol were used as markers of dopamine D1 receptors, D2 receptors and uptake sites, respectively. A significant reduction in [3H] SCH23390 binding was found in the striatum from 48 h after ischemia. In contrast, during the recirculation periods, [3H]nemonapride and [3H]mazindol binding was mostly unaffected in this region which was the most vulnerable to ischemia. The frontal cortex, where ischemic neuronal damage was mild, also showed no significant changes in [3H]SCH23390, [3H]nemonapride and [3H]mazindol binding after ischemia. Thus, cerebral ischemia that was associated with cell loss in the striatum resulted in a selective reduction of dopamine D1 receptors and not D2 receptors. No changes in dopamine D1 or D2 receptors were observed in frontal cortex. If massive dopamine release occurs with cerebral ischemia, it is not reflected by modification in the number of uptake sites located on dopamine terminals.

Different age-related changes in NMDA and glycine receptors in the rat brain.

We investigated the age-related changes of N-methyl-d-aspartate (NMDA) and strychnine-insensitive glycine receptors in the brain from Fischer rats aged 3 weeks (immature), 6 months (adult), 12 months (mature), 18 months (middle-aged) and 24 months (aged) using receptor autoradiography. [(3)H]MK-801 and [(3)H]glycine were used to label the NMDA receptor and the glycine receptor, respectively. In immature rats, [(3)H]MK-801 binding showed a significant decline only in the nucleus accumbens, whereas [(3)H]glycine binding exhibited a significant increase in the frontal cortex, parietal cortex, striatum and thalamus as compared with young rats. In mature, middle-aged and aged rats, [(3)H]MK-801 binding showed no significant change in the brain. In contrast, [(3)H]glycine binding showed a conspicuous reduction in the striatum and hippocampal CA3 sector and thalamus from mature rats. Thereafter, the age-related reduction in [(3)H]glycine binding was observed in all brain areas of middle-aged and aged rats. These results demonstrate that the glycine receptor in the rat brain is far more susceptible to aging processes than the NMDA receptor. Furthermore, they suggest the conspicuous differences in the developmental pattern between NMDA and glycine receptors in the rat brain after birth. These findings suggest that glycine receptor in the brain is primarily and severely affected in aging processes and this may lead to age-related neurological deficits.

Effect of cerebral ischemia on dopamine receptors and uptake sites in the gerbil hippocampus

Dopamine D1 and D2 receptors and uptake sites were studied in the gerbil hippocampus, parietal cortex and thalamus 1 h to 7 days after 10 min of cerebral ischemia using the occlusion of bilateral common carotid arteries. [3H]SCH23390 ([N-methyl-3H]R[+]-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-7-ol-be nzazepine) and [3H]mazindol were used as markers of dopamine D1 receptors and uptake sites, respectively. [3H]Nemonapride was used to label dopamine D2 receptors. No obvious alteration in [3H]SCH23390 and [3H]mazindol binding was found in the hippocampus up to 48 h after ischemia. These bindings showed a significant reduction in the hippocampus after 7 days of recirculation. In contrast, [3H]nemonapride binding was unaffected in the hippocampus during the recirculation periods. The parietal cortex and thalamus also exhibited no significant changes in [3H]SCH23390, [3H]nemonapride and [3H]mazindol binding after ischemia. MAP2 (microtubule-associated protein 2) immunoreactivity was unchanged in all regions up to 48 h after ischemia. Thereafter, a marked loss of MAP2-immunoreactive neurons was observed in the hippocampal CA1 and CA3 neurons 7 days after recirculation. These findings were consistent with histological observations with cresyl violet staining. Our results demonstrate that dopamine D1 receptors and dopamine uptake sites in the hippocampus are susceptible to cerebral ischemia, whereas dopamine D2 receptors in this region are particularly resistant. Furthermore, these findings suggest that dopamine transmission may not be major factor in producing ischemic hippocampal damage.

Postischemic changes of [3H]nimodipine and [3H]ryanodine binding in the gerbil striatum and hippocampus

Sequential alterations of [3H]nimodipine and [3H]ryanodine binding in gerbils were investigated in selectively vulnerable regions, such as the striatum and hippocampus, 1 h to 7 days after 10 min of transient cerebral ischemia. [3H]Nimodipine binding showed no significant changes in the striatum and hippocampus up to 48 h after ischemia. Seven days after ischemia, however, a severe reduction in [3H]nimodipine binding was observed in the dorsolateral striatum, hippocampal CA1 (stratum oriens, stratum pyramidale and stratum radiatum) and hippocampal CA3 sector. On the other hand, [3H]ryanodine binding showed a significant increase in the hippocampus 1 h after ischemia. Five hours after ischemia, a significant reduction in [3H]ryanodine binding was observed only in the hippocampal CA1 sector. Thereafter, the striatum and hippocampus showed no significant alterations in [3H]ryanodine binding up to 48 h after ischemia. After 7 days, a marked reduction in [3H]ryanodine binding was observed in the striatum and hippocampus which were particularly vulnerable to ischemia. These results demonstrate that postischemic alteration in [3H]nimodipine and [3H]ryanodine binding is produced with different processes in the hippocampus. They also suggest that the mechanism for striatal cell damage caused by transient cerebral ischemia may, at least in part, differ from that for hippocampal neuronal damage. Furthermore, our findings suggest that abnormal calcium release from intracellular stores may play a pivotal role in the development of hippocampal neuronal damage.

Effects of vinconate on neurotransmitter receptor systems in aged rat brain

We investigated the effects of age and (±)-methyl-3-ethyl-2,3,3a,4-tetrahydro-1 H-indolo[3,2,1-de][1,5]naphthyridine-6-carboxylate hydrochloride (vinconate), an indolonaphthyridine derivative, on neurotransmitter receptor systems in the rat brain using quantitative receptor autoradiography. [(3)H]Quinuclidinyl benzilate (QNB), [(3)H]hemicholinium-3 (HC) and [(3)H]muscimol were used to label acetylcholine receptors, high-affinity choline uptake sites and γ-aminobutyric acid(A) (GABA(A)) receptors, respectively. [(3)H]QNB, [(3)H]HC and [(3)H]muscimol binding decreased in any brain areas of 24-month-old (aged) rats in comparison with 6-month-old (adult) animals. Chronic treatment with vinconate (10 and 30 mg/kg, i.p., once a day for 4 weeks) partly ameliorated the reduction in [(3)H]QNB, [(3)H]HC and [(3)H]muscimol biding in aged rat brains. This effect was especially noted in [(3)H]muscimol binding. The results suggest that vinconate may have beneficial effects on age-related changes in neurotransmitter receptor systems.