Mitsuko Okada

The chronic administration of docosahexaenoic acid reduces the spatial cognitive deficit following transient forebrain ischemia in rats

The purpose of this study was to investigate whether chronic administration of docosahexaenoic acid is able to reduce spatial cognitive deficit following transient ischemia in rats. In addition, we investigated whether the chronic treatment of docosahexaenoic acid is able to protect the hippocampal neuronal damage induced by either hypoxia in vitro or cerebral ischemia in vivo. A chronic administration of 200 mg/kg/day docosahexaenoic acid over 21 days did not affect the content of docosahexaenoic acid in the hippocampus, but did tend to increase it in the frontal cortex. On the other hand, this chronic administration decreased the content of arachidonic acid significantly both in the hippocampus and the frontal cortex. Under hypoxic conditions, the onset of the increase in the NADH fluorescence in the hippocampal slice was made significantly slower relative to the control by the chronic administration of docosahexaenoic acid. Rats were subjected to 10 min of transient forebrain ischemia by the method of four-vessel occlusion and were tested in a radial eight-arm maze task after cerebral reperfusion. Docosahexaenoic acid was administered either once 1 h before occlusion or daily for 21 days before occlusion. The single treatment of docosahexaenoic acid (1, 10, 100 or 200 mg/kg) did not significantly affect any aspect of the spatial learning deficit following occlusion. On the other hand, chronic treatment with docosahexaenoic acid (10, 100 or 200 mg/kg/day) significantly improved the spatial learning deficit following occlusion. A comparison of the neuronal densities in the hippocampal CA1 region of the chronically docosahexaenoic acid-treated (200 mg/kg/day) rats with those of the ischemic control revealed a significant neuronal preservation. From these results, it appears that chronic administration of docosahexaenoic acid may be valuable in ameliorating the spatial cognitive deficit induced by transient forebrain ischemia. In addition, docosahexaenoic acid might contribute to the protection of hippocampal neuronal damage caused by either hypoxia or ischemia.

Modification of human 5-HT 2C receptor function by Cys23Ser, an abundant, naturally occurring amino-acid substitution

A human serotonin (5-HT)2C receptor gene polymorphism leads to the substitution of cysteine for serine at codon 23 (Cys23Ser); the frequency of the Ser23 allele in unrelated Caucasians is approximately 0.13. In the present study, we assessed whether Cys23Ser could affect receptor function. The two alleles were functionally compared following expression in COS-7 cells. The constitutive activity of the receptor in an in situ reconstitution system was also evaluated following expression of each allele in Sf9 cells. Using radioligands, Ser23-expressed membranes showed reduced high-affinity binding to meta-chlorophenylpiperazine (m-CPP) and 5-HT. Although the amplitude of the 5-HT-induced intracellular Ca2+ peak did not differ between the alleles, Ser23 required higher 5-HT concentrations to elicit the same response. These differences might be due to more extensive desensitization in the Ser23 form. In the in situ reconstitution system, the 5-HT2C receptor displayed considerable constitutive activity, with the Ser23 allele being significantly higher in this regard than the Cys23 form. After prolonged serum deprivation in order to resensitize the receptor, four of the 15 cells expressing Ser23 showed abnormally higher m-CPP-induced sensitivity of the Ca2+ response. These results indicate that the Ser23 allele may be constitutively more active than Cys23. Thus, Ser23 appears to be an abundant candidate allele capable of directly influencing inter-individual variation in behavior, susceptibility to mental disorder, and response to drugs including atypical antipsychotic and some antidepressant drugs that are potent 5-HT2C inverse agonists or antagonists.

Long-term spatial cognitive impairment following middle cerebral artery occlusion in rats. A behavioral study.

Behavioral changes in the chronic phase of permanent occlusion of the right middle cerebral artery (MCA) in rats were investigated. One month after MCA occlusion, 23 rats were unable and 7 rats were able to solve a radial 8-arm maze task during a 1-month period. Three months after occlusion, 19 MCA-occluded rats failed to solve the task successfully again during at least a 1-month period (the cognitively impaired rats), and 11 MCA-occluded rats were able to solve it (the cognitively unimpaired rats). When a delay of 60 min was imposed for this task, five cognitively unimpaired rats failed to solve it. The locomotor activity of the cognitively impaired rats increased significantly 2 months after occlusion, and this increase showed good correlation with spatial cognitive deficit. However, the mean time a rat spent at each arm remained unchanged among the cognitively impaired, unimpaired, and sham-operated rats. There was no significant difference in the ratio between the cognitively impaired and unimpaired rats for disturbed motor coordination. These results suggest that MCA occlusion is capable of producing long-term spatial cognitive disturbance in rats. In addition, this spatial cognitive deficit does not seem to be primarily due to hypermotility or a disturbance in motor coordination.

Long-Term Spatial Cognitive Impairment after Middle Cerebral Artery Occlusion in Rats: No Involvement of the Hippocampus

The behavioral and neurochemical changes in the chronic phase of permanent occlusion of the right middle cerebral artery (MCA) in rats were investigated. One month after MCA occlusion, 23 rats were unable to solve a radial eight-arm maze task during an entire 1-month period, whereas seven rats were able to solve this task. Three months after occlusion, 19 MCA-occluded rats failed to solve the task successfully again for at least 1 month (the cognitively impaired rats), whereas 11 MCA-occluded rats were able to solve it (the cognitively unimpaired rats). The rats that underwent behavioral testing were examined for any changes in the acetylcholine (ACh) levels in the hippocampus using HPLC with electrochemical detection or the formation of long-term potentiation (LTP) in the population spike of the hippocampal CA1 field. The immunohistochemical distribution of either the microtubule-associated protein 2 (MAP2) or glial fibrillary acidic protein (GFAP) in the hippocampus of the cognitively impaired rats was also studied. In the cognitively impaired rats, neither the suppression of the induction of LTP, nor the degradation of MAP2, nor the increase in the GFAP immunoreactivity was observed in the hippocampus. The levels of ACh in the hippocampus did not change significantly among the cognitively impaired, unimpaired, and the sham-operated rats. These results suggest that MCA occlusion is capable of producing long-term spatial cognitive disturbance in rats without any evidence of neurobiological damage in the hippocampus.

Recombinant human serotonin 5A receptors stably expressed in C6 glioma cells couple to multiple signal transduction pathways

Human serotonin 5A (5‐HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5‐HT5A receptors‐expressing cells, accumulation of cAMP by forskolin was inhibited by 5‐HT as reported previously. Pertussis toxin‐sensitive inhibition of ADP‐ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine‐sensitive Ca2+ mobilization. On the other hand, 5‐HT‐induced outward currents were observed using the patch‐clamp technique in whole‐cell configuration. The 5‐HT‐induced outward current was observed in 84% of the patched 5‐HT5A receptor‐expressing cells and was concentration‐dependent. The 5‐HT‐induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5‐HT‐induced current was significantly attenuated by 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5‐HT‐induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5‐HT‐induced current. IP3 showed a transient increase after application of 5‐HT in 5‐HT5A receptor‐expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP‐ribosyl cyclase activity, 5‐HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3‐sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function.

The facilitating and suppressing effects of Δ9-tetrahydrocannabinol on the rise in intrasynaptosomal Ca2+ concentration in rats

The effects of delta 9-tetrahydrocannabinol (delta 9-THC) on the rise in intracellular Ca2+ concentrations ([Ca2+]i) after stimulation with 15 mM or 29 mM K+ in rat whole brain synaptosomes were examined. A fluorescent chelating agent, Rhod-2, was employed to monitor any alterations of K(+)-evoked [Ca2+]i. Pretreatment with 10(-10) M delta 9-THC for 3 min enhanced K(+)-evoked [Ca2+]i significantly, while 10(-9), 10(-8) or 5 x 10(-8) M delta 9-THC significantly inhibited the K(+)-evoked [Ca2+]i. These results suggest that delta 9-THC had a biphasic effect on the K(+)-evoked Ca2+ response in rat brain synaptosomes.

Is the Augmentation of K+-Evoked Intrasynaptosomal Ca2+ Concentration Due to the Influx of Ca2+ in Rat Brain Synaptosomes?

Abstract: Intraterminal free Ca2+ concentration modulates the subsequent release of neurotransmitters. Depolarization of synaptosomes with 29 mM K+ augments cytosolic free Ca2+ concentration, which is triphasic, the peak times being at 10, 60, and 180 s. We examined the characteristics of each elevation of cytosolic free Ca2+ concentration in rat brain synaptosomes which had been preincubated for 3 min with a Ca2+‐channel blocker, such as La3+, diltiazem, nifedipine, or verapamil, and under conditions of hypoxia or acidosis. The concentration of free Ca2+ in the quin‐2‐loaded rat brain synaptosomes was detected fluorometrically. All these elevations were suppressed in the presence of 200 μM EGTA or 100 μM La3+. At the first phase, the elevation of cytosolic free Ca2+ concentration with high K+ stimuli was significantly inhibited by La3+ (20 μM) or by acidosis (pH 6.7). On the other hand, diltiazem, which is a more potent blocker of the release of Ca2+ from the mitochondria, inhibited the increasing cytosolic free Ca2+ concentration at the third phase in a concentration‐dependent manner. Hypoxia also showed inhibition at the third phase. These results suggest that the augmentation of high K+‐evoked cytosolic free Ca2+ concentration may be due to the influx of extracellular Ca2+. The increase in cytosolic free Ca2+ concentration at the third phase is no doubt linked to the mitochondrial function.

How does prolonged caloric restriction ameliorate age-related impairment of long-term potentiation in the hippocampus?

Prolonged dietary restriction has been reported to suppress age-induced phenomena. In order to investigate how prolonged caloric restriction reduces age-related deterioration of hippocampal synaptic transmission, we compared the levels of major hippocampal polyunsaturated fatty acids, arachidonic acid and docosahexaenoic acid between 4- and 26-month-old rats. The Ca(2+) responses upon perfusion of NMDA or 30 mM K(+) between 4- and 26-month-old rats with prolonged dietary restriction were also compared using the fluorescent probe Fura-2. A decrease in membrane arachidonic acid is thought to be a major causal factor in the age-related impairment of long-term potentiation. Long-term caloric restriction seems to increase arachidonic acid levels regardless of age. However, there is no significant difference of hippocampal arachidonic acid levels between in freely feeding 4- and 26-month-old rats. Similar results were obtained from the measurement of hippocampal docosahexaenoic acid levels. Under caloric restriction, the 500 microM N-methyl-D-aspartate-induced Ca(2+) response was greatly reduced by aging, while the 30 mM K(+)-induced Ca(2+) response was not affected. In our preliminary data, the amplitude of the population spike after tetanic stimulation did not differ between 4- and 26-month-old rats under caloric restriction, while 50 microM of 2-amino-5-phosphonovaleric acid, a N-methyl-D-aspartate antagonist, markedly inhibited a potentiation of the population spike in 4-month-old rats, but with negligible inhibition in 26-month-old rats. From these results, an age-related impairment of hippocampal excitatory synaptic transmission may not be solely due to the reduction of membrane arachidonic acid. Caloric restriction might prevent age-related reduction in hippocampal synaptic transmission by enhancing non-N-methyl-D-aspartate mechanisms.

Protective effect of MK-801 on the anoxia-aglycemia induced damage in the fluorocitrate-treated hippocampal slice of the rat

We investigated electrophysiological responses induced by ischemia-like insult (anoxia and aglycemia, AA) in the rat hippocampal CA1 pyramidal cells in an in vitro slice preparation devoid of glial metabolism. In the slice treated with fluorocitrate (100 microM), a glia-specific metabolic inhibitor, 10 min AA induced hyperexcitation as evidenced by an appearance of multiple population spikes evoked by stimulation of the Schaffer collateral/commissural pathway in the CA1 region prior to elimination of the response. Readministration of oxygen and glucose failed to restore the population spike amplitude. Intracellular recordings revealed that 10 min AA induced slow EPSPs with relative long duration. The induction of the slow EPSPs was followed by a rapid membrane depolarization with a large amplitude. When the fluorocitrate-treated slice was exposed to MK-801 (10 microM), a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, 10 min AA failed to induce either the hyperexcitation of synaptic responses or the rapid depolarization. Furthermore, synaptic responses were fully restored after readministration of oxygen and glucose. In contrast, neither the synaptic hyperexcitation nor the rapid depolarization was observed during 10 min AA in the hippocampal CA1 pyramidal cells of the control slice. In addition, an irreversible synaptic failure associated with AA was not induced in the control slice. These results strongly suggest that fluorocitrate increases NMDA receptor-dependent AA-induced damage in the hippocampal slice by interfering glial spatial buffering of K+.

Relationship of calcium and adenylate cyclase messenger systems in rat brain synaptosomes.

The effects of cyclic AMP on the rise in cytosolic free calcium concentration, [Ca2+]i, after stimulation with 15 mM K+ in rat brain synaptosomes were investigated. The fluorescent chelating agent Quin-2 was employed to monitor alterations of K+-evoked [Ca2+]i. Under normoxic conditions, clonidine (1, 10 microM), an alpha 2-adrenoceptor agonist, decreased the 15 mM K+-evoked [Ca2+]i. Although yohimbine (1, 10 microM), an alpha 2-adrenoceptor antagonist, had little or no effect on K+-evoked [Ca2+]i, the inhibitory effects of clonidine were blocked by yohimbine. 8-Bromo cyclic AMP, a cyclic AMP analogue, (50-500 microM), increased K+-evoked [Ca2+]i in a dose-dependent manner. The addition of cyclic AMP analogues subsequent to clonidine treatment reversed the clonidine-induced suppression of K+-evoked [Ca2+]i. On the other hand, under hypoxic conditions, K+-evoked [Ca2+]i was reduced by about 50-60%. 8-Bromo cyclic AMP and the adenylate cyclase activators, yohimbine (1-10 microM) and isoproterenol, a beta-adrenoceptor agonist, (0.1-10 microM), transiently reversed the reduction of the K+-evoked [Ca2+]i caused by hypoxia. These results indicate that the activation of alpha 2-adrenoceptor produces a rapid, sustained decrease in [Ca2+]i which may be due to a decrease in the levels of intracellular cyclic AMP. In addition, the increase in cellular levels of cyclic AMP reversed the reduction of the Ca2+ response to high K+ stimulation caused by hypoxia. If this is so, there is the possibility that increased cyclic AMP might improve the hypoxic damage.