Ken-ichi Ueno

Consecutive evaluation of nitric oxide production after transient cerebral ischemia in the rat hippocampus using in vivo brain microdialysis

The time-course effects of transient cerebral ischemia on nitric oxide (NO) formation in the rat hippocampus were evaluated by the consecutive determination of oxidative NO metabolites (NO2- and NO3-), using brain microdialysis under the freely moving condition. Bilateral carotid artery occlusion (CAO; 2-vessel occlusion, 2VO; 10 and 20 min) and combined vertebral artery occlusion (4VO; 10 min) produced a transient increase in hippocampal NO2- and NO3- levels, according to the duration and degree of ischemic insults. In addition, 4VO produced a gradual increase in hippocampal NO2- and NO3- levels over a 24 h period after reperfusion, which was abolished by an inducible NO synthase inhibitor, aminoguanidine (10 mg/kg, intraperitoneally). These findings suggest that the dynamic changes in oxidative NO metabolite levels reflect NO production following transient cerebral ischemia, which is possibly mediated in part by an inducible NO synthase, in the rat hippocampus.

Aminoguanidine prevented the impairment of learning behavior and hippocampal long-term potentiation following transient cerebral ischemia

The present study was performed to investigate whether increases in nitric oxide (NO) production via inducible nitric oxide synthase (iNOS) were involved in impairment of learning behavior and hippocampal long-term potentiation (LTP) following transient ischemia. Rats with four-vessel occlusion (4-VO) were used as an ischemic model. One week after permanent occlusion of the vertebral arteries, the common carotid arteries were clamped for 10 min under halothane anesthesia. Aminoguanidine (10 mg/kg i.p.), a relatively selective iNOS inhibitor, or saline was administered 30 min before common carotid arteries occlusion, and every 24-h for 4 days. We investigated whether hippocampal NO production was increased by ischemic insult using microdialysis on days 1, 4 and 7 after 4-VO. On days 1 and 4 after 4-VO, increases in NO production were observed, and this effect was inhibited by aminoguanidine. Four days after 4-VO, rats were subjected to the Y-maze test and contextual fear conditioning. Ischemic insults impaired learning behavior, and aminoguanidine ameliorated the impairment induced by 4-VO. Four days after 4-VO, the changes in the population spike amplitude were recorded as an index of LTP in Schaffer collateral-CA1 (carotid artery 1), the mossy fiber-CA3 and the perforant path-dentate gyrus synapses. LTP was significantly inhibited by 4-VO, except in mossy fiber-CA3 synapses. Pretreatment with aminoguanidine prevented the reduction of LTP in perforant path-dentate gyrus, but not in Schaffer collateral-CA1 synapses. These results suggest that post-ischemic increase in NO production via iNOS impaired the learning behavior. There is an association between behavioral performance and LTP formation in perforant path-dentate gyrus synapses, but neither in Schaffer collateral-CA1 nor in mossy fiber-CA3 synapses.

Changes in synaptic plasticity in the rat hippocampo-medial prefrontal cortex pathway induced by repeated treatments with fluvoxamine

The present studies were conducted to examine the effects of single and repeated treatments with fluvoxamine, which is a selective serotonin reuptake inhibitor (SSRI), on the synaptic efficacy and synaptic plasticity in the rat hippocampo-medial prefrontal cortex (mPFC) pathway in vivo. It has been reported that the projections arising from the hippocampal structures to the mPFC are involved in the execution of higher cognitive functions in rats. The evoked potentials were recorded in the mPFC by stimulation of the CA1/subicular region of the ventral hippocampus in halothane-anesthetized rats. Single administration of fluvoxamine (10 and 30 mg/kg, i.p.) enhanced synaptic efficacy in the hippocampo-mPFC pathway in a dose-dependent manner. Although repeated treatments with fluvoxamine (30 mg/kg, i.p. after 30 mg/kg/dayx21 days, p.o.) caused an enhancement of synaptic efficacy, there was no significant difference between single and repeated treatments. The input/output characteristics showed hypersensitivity to stimulation intensity in the group with repeated fluvoxamine treatments. The establishment of long-term potentiation (LTP) in the hippocampo-mPFC pathway after a single administration of fluvoxamine was not different from that in the saline-injected group. On the other hand, the hippocampo-mPFC LTP was significantly augmented by repeated treatments with fluvoxamine when compared to a single treatment. These findings suggest that the serotonergic system could modulate the synaptic plasticity at hippocampal-mPFC synapses. The present study, furthermore, suggests that the enhancement of LTP in the hippocampo-mPFC pathway produced by repeated treatments with fluvoxamine may be implicated in the SSRI-induced therapeutic effect on psychiatric disorders.

Different effects of anxiolytic agents, diazepam and 5-HT1A agonist tandospirone, on hippocampal long-term potentiation in vivo

Benzodiazepines and 5-HT(1A) agonists have been widely used as anxiolytic agents. Some clinical reports document that 5-HT(1A) agonists induce memory impairment to a lesser degree than diazepam. In the present study, we compared the effects of diazepam and 5-HT(1A) agonist, tandospirone, on hippocampal long-term potentiation (LTP) in Schaffer collateral-CA1, mossy fiber-CA3 and perforant path-dentate gyrus synapses. In the diazepam-injected group, the reduction in LTP was observed in all three types of synapses, although the effective dose differed among these. In the tandospirone-injected group, no reduction in LTP was observed except in Schaffer-CA1 synapses. In addition, population spike amplitude was potentiated by tandospirone in mossy fiber-CA3 synapses in a dose-dependent manner. Thus, there was a discrepancy in the effects on hippocampal LTP between diazepam and tandospirone, possibly reflecting the reported clinical properties of these drugs, in that 5-HT(1A) partial agonists do not affect learning and memory, whereas diazepam impairs memory function.

Effects of idazoxan on dopamine release in the prefrontal cortex of freely moving rats

To clarify the involvement of dopaminergic neuronal systems in anxiety or fear, the present study was undertaken to elucidate the effect of an anxiogenic agent, idazoxan, a selective alpha2-adrenoceptor antagonist, on dopamine release from the rat prefrontal cortex by use of in vivo microdialysis. Systemic administration of idazoxan (0.25 mg/kg, i.p.) produced significant increases in extracellular levels of dopamine. The maximum response of the facilitatory effect of dopamine release was 241.5%, which was detected 80 min after the injection of idazoxan. Idazoxan-induced (0.25 mg/kg, i.p.) increases in dopamine release were prevented by an established anxiolytic agent, diazepam (0.5 mg/kg, i.p.) and a putative anxiolytic agent tropisetron (100 microg/kg, i.p.). These results suggest that the excessive dopaminergic neuronal activity in the rat prefrontal cortex is related to idazoxan-induced anxiogenic effects. The idazoxan-induced (0.25 mg/kg, i.p.) enhancement of dopamine release was further prevented by pretreatment with serotonin (5-hydroxytryptamine; 5-HT) neurotoxin, 5,7-dihydroxytryptamine (200 microg/kg, i.c.v.). The basal output of dopamine release was not altered in 5-HT lesioned rats. These findings indicate that intact serotonergic neurons are required for the facilitatory effects of idazoxan on dopamine release. In other words, the functional interaction between dopaminergic and serotonergic neuronal systems in the rat prefrontal cortex might be involved in anxiety or fear.

The serotonergic modulation of synaptic plasticity in the rat hippocampo-medial prefrontal cortex pathway.

The ability of the serotonergic (5-HTergic) system to affect the hippocampo-medial prefrontal cortex (mPFC) synaptic properties was examined in rats with lesions of 5-HTergic neurons. Intracerebroventricular injections of 5,7-dihydroxytryptamine (5,7-DHT) resulted in selective depletion of 5-HT and 5-hydroxyindoleacetic acid in the cerebral cortex, hippocampus and raphe regions. The 5,7-DHT-lesioned rat had no changes in basal synaptic transmission in the hippocampo-mPFC pathway. Conversely, we observed the augmentation of short-term synaptic plasticity, i.e. paired-pulse facilitation, when compared with sham-operated rats in this pathway. The magnitude of long-term potentiation (LTP) was significantly augmented in 5,7-DHT-lesioned rats. This augmentation of hippocampo-mPFC LTP had a significant correlation with the degree of cortical 5-HT levels. These results suggest that the 5-HTergic system may modulate plastic properties at the hippocampal-mPFC synapses in vivo.

An incomplete cerebral ischemia produced a delayed dysfunction in the rat hippocampal system

We investigated whether functional changes occur with incomplete cerebral ischemia which do not lead to neural cell death. If functional changes are recognized, it is necessary to clarify whether they occur immediately after ischemia or after a lag of a few days similar to the pathological changes. Long-term potentiation (LTP) in both the Schaffer collateral-CA1 and the perforant path-dentate gyrus synapses in halothane-anesthetized rats were examined 1 day and 4 days after 10 min clamping of the bilateral common carotid arteries. LTP was substantially attenuated after clamping of the bilateral common carotid arteries. In Schaffer-CA1 synapses, the inhibition of LTP was significant on both the 1 day and 4 days after-clamping group. In perforant path-dentate gyrus synapses, LTP was significantly inhibited on only the 4 days after-clamping group. These results suggest that functional damages may occur with incomplete ischemia without any histological damages. In the 1 day after-clamping group, LTP was reduced, but the changes in LTP differed from the inhibition of the 4 days after-clamping group. Therefore, a so-called delayed dysfunction might exist in the hippocampal neurons, despite absence of pathological changes.

Age-related changes in cardiac expression of VEGF and its angiogenic receptor KDR in stroke-prone spontaneously hypertensive rats

We examined the age-related changes in cardiac expression of angiogenic molecules during the development of cardiac remodeling in stroke-prone spontaneously hypertensive rats (SHRSP) in comparison with those in Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Vascular endothelial growth factor (VEGF) was highly upregulated in SHRSP aged 20 weeks compared with the same age of WKY, but it was downregulated at 40 weeks. On the other hand, KDR, an angiogenic receptor of VEGF, and endothelial nitric oxide synthase, which is important in the VEGF-mediated angiogenic pathway, were markedly downregulated in SHRSP from 20 weeks of age. Such age-related changes in their expression levels seen in SHRSP were quite different from those in SHR. In both SHR and SHRSP, transforming growth factor-β1 (TGF-β1) expression was increased with age, although SHRSP showed more marked upregulation. Cardiac remodeling in SHRSP was characterized by decreased coronary capillary density, cardiomyocyte hypertrophy, and cardiac fibrosis. We conclude that, in addition to overexpression of TGF-β1, which appears to play a pivotal role in promoting cardiac hypertrophy and fibrosis, a defect of the VEGF-KDR system could result in impaired physiologic coronary angiogenesis in SHRSP, contributing to cardiac deteroration associated with myocardial ischemia in this malignant hypertensive model.

Characterization of regional cerebral blood flow and expression of angiogenic growth factors in the frontal cortex of juvenile male SHRSP and SHR

Attention-deficit/hyperactivity disorder (AD/HD) is a common pediatric behavioral disorder associated with male preponderance and reduction of regional cerebral blood flow (rCBF). However, lack of an appropriate animal model exhibiting appropriate AD/HD symptoms stands in the way of studying mechanism(s) underlying reduced rCBF and male preponderance. Our group has been investigating the suitability of juvenile male stroke-prone spontaneously hypertensive rats (SHRSP), a substrain of the commonly used AD/HD animal model SHR, as a model for AD/HD because, unlike SHR, SHRSP displays cognitive impairment and male preponderance. Our more recent studies revealed alterations in the synthesis of sex steroid hormones and angiogenic factors in the frontal cortex of male SHRSP compared to the genetic control WKY. Based on these observations, the present study utilizes laser-Doppler flowmetry, histochemistry, enzyme immunoassay, immunoblotting, and real-time PCR to characterize and compare the patterns of regional cerebral blood flow and synthesis of angiogenic molecules [basic fibroblast growth factor; nitric oxide synthase isoforms (endothelial, neuronal and inducible); vascular endothelial growth factor (VEGF) and its signaling molecules (VEGF receptors, phosphorylated Akt, endothelial nitric oxide synthase eNOS] between male SHRSP and SHR. Overall, consistent with our previous data showing alteration in VEGF/Akt/NO signaling, there was a marked reduction in the profile of rCBF (35%) and angiogenic factors of SHRSP, compared to age-matched genetic control Wistar-Kyoto rats (WKY) and SHR. We conclude that, unlike SHR, the profiles of rCBF and angiogenic factors in SHRSP are altered in juvenile male. Thus, SHRSP appears to be a more suitable animal model for studying changes in rCBF in AD/HD.

Effects of an interleukin-1β analogue [Lys-d-Pro-Thr], on incomplete cerebral ischemia-induced inhibition of long-term potentiation in rat hippocampal neurons in vivo

Although interleukin-1beta (IL-1beta) has recently been implicated in neuronal cell death in vitro and in vivo after global forebrain ischemia, the role of IL-1beta in the functional injuries, i.e. impairment of synaptic transmission, after cerebral ischemia that does not cause neuronal death in the nervous system remains unknown. To address this question, we investigated the effect of short-term incomplete ischemia without apparent neural death on hippocampal long-term potentiation (LTP) in anesthetized rats, and examined the possible role of IL-1beta as an intermediary in this effect. Short-term incomplete cerebral ischemia (10 min) was induced in halothane-anesthetized rats by bilaterally clamping the common carotid arteries. Four days after ischemia, functional injuries in neuronal transmission in the hippocampal formation were observed without significant changes in pathological studies such as neuronal cell death. The LTP elicited in both Shaffer-CA1 synapses and perforant path-dentate gyrus synapses was significantly inhibited by the short-term incomplete ischemia. This inhibition of LTP was blocked by IL-1beta tripeptide antagonist (Lys-D-Pro-Thr), suggesting that the inhibitory effect of mild ischemia on synaptic potentials and LTP may be mediated by the generation of IL-1beta. These findings have important implications for the role of IL-1beta in not only neuronal cell death but also functional injuries without cell loss, perhaps elicited by transient cerebral ischemia.