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H Ishimaru

Publications and source records attributed to H Ishimaru.

At least 55 records · Page 3Linked to original sources
PubMed1997

Comprehensive analysis of neurotransmitters and their metabolites including acetylcholine and choline in rat brain nuclei.

We have designed this method for the comprehensive and adequate analysis of neurotransmitters and metabolites including acetylcholine (ACh) and choline in specific brain nuclei. In physiological and pharmacological studies, the role of specific neurotransmitters in the central nervous system (CNS) has often been studied by microinjection of their agonist and antagonist into the target regions, with the action and role of the transmitter in question being deduced in a relatively straightforward manner from the results. However, none of these studies have investigated fluctuation in neurotransmitter and metabolite contents in response to a variety of stimuli in vivo. No comprehensive analysis of neurotransmitters in small specific area in the CNS has been undertaken due to the difficulties of proper analysis of ACh and choline. Different types of neuronal systems in the CNS affect each other. Thus, it is not necessarily clear that different types of neurons do in fact respond to an administered agonist and antagonist. For example, dopaminergic neuron mediated control of cholinergic interneuron in the striatum and dopaminergic regulation of cortical ACh release have been documented. Similarly, the cholinergic system affects dopaminergic and noradrenergic neurons. Intraventricular administration of an ACh esterase inhibitor, neostigmine, increases dopamine and noradrenaline release in the hypothalamus. So, an adequate comprehensive analysis of the transmitters and the metabolites in the same tissue sample provides valuable aid to clarify what types of neurons truly respond to the administered drugs and the excitation of physiological events. For an appropriate analysis we employed a microwave device, which made the estimation of ACh and choline possible. In addition to this, we employed a punch technique for the microdissection of the specific nuclei and area from the brain slice.

Acetylcholine↗
PubMed1997

Relations between the extracellular concentrations of choline and acetylcholine in rat striatum.

Changes in extracellular levels of acetylcholine (ACh) and choline (Ch) in the striatum of rats were examined by in vivo microdialysis after intraperitoneal injections of drugs. A dopamine D2 antagonist, sulpiride (20 mg/kg), and a muscarinic antagonist, atropine (3.5 mg/ kg), increased ACh levels and decreased Ch levels. On the contrary, the D2 agonist (+/-)-2-(N-phenylethyl-N-propyl)amino-5-hydroxytetralin (N-434; 5 mg/kg) and an anesthetic, pentobarbital (50 mg/kg), decreased ACh levels and increased Ch levels. Perfusion of 10 microM hemicholinium-3 (HC-3), a Ch uptake inhibitor, through the striatum induced a complete inhibition of ACh release and increased Ch levels in all drug-treated groups. The degree of relative increase in the level of Ch induced by HC-3 differed among the drug-pretreated groups; compared with the control group, the relative increase was larger in the sulpiride- and atropine-treated groups and smaller in the N-434 and pentobarbital-treated groups. Thus, we demonstrated reciprocal relations between extracellular concentrations of Ch and ACh after treatments by drugs. The data suggest that in the striatum, which is rich in cholinergic innervation, the extracellular Ch concentration is to a large extent determined by activity of the cholinergic transmission reflected in high-affinity choline uptake.

Acetylcholine↗
PubMed1996

Functional expression of a recombinant unitary glutamate receptor from Xenopus, which contains N-methyl-D-aspartate (NMDA) and non-NMDA receptor subunits.

A cDNA encoding a 100-kDa subunit (XenNR1) of the N-methyl-D-aspartate (NMDA) glutamate receptor type has been cloned from Xenopus central nervous system. When XenNR1 is coexpressed in a mammalian cell line with a recently cloned 51-kDa non-NMDA receptor subunit (XenU1), also from Xenopus, it forms a functional unitary receptor exhibiting the pharmacological properties characteristic of both NMDA and non-NMDA receptors. Firstly, XenU1 can replace NR2 subunits, in complementing XenNR1 to introduce the ligand binding properties of a complete NMDA receptor. Second, responses to both NMDA and non-NMDA receptor agonists and antagonists were obtained in patch-clamp recordings from the cotransfected cells, but no significant responses were recorded when the cells were singly transfected. Third, from solubilized cell membranes from the cotransfected cells, an antibody to the NR1 subunit coprecipitated the binding sites of the non-NMDA receptor subunit. The unitary glutamate receptor has a unique set of properties that denote intersubunit interaction, including a glycine requirement for the responses to non-NMDA as well as to NMDA receptor agonists and voltage-dependent block by Mg2+ of the non-NMDA agonist responses.

Amino Acid Sequence↗
PubMed1996

Accumulation of apolipoprotein E and beta-amyloid-like protein in a trace of the hippocampal CA1 pyramidal cell layer after ischaemic delayed neuronal death.

We found that apolipoprotein E (apo E) accumulates in a trace of the hippocampal CA1 pyramidal cell layer within 6 months after 5 min ischaemia. Intense methenamine-silver (M-S) stainings were seen in the entire CA1 subfield 3 months after ischaemia. The M-S staining pattern may imply the appearance of soluble A beta-like proteins. The apo E-positive trace was also positively stained with anti-beta-amyloid (A beta) protein antibodies, but not with anti-beta-amyloid protein precursor (APP) antibodies. These results suggest that A beta-like protein accumulates in ischaemic brain after neuronal cell death. Adjacent reactive astrocytes showed both apo E- and A beta-immunoreactivities. These astrocytes may be involved in the clearance of apo E- and A beta-positive material. We presume that the coincident distribution pattern for apo E- and A beta-immunoreactivity implies formation of the insoluble and stable A beta-apo E complex which is known to exist in the brain of individuals with Alzheimer's disease. The ischaemic model may be useful in studying apo E and A beta deposition in the brain.

Amyloid beta-Peptides↗
PubMed1996

Effects of hepatic nerve stimulation on blood glucose and glycogenolysis in rat liver: studies with in vivo microdialysis.

In vivo microdialysis was applied to investigate the effects of hepatic nerve stimulation on glycogenolysis in rat liver under anesthesia. We analyzed the norepinephrine (NE) outflow and glucose output from the liver through the measurement of NE and glucose in the microdialysis dialyzate, as well as the plasma glucose level. Stimulation of the hepatic nerves (10 Hz, 20 V, 2 ms, 20 s every minute) increased NE outflow and glucose output from the liver. The blood glucose level increased by 1.5-1.6 times over the basal level at the end of the 10 min intermittent stimulation. Bilateral adrenalectomy and pancreatectomy did not abolish the glycogenolysis that was induced by the nerve stimulation. Phentolamine an alpha-antagonist, reduced the effects of nerve stimulation on the glucose output and the plasma glucose level. Phentolamine caused an increase in the NE outflow. Quinacline, an inhibitor of phospholipase A2, inhibited the glycogenolytic nerve effects without any inhibition of the NE outflow. These data show that hepatic nerve stimulation produces glycogenolysis via alpha-adrenergic mechanism and partly mediated by eicosanoids, and that microdialysis is a useful and simple method for the study of liver metabolism in physiological conditions.

Adrenalectomy↗
PubMed1996

Hypothalamic cholinergic activity associated with 2-deoxyglucose-induced hyperglycemia.

In order to clarify the role of the hypothalamic cholinergic system in the regulation of peripheral glucose metabolism, we investigated hypothalamic cholinergic activity after administration of 2-deoxyglucose (2-DG). Intravenous administration of 2-DG (500 mg/kg) caused marked hyperglycemia; the level of plasma glucose increased by 2.1 times over the initial levels at 20 min. For evaluation of the cholinergic activity, we analyzed extracellular levels of acetylcholine (ACh) and choline using brain microdialysis, as well as measuring their tissue levels in the ventromedial hypothalamic nucleus (VMH) and lateral hypothalamus (LH) of rats killed by microwave. In the microdialysis perfusate, extracellular levels of ACh and the metabolite choline were increased by 2-DG administration. In the tissue, a dose dependent decrease in the ACh content and a corresponding increase in the choline content were observed in both hypothalamic nuclei 20 min after administration of 2-DG. These data show that cholinergic activity is increased after 2-DG administration. Both the plasma glucose increment and the fluctuation of ACh and choline content were reduced in pentobarbital anesthetized rats. In 6-hydroxydopamine (6-OHDA) pretreated rats, the hypothalamic content of norepinephrine (NE) was reduced to one-third of that in controls, but there was no significant effect on the hyperglycemia or increase in hypothalamic tissue choline levels following 2-DG. Our results suggest the involvement of the hypothalamic cholinergic system in 2-DC-induced hyperglycemia.

Acetylcholine↗
PubMed1996

Activation of iron handling system within the gerbil hippocampus after cerebral ischemia.

Analyzing the distribution pattern of transferrin (Tf) and ferritin, we investigated the changes in iron metabolism related proteins in the process of neuronal death induced by 5 min ischemia. In the control animals, Tf immunoreactivity was localized in the oligodendrocytes. Ferritin was distributed in both neurons and gliacytes, particularly microglia. In parallel with the delayed neuronal death, Tf-positive atrophied neurons and numerous ferritin-positive gliacytes appeared in the CA1 subfield of the hippocampus 4 days after ischemia, when glia fibrillary acidic protein (GFAP)-positive astrocytes also appeared throughout the hippocampal structure. A considerable number of ferritin-positive phagocytes (reactive microglia) appeared in the stratum pyramidale from the seventh day. Our data show clearly that the mobilization of Tf and ferritin-positive phagocytes are linked with the degeneration of neurons induced by cerebral ischemia. These events may suggest an activation of iron handling system under the postischemic condition.

Animals↗
PubMed1996

Cystatin C and apolipoprotein E immunoreactivities in CA1 neurons in ischemic gerbil hippocampus.

The distribution patterns of cystatin C and apolipoprotein E (apo E) were studied immunocytochemically in the gerbil hippocampus before and after 5 min ischemia. In the controls, cystatin C was distributed mainly in astrocytes. In addition, a large number of dots positive for cystatin C were observed around the outlines of neuronal perikarya in the CA1 subfields. One day after ischemia, cystatin C-positive stainings outlining neuronal cell bodies disappeared. On the fourth day, intense stainings for cystatin C appeared in atrophied pyramidal neurons and these stainings in neurons disappeared by the 14th day. A remarkable increase in the number of cystatin C-positive astrocytes occurred on the fourth day and thereafter these spread over the whole of the CA1 subfield. Apo E was also distributed in astrocytes in the control specimens. From the fourth day, extra- and/or intracellular distribution of apo E-immunoreactivities was noted in the stratum pyramidale. Apo E-positive astrocytes disappeared transiently on the fourth day and then reappeared and increased remarkably by the 14th day. These findings indicate that cystatin C and apo E are involved in the degeneration process of brain neuronal cells.

Animals↗
PubMed1996

A unitary non-NMDA receptor short subunit from Xenopus: DNA cloning and expression.

A high-affinity homomeric, non-NMDA glutamate receptor was previously purified from the amphibian Xenopus laevis. We have obtained nine peptide sequences from its subunit, applied in cDNA cloning. The cDNA encodes a subunit (XenU1) containing all nine sequences. The 51,600-dalton mature subunit has four hydrophobic domains homologous to the four in the C-terminal half of mammalian non-NMDA receptor subunits. Transient expression in COS cells showed 1:1 binding (at Bmax) of [3H] kainate (KD = 9.1 nM) and of [3H] AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid; KD = 62 nM). The competitive binding series domoate > kainate > AMPA > NBQX > glutamate was established (where NBQX is 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo (f) quinoxaline). Each agonist shows the same KI value against [3H] kainate and [3H] AMPA binding, suggesting a common agonist site, but two conformations thereof are distinguishable by their different affinities for the antagonist NBQX and by the allosteric effect of thiocyanate anion (greatly potentiating AMPA binding, inert with kainate). XenU1 is exceptional among non-NMDA receptor subunits because it lacks most of the large N-terminal domain found in those of mammals and it has high affinity for both kainate and AMPA. It differs from the similarly-short "kainate-binding proteins" (KBPs), in binding AMPA and in forming glutamate receptor channels when the native protein is reconstituted. Moreover, whereas a full-length kainate receptor of mammals, GluR6, is shown here (from a partial cDNA sequence) to exist also in Xenopus, with approximately 97% sequence identity to rat GluR6, XenU1 is much less homologous to any rat kainate or AMPA receptor and also to the KBPs, even from another amphibian, Rana. Another difference is that a potential concensus sequence ("EF hand") for Ca2+ binding is present in the N-terminal domain of XenU1, but not in the chicken (glial) KBP. XenU1 is deduced to be in a new family of non-NMDA receptors.

Amino Acid Sequence↗
PubMed1995

Pentobarbital protects against CA1 pyramidal cell death but not dysfunction of hippocampal cholinergic neurons following transient ischemia.

Effects of pentobarbital on the release of acetylcholine (ACh), the area of CA1 pyramidal cell soma and the immunoreactivity of choline acetyltransferase (ChAT) in the hippocampus following ischemia were investigated. Five minute ischemia significantly decreased the KCl-, atropine-induced and basal release of ACh and the area of CA1 pyramidal cell soma in the hippocampus. Moreover, ChAT immunoreactivity, a marker of pre-synaptic terminal survival in the cholinergic neurons, was lowered 14 days after ischemia-recirculation. Although treatment with pentobarbital (50 mg/kg) 30 min before ischemia provided complete protection against hippocampal CA1 pyramidal cell death, pentobarbital failed to improve the decrements of ACh release and the low ChAT immunoreactivity over the test period. Our study thus showed discrepancies between pre-synaptic neurochemical estimation and post-synaptic morphological observation of the effect of pentobarbital on ischemic damage.

Acetylcholine↗
PubMed1995

Immunohistochemical and neurochemical studies of hippocampal cholinergic neurones after ischaemia.

We investigated alterations in cholinergic neurones in the gerbil hippocampus after ischaemia. The cholinergic function of acetylcholine (ACh) release fluctuated over the test period. Choline acetyltransferase (ChAT) immunoreactivity decreased slightly on day 1 and no ChAT immunoreactivity was observed on or after day 4 after ischaemia. Since ChAT immunoreactivity is a marker of cholinergic terminal survival, post-ischaemic cholinergic dysfunction on and after day 4 was accompanied by the destruction of terminals. However, dysfunction of the cholinergic system without destruction of the terminals is possible since mild ischaemia decreases ACh release in spite of retaining intact ChAT immunoreactivity. In the morphological study, delayed neuronal death in the stratum pyramidale was observed from day 4. The present study shows that presynaptic cholinergic dysfunction occurs in the early stage prior to pyramidal cell death.

Acetylcholine↗
PubMed1995

Decrease of norepinephrine and preservation of acetylcholine in the hypothalamus of VMH obese rats.

We investigated to find which types of neuronal disturbance in the hypothalamus are responsible for ventromedial hypothalamic nucleus (VMH) lesion-induced development of obesity. We found that in VMH-lesioned obese rats, the contents of norepinephrine (NE) and dopamine in the hypothalamus were selectively decreased, but that the serotonin and acetylcholine levels were unchanged from those in sham controls. Also, the content of NE in the lateral portion of the hypothalamus was decreased. Our results show that disturbance of the hypothalamic noradrenergic and dopaminergic neurons, but not of the serotonergic or cholinergic neurons, contributes to the development of VMH lesion-induced obesity.

Acetylcholine↗
PubMed1994

Increase of hypothalamic cholinergic activity in 2-deoxyglucose hyperglycemia.

Under inducement of hyperglycemia by intravenous administration of 2-deoxyglucose (2-DG), the contents of choline and acetylcholine (ACh) in the ventromedial hypothalamic nucleus (VMH), lateral hypothalamus (LH) and paraventricular nucleus (PVN) were analyzed after a microdissection of the microwave treated brains. After the administration of 2-DG, the content of choline in those hypothalamic nuclei increased markedly, and the ACh decreased in both the LH and the PVN. The increment of plasma glucose was reduced in the adrenodemedullectomized rats, and simultaneously the increment of the choline in the LH further augmented. These results suggest the contribution of hypothalamic cholinergic neurons in 2-DG-induced hyperglycemia.

Acetylcholine↗
PubMed1994

Hypothalamic cholinergic and noradrenergic neurons in hyperglycemia induced by 2-deoxyglucose.

To investigate the contribution of the hypothalamic cholinergic and noradrenergic neurons in 2-deoxyglucose (2-DG) induced hyperglycemia, we after microwave irradiation analyzed the contents of the neurotransmitters and the metabolites in the microdissected hypothalamic nucleus, ventromedial hypothalamic nucleus (VMH), paraventricular nucleus (PVN) and lateral hypothalamus (LH). A dose dependent decrease in the acetylcholine (ACh) content and a corresponding increase in the choline content was observed in those hypothalamic nuclei 20 min after intravenous administration of 250 or 500 mg/kg 2-DG. The norepinephrine content decreased in 500 mg/kg 2-DG group but did not change significantly in the 250 mg/kg group. These results suggest the involvement and importance of the hypothalamic cholinergic system in 2-DG induced hyperglycemia and, furthermore, that the hyperglycemic response can not be solely attributed to the noradrenergic system.

Acetylcholine↗
PubMed1994

Temporal changes in extracellular acetylcholine and CA1 pyramidal cells in gerbil hippocampus following transient cerebral ischemia.

Temporal changes in cholinergic functions following transient cerebral ischemia (10 min) were studied in the hippocampus of awake unrestrained gerbils using in vivo microdialysis. These data were compared with the results for temporal change in the area of each CA1 cell soma, measured with a microcomputer imaging device. KCl-induced release of acetylcholine (ACh) tended to be lower within 1 day after recirculation, and was significantly lower on the 4th, 7th and 14th days. Atropine-induced release of ACh gradually decreased over the test period. In histological estimation, no differences were observed within the 1st day, but a significant decrease of the area of CA1 cell soma was observed from the 4th to 14th days. Moreover, ischemia over 2 min decreased KCl- and atropine-induced ACh release on the 14th day without significant changes of hippocampal CA1 pyramidal cell. From these results, it is clear that ischemia produced dysfunction of hippocampal cholinergic neurons, and that dysfunction of the hippocampal cholinergic system following transient ischemia precedes pyramidal cell damage in the hippocampal CA1 subfield.

Acetylcholine↗
PubMed1994

Effect of transient cerebral ischaemia on acetylcholine release in the gerbil hippocampus.

To clarify the relationship between presynaptic cholinergic dysfunction and postsynaptic cell death in the hippocampus, extracellular levels of acetylcholine (ACh) were assayed and CA1 pyramidal cells were histologically investigated in gerbils which had undergone 2, 5 and 10 min ischaemia. It was found that the KCl- and atropine-induced release of ACh, an index of the functioning cholinergic system at the presynaptic terminals, was significantly lower in the ischaemic groups than in control groups. The hippocampal CA1 pyramidal cell area of the 5 and 10 min ischaemic animals was also significantly decreased, but the 2 min ischaemia caused no cell damage. These findings indicate that the presynaptic terminals of the cholinergic neurone are vulnerable to ischaemic insult and that cholinergic dysfunction precedes postsynaptic CA1 pyramidal cell death in the hippocampus.

Acetylcholine↗