Biomedical subjects

H Kiyama

Publications and source records attributed to H Kiyama.

At least 163 records · Page 9Linked to original sources

PubMed1991

Differential effects of acute dopaminergic D1 and D2 receptor antagonists on proneurotensin mRNA expression in rat striatum.

The effect of acute dopamine (DA) antagonist treatment on neuronal proneurotensin (NT) mRNA was investigated in the rat striatum using a technique of non-radioactive in situ hybridisation. Adult Wistar rats were given a single intraperitoneal injection of either raclopride (D2 antagonist), SCH 23390 (D1 antagonist) or its inactive isomer SCH 23388 and left to survive for 3 h. Their brains were rapidly removed and striatal sections processed for in situ hybridisation using an alkaline phosphatase (AP) labelled oligonucleotide specific for NT mRNA. Blockade of the DA D2 receptors by a single injection of raclopride resulted in an increase in the number of NT mRNA containing cells in the dorsal lateral rim of the striatum adjacent to the corpus callosum. In contrast, no such increase was observed following blockade of the DA D1 receptors with SCH 23390. These findings demonstrate that NT mRNA expression is differentially regulated in the adult rat striatum by selective D1 and D2 antagonists.

Animals↗

PubMed1991

Colchicine-induced expression of proneurotensin mRNA in rat striatum and hypothalamus.

The effect of colchicine treatment on proneurotensin mRNA expression was examined using a sensitive non-radioactive in situ hybridization histochemistry technique with an enzyme-labelled antisense oligonucleotide probe. Intraventricular injection of colchicine rapidly induced strong neurotensin mRNA expression in a number of neurons in the dorsomedial part of the caudate putamen, and in the paraventricular, and arcuate nuclei while an induction of neurotensin mRNA was not observed in vehicle (saline)-injected animals. Neurotensin mRNA was not induced significantly in other periventricular areas such as the medial preoptic area or the lateral septum. These results indicate that colchicine can itself rapidly induce neurotensin mRNA expression and that this induction is specific to certain cell populations in the vicinity of the ventricles. These results demonstrate that colchicine should be used with caution in immunohistochemical studies demonstrating the normal distribution of peptide immunoreactivity or mRNA.

Animals↗

PubMed1991

Co-expression of cholecystokinin mRNA and tyrosine hydroxylase mRNA in populations of rat substantia nigra cells; a study using a combined radioactive and non-radioactive in situ hybridization procedure.

Improvements in the sensitivity of non-radioactive in situ hybridization histochemistry methods for detection of mRNA now make it feasible to combine the use of non-radioactive and radioactive in situ methods to visualize two mRNAs on the same tissue section. The method reported here allows the simultaneous detection of two mRNAs in one cell and therefore is ideally suited to the studies of co-expression. Here we demonstrate the co-expression of tyrosine hydroxylase (TH) mRNA and cholecystokinin (CCK) mRNA in the ventral mesencephalic dopaminergic neurones of the rat. The distribution of dopaminergic neurones containing both TH and CCK transcripts suggests, on the basis of earlier anatomical studies that these CCK/TH-containing doubled-labelled cells project mainly to the striatal matrix. Dopamine neurones believed to project to the patch compartment did not contain CCK mRNA.

Animals↗

PubMed1991

Dopaminergic D1 and D2 receptor antagonists decrease prosomatostatin mRNA expression in rat striatum.

The effect of acute dopamine receptor antagonist treatment on cellular prosomatostatin mRNA expression was investigated in the adult rat striatum using the technique of non-radioactive in situ hybridization. Adult female Wistar rats were given a single intraperitoneal injection of either raclopride (D2 antagonist), SCH 23390 (D1 antagonist) or the D1 (S) enantiomer SCH 23388. Animals were killed either 1, 3 or 9 h following the single i.p. injection and their brains rapidly removed. Striatal sections were then processed for in situ hybridization using an alkaline phosphatase-labelled oligonucleotide probe complementary to a portion of the rat somatostatin cDNA. Blockade of dopamine D1 and D2 receptors resulted in a significant decrease in the cellular content of prosomatostatin mRNA. However, no change in the number of prosomatostatin mRNA containing striatal cells was observed following any of the treatments at any time point. These findings demonstrate that the cellular content of prosomatostatin mRNA in the adult rat striatum is influenced by selective dopamine D1 and D2 receptor antagonists. Further, these findings are consistent with a functional interaction between dopamine and somatostatin in the rat striatum.

Animals↗

PubMed1991

Developmental control over vitamin-D-induced calbindin gene expression during early differentiation of chicken jejunal enterocytes.

In situ hybridization and immunocytochemical techniques have been used to examine the distribution of vitamin-D-induced calbindin mRNA and calbindin protein in enterocytes lining the crypts and villi of chicken small intestine. Basal villus enterocytes contained approximately twice as much calbindin but over three times as much calbindin mRNA compared to values found in basal crypt and upper villus enterocytes, all values being measured 2 days after vitamin D injection into D-deficient chickens. Virtually no calbindin mRNA was detected in tissues taken from control D-deficient birds. Direct proportionality found between calbindin mRNA and calbindin content in enterocytes of basal crypt, mid and upper villus suggests pre-translational control over calbindin synthesis. The implications of possible inefficient translation of calbindin mRNA in basal villus enterocytes are discussed. Present methods of analysis provide a novel way to study mechanisms controlling gene expression throughout the whole process of enterocyte differentiation.

Animals↗

PubMed1991

Electrical activation and c-fos mRNA expression in rat neurosecretory neurones after systemic administration of cholecystokinin.

1. The expression of c-fos mRNA in the rat hypothalamus was examined by in situ hybridization following systemic administration of cholecystokinin (CCK), a procedure known to activate magnocellular oxytocin neurons but not magnocellular vasopressin neurones. 2. Conscious male rats were given a single I.P. injection of 50 micrograms/kg CCK, c-fos mRNA signal was apparent in the supraoptic and paraventricular nuclei in rats killed 10 min after injection but not in uninjected or saline-(vehicle) injected rats. The density of c-fos mRNA at both sites was further elevated in rats killed 30 min or 60 min following injection, and was absent in rats killed 4 h after injection. 3. In the paraventricular nucleus the most dense expression of c-fos mRNA following CCK administration was in the medial, mainly parvocellular portion of the nucleus, in an area corresponding to the distribution of corticotrophin-releasing factor mRNA determined by in situ hybridization in adjacent sections. 4. The I.P. injection of CCK increased plasma oxytocin concentrations, measured by specific radioimmunoassay from 13 +/- 5 pg/ml in control rats to 107 +/- 9 pg/ml in the rats killed 10 min after injection, a similar response to that observed previously in urethane-anaesthetized rats. 5. In each of six urethane-anaesthetized rats, recordings were made from single neurones in the supraoptic nucleus, identified antidronomically as projecting to the posterior pituitary and identified electrophysiologically as putative oxytocin neurones. Following I.P. injection of 50 micrograms/kg CCK, the neurones increased their firing rate by a mean of 1.3 +/- 0.2 spikes/s averaged over the 10 min following injection. 6. From the appearance of c-fos mRNA in supraoptic neurones following CCK administration we conclude that this message is expressed in magnocellular oxytocin neurones, since vasopressin neuronal activity and vasopressin release is known to be unaffected by this stimulus, and since the supraoptic nucleus contains essentially only oxytocin neurones and vasopressin neurones. 7. We conclude that c-fos mRNA expression can be induced in supraoptic oxytocin neurones following brief and modest episodes of electrical activation, suggesting that c-fos may be involved in the gene regulation of these neurones under physiological conditions.

Action Potentials↗

PubMed1991

An in situ hybridization histochemistry method for the use of alkaline phosphatase-labeled oligonucleotide probes in small intestine.

We have developed a method of non-radioactive in situ hybridization histochemistry using alkaline phosphatase-labeled oligonucleotide probes to detect gene expression in the intestine. Because the intestine contains a large amount of endogenous alkaline phosphatase activity, mild acid pretreatment of the tissue sections was required to inactivate the alkaline phosphatase. Acid pre-treatment dramatically reduced the endogenous activity without affecting the efficiency of hybridization or the probe's ability to reveal a positive mRNA signal. Furthermore, the addition of polyvinyl alcohol to the substrate solution helped to keep the background staining low without adversely affecting the intensity of the signal. The current protocol allows rapid and sensitive detection of sites of gene expression in intestinal tissue.

Alkaline Phosphatase↗

PubMed1990

Localization of two calcium binding proteins, calbindin (28 kD) and parvalbumin (12 kD), in the vertebrate retina.

We used immunocytochemistry to locate two calcium binding proteins, calbindin (CaB) and parvalbumin (PV), in the retina of goldfish, frog, chick, rat, guinea pig, dog, and man. The location of CaB depended on the type of dominant photoreceptor cells in birds and mammals. In cone-dominant retinas such as those of the chick, CaB-like immunoreactivity was found in the cones, cone bipolars, and ganglion cells. Amacrine cells 5-12 microns across were also labeled. In rod-dominant retinas, such as those of the rat, guinea pig, and dog, horizontal cells, small amacrine cells (about 6 microns across), and cells in the ganglion cell layer were labeled. In the human retina, which has both cones and rods in abundance, cones, cone bipolars, ganglion cells, horizontal cells, and small and large amacrine cells were labeled. In the frog and goldfish, the level of CaB-like immunoreactivity was low. In the frog, a few cones, amacrine cells, and cells in the ganglion cell layer were labeled. No immunoreactive structures were seen in the goldfish retina. PV-like immunoreactivity was found in chicks, rats, and dogs. No such immunoreactive structures were seen in the other species. In the chick, only amacrine cells were labeled. In the rat, amacrine cells and several displaced amacrine cells were labeled. In the dog, in addition to amacrine cells and displaced amacrine cells, horizontal cells were strongly labeled. Thus, PV-like immunoreactivity was found in those elements relating to the modulation of the main pathway of the visual transmission system.

Animals↗

PubMed1990

Calbindin D28K as a marker for the degeneration of the striatonigral pathway in Huntington's disease.

An antibody raised against the chick calcium-binding protein calbindin D28K was used in immunohistochemical studies of normal post mortem human brain and the brains of individuals with Huntington's disease. Calbindin D28K immunoreactivity in the caudate nucleus and putamen coincided with the distribution of areas of acetylcholinesterase staining termed the matrix. In the matrix, calbindin D28K immunoreactivity was present in medium-sized neurons, the major neuronal population, however a further minor population of more strongly stained large neurons was detected. In Huntington's disease there was a dramatic loss of the majority of matrix calbindin D28K immunoreactive neurons and a parallel loss of calbindin D28K immunoreactivity from the substantia nigra. In contrast to the medium-sized calbindin D28K immunoreactive neurons which degenerate in Huntington's disease, the larger immunoreactive neurons were relatively preserved.

Acetylcholinesterase↗

PubMed1990

Postnatal development of preproenkephalin mRNA containing neurons in the rat lower brainstem.

Postnatal developmental changes of preproenkephalin (PPE) gene expression in rat brainstem neurons were studied by in situ hybridization histochemistry. On the basis of PPE mRNA expression, brainstem neurons were categorized into three types: 1) type I neurons were characterized by constant or increasing expression of PPE mRNA during postnatal development; 2) type II neurons started to express PPE mRNA several days after birth and continued to do so thereafter; and 3) type III neurons showed transient expression of PPE mRNA or stopped expressing the mRNA during early postnatal development. Type I PPE neurons were observed in diverse brainstem structures including the mesencephalic and pontine central gray matter, various reticular and raphe nuclei, the ventral tegmental area of Tsai, the interpeduncular nucleus, the nucleus of the brachium of the inferior colliculus, the ventral and dorsal tegmental nuclei of Gudden, the sphenoid nucleus, the laterodorsal tegmental nucleus, Barrington's nucleus, the parabrachial region, the lateral lemniscus and its related nuclei, the trapezoid nucleus, the rostral and ventromedial periolivary nuclei, the mesencephalic trigeminal and principal sensory trigeminal nuclei, the locus coeruleus, the subcoeruleus nucleus, the medial and spinal vestibular nuclei, the dorsal and ventral cochlear nuclei, the medial and lateral cerebellar nuclei, the Roller nucleus, and the intermedius nucleus of the medulla. Type II PPE neurons were found in the superior colliculus, the inferior colliculus, the central part of the dorsal tegmental nucleus, and as Golgi neurons in the granular layer of the cerebellum. Type III PPE neurons were located in the substantia nigra, the red nucleus, the superior olive, the motor trigeminal nucleus, the facial nucleus, the inferior olive, the dorsal motor nucleus of the vagus, and the hypoglossal nucleus. Such region-specific expression of the PPE gene during postnatal ontogeny suggests that rat brainstem PPE neurons may be involved in a variety of developmental events, such as cell proliferation, differentiation, and migration.

Aging↗

PubMed1990

Effects of constant light and darkness on the intrapineal neurons of golden hamsters, stained for tyrosine hydroxylase. A morphometric analysis.

Tyrosine hydroxylase (TH)-positive neurons (TH neurons) were found in the pineal gland of golden hamsters. To examine possible relations between TH neurons and environmental light, we kept male animals under constant light (LL) and darkness (DD) for a week, and morphometrically compared the number, size, and immunoreactivity of TH neurons with those of control animals kept under 12L/12D (LD), using an image processor, Nexus 6400. In LL animals, the number of TH neurons/mm2 of pineal tissue and each cell area were decreased, and immunoreactivity to TH was less than in LD animals. In DD animals, the number of TH neurons and each cell area were increased, and immunoreactivity decreased slightly. These data suggested that environmental light affected the TH neurons, and the amount of TH in the neurons would be decreased by LL, but increased by DD.

Animals↗

PubMed1990

Recent progress in the use of the technique of non-radioactive in situ hybridization histochemistry: new tools for molecular neurobiology.

Recent developments in DNA and oligonucleotide chemistry have made it possible to modify nucleotides and link quite complex molecules to the modified nucleotides. These advancements in DNA chemistry provide a number of possibilities for labelling oligonucleotide probes for DNA or RNA detection by non-radioactive methods. Over the years a number of non-radioactive detection systems for mRNA or chromosomal DNA have been developed. As reporter molecules, biotin, acetylaminofluorene, dinitrophenol, digoxigenin, sulfonized nucleotides, and mercury have been used and may be detected with a variety of high-affinity detectors, e.g. avidin (in the case of biotin) or antibodies specific to digoxigenin. These various 'indirect methods' of detection have used a number of chemical amplification procedures in attempts to improve their sensitivity. However, the sensitivity of these methods is often less than that of conventional radioactive methods. A sensitive non-radioactive technique would have a number of advantages over the complex and specialized radioactive in situ hybridization methods. In our laboratory we have recently found that simple enzyme-labelled probes provide excellent sensitivity (equivalent to that found with radioactive methods) and substantially improved cellular resolution. In this article, we describe the principle of the method and illustrate some applications of this novel non-radioactive in situ method.

Animals↗

PubMed1990

Sensitive non-radioisotopic in situ hybridization histochemistry: demonstration of tyrosine hydroxylase gene expression in rat brain and adrenal.

An alkaline phosphatase-labelled anti-sense oligonucleotide probe specific for tyrosine hydroxylase (TOH) mRNA has been used for visualisation of TOH mRNA in the rat brain and adrenal gland. Both ribonuclease pre-treatment and the use of excess non-labelled probe abolished the specific hybridization signal. Furthermore the TOH mRNA-positive signal was only found in cells known from earlier studies to react with anti-TOH antibodies. To determine if the alkaline phosphatase-labelled probe could be used in a semiquantitative manner for measurement of the density of TOH mRNA signal, we used reserpine pre-treatment which induces TOH mRNA expression. The results revealed a significant increase in TOH mRNA signal in locus coeruleus and substantia nigra neurons, and in adrenal medulla chromaffin cells. The increased signal in these areas agreed with the increase in TOH mRNA signal previously observed by Northern analysis and suggests that this type of alkaline phosphatase-labelled probe allows sensitive detection of changes in TOH gene expression.

Adrenal Glands↗

PubMed1990

Distribution of somatostatin mRNA in the rat nervous system as visualized by a novel non-radioactive in situ hybridization histochemistry procedure.

The cellular localization of preprosomatostatin mRNA in the rat brain and sensory ganglia has been examined in detail using a newly developed highly sensitive non-radioactive in situ hybridization histochemistry procedure. An alkaline phosphatase labelled anti-sense 30mer oligodeoxynucleotide probe was used for detection of somatostatin mRNA. This probe readily demonstrated somatostatin gene expression throughout the rat CNS with very high contrast and good cellular localization. As a result, we visualized numerous somatostatin mRNA-positive cells in many CNS areas which had previously not been shown to contain a mRNA signal. This method detected a number of somatostatin mRNA-positive cells, in the mitral cell layer of accessory olfactory bulb, the glomerular layer of the main olfactory bulb, the dorsal part of the lateral septum, superficial gray layer of superior colliculus, inferior colliculus, anterior ventral cochlear nucleus, granular layer and Purkinje cell layer of cerebellum, and substantia gelatinosa of medulla and spinal cord, all areas where signal detection using radiolabelled in situ probes has previously been rather difficult. The principle advantages of the present method include the very precise cellular resolution of signal, the rapid reaction time and low background. The sensitivity of the present method seems to be at least equivalent to most immunocytochemical procedures and more sensitive than most isotopic in situ hybridization methods.

Animals↗

PubMed1990

Adenosine 3',5'-cyclic monophosphate enhances dopamine accumulation in rat hypothalamic cell culture containing dopaminergic neurons.

The regulation of dopamine accumulation in cultured rat hypothalamic cells by adenosine 3',5'-cyclic monophosphate (cAMP) was investigated in cultures of newborn rat hypothalamic cells. Both dibutyryl cAMP, (Bu)2-cAMP, and forskolin enhanced [3H]dopamine accumulation in a dose-dependent manner. cAMP also enhanced [3H]dopamine accumulation, but to a lesser degree. Neither n-butyrate nor adenosine alone enhanced [3H]dopamine accumulation. (Bu)2-cAMP had no effect on basal efflux of [3H]radioactivity. The effect of (Bu)2-cAMP appeared on day 5 of culture, reached a maximum on day 6, and then rapidly decreased. These results suggest that dopamine uptake by cultured rat hypothalamic cells is regulated by intracellular cAMP.

Adenosine↗

PubMed1990

[Reconstruction of radicular arteries in the total replacement of thoracoabdominal aorta].

A technique of reconstructing low intercostal, high lumbar arteries and Adamkiewicz artery is described to prevent paraplegia following operations on the thoracoabdominal aorta. A graft with a side branch is anastomosed proximally and a side branch to the distal thoracic aorta after reinforcement of the stumps under the partial bypass. Applying a clamp on the main graft, flow through the side branch and true lumen of the aneurysm is reestablished and bypass is terminated. The main graft, extended with a Y-graft, is anastomosed to common iliac arteries after proximal suture-ligation to perfuse the lower extremities. Splanchnic arteries are reconstructed according to DeBakey's technique which is followed by trimming of the thoracoabdominal aorta incising longitudinally with a clamp applied on the 1st side branch. Edges of the trimmed aneurysm are sutured to the slit of the graft from the right common iliac artery reconstructing intercostal, lumbar arteries and Adamkiewicz artery from the level of L2 upwards to T4 or more. As a final step, the graft is sutured to the 1st side branch allowing perfusion distally to the right lower extremity and the main graft to the splanchnic arteries and left lower extremity after division of a Y-graft right branch. The technique minimizes ischemic time for reconstruction of the aortic branches and enables complete reconstruction of the radicular arteries.

Aorta, Abdominal↗

PubMed1989

Coexistence of enkephalin and somatostatin in the chicken retina.

A double immunostaining method was used in the present study to demonstrate that enkephalin (ENK) and somatostatin (SOM) coexist within single amacrine cells of the chicken retina. The ratio of double-labeled cells to either ENK- or SOM-bearing cells was more than 90%, indicating that retinal ENK and SOM are most likely to be found together in the same amacrine cell. This rate of colocalization is the highest yet observed among any neurotransmitters and modulators that coexist.

Animals↗

PubMed1989

Postnatal ontogeny of POMC gene expression in the rat pituitary: an analysis by in situ hybridization histochemistry.

Postnatal development of proopiomelanocortin gene expression in the rat pituitary was examined using in situ hybridization histochemistry. In adult rats, a very high density of hybridization signals was seen in the intermediate lobe of the pituitary, while only a moderate density occurred in the anterior lobe. No evidence of hybridization was detected in the posterior lobe. At birth, both the intermediate and anterior lobes had low to moderate frequencies of hybridization signals but a rapid rise to moderate density was noted by the 8th postnatal day. Radioactive labelling in the intermediate lobe continued to increase sharply with age to reach a plateau at postnatal day 28, while hybridization signals in the anterior lobe levelled off at postnatal day 8 with no subsequent rise in density.

Aging↗