Biomedical subjects

R Shigemoto

Publications and source records attributed to R Shigemoto.

At least 91 records · Page 5Linked to original sources

PubMed1994

Immunohistochemical localization of substance P receptor in the superior colliculus. A light and electron microscope study in the rat.

The superficial layers of the superior colliculus (SC) have been known to contain many axons showing substance P-like immunoreactivity (SP-LI). We, therefore, immunohistochemically examined the distribution of SP receptor (SPR) in the superficial layers of the SC in the rat by using a specific antibody against SPR. The majority of SC neurons with SPR-LI were distributed in the zonal and the superficial gray layers, the rest of them were in the optic layer. Electron microscopy revealed that SPR-immunoreaction products in SC neurons were distributed not only in postsynaptic sites, but also in non-synaptic regions of perikaryal and dendritic profiles.

Animals↗

PubMed1994

Molecular characterization of a new metabotropic glutamate receptor mGluR7 coupled to inhibitory cyclic AMP signal transduction.

A cDNA clone for a new rat metabotropic glutamate receptor termed mGluR7 was isolated through polymerase chain reaction-mediated DNA amplification by using primer sequences conserved among the metabotropic receptor (mGluR) family and by the subsequent screening of a rat forebrain cDNA library. The cloned mGluR7 subtype consists of 915 amino acid residues and exhibits a structural architecture common to the mGluR family with a large extracellular domain preceding the seven putative membrane-spanning domains. mGluR7 shows the highest sequence similarity to mGluR4 and mGluR6 among the members of the mGluR family. Similar to mGluR4 and mGluR6, mGluR7 inhibits forskolin-stimulated cyclic AMP accumulation in response to agonist interaction and potently reacts with L-2-amino-4-phosphonobutyrate and L-serine-O-phosphate in Chinese hamster ovary cells transfected with the cloned cDNA. RNA blot and in situ hybridization analyses of mGluR7 mRNA indicated that it is widely expressed in many neuronal cells of the central nervous system and is thus different from the more limitedly expressed mGluR4 or mGluR6 mRNA. mGluR7 together with mGluR4 thus corresponds to the putative L-2-amino-4-phosphonobutyrate receptor which plays an important role in modulation of glutamate transmission in the central nervous system.

Amino Acid Sequence↗

PubMed1994

Molecular diversity of glutamate receptors and their physiological functions.

Glutamate receptors play an important role in many integrative brain functions and in neuronal development. We report the molecular diversity of NMDA receptors and metabotropic glutamate receptors on the basis of our studies of molecular cloning and characterization of the diverse members of these receptors. The NMDA receptors consist of two distinct types of subunits. NMDAR1 possesses all properties characteristic of the NMDA receptor-channel complex, whereas the four NMDAR2 subunits, termed NMDAR2A-2D, show no channel activity but potentiate the NMDAR1 activity and confer functional variability by different heteromeric formations. The NMDA receptor subunits are considerably divergent from the other ligand-gated ion channels, and the structural architecture of these subunits remains elusive. The mGluRs form a family of at least seven different subtypes termed mGluR1-mGluR7. These receptor subtypes have, seven transmembrane segments and possess a large extracellular domain at their N-terminal regions. The seven mGluR subtypes are classified into three subgroups according to their sequence similarities, signal transduction mechanisms and agonist selectivities: mGluR1/mGluR5, mGluR2/mGluR3 and mGluR4/mGluR6/mGluR7. On the basis of our knowledge of the molecular diversity of the NMDA receptors and mGluRs, we have studied the physiological roles of individual receptor subunits or subtypes. We have shown that K(+)-induced depolarization or NMDA treatment in primary cultures of neonatal cerebellar granule cells induces the functional NMDA receptor and specifically up-regulates NMDAR2A mRNA among the multiple NMDA receptor subunits through the increase in resting intracellular Ca2+ concentrations. Our study demonstrates that the regulation of the specific NMDA receptor subunit mRNA governs the NMDA receptor induction that is thought to play an important role in granule cell survival and death. Analysis of an agonist selectivity and an expression pattern of mGluR6 has indicated that mGluR6 is responsible for synaptic neurotransmission from photoreceptor cells to ON-bipolar cells in the visual system. We have also investigated the function of mGluR2 in granule cells of the accessory olfactory bulb by combining immunoelectron-microscopic analysis with slice-patch recordings on the basis of the identification of a new agonist selective for this receptor subtype. Our results demonstrate that mGluR2 is present at the presynaptic site of granule cells and modulates inhibitory GABA transmission from granule cells to mitral cells. This finding indicates that the mGluR2 activation relieves excited mitral cells from GABA inhibition but maintains the lateral inhibition of unexcited mitral cells, thus resulting in enhancement of the signal-to-noise ratio between the excited mitral cells and their neighboring unexcited mitral cells.

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PubMed1994

Morphological and chemical characteristics of substance P receptor-immunoreactive neurons in the rat neocortex.

Substance P receptor-expressing neurons in the rat cerebral neocortex were examined by single- and double-immunolabeling methods with an affinity-purified specific antibody to substance P receptor. Substance P receptor immunoreactivity was observed exclusively in non-pyramidal neurons. About a quarter of these substance P receptor-positive neocortical neurons showed intense immunoreactivity, and the other three quarters displayed weak substance P receptor immunoreactivity. The neurons showing intense substance P receptor immunoreactivity were large multipolar cells with a few long aspiny or sparsely-spiny dendrites, and were scattered throughout the neocortical layers except for layer I, and also in the underlying white matter. The weakly immunoreactive neurons were medium-sized multipolar cells with oval to round somata and aspiny varicose dendrites, and were distributed in all cortical layers with a bias to layers II-III and the superficial part of layer V. The double-immunofluorescence study revealed that almost all substance P receptor-positive neurons were immunoreactive for GABA, but negative for glutaminase. Substance P receptor immunoreactivity in GABAergic neocortical neurons were further examined by the double-immunofluorescence method with antibodies to markers for subgroups of GABAergic neurons. Somatostatin immunoreactivity was found in 89% of neurons with intense substance P receptor immunoreactivity, and in 1.5% of neurons with weak substance P receptor immunoreactivity. Neuropeptide Y immunoreactivity was also observed in 92% of neurons with intense immunoreactivity for substance P receptor, and in 1.6% of neurons with weak immunoreactivity for substance P receptor. In contrast, parvalbumin immunoreactivity was seen in 1.3% of neurons with intense substance P receptor immunoreactivity, and in 59% of weak substance P receptor immunoreactivity. Calbindin D28k immunoreactivity was found in 12 and 19% of neurons, respectively, with weak and intense immunoreactivities for substance P receptor. Virtually no cells showing substance P receptor immunoreactivity displayed immunoreactivity for vasoactive intestinal polypeptide or choline acetyltransferase. These results indicate that the neocortical neurons expressing substance P receptor constitute a subpopulation of GABAergic non-pyramidal cells, and are segregated into neurons with intense immunoreactivity and those with weak immunoreactivity for substance P receptor; the vast majority of neurons with intense substance P receptor immunoreactivity contain somatostatin and neuropeptide Y, and the majority of neurons with weak substance P receptor immunoreactivity have parvalbumin.

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PubMed1994

Distribution of the messenger RNA for the prostaglandin E receptor subtype EP3 in the mouse nervous system.

Distribution of the messenger RNA for the prostaglandin E receptor subtype EP3 was investigated by in situ hybridization in the nervous system of the mouse. The hybridization signals for EP3 were widely distributed in the brain and sensory ganglia and specifically localized to neurons. In the dorsal root and trigeminal ganglia, about half of the neurons were labeled intensely. In the brain, intensely labeled neurons were found in Ammon's horn, the preoptic nuclei, lateral hypothalamic area, dorsomedial hypothalamic nucleus, lateral mammillary nucleus, entopeduncular nucleus, substantia nigra pars compacta, locus coeruleus and raphe nuclei. Moderately labeled neurons were seen in the mitral cell layer of the main olfactory bulb, layer V of the entorhinal and parasubicular cortices, layers V and VI of the cerebral neocortex, nuclei of the diagonal band, magnocellular preoptic nucleus, globus pallidus and lateral parabrachial nucleus. In the thalamus, moderately labeled neurons were distributed in the anterior, ventromedial, laterodorsal, paraventricular and central medial nuclei. Based on these distributions, we suggest that EP3 not only mediates prostaglandin E2 signals evoked by blood-borne cytokines in the areas poor in the blood-brain barrier, but also responds to those formed intrinsically within the brain to modulate various neuronal activities. Possible EP3 actions are discussed in relation to the reported neuronal activities of prostaglandin E2 in the brain.

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PubMed1994

Antibodies inactivating mGluR1 metabotropic glutamate receptor block long-term depression in cultured Purkinje cells.

Antibodies were raised against two distinct extracellular sequences of the rat mGluR1 metabotropic glutamate receptor expressed as bacterial fusion proteins. Both antibodies specifically reacted with mGluR1 in the rat cerebellum and inhibited the mGluR1 activity as assessed by the analysis of glutamate-stimulated inositol phosphate formation in CHO cells expressing mGluR1. Using these antibodies, we examined the role of mGluR1 in the induction of long-term depression in cultured Purkinje cells. In voltage-clamped Purkinje cells, current induced by iontophoretically applied glutamate was persistently depressed by depolarization of the Purkinje cells in conjunction with the glutamate application. The mGluR1 antibodies completely blocked the depression of glutamate-induced current. The results indicate that activation of mGluR1 is necessary for the induction of cerebellar long-term depression and that these mGluR1 antibodies can be used as selective antagonists.

Animals↗

PubMed1994

Immunohistochemical localization of metabotropic glutamate receptors, mGluR2 and mGluR3, in rat cerebellar cortex.

The distribution of the metabotropic glutamate receptors mGluR2 and mGluR3 was immunohistochemically examined in the rat cerebellar cortex at both light and electron microscope levels. An antibody was raised against a fusion protein containing a C-terminal portion of mGluR2. On immunoblot, the antibody reacted with both mGluR2 and mGluR3 in rat brain. mGluR2/3 immunoreactivity was expressed in cell bodies, dendrites, and axon terminals of Golgi cells, as well as in presumed glial processes. Golgi axon terminals with mGluR2/3 immunoreactivity were often encountered in the vicinity of glutamatergic mossy fiber terminals. The results suggest that transmitter glutamate may exert control influences upon Golgi cells not only through dendritic mGluR2/3, but also through axonal mGluR2/3.

Animals↗

PubMed1994

Distinct cellular localization of mRNAs for three subtypes of prostaglandin E receptor in kidney.

Distribution of the mRNAs for three subtypes of prostaglandin E (PGE) receptors in the mouse kidney was investigated by in situ hybridization. The mRNA for EP1 subtype, which is coupled to Ca2+ mobilization, was specifically localized to the collecting ducts from the cortex to the papilla. The mRNA for EP2 subtype, which is linked to stimulation of adenylate cyclase, was localized to the glomeruli. The mRNA for EP3 subtype, which is coupled to inhibition of adenylate cyclase, was located densely in the tubules in the outer medulla and in the distal tubules in the cortex. These results exhibit distinct cellular localization of three subtypes of PGE receptor in the kidney and suggest that PGE2 exerts multiple functions via these subtypes expressed in different segments of the nephron.

Adenylyl Cyclases↗

PubMed1993

Substance P receptor-immunoreactive neurons in the rat neostriatum are segregated into somatostatinergic and cholinergic aspiny neurons.

Immunochemical characteristics of neostriatal neurons producing substance P receptor (SPR) were examined in adult rats by double- and triple-immunofluorescence methods. In the neostriatum, SPR immunoreactivity was detected in large and medium-sized aspiny neurons. Virtually all SPR-immunoreactive neurons in the neostriatum contained somatostatin (SS) or choline acetyltransferase (ChAT), but not parvalbumin. All SS- and ChAT-immunoreactive neurons in the neostriatum showed SPR immunoreactivity. The co-existence of SS and ChAT was, however, not found in single neurons expressing SPR immunoreactivity. The present results indicate that neostriatal neurons immunoreactive for SPR are segregated into 2 groups: (1) medium-sized, spiny somatostatinergic, and (2) large, aspiny cholinergic neurons.

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PubMed1993

Role of a metabotropic glutamate receptor in synaptic modulation in the accessory olfactory bulb.

Various functions of glutamate transmission are mediated by both ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors (mGluRs) consists of at least six different subtypes that are classified into three subgroups, mGluR1/mGluR5, mGluR2/mGluR3, and mGluR4/mGluR6 (refs 1-5), but their physiological roles are largely unknown. Here we report the identification of a very potent agonist for mGluR2/mGluR3, DCG-IV, and the specific localization of mGluR2 in granule cell dendrites that form dendrodendritic synapses with mitral cells in the accessory olfactory bulb. Using the DCG-IV agonist for mGluR2 in combination with slice patch-recording, we demonstrate that the granule cell mGluR2 presynaptically suppresses inhibitory GABA (gamma-aminobutyrate) transmission to the mitral cell. Our results indicate that mGluR2 in granule cells plays an important role in the persistent excitation of olfactory sensory transmission in the accessory olfactory bulb by relieving mitral cells from the GABA inhibition.

Animals↗

PubMed1993

Immunohistochemical localization of a metabotropic glutamate receptor, mGluR5, in the rat brain.

A trpE-fusion protein containing a C-terminal sequence of a rat metabotropic glutamate receptor, mGluR5, was used to produce an antibody. On immunoblot, the antibody specifically reacted with mGluR5 expressed in mammalian cells and rat brain. Immunohistochemical analysis revealed intense mGluR5-like immunoreactivity (LI) in the olfactory bulb, anterior olfactory nuclei, olfactory tubercle, cerebral cortex, hippocampus, lateral septum, striatum, nucleus accumbens, inferior colliculus, and spinal trigeminal nuclei. The distribution pattern of mGluR5-LI corresponds very well with that of mGluR5 mRNA. Electron microscope analysis of the striatum revealed dense accumulation of immunoreaction products in dendrites which were often provided with asymmetrical synapses. These results suggest that mGluR5 is predominantly located in postsynaptic elements.

Animals↗

PubMed1993

Distribution of the mRNA for a metabotropic glutamate receptor (mGluR3) in the rat brain: an in situ hybridization study.

Distribution of the mRNA for a metabotropic glutamate receptor, mGluR3, which is coupled to the inhibitory cAMP cascade, was examined in the central nervous system of the adult albino rat by in situ hybridization. The hybridization signals of mGluR3 were detected not only on neuronal cells but also on many glial cells throughout the brain and spinal cord. In the neuronal cells, prominent expression of mGluR3 mRNA was seen in the thalamic reticular nucleus. Moderately labeled neurons were seen in the anterior olfactory nucleus, cerebral neo- and mesocortical regions, lateral amygdaloid nucleus, ventral part of the basolateral amygdaloid nucleus, dorsal endopiriform nucleus, supraoptic nucleus, superficial layers of the superior colliculus, inferior colliculus, interpeduncular nucleus, superior olivary nuclei, and Golgi cells in the cerebellar cortex. Weakly labeled neurons were observed in the striatum, nucleus accumbens, ventral pallidum, globus pallidus, entopeduncular nucleus, lateral hypothalamic area, hypothalamic paraventricular nucleus, medial habenular nucleus, anterior pretectal nucleus, Barrington's nucleus, Nucleus O, paragenual nucleus, trigeminal sensory complex, cochlear nuclei, dorsal motor nucleus of the trigeminal nerve, dorsal cap of the inferior olive, spinal dorsal horn, and lamina X of the spinal cord. The stellate cells in the cerebellar cortex, and neurons in the deep cerebellar nuclei were also labeled weakly. The granule cell layer of the dentate gyrus, as a whole, appeared to be labeled intensely, but each of the granule cells was labeled only weakly. No significant labeling was detected in the mitral and tufted cells in the olfactory bulb, hippocampal pyramidal cells, Purkinje and granule cells in the cerebellar cortex, or somatic motoneurons. The distribution of mGluR3 mRNA in particular neurons and glial cells indicates specific roles of mGluR3 in the glutamatergic system of the central nervous system.

Animals↗

PubMed1993

Molecular characterization of a novel retinal metabotropic glutamate receptor mGluR6 with a high agonist selectivity for L-2-amino-4-phosphonobutyrate.

A cDNA clone for a new metabotropic glutamate receptor, termed mGluR6, was isolated from a rat retinal cDNA library by cross-hybridization with the previously isolated cDNA clone for a metabotropic glutamate receptor. The cloned mGluR6 subtype consists of 871 amino acid residues and exhibits a structural architecture common to the metabotropic receptor family, possessing a large extracellular domain preceding the seven putative membrane-spanning domains. mGluR6 shows the highest sequence similarity to mGluR4 among the metabotropic receptor subtypes and inhibits the forskolin-stimulated cyclic AMP accumulation in Chinese hamster ovary cells transfected with the cloned cDNA. mGluR6 potently reacts with L-2-amino-4-phosphonobutyrate (L-AP4) and L-serine-O-phosphate, and the potencies of these compounds are one order of magnitude greater than that of L-glutamate. Blot and in situ hybridization analyses indicated that mGluR6 mRNA is restrictedly expressed in the inner nuclear layer of the retina where ON-bipolar cells are distributed. The metabotropic receptor that responds strongly to L-AP4 and L-serine-O-phosphate in ON-bipolar cells is known to mediate glutamate synaptic transmission between photoreceptor cells and ON-bipolar cells. On the basis of the agonist selectivity of mGluR6 and its specific expression in retinal cells, the physiological role of this receptor subtype in the visual system is discussed.

Amino Acid Sequence↗

PubMed1993

Immunocytochemical localization of rat substance P receptor in the striatum.

A trp E fusion protein containing a C-terminal portion of the rat substance P receptor (SPR) was expressed in bacteria and used to produce an antibody. The antibody specifically reacted with SPR expressed in a mammalian cell line and rat striatum. Light and electron microscope analyses of the rat striatum revealed intense SPR-like immunoreactivity in neuronal somata and dendrites. These immunoreactive neurons constituted approximately 3% of the total population of striatal neurons; they were putative interneurons of large and medium-sized aspiny type.

Animals↗

PubMed1993

Molecular characterization of the family of the N-methyl-D-aspartate receptor subunits.

cDNA clones for four different N-methyl-D-aspartate (NMDA) receptor subunits (NMDAR2A-NMDAR2D) were isolated through polymerase chain reactions followed by molecular screening of a rat brain cDNA library. These subunits are only about 15% identical with the key subunit of the NMDA receptor (NMDAR1) but are highly homologous (approximately 50% homology) with one another. They also commonly possess large hydrophilic domains at both amino- and carboxyl-terminal sides of the four putative transmembrane segments. NMDAR2A and NMDAR2C expressed individually in Xenopus oocytes showed no electrophysiological response to agonists. However, these subunits in combined expression with NMDAR1 markedly potentiated the NMDAR1 activity and produced functional variability in the affinity of agonists, the effectiveness of antagonists, and the sensitivity to Mg2+ blockade. Thus, NMDAR1 is essential for the function of the NMDA receptor, and multiple NMDAR2 subunits potentiate and differentiate the function of the NMDA receptor by forming different heteromeric configurations with NMDAR1. Northern blotting and in situ hybridization analyses revealed that the expressions of individual mRNAs for the NMDAR2 subunits overlap in some brain regions but are also specialized in many other regions. This investigation demonstrates the anatomical and functional differences of the NMDAR2 subunits, which provide the molecular basis for the functional diversity of the NMDA receptor.

Amino Acid Sequence↗

PubMed1993

Distribution of the messenger RNA for a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat.

Distribution of the messenger RNA for a metabotropic glutamate receptor, mGluR2, which is coupled to the inhibitory cyclic AMP cascade, was investigated in the central nervous system of the adult rat by in situ hybridization. Transcripts of mGluR2 were specifically localized to neuronal cells of the brain. Although the hybridization signals were widely distributed in the brain, the most prominent expression of mGluR2 messenger RNA was seen in Golgi cells of the cerebellum. Marked expression of mGluR2 messenger RNA was further observed in the mitral cells of the accessory olfactory bulb, neurons in the external part of the anterior olfactory nucleus, and pyramidal neurons in the entorhinal and parasubicular cortical regions. The granule cells of the accessory olfactory bulb, and many pyramidal and non-pyramidal neurons in the neocortical, cingulate, retrosplenial and subicular cortices, were moderately labeled. All of the granule cells in the dentate gyrus were also labeled moderately, whereas no significant hybridization signals were detected in Ammon's horn. In the basal forebrain regions, moderately labeled neurons were distributed in the triangular septal nucleus, in the lateral, basolateral and basomedial amygdaloid nuclei, and in the medial mammillary nucleus. Weakly labeled neurons were sparsely scattered in the striatum, globus pallidus, ventral pallidum and claustrum. The subthalamic nucleus was also labeled weakly. No significant labeling was found in the entopeduncular nucleus and substantia nigra. In the thalamus, moderately labeled neurons were distributed in the anterodorsal, anteromedial, ventromedial, intralaminar and midline nuclei; the ventrolateral part of the anteroventral nucleus and the rostral pole of the ventrolateral nucleus also contained moderately labeled neurons. No significant labeling was found in the thalamic reticular, submedius, ventroposterior, lateral geniculate and medial geniculate nuclei. In the lower brainstem, labeling was generally weak. No significant hybridization signals were found in the spinal cord. Some neurons in the inner part of the inner nuclear layer of the retina and some retinal ganglion cells were labeled moderately. The pattern of distribution of mGluR2 messenger RNA revealed in the present study indicates specific roles of mGluR2 in the glutamatergic system in the brain.

Animals↗

PubMed1993

Molecular cloning and tissue distribution of a receptor for pituitary adenylate cyclase-activating polypeptide.

Pituitary adenylate cyclase-activating polypeptide (PA-CAP) is a polypeptide hormone related to vasoactive intestinal polypeptide (VIP). Rat PACAP receptor cDNA was isolated from a brain cDNA library by cross-hybridization with rat VIP receptor cDNA. The recombinant PACAP receptor expressed in COS cells bound PACAP with about 1000 times higher affinity than VIP, and PACAP stimulated adenylate cyclase through the cloned PACAP receptor. The rat PACAP receptor consists of 495 amino acids, contains seven transmembrane segments, and has a significant similarity with other Gs-coupled receptors, such as VIP, glucagon, and secretin receptors. PACAP receptor mRNA was abundantly expressed in the brain, but not in the peripheral tissues except for the adrenal gland. In situ hybridization revealed a high level of expression of PACAP receptor mRNA in the hippocampal dentate gyrus, olfactory bulb, and cerebellar cortex.

Amino Acid Sequence↗

PubMed1993

Localization of endothelin receptor messenger ribonucleic acid in the rat ovary and fallopian tube by in situ hybridization.

Rat mRNAs encoding two subtypes of the endothelin (ET) receptor (ET(A) and ETB) were studied in the rat ovary and fallopian tube by means of Northern blotting and in situ hybridization. The mRNA transcripts for the endothelin-1-specific type receptor (ET(A)) in pooled RNA from the ovary and fallopian tube were 4.2 and 5.2 kilonucleotides, and that for the nonselective type receptor (ETB) was 4.7 kilonucleotides; these were similar to transcripts for endothelin receptors from other tissues. ET(A) mRNA expression was abundant in the muscle cell layer of the fallopian tube, but low in the ovary. On the other hand, ETB mRNA was abundant in the granulosa cells in the developing follicles, but low in atretic follicles and absent in the fallopian tube. These results demonstrated that the mRNAs for the two subtypes of the rat endothelin receptor have different expression profiles in the ovary and fallopian tube. ETs may mainly affect the granulosa cells in the dominant follicles as well as the muscle cells of the fallopian tube through ETB and ET(A), respectively.

Animals↗