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R Shigemoto

Publications and source records attributed to R Shigemoto.

At least 37 records · Page 2Linked to original sources
PubMed2000

Relationship between neurokinin-1 receptor and substance P in the striatum: light and electron microscopic immunohistochemical study in the rat.

The synaptic relationship between substance P (SP) and its receptor, i.e., neurokinin-1 receptor (NK1R), was examined in the striatum of the rat by confocal laser-scanning microscopy and electron microscopy. For confocal laser-scanning microscopy, triple-immunofluorescence histochemistry was performed to label NK1R, SP, and vesicular acetylcholine transporter (a specific marker for cholinergic neurons). In electron microscopic double-immunolabeling study, immunoreactivity for NK1R was detected with the silver-intensified gold method, while immunoreactivity for SP was detected with peroxidase immunohistochemistry. Simultaneous immunolabeling of NK1R and SP revealed significant mismatch at the synaptic level: although some SP-immunopositive axon terminals were in synaptic contact with NK1R-immunopositive sites of plasma membrane, NK1R-immunoreactivity was observed at both synaptic and non-synaptic sites of plasma membrane. Thus, SP released from the sites remote from NK1Rs might diffuse in the extracellular fluid to act, as a paracrine neurotransmitter, on NK1Rs distant from its releasing site. SP neurotransmission in the striatum might occur not only synaptically but also extrasynaptically. The SP-NK1R system might constitute an association system within the striatum.

Animals↗
PubMed2000

The relationship between neurokinin-1 receptor and substance P in the medullary dorsal horn: a light and electron microscopic immunohistochemical study in the rat.

The synaptic relationship between substance P (SP) and its receptor, i.e. neurokinin-1 receptor (NK1R), was examined in the superficial laminae of the caudal subnucleus of the spinal trigeminal nucleus (medullary dorsal horn; MDH) of the rat. For confocal laser-scanning microscopy, double-immunofluorescence histochemistry for NK1 and SP was performed. In electron microscopic double-immunolabeling study, immunoreactivity for NK1R was detected with the silver-intensified gold method, while immunoreactivity for SP was detected with peroxidase immunohistochemistry. SP-immunoreactive axon terminals were observed to be in synaptic (mostly asymmetric) contact with NK1R-immunoreactive neuronal profiles in lamina I and lamina IIo. Although some SP-immunoreactive axon terminals were in synaptic contact with NK1R-immunoreactive sites of plasma membranes, NK1R-immunoreactivity was observed at both synaptic and non-synaptic sites of plasma membrane. Thus, SP released from axon terminals might not only act on NK1Rs facing the SP-containing axon terminals, but also diffuse in the extracellular fluid for distances larger than the synaptic cleft to act on NK1Rs at some distances from the synaptic sites.

Animals↗
PubMed2000

Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site.

Aggregation of neurotransmitter receptors at pre- and postsynaptic structures is crucial for efficient neuronal communication. In contrast to the wealth of information about postsynaptic specializations, little is known about the molecular organization of presynaptic membrane proteins. We show here that the metabotropic glutamate receptor mGluR7a, which localizes specifically to presynaptic active zones, interacts in vitro and in vivo with PICK1. Coexpression in heterologous systems induces coclustering dependent upon the extreme C terminus of mGluR7a and the PDZ domain of PICK1. mGluR7a and PICK1 localize to excitatory synapses in hippocampal neurons. Furthermore, whereas transfected mGluR7a clusters at presynaptic sites, mGluR7adelta3 lacking the PICK1 binding site targets to axons but does not cluster. These results suggest that PICK1 is a component of the presynaptic machinery involved in mGluR7a aggregation and in modulation of glutamate neurotransmission.

Animals↗
PubMed1999

Spatial distribution of GABA(B)R1 receptor mRNA and binding sites in the rat brain.

A gamma-aminobutyric acid (GABA)(B) receptor (named GABA(B)R1) has been recently cloned in the rat and human brain and two variants generated by alternative RNA splicing were identified. In the present study, we addressed the question as to whether these variants contribute to the diversity of GABA(B) receptor-mediated physiological responses and constitute real receptor subtypes with distinct functions. To this aim, we have mapped the GABA(B)R1 (R1a) and GABA(B)R1b (R1b) transcript distribution in the rat brain using in situ hybridization. We have compared the mRNA distribution with the distribution of [(3)H]CGP54626-labeled binding GABA(B)R1 receptor sites as assessed in adjacent cryosections by quantitative autoradiography. We found that GABA(B) receptor transcripts and binding sites are expressed in the brain in almost all neuronal cell populations. Expression in glial cells, if any, is marginal. We observed a good parallelism between GABA(B)R1 mRNA transcripts and binding sites in broad neuroanatomical entities with highest densities in hippocampus, thalamic nuclei, and cerebellum. By contrast, R1a and R1b transcripts exhibit marked differences in their regional and cellular distribution pattern. A typical example is the cerebellum with an almost exclusive expression of R1b in the Purkinje cells and of R1a in the granule, stellate, and basket cells. Data pointing at a pre- versus postsynaptic localization for R1a and R1b, respectively, at some neuronal sites are presented.

Animals↗
PubMed1999

The distribution of substance P receptor (NK1)-like immunoreactive neurons in the newborn and adult human spinal cord.

Substance P receptor (i.e. NK1)-like immunoreactive (SPR-LI) neurons were observed in the newborn and adult human spinal cord. Substance P receptor-like immunoreactive neuronal cell bodies were seen most frequently in lamina I, and were scattered throughout the remaining laminae of the dorsal horn and the area around the central canal. Some neurons in the intermediolateral nucleus also showed weak immunoreactivity. The pattern of distribution of SPR-LI neurons in the adult spinal cord was essentially the same as that in the newborn spinal cord. However, SPR-LI neurons cell bodies were seen much more frequently in the newborn than in the adult dorsal horn, especially in lamina II.

Adult↗
PubMed1999

NK-1 receptor immunoreactivity in distinct morphological types of lamina I neurons of the primate spinal cord.

In cat and monkey, lamina I cells can be classified into three basic morphological types (fusiform, pyramidal, and multipolar), and recent intracellular labeling evidence in the cat indicates that fusiform and multipolar lamina I cells are two different types of nociceptive cells, whereas pyramidal cells are innocuous thermoreceptive-specific. Because earlier observations indicated that only nociceptive dorsal horn neurons respond to substance P (SP), we examined which morphological types of lamina I neurons express receptors for SP (NK-1r). We categorized NK-1r-immunoreactive (IR) lamina I neurons in serial horizontal sections from the cervical and lumbar enlargements of four monkeys. Consistent results were obtained by two independent teams of observers. Nearly all NK-1r-IR cells were fusiform (42%) or multipolar (43%), but only 6% were pyramidal (with 9% unclassified). We obtained similar findings in three monkeys in which we used double-labeling immunocytochemistry to identify NK-1r-IR and spinothalamic lamina I neurons retrogradely labeled with cholera toxin subunit b from the thalamus; most NK-1r-IR lamina I spinothalamic neurons were fusiform (48%) or multipolar (33%), and only 10% were pyramidal. In contrast, most (approximately 75%) pyramidal and some (approximately 25%) fusiform and multipolar lamina I spinothalamic neurons did not display NK-1r immunoreactivity. These data indicate that most fusiform and multipolar lamina I neurons in the monkey can express NK-1r, consistent with the idea that both types are nociceptive, whereas only a small proportion of lamina I pyramidal cells express this receptor, consistent with the previous finding that they are non-nociceptive. However, these findings also indicate that not all nociceptive lamina I neurons express receptors for SP.

Animals↗
PubMed1999

Metabotropic glutamate receptor subtype 7 ablation causes deficit in fear response and conditioned taste aversion.

Metabotropic glutamate receptors (mGluRs) consist of eight different subtypes and exert their effects on second messengers and ion channels via G-proteins. The function of individual mGluR subtypes in the CNS, however, largely remains to be clarified. We examined the fear response of freezing after electric shock in wild-type and mGluR7(-/-) knockout littermates. Wild-type mice displayed freezing immediately after and 1 d after footshock. In comparison, mGluR7(-/-) knockout mice showed significantly reduced levels in both immediate postshock and delayed freezing responses. However, the knockout mice exhibited no abnormalities in pain sensitivity and locomotor activity. To further examine amygdala-dependent behavior, we performed conditioned taste aversion (CTA) experiments. In wild-type mice, the administration of saccharin followed by intraperitoneal injection of the malaise-inducing agent LiCl resulted in an association between saccharin and LiCl. This association caused strong CTA toward saccharin. In contrast, mGluR7(-/-) knockout mice failed to associate between the taste and the negative reinforcer in CTA experiments. Again, the knockout mice showed no abnormalities in taste preference and in the sensitivity to LiCl toxicity. These results indicate that mGluR7 deficiency causes an impairment of two distinct amygdala-dependent behavioral paradigms. Immunohistochemical and immunoelectron-microscopic analyses showed that mGluR7 is highly expressed in amygdala and preferentially localized at the presynaptic axon terminals of glutamatergic neurons. Together, these findings strongly suggest that mGluR7 is involved in neural processes subserving amygdala-dependent averse responses.

Animals↗
PubMed1999

Smooth muscle cells are the site of neurokinin-1 receptor localization in the arterial supply of the rat sciatic nerve.

The occurrence and distribution of the preferred receptor for the neuropeptide, substance P (SP), the neurokinin-1 receptor (NK1R) was investigated in the vascular supply of the rat sciatic nerve. Messenger RNA for NK1R was demonstrated by RT-PCR in the epineurial layer where the majority of small arteries and arterioles feeding the endoneurial vasculature are located. Immunoreactivity to NK1 R-protein was localized on the smooth muscle cells of these arterial vessels by means of immunofluorescence using a polyclonal NK1R antiserum. This muscular localization of NK1R explains the previously reported [Zochodne, D.W. and Ho, L.T., J. Physiol., 444 (1991) 615-630] moderate vasoconstrictor rather than vasodilator effects of SP in this vascular bed.

Animals↗
PubMed1999

Modulation of synaptic transmission and differential localisation of mGlus in cultured hippocampal autapses.

Metabotropic glutamate receptors (mGlus) are known to modulate synaptic transmission in various pathways of the central nervous system, but the exact mechanisms by which this modulation occurs remain unclear. Here we utilise electrophysiological and immunocytochemical techniques on cultured autaptic hippocampal neurones to investigate the mechanism of action and distribution of mGlus. Agonists at all three groups of mGlus depressed glutamatergic transmission, whereas only agonists at group I mGlus depressed GABAergic transmission. Agonists at all mGlus failed to modulate Ca2+ and K+ channels in glutamatergic autapses whereas an agonist at group III mGlus did depress the frequency of miniature excitatory postsynaptic currents (mEPSCs). Agonists failed to modulate Ca2+ or K+ channels and miniature inhibitory postsynaptic currents (mIPSCs) in GABAergic autapses. Distribution studies using selective antibodies revealed punctate staining for group III mGlus that co-localised with the synaptic marker, synaptophysin. Staining for the remaining mGlus was more diffuse throughout the soma and processes with little co-localisation with synaptophysin. The distribution of the group III receptors is consistent with the direct 'downstream' modulation of mEPSCs, although the exact mechanism of action for the remaining receptors remains unclear.

Animals↗
PubMed1999

mGluR7-like receptor and GABA(B) receptor activation enhance neurotoxic effects of N-methyl-D-aspartate in cultured mouse striatal GABAergic neurones.

Presynaptic metabotropic glutamate receptors (mGluRs) of group III constitute possible targets for putative neuroprotective drugs acting against glutamate excitotoxic insults. Indeed, in glutamatergic cerebellar granule neurones in culture, high concentrations of L-2-amino-4-phosphonobutyrate (L-AP4, above 0.3 mM, thus activating mGluR7) inhibit NMDA-induced cell death. In contrast, in striatal cultures which are enriched in GABAergic neurones, we show that high concentrations of L-AP4 increased neuronal death in control as well as in NMDA-stimulated cultures. Moreover, similar results were obtained with the GABA(B)R agonist. baclofen. Both the neuroprotective effects in cerebellar granule cells and the neurotoxic effects in striatal neurones were mediated via Gi-Go-coupled mGluRs, suggesting that these effects were probably mediated by mGluR7a or b and GABA(B)R expressed in these neurones. In striatal neurones, we found that L-AP4 and baclofen inhibited both basal and NMDA-stimulated GABA release. These inhibitions of GABA release may be responsible for the increase in basal and NMDA-stimulated neuronal death. Indeed, blockade of GABA(A) receptors with bicuculline increased neuronal death of control and NMDA-treated striatal cultures. Taken together, these results suggest that L-AP4 and baclofen, via mGluR7 and GABA(B)R, reduced the neuroprotective effect of GABA present in striatal cultures acting via GABA(A) receptors. Although caution must be taken when extrapolating from in vitro to in vivo situations, the present experiments and the recent observations that mGluR7 and GABA(B)R are expressed in heterologous synapses, should be taken into consideration when evaluating the neuroprotective action of future mGluR7 specific agonists or GABA(B)R specific antagonists.

Animals↗
PubMed1999

mGluR7-like metabotropic glutamate receptors inhibit NMDA-mediated excitotoxicity in cultured mouse cerebellar granule neurons.

Glutamate-induced glutamate release may be involved in the delayed neuronal death induced by N-methyl-D-aspartate (NMDA). In order to examine a possible modulatory effect of the presynaptic group III mGluRs on glutamate excitotoxicity, the effect of L-2-amino-4-phosphonobutyrate (L-AP4) was examined on NMDA-induced delayed death of mouse cerebellar granule neurons in culture. We found that L-AP4, at high concentration (in the millimolar range), inhibited in a non-competitive manner the NMDA-induced toxicity. This effect was mimicked by high concentration of L-serine-o-phosphate (L-SOP), and was inhibited by pertussis toxin (PTX) indicating the involvement of a Gi/o protein. This suggests the involvement of mGluR7 in the L-AP4 effect, and this was consistent with the detection of both mGluR7 protein and mRNA in these cultured neurons. To examine the mechanism of the L-AP4-induced protection from excitotoxic damage, the effect of L-AP4 on glutamate release was examined. L-AP4 (> or = 1 mM) noncompetitively inhibited by more than 60% the glutamate release induced by NMDA during the insult. We also observed that the 10-min NMDA receptor stimulation resulted in a dramatic increase in the extracellular glutamate concentration reaching 6000% of the control value 24 h after the insult. This large increase was also inhibited when NMDA was applied in the presence of > or = 1 mM L-AP4. Part of the L-AP4-induced protection from excitotoxic damage of granule neurons may therefore result from the inhibition of the vicious cycle: dying cells release glutamate, glutamate induced cell death. The present results add to the hypothesis that presynaptic mGluRs, probably mGluR7, may be the targets of drugs decreasing glutamate release and then neuronal death observed in some pathological situations.

Animals↗
PubMed1998

GABA(B)-receptor subtypes assemble into functional heteromeric complexes.

B-type receptors for the neurotransmitter GABA (gamma-aminobutyric acid) inhibit neuronal activity through G-protein-coupled second-messenger systems, which regulate the release of neurotransmitters and the activity of ion channels and adenylyl cyclase. Physiological and biochemical studies show that there are differences in drug efficiencies at different GABA(B) receptors, so it is expected that GABA(B)-receptor (GABA(B)R) subtypes exist. Two GABA(B)-receptor splice variants have been cloned (GABA(B)R1a and GABA(B)R1b), but native GABA(B) receptors and recombinant receptors showed unexplained differences in agonist-binding potencies. Moreover, the activation of presumed effector ion channels in heterologous cells expressing the recombinant receptors proved difficult. Here we describe a new GABA(B) receptor subtype, GABA(B)R2, which does not bind available GABA(B) antagonists with measurable potency. GABA(B)R1a, GABA(B)R1b and GABA(B)R2 alone do not activate Kir3-type potassium channels efficiently, but co-expression of these receptors yields a robust coupling to activation of Kir3 channels. We provide evidence for the assembly of heteromeric GABA(B) receptors in vivo and show that GABA(B)R2 and GABA(B)R1a/b proteins immunoprecipitate and localize together at dendritic spines. The heteromeric receptor complexes exhibit a significant increase in agonist- and partial-agonist-binding potencies as compared with individual receptors and probably represent the predominant native GABA(B) receptor. Heteromeric assembly among G-protein-coupled receptors has not, to our knowledge, been described before.

Amino Acid Sequence↗
PubMed1998

Cellular and subcellular distribution of substance P receptor immunoreactivity in the dorsal vagal complex of the rat and cat: a light and electron microscope study.

Immunoreactivity for the substance P receptor (NK1 receptor) has been investigated by light and electron microscopy in the dorsal vagal complexes of adult rats and cats. The general pattern of NK1 immunoreactivity was similar for both rat and cat. Numerous NK1-immunoreactive neurons were present in the area postrema, the nucleus of the solitary tract, and the dorsal motor nucleus of the vagus nerve. The density of labelled neurons differed between the subnuclei of the nucleus of the solitary tract. Overall, the efferent neurons of the dorsal motor nucleus of the vagus nerve highly expressed NK1 when compared to neurons in the nucleus of the solitary tract. The results are discussed with reference to the viscerotopic organisation of the dorsal vagal complex. Ultrastructural analysis demonstrated that NK1 immunoreactivity was present only at the membrane surface of somatic and dendritic profiles of neurons. No labelling was found in axon terminals, axons, or glial processes. NK1 immunoreactivity, as revealed by a preembedding immunogold technique in serial ultrathin sections, was preferentially located at nonsynaptic sites. A semiquantitative study suggested that the density of NK1 receptors is statistically higher at membrane sites free of any contact (synaptic or not) with axon terminals. The subcellular localisation of NK1 immunoreactivity was similar for neurons of both rat and cat. These results suggest that in the dorsal vagal complex, substance P might act on NK1 receptors through a process of volume transmission.

Animals↗
PubMed1998

Ablation of cerebellar Golgi cells disrupts synaptic integration involving GABA inhibition and NMDA receptor activation in motor coordination.

The role of inhibitory Golgi cells in cerebellar function was investigated by selectively ablating Golgi cells expressing human interleukin-2 receptor alpha subunit in transgenic mice, using the immunotoxin-mediated cell targeting technique. Golgi cell disruption caused severe acute motor disorders. These mice showed gradual recovery but retained a continuing inability to perform compound movements. Optical and electrical recordings combined with immunocytological analysis indicated that elimination of Golgi cells not only reduces GABA-mediated inhibition but also attenuates functional NMDA receptors in granule cells. These results demonstrate that synaptic integration involving both GABA inhibition and NMDA receptor activation is essential for compound motor coordination. Furthermore, this integration can adapt after Golgi cell elimination so as not to evoke overexcitation by the reduction of NMDA receptors.

Animals↗
PubMed1998

Metabotropic glutamate receptor subtypes in axon terminals of projection fibers from the main and accessory olfactory bulbs: a light and electron microscopic immunohistochemical study in the rat.

Localization of metabotropic glutamate receptor subtypes, mGluR1, mGluR1alpha, mGluR2/3, mGluR4a, mGluR5, mGluR7a, mGluR7b, and mGluR8, was examined in some of the target areas of projection fibers from the main and accessory olfactory bulbs (MOB and AOB) by using subtype-specific antibodies. The superficial layer of the olfactory tubercle and layer Ia of the piriform cortex, the target areas of MOB, showed marked mGluR1-, mGluR5-, mGluR7a-, and mGluR8-like immunoreactivities (-LI), and rather weak mGluR2/3-LI. The periamygdaloid cortical region including the target areas of both MOB and AOB showed intense mGluR2/3-LI as well as marked mGluR1-, mGluR5-, mGluR7a-, and mGluR8-LI. No significant mGluR1alpha-, mGluR4a-, or mGluR7b-LI was seen in these regions. After transection of the lateral olfactory tract, mGluR2/3-, mGluR7a-, and mGluR8-LI were reduced markedly in the target regions on the side ipsilateral to the transection; no significant changes were detected in mGluR1- or mGluR5-LI. Double labeling experiments indicated light and electron microscopically colocalization of mGluR7a- and mGluR8-LI in axon terminals on dendritic shafts of presumed interneurons in the superficial layer of the olfactory tubercle and layer Ia of the piriform cortex. Electron microscopically mGluR2/3-LI was seen in preterminal and terminal portions of axons, whereas mGluR7a- and mGluR8-LI were associated with presynaptic membrane specialization. Immunolabeled axon terminals were filled with round synaptic vesicles and constituted asymmetric synapses with dendritic profiles. The results suggest that glutamate release from axon terminals of projection fibers from MOB and AOB is regulated presynaptically and differentially through mGluR2/3, mGluR7a, and/or mGluR8.

Animals↗
PubMed1998

Immunohistochemical localization of metabotropic glutamate receptors, mGluR7a and mGluR7b, in the central nervous system of the adult rat and mouse: a light and electron microscopic study.

The distributions of two alternative splicing variants of metabotropic glutamate receptor mGluR7, mGluR7a and mGluR7b, were examined immunohistochemically in the rat and mouse by using variant-specific antibodies raised against C-terminal portions of rat mGluR7a and human mGluR7b. Many regions throughout the central nervous system (CNS) showed mGluR7-like immunoreactivities (LI). The distribution patterns of mGluR7-LI in the rat were substantially the same as those in the mouse, although some species differences were observed in a few regions. Intense mGluR7a-LI was seen in the main and accessory olfactory bulbs, anterior olfactory nucleus, islands of Calleja, superficial layers of the olfactory tubercle, piriform cortex and entorhinal cortex, periamygdaloid cortex, amygdalohippocampal area, hippocampus, layer I of the neocortical regions, globus pallidus, superficial layers of the superior colliculus, locus coeruleus, and superficial layers of the medullary and spinal dorsal horns. The distribution of mGluR7b was more restricted. It was intense in the islands of Calleja, substantia innominata, hippocampus, ventral pallidum, and globus pallidus. The medial habenular nucleus also showed intense mGluR7a-LI in the rat but not in the mouse. For both mGluR7a- and mGluR7b-LI, localization in the active zones of presynaptic axon terminals was confirmed electron microscopically at synapses of both the asymmetrical and symmetrical types. It is noteworthy that mGluR7a-LI is seen preferentially in relay nuclei of the sensory pathways and that both mGluR7a- and mGluR7b-LI are observed not only in presumed glutamatergic axon terminals, but also in non-glutamatergic axon terminals including presumed inhibitory ones. Thus, mGluR7 may play roles not only as an autoreceptor in glutamatergic axon terminals, but also as a presynaptic heteroreceptor in non-glutamatergic axon terminals in various CNS regions.

Amygdala↗
PubMed1998

Substance P receptor (NK1)-immunoreactive neurons projecting to the periaqueductal gray: distribution in the spinal trigeminal nucleus and the spinal cord of the rat.

Substance P receptor (SPR)-immunoreactive neurons projecting to the periaqueductal gray (PAG) were examined in the rat spinal trigeminal nucleus and spinal cord by a retrograde tracing method combined with immunofluorescence histochemistry. After injection of Fluoro-gold (FG) into the PAG, SPR-immunoreactive neurons labeled with FG were observed mainly in the lateral spinal nucleus and lamina I of the medullary and spinal dorsal horns and additionally in laminae V and X of the spinal cord.

Animals↗