[Show abstract][Hide abstract] ABSTRACT: GABA(A) receptors are pentamers in structure and are mainly composed of alpha, beta and gamma subunits. These receptors are known to function as chloride channels. We observed alpha5, beta1 and gamma3 subunit immunoreactivity in the mouse testes, specifically in the cytoplasm surrounding the nucleus in the spermatocytes and spermatids. In the current study, alpha1 subunit immunoreactivity was located in the nucleus of spermatogonia, spermatocytes and round spermatids. Immunoelectron microscopy revealed that the alpha1 subunit was localized within the nucleus of pachytene and diplotene spermatocytes in the area of condensed chromatin rather than extended chromatin. Protein sequence analysis revealed that the alpha1 subunit included DM DNA binding domains that were related to transcription factors involved in testicular differentiation in adult mice. These findings suggest that the alpha1 subunit may undertake a gene transcription function during the maturation of germ cells. a1 immunoreactivity was also detected within the mitochondria of spermatocytes and in the acrosome of round and elongated spermatids. Although the precise physiological role of the GABA(A) receptor alpha1 subunit in mitochondria remains unknown, we hypothesize that its function in the acrosome may be related to the acrosome reaction during fertilization or during spermatogenesis.
No preview · Article · Oct 2010 · Histology and histopathology
[Show abstract][Hide abstract] ABSTRACT: The neostriatum, which possesses a mosaic organization consisting of patch and matrix compartments, receives glutamatergic excitatory afferents from the cerebral cortex and thalamus. Differences in the synaptic organization of these striatopetal afferents between the patch and matrix compartments were examined in the rat using confocal laser scanning and electron microscopes. Thalamostriatal terminals immunopositive for vesicular glutamate transporter (VGluT) 2 were less dense in the patch than in the matrix compartment, although the density of VGluT1-immunopositive corticostriatal terminals was almost evenly distributed in both the compartments. Quantitative analysis of ultrastructural images revealed that 84% of VGluT2-positive synapses in the patch compartment were formed with dendritic spines, whereas 70% in the matrix compartment were made with dendritic shafts. By contrast, VGluT1-positive terminals display a similar preference for specific synaptic targets in both compartments: about 80% made synapses with dendritic spines. In addition, VGluT2-positive axospinous synapses in the patch compartment were larger than the VGluT1-positive axospinous synapses in both compartments. As axospinous synapses are generally found in neuronal connections showing high synaptic plasticity, the present findings suggest that the thalamostriatal connection requires higher synaptic plasticity in the patch compartment than in the matrix compartment.
No preview · Article · Dec 2006 · European Journal of Neuroscience
[Show abstract][Hide abstract] ABSTRACT: The GABAergic system, a major inhibitory regulator in the central nervous system, may also play important roles in peripheral nonneuronal tissues and cells. Recent studies showed that GABAB receptor is expressed in testis and sperm. To understand the role of the GABAergic system in spermiogenesis, we examined cellular localization of GABA and GABAB receptor subunits in rat spermatids by immunocytochemistry. Immunoreactivity for GABA was detected around acrosomal granules of spermatids during the Golgi and cap phases. GABAB1 immunoreactivity was observed in the acrosomal vesicle of spermatids in Golgi phase, and during cap phase, this reactivity expanded to the entire region of the acrosome covering the nuclear membrane. The level of reactivity decreased gradually with maturation of spermatids. In contrast, GABAB2 immunoreactivity was not observed in spermatids during Golgi phase but was detected in the equatorial region during cap phase. Both GABA immunoreactivity and GABAB2 immunoreactivity were transferred to the residual cytoplasm during the release of spermatozoa. Electron microscopic immunocytochemistry revealed that, during cap phase, GABA and GABAB1 were distributed within the whole acrosomal vesicle but not in the acrosomal granule. GABAB2 immunoreactivity was observed in the narrow space between the inner acrosomal and nuclear membrane and was limited to the equatorial region of the spermatid head. These results indicate that the GABAergic system might be involved in regulation of spermiogenesis.
Preview · Article · Jul 2005 · Journal of Andrology
[Show abstract][Hide abstract] ABSTRACT: The neostriatum is known to receive glutamatergic projections from the cerebral cortex and thalamic nuclei. Vesicular glutamate transporters 1 and 2 (VGluT1 and VGluT2) are located on axon terminals of corticostriatal and thalamostriatal afferents, respectively, whereas VGluT3 is found in axon terminals of cholinergic interneurons in the neostriatum. In the present study, the postsynaptic localization of ionotropic glutamate receptors was examined in rat neostriatum by the postembedding immunogold method for double labelling of VGluT and glutamate receptors. Immunoreactive gold particles for AMPA receptor subunits GluR1 and GluR2/3 were frequently found not only on postsynaptic but also on presynaptic profiles immunopositive for VGluT1 and VGluT2 in the neostriatum, and GluR4-immunoreactive particles were observed on postsynaptic and presynaptic profiles positive for VGluT1. Quantitative analysis revealed that 27-45% of GluR1-, GluR2-, GluR2/3- and GluR4-immunopositive particles found in VGluT1- or VGluT2-positive synaptic structures in the neostriatum were associated with the presynaptic profiles of VGluT-positive axons. In contrast, VGluT-positive presynaptic profiles in the neostriatum showed almost no immunoreactivity for NMDA receptor subunits NR1 or NR2A/B. Furthermore, almost no GluR2/3-immunopositive particles were observed in presynaptic profiles of VGluT3-positive (cholinergic) terminals that made asymmetric synapses in the neostriatum, or in those of VGluT1- or VGluT2-positive terminals in the neocortex. The present results indicate that AMPA receptor subunits but not NMDA receptor subunits are located on axon terminals of corticostriatal and thalamostriatal afferents, and suggest that glutamate released from these axon terminals controls the activity of the terminals through the presynaptic AMPA autoreceptors.
Full-text · Article · Jan 2005 · European Journal of Neuroscience
[Show abstract][Hide abstract] ABSTRACT: To clarify which vesicular glutamate transporter (VGluT) is used by excitatory axon terminals of the retinofugal system, we examined immunoreactivities and mRNA signals for VGluT1 and VGluT2 in the rat retina and compared immunoreactivities for VGluT1 and VGluT2 in the retinorecipient regions using double immunofluorescence method, anterograde tracing, and immunoelectron microscopy. Furthermore, the changes of VGluT1 and VGluT2 immunoreactivities were studied after eyeball enucleation. Intense immunoreactivity and mRNA signal for VGluT2, but not for VGluT1 immunoreactivity, were observed in most perikarya of ganglion cells in the retina. Immunoelectron microscopy revealed that VGluT1- and VGluT2-immunolabeled terminals made asymmetrical synapses, suggesting that they were excitatory synapses, and that VGluT1-immunolabeled terminals were smaller than VGluT2-labeled ones in many retinorecipient regions, such as the dorsal lateral geniculate nucleus (LGd) and superior colliculus (SC). Double immunofluorescence study further revealed that almost no VGluT2 immunoreactivity was colocalized with VGluT1 in the retinorecipient regions. After wheat germ agglutinin (WGA) injection into the eyeballs, WGA immunoreactivity was colocalized in the single axon terminals of LGd and SC with VGluT2 but not VGluT1 immunoreactivity. After unilateral enucleation, VGluT2 immunoreactivity in the LGd, SC, nucleus of the optic tract, and nuclei of the accessory optic tract in the contralateral side of the enucleated eye was clearly decreased. Although only a small change of VGluT2 immunoreactivity was observed in the contra- and ipsilateral suprachiasmatic nuclei, olivary pretectal nucleus, anterior pretectal nucleus, and posterior pretectal nucleus, moderate reduction of VGluT2 was found in these regions after bilateral enucleation. On the other hand, almost no change in VGluT1 immunoreactivity was found in the structures examined in the present enucleation study. Thus, the present results support the notion that the retinofugal pathways are glutamatergic, and indicate that VGluT2, but not VGluT1, is employed for accumulating glutamate into synaptic vesicles of retinofugal axons.
Full-text · Article · Oct 2003 · The Journal of Comparative Neurology
[Show abstract][Hide abstract] ABSTRACT: The supratrigeminal region (STR), a reticular zone capping the motor trigeminal nucleus (Tm), contains gamma-aminobutyric acid (GABA)ergic and glycinergic neurons which send axons to the contralateral Tm (J. Comp. Neurol. 373 (1996) 498). In the present study we observed that some single synaptic terminals upon Tm motoneurons showed immunoreactivities (IRs) for both glutamic acid decarboxylase (GAD) and glycine transporter 2 (GlyT2). After injecting biotinylated dextran amine (BDA) into the STR, we further observed in the Tm contralateral to the BDA injection that some BDA-labeled axon terminals in close contact with Tm motoneurons showed both GAD- and GlyT2-IRs. Thus, the STR was indicated to send GABAergic/glycinergic axon terminals contralaterally to Tm motoneurons.
Full-text · Article · Oct 2002 · Neuroscience Letters
[Show abstract][Hide abstract] ABSTRACT: We examined immunoreactivities for gamma-aminobutyric acidB-receptor (GABA(B)R) subtypes, GABA(B)R1 and GABA(B)R2, in the mesencephalic trigeminal nucleus neurons (MTN neurons) of the rat. Immunoreactivity for GABA(B)R1 was prominent in cell bodies of MTN, whereas that for GABA(B)R2 was very weak, if existed. For electron microscopy, the immunogold-silver method for GABA(B)R1 was combined with the immunoperoxidase method for glutamic acid decarboxylase (GAD: the synthetic enzyme of GABA). Immunogold-silver particles indicating GABA(B)R1 immunoreactivity were distributed widely in the cytoplasm of the cell bodies postsynaptic to GAD-immunoreactive axon terminals, but were rarely associated with synaptic membrane specialization or extrasynaptic sites of plasma membrane. It has been indicated that GABA(B)R1 may not be transported to plasma membrane when no GABA(B)R2 exists. Thus, it was presumed that GABA(B)R1 in the cell body of the rat MTN neurons might not be involved in the synaptic transmission.
Full-text · Article · Dec 2001 · Neuroscience Letters
[Show abstract][Hide abstract] ABSTRACT: We examined protein kinase C gamma-immunoreactivity (PKCgamma-IR) in the substantia gelatinosa (SG) of the rat medullary dorsal horn (MDH). The density of PKCgamma-IR in the MDH was most intense in the SG. The number of neurons with PKCgamma-IR were also much larger in the SG than in the other layers of the MDH. Double-immunohistochemical studies indicated light and electron microscopically that substance P-containing fibers and I-B4 (isolectin from Bandeiraea simplicifolia)-labeled fibers made synapses on SG neurons with PKCgamma-IR, indicating that SG neurons with PKCgamma might receive nociceptive primary afferent fibers. The results support the notion that PKCgamma in the MDH may contribute to the regulation of the nociception.
Full-text · Article · Nov 2001 · Neuroscience Letters
[Show abstract][Hide abstract] ABSTRACT: The metabotropic glutamate receptors (mGluRs) have distinct distribution patterns in the CNS but subtypes within group I or group III mGluRs share similar ultrastructural localization relative to neurotransmitter release sites: group I mGluRs are concentrated in an annulus surrounding the edge of the postsynaptic density, whereas group III mGluRs are concentrated in the presynaptic active zone. One of the group II subtypes, mGluR2, is expressed in both pre- and postsynaptic elements, having no close association with synapses. In order to determine if such a distribution is common to another group II subtype, mGluR3, an antibody was raised against a carboxy-terminus of mGluR3 and used for light and electron microscopic immunohistochemistry in the mouse CNS. The antibody reacted strongly with mGluR3, but it also reacted, though only weakly, with mGluR2. Therefore, to examine mGluR3-selective distribution, we used mGluR2-deficient mice as well as wild-type mice.
[Show abstract][Hide abstract] ABSTRACT: We applied high-resolution laser-scanning microscopy, electron microscopy, and non-radioactive in situ hybridization histochemistry to determine the cellular and intracellular localization of lipocalin-type prostaglandin D synthase, the major brain-derived protein component of cerebrospinal fluid, and its mRNA in leptomeninges, choroid plexus, and parenchyma of the adult rat brain. Both immunoreactivity and mRNA for prostaglandin D synthase were located in arachnoid barrier cells, arachnoid trabecular cells, and arachnoid pia mater cells. Furthermore, meningeal macrophages and perivascular microglial cells, identified by use of ED2 antibody, were immunopositive for prostaglandin D synthase. In the arachnoid trabecular cells, the immunoreactivity for prostaglandin D synthase was located in the nuclear envelope, Golgi apparatus, and secretory vesicles, indicating the active production and secretion of prostaglandin D synthase. In the meningeal macrophages, prostaglandin D synthase was not found around the nucleus but in lysosomes in the cytoplasm, pointing to an uptake of the protein from the cerebrospinal fluid. Furthermore, the existence of meningeal cyclooxygenase (COX) -1 and COX-2 was investigated by Western blot, Northern blot, and reverse transcriptase-polymerase chain reaction (RT-PCR), and the colocalization of COX-2 and prostaglandin D synthase was demonstrated in virtually all cells of the leptomeninges, choroid plexus epithelial cells, and perivascular microglial cells, suggesting that these cells synthesize prostaglandin D(2) actively. Alternatively, oligodendrocytes showed prostaglandin D synthase immunoreactivity without detectable COX-2. The localization of lipocalin-type prostaglandin D synthase in meningeal cells and its colocalization with COX-2 provide evidence for its function as a prostaglandin D(2)-producing enzyme.
No preview · Article · Jan 2001 · The Journal of Comparative Neurology
[Show abstract][Hide abstract] ABSTRACT: Previously, we produced the whole extracellular region of metabotropic glutamate receptor subtype 1 (mGluR1) in a soluble form. The soluble receptor retained a ligand affinity comparable with that of the full-length membrane-bound receptor and formed a disulfide-linked dimer. Here, we have identified a cysteine residue responsible for the intermolecular disulfide bond and determined domain organization of the extracellular region of mGluR1. A mutant, C140A, was a monomer under nonreduced conditions by SDS-polyacrylamide gel electrophoresis; however, C140A was eluted at the position similar to that of mGluR113, the wild type soluble receptor, by size exclusion column chromatography. Furthermore, C140A bound a ligand, [(3)H]quisqualate, with an affinity similar to that obtained by mGluR113. Oocytes injected with RNA for full-length mGluR1 containing C140A mutation showed responses to ligands at magnitudes similar to those with wild type full-length RNA. Thus, elimination of the disulfide linkage did not perturb the dimer formation and ligand signaling, suggesting that cryptic dimer interface(s) possibly exist in mGluR1. Limited proteolysis of the whole extracellular fragment (residue 33-592) revealed two trypsin-sensitive sites, after the residues Arg(139) and Arg(521). A 15-kDa NH(2)-terminal proteolytic fragment (residue 33-139) was associated with the downstream part after the digestion. Arg(521) was located before a cysteine-rich stretch preceding the transmembrane region. A new shorter soluble receptor (residue 33-522) lacking the cysteine-rich region was designed based on the protease-sensitive boundary. The purified receptor protein gave a K(d) value of 58.1 +/- 0.84 nm, which is compatible to a reported value of the full-length receptor. The B(max) value was 7.06 +/- 0. 82 nmol/mg of protein. These results indicated that the ligand-binding specificity of mGluR1 is confined to the NH(2)-terminal 490-amino acid region of the mature protein.
Preview · Article · Oct 2000 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: gamma-Aminobutyric acid (GABA)ergic and glycinergic neurons were examined light- and electron-microscopically in laminae I and II of the medullary dorsal horn (MDH, i.e. spinal trigeminal nucleus caudalis in the rat). The majority of GABA- and glycine (Gly)-immunoreactive (-ir) neurons showed both GABA- and Gly-immunoreactivities (-IRs). Noxious stimulation (subcutaneous injection of formalin into perioral regions) induced Fos-IR in some of GABA- and Gly-ir neurons. GABA- and Gly-ir neuronal profiles were postsynaptic to substance P-ir axon terminals. These results suggest that nociceptive information being carried by primary afferent SP-fibers may be relayed directly to GABAergic and glycinergic neurons in laminae I and II of the MDH.
Full-text · Article · Aug 2000 · Neuroscience Letters
[Show abstract][Hide abstract] ABSTRACT: Connections of layer III pyramidal neurons to corticospinal neurons of layer V and corticothalamic neurons of layer VI in the rat primary motor cortex were examined in brain slices by combining intracellular staining with Golgi-like retrograde labeling of corticofugal neurons. Forty layer III pyramidal neurons stained intracellularly were of the regular-spiking type, showed immunoreactivity for glutaminase, and emitted axon collaterals arborizing locally in layers II/III and/or V. Nine of them were reconstructed for morphologic analysis; 15.2% or 3.8% of varicosities of axon collaterals of the reconstructed neurons were apposed to dendrites of corticospinal or corticothalamic neurons, respectively. By confocal laser scanning and electron microscopy, some of these appositions were revealed to make synapses. These findings suggest that corticospinal neurons receive information from the superficial cortical layers four times more frequently than corticothalamic neurons. The connections were further examined by intracellular recording of excitatory postsynaptic potential (EPSP) that were evoked in layer V and layer VI pyramidal neurons by stimulation of layer II/III. EPSPs evoked in layer V pyramidal neurons showed short and constant onset latencies, suggesting their monosynaptic nature. In contrast, most EPSPs evoked in layer VI pyramidal neurons had long onset latencies, showed double-shock facilitation of onset latency, and were largely suppressed by an N-methyl-D-aspartic acid receptor blocker, suggesting that they were polysynaptic. The results suggest that information from the superficial cortical layers is transferred directly and efficiently to corticospinal neurons in layer V and thereby exerts an important influence on cortical motor output. Corticothalamic neurons are, in contrast, considered relatively independent of, or indirectly related to, information processing of the superficial cortical layers.
Full-text · Article · Aug 2000 · The Journal of Comparative Neurology
[Show abstract][Hide abstract] ABSTRACT: A cDNA encoding a proline (Pro) transporter (ProT) was isolated and characterized from a cDNA library prepared from 14-d-old seedlings of Oryza sativa cv. Akibare. The deduced amino acid sequence of the rice ProT protein (OsProT) had 68.8% homology to the ProT protein 1 from Arabidopsis thaliana and 59.6% homology to that from Lycopersicon esculentum. Northern blot analysis revealed that the gene for OsProT (OsProT) was expressed in all organs examined, comparatively strongly in leaf sheath and stem. Salt treatment did not induce expression of OsProT but strongly induced expression of the gene for delta1-pyrroline-5-carboxylate synthetase (P5CS), a key enzyme in Pro biosynthesis. Southern blot analysis revealed that OsProT has a gene family. OsProT specifically transported L-Pro in a transport assay using Xenopus laevis oocytes.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · May 2000 · Neuroscience Research
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Apr 2000 · The Journal of Comparative Neurology