Noboru Mizuno

Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: cobalt-glucose oxidase method

Since the horseradish peroxidase (HRP) method was first applied to the peripheral nervous system by Kristensson and Olsson 12 and subsequently to the central nervous system by LaVail and LaVai116, it has become one of the most common experimental methods for demonstrating neuronal connectivity in the nervous system (for review see refs. 10 and 15). Previous studies have shown that the light microscopical procedure for demonstrating HRP can also be used for electron microscopical visualization of the enzyme, not only of the enzyme transported retrogradely to neuronal somata and dendrites3,5,8,1a,14,17,19,26, 28, but also of that transported anterogradely to axon terminals2,4,s,9,17,22-za,z6, zS. In an attempt to increase the sensitivity of the HRP method and to improve the reliability of tracing neuronal connections using retrograde and anterograde transport of HRP in combination with electron microscopy, the cobalt method of Adams 1 and the coupled oxidation reaction utilized by Lundquist and Josefsson 18 for the determination of low levels of tissue peroxidase were applied to the histochemical visualization of HRP. The coupled oxidation method, utilizing a continuous supply of hydrogen peroxide formed in the glucose oxidase (GOD) reaction, has been shown to be at least I0 times more sensitive than other colorimetric peroxidase assays 18, and also to be applicable to electron microscopical demonstration of HRP 27. In the present study, the coupled oxidation method, when applied to the histochemical demonstration of HRP in combination with cobalt method 1, was found to give excellent electron microscopical pictures of the electron dense reaction product. The experiments were performed in more than 50 adult cats anesthetized with i.p. Nembutal (35 mg/kg). For observation of the retrograde transport of HRP, 0.1-0.5 /~1 of 2 5 ~ HRP (Toyobo Grade-I-C, RZ: 3.4) was injected, in different experiments, into the masticatory muscles 2z, perineal muscles H, or wall of the urinary bladder 25. For detection of HRP transported anterogradely to axon terminals, HRP was injected in a variety of brain areas such as the cerebral cortical areas 21 and the motor trigeminal nucleus23; a single injection of 0.01~3.1 ~1 of 50 % HRP dissolved in

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 Ammons 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.

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.

Developmentally regulated postsynaptic localization of a metabotropic glutamate receptor in rat rod bipolar cells

The retinal bipolar cell receiving glutamate transmission from photoreceptors mediates a key process in segregating visual signals into ON center and OFF center pathways. This transmission involves a G protein-coupled metabotropic glutamate receptor (mGluR). Immunocytochemical and immunoelectron microscopic studies indicate that restricted localization of a specific mGluR subtype, mGluR6, at the postsynaptic site of the rat rod bipolar cell. This specialization is developmentally regulated: mGluR6 is initially distributed in both the soma and dendrites and is finally concentrated on the postsynaptic site. The mGluR6 localization is reversed when photoreceptors degenerate in the mutant rat with retinal dystrophy. Evidence is thus presented indicating specialized, developmentally regulated receptor distribution in the central nervous system and the crucial role of mGluR6 in photoreceptor-bipolar cell synaptic transmission.

IMMUNOHISTOCHEMICAL LOCALIZATION OF MU -OPIOID RECEPTORS IN THE CENTRAL NERVOUS SYSTEM OF THE RAT

Of the three major types of opioid receptors (μ, δ κ) in the nervous system, μ‐opioid receptor shows the highest affinity for morphine that exerts powerful effects on nociceptive, autonomic, and psychological functions. So far, at least two isoforms of μ‐opioid receptors have been cloned from rat brain. The present study attempted to examine immunohistochemically the distribution of μ‐opioid receptors in the rat central nervous system with two kinds of antibodies to recently cloned μ‐opioid receptors (MOR1 and MOR1B). One antibody recognized a specific site for MOR1, and the other bound to a common site for MOR1 and MOR1B.

Distribution of metabotropic glutamate receptor mGluR3 in the mouse CNS: differential location relative to pre- and postsynaptic sites

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. Strong immunoreactivity for mGluR3 was found in the cerebral cortex, striatum, dentate gyrus of the hippocampus, olfactory tubercle, lateral septal nucleus, lateral and basolateral amygdaloid nuclei, and nucleus of the lateral olfactory tract. Pre-embedding immunoperoxidase and immunogold methods revealed mGluR3 labeling in both presynaptic and postsynaptic elements, and also in glial profiles. Double labeling revealed that the vast majority of mGluR3 in presynaptic elements is not closely associated with glutamate and GABA release sites in the striatum and thalamus, respectively. However, in the spines of the dentate granule cells, the highest receptor density was found in perisynaptic sites (20% of immunogold particles within 60 nm from the edge of postsynaptic membrane specialization) followed by a decreasing receptor density away from the synapses (to approximately 5% of particles per 60 nm). Furthermore, 19% of immunogold particles were located in asymmetrical postsynaptic specialization, indicating an association of mGluR3 to glutamatergic synapses. The present results indicate that the localization of mGluR3 is rather similar to that of group I mGluRs in the postsynaptic elements, suggesting a unique functional role of mGluR3 in glutamatergic neurotransmission in the CNS.

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. J. Comp. Neurol. 393:332–352, 1998.

Cloning and sequence analysis of cDNA for rat corticotropin-releasing factor precursor.

DNA complementary to the rat hypothalamic mRNA coding for the corticotropin‐releasing factor precursor (prepro‐CRF) has been cloned by screening a cDNA library with a human genomic DNA probe. Nucleotide sequence analysis of the cloned cDNA has revealed that rat prepro‐CRF consists of 187 amino acid residues including a putative signal peptide. The CRF and putative signal peptide regions are more highly conserved among rat, human and ovine prepro‐CRF than is the cryptic portion.

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.

Distribution of a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat and mouse: an immunohistochemical study with a monoclonal antibody

The distribution of a metabotropic glutamate receptor mGluR2 in the central nervous system was immunohistochemically examined in the rat and mouse with a monoclonal antibody raised against an N-terminal sequence of rat mGluR2 (amino acid residues 87-134). Neuronal cell bodies with mGluR2-like immunoreactivity (mGluR2-LI) were clearly shown in the horizontal cells of Cajal in the cerebral cortex, neurons in the triangular septal nucleus and medial mammillary nucleus, Golgi cells and the unipolar brush cells in the cerebellar cortex, and Golgi-like and unipolar brush-like cells in the cochlear nucleus. Neuropil was intensely immunostained in the accessory olfactory bulb, bed nucleus of the accessory olfactory tract, neocortex, cingulate cortex, retrosplenial cortex, subicular and entorhinal cortices, stratum lacunosum-moleculare of CA1 and CA3, molecular layer of the dentate gyrus, periamygdaloid cortex, basolateral amygdaloid nucleus, bed nucleus of the anterior commissure, caudate-putamen, accumbens nucleus, thalamic reticular nucleus, anteroventral and paraventricular thalamic nuclei, granular layer of the cerebellar cortex, anterior and ventral tegmental nuclei, granular layer of the cochlear nucleus, and parvicellular part of the lateral reticular nucleus. Many axons in the white matter and fiber bundles were also immunostained. No glial cells with mGluR2-LI were found. No particular species differences were found in the distribution pattern of mGluR2-LI between the rat and mouse. The results indicate that mGluR2 is expressed not only in somato-dendritic domain, but also in axonal domain of excitatory and inhibitory neurons.