Yuzo Murata

Afferent and efferent connections of the medial preoptic area in the rat: A WGA-HRP study

Afferent and efferent connections of the medial preoptic area including medial preoptic nucleus (MP) and periventricular area at the MP level were examined using WGA-HRP as a marker. Injections were performed by insertion of micropipette containing (1) small amount of HRP powder or (2) dryed HRP solution for 24 to 48 hr until the fixation or for 5 min respectively. Dorsal and ventral approaches of injection micropipettes were performed and the results were compared. Previously reported reciprocal connections with lateral septum, bed nucleus of the stria terminalis, medial amygdaloid nucleus, lateral hypothalamic nucleus, paraventricular hypothalamic nucleus, ventromedial hypothalamic nucleus, arcuate nucleus, supramammillary nucleus, central gray at the mesencephalon, raphe dorsalis, raphe medianus, and lateral parabrachial nucleus have been confirmed. In addition, we found reciprocal connections with septo-hypothalamic nucleus, amygdalo-hipocampal nucleus, subiculum, parafascicular thalamic nucleus, posterior thalamic nucleus at the caudo-ventral subdivision, median preoptic nucleus, lateral preoptic nucleus, anterior hypothalamic nucleus, periventricular area at the caudal hypothalamic level, dorsomedial hypothalamic nucleus, posterior hypothalamic nucleus, dorsal and ventral premammillary nucleus, lateral mammillary nucleus, peripeduncular nucleus, periventricular gray, ventral tegmental area, interpeduncular nucleus, nucleus raphe pontis, nucleus raphe magnus, pedunculo-pontine tegmental nucleus, gigantocellular reticular nucleus and solitary tract nucleus. The areas which had only efferent connections from MP were accumbens, caudate putamen, ventral pallidum, substantia innominata, lateral habenular nucleus, paratenial thalamic nucleus, paraventricular thalamic nucleus, mediodorsal thalamic nucleus, reuniens thalamic nucleus, median eminence, medial mammillary nucleus, subthalamic nucleus, pars compacta of substantia nigra, oculomotor nucleus, red nucleus, laterodorsal tegmental nucleus, reticular tegmental nucleus, cuneiform nucleus, nucleus locus coeruleus, and dorsal motor nucleus of vagus among which substantia innominata and median eminence were previously reported. Efferent connections to the nucleus of Darkschewitsch, interstitial nucleus of Cajal, dorsal tegmental nucleus, ventral tegmental nucleus, vestibular nuclei, nucleus raphe obsculus were very weak or abscent in the ventral approach while they were observed in dorsal approach. Previously reported afferent connections from dorsal tegmental nucleus, cuneiform nucleus, and nucleus locus ceruleus were not detected in this study.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell‐type‐specific excitatory and inhibitory circuits involving primary afferents in the substantia gelatinosa of the rat spinal dorsal horn in vitro

The substantia gelatinosa (SG) of the spinal dorsal horn shows significant morphological heterogeneity and receives primary afferent input predominantly from Aδ‐ and C‐fibres. Despite numerous anatomical and physiological studies, correlation between morphology and functional connectivity, particularly in terms of inhibitory inputs, remains elusive. To compare excitatory and inhibitory synaptic inputs on individual SG neurones with morphology, we performed whole‐cell recordings with Neurobiotin‐filled‐pipettes in horizontal slices from adult rat spinal cord with attached dorsal roots. Based on dendritic arborization patterns, four major cell types were confirmed: islet, central, radial and vertical cells. Dorsal root stimulation revealed that each class was associated with characteristic synaptic inputs. Islet and central cells had monosynaptic excitatory inputs exclusively from C‐afferents. Islet cells received primary‐afferent‐evoked inhibitory inputs only from Aδ‐fibres, while those of central cells were mediated by both Aδ‐ and C‐fibres. In contrast, radial and vertical cells had monosynaptic excitatory inputs from both Aδ‐ and C‐fibres and inhibitory inputs mediated by both fibre types. We further characterized the neurochemical nature of these inhibitory synaptic inputs. The majority of islet, central and vertical cells exhibited GABAergic inhibitory inputs, while almost all radial cells also possessed glycinergic inputs. The present study demonstrates that SG neurones have distinct patterns of excitatory and inhibitory inputs that are related to their morphology. The neurotransmitters responsible for inhibitory inputs to individual SG neurones are also characteristic for different morphological classes. These results make it possible to identify primary afferent circuits associated with particular types of SG neurone.

Convergence of afferents from the SLN and GPN in cat medullary swallowing neurons

We demonstrated the convergence of information from the pharyngeal and laryngeal mucosa, transmitted by the glossopharyngeal nerve (GPN) and superior laryngeal nerve (SLN), in the nucleus of the tractus solitarius (NTS). First, the distribution of terminals of the GPN and SLN in the NTS was examined by an HPR tracing technique in cats, and the synapse formation of these neurons with NTS neurons was demonstrated by electron microscopy. The HRP-labeled SLN and GPN terminals were localized in a small area of the interstitial subnucleus of the NTS, slightly rostral to the obex, forming synapses with NTS neurons. Next, using extracellular recording in anesthetized cats, we determined whether or not swallowing-related neurons in the medulla oblongata receive peripheral inputs. Convergence of peripheral sensory inputs from the SLN and GPN was observed in more than 80% of the NTS cells. These results suggest that the NTS is not only a sensory-relay nucleus but also integrates information necessary for eliciting protective reflexes of the upper airway, such as swallowing.

A correlative quantitative study comparing the nerve fibers in the cervical sympathetic trunk and the locus of the somata from which they originate in the rat

Correlative quantitative analyses were performed on the rat comparing the number of fibers in the cervical sympathetic trunk (CST) and the horseradish peroxidase (HRP)-labeled neurons in the superior cervical sympathetic ganglion (SCG), stellate ganglion, as well as in the spinal cord. The total number of nerve fibers in the left CST was 4180 +/- 169 (mean +/- S.E.M.) among which 92 +/- 3 (mean +/- S.E.M.) were myelinated. The diameter of unmyelinated fibers was 0.68 +/- 0.22 (mean +/- S.D.) microns and showed single-peaked distribution. After the application of HRP to the proximal cut end of the CST, labeled neurons were found in the stellate ganglion as well as in the ipsilateral spinal cord from C7 to T4 segments. The total number of HRP-labeled neurons in the spinal cord was 1334 +/- 45 (mean +/- S.E.M.) with the range between 844 and 1808. Ninety-nine percent of labeled neurons were located in the intermediolateral column and in the lateral funiculus while 1% were in the intercalated region and central autonomic area. Labeled neurons were encountered only sporadically in the dorsal root ganglia (DRG) from C8 to T3 level. After the application of HRP to the distal cut end of the CST, about 200 labeled neurons were observed in the caudal part of the SCG. The present results were discussed with special reference to the organization of the CST of the rat.

Architecture and synaptic relationships in the intermediolateral nucleus of the thoracic spinal cord of the rat: HRP labelling, catecholamine histochemistry and electron microscopic studies

SummaryThe organization of the intermediolateral nucleus (IML) of the thoracic spinal cord was examined using glyoxylic acid-induced fluorescence histochemistry, retrograde horseradish peroxidase (HRP) labelling and electron microscopy. In serial sections of T2, it was found that the distribution of catecholamine nerve terminals was intimately related to the neuronal perikarya of IML. Potassium permanganate fixation and 5-hydroxydopamine treatment revealed small dense-cored vesicles in axon varicosities with or without synaptic specializations. A gelatinous region, composed of small diameter dendrites and unmyelinated axons, formed a narrow longitudinal bundle in the centre of the nucleus. The population of the axon varicosities in the IML was 0.17 ± 0.02/μm2 in 75 nm sections. The average size of the axon varicosities with flat synaptic vesicles was 1.44 ± 0.05 μm2 and that of varicosities with spherical vesicles was 0.97 ± 0.02 μm2. After HRP injection into the superior cervical ganglion, ipsilateral IML neurons were labelled in T1–T3 segments of the spinal cord. Axon varicosities with flat and others with spherical synaptic vesicles synapsed on the dendrites labelled by HRP. Among axon varicosities synapsing on the preganglionic sympathetic neurons, 74.8 ± 7.1% at axo-somatic synapses and 46.0 ± 6.7% at synapses on proximal dendrites contained flat synaptic vesicles.

Formation of synaptic graft-host connections by noradrenergic locus coeruleus neurons transplanted into the adult rat hippocampus

Transplants of cell suspension obtained from the locus coeruleus region of 13- to 14-day-old rat fetuses were implanted into the hippocampal formation of intact adult rats or rats from which the noradrenergic afferents to the hippocampus had been removed by bilateral 6-hydroxydopamine (6-OHDA) injections into the dorsal tegmental noradrenergic bundle. The growth noradrenergic axons into the host hippocampus from the implant was studied at 4-8 months after surgery by immunohistochemistry using antisera raised against tyrosine hydroxylase or noradrenaline. In the animals with an intact noradrenergic system the host noradrenergic afferents were removed by bilateral dorsal bundle lesions 2 weeks before sacrifice. Fine axon-like fibers (diameter about 0.3 micron) and thick dendrite-like fibers (diameter about 1.3 micron), labeled immunohistochemically, were abundant and spread far from the graft. By electron microscopy, immunolabeled axon-like fibers formed mostly symmetrical synaptic contacts with nonlabeled spines and shafts of dendrites in the host. Labeled dendrite-like fibers of presumed graft origin penetrated deep into the host neuropil and received abundant afferents from nonlabeled axon terminals. The extent of graft-derived noradrenergic axons and the synapses established with the host hippocampal neurons were similar in the chronically denervated animals and in the animals where the intrinsic noradrenergic afferents had been left intact until 2 weeks before sacrifice. The results show that implanted embryonic noradrenergic neurons are able to innervate the hippocampus in both the presence and the absence of an intact intrinsic noradrenergic innervation and that the ingrowing axons form abundant synaptic connections with the host hippocampal neurons under both conditions. Dendritic processes from the grafted noradrenergic neurons that extend deep into the host tissue may receive a reciprocal synaptic host afferent input.

High Resolution Electron Microscopy of Crystal and Defect Structures of the High-Tc Superconductor Ba2YCu3O7-δ

Structural study of the superconducting oxide Ba2YCu3O7-δ has been made at room temperature by high resolution electron microscopy. The orthorhombic structure was confirmed by the direct imaging of metal atoms. Planar defects with stacking sequence of metal-oxygen planes in the La2CuO4 type of structure were observed. Depending on the selected arca in electron diffraction, the axial ratio b/a was found to vary in a range from 1.003 to 1.018. Black and white contrasts appearing alternately along the twin boundaries are discussed in relation to the lattice distortion and the oxygen deficiency in the Cu-O layer.

Substance P-like immunoreactive axon varicosities with synaptic junctions in the nucleus tractus solitarius of the rat.

Substance P-like immunoreactive axon varicosities form both Grays type I and type II synaptic junctions to the dendrites and somata in the nucleus of the tractus solitarius of the rat. Peroxidase anti-peroxidase reaction products are observed mainly on the large dense-cored vesicles and in the axoplasm preferentially surrounding small clear vesicles. Labeled axon varicosities are filled with small clear vesicles of 50 nm in diameter.

Developing patterns of nitric oxide synthesizing neurons in the rat striatum: histochemical analysis

The prenatal and postnatal development of NADPH-diaphorase (NADPH-d)/neuronal nitric oxide synthase (nNOS) positive neurons was studied in the striatum of rats. NADPH-d was demonstrated enzyme histochemically and nNOS immunohistochemically using a polyclonal antibody. NADPH-d neurons appeared in the ventrolateral part of the striatum on embryonic day 18 (E18). Thereafter, the number of NADPH-d neurons increased and began to distribute homogeneously in the striatum. The density of NADPH-d neurons became highest at postnatal day 5 (P5) and then decreased as the volume of the striatum continued to increase. The number of NADPH-d neurons reached its peak around 3-4 weeks after birth. The sizes of NADPH-d neurons were measured. The NADPH-d neurons grew larger until P14 (mean area 260 microm(2)) and became smaller thereafter (mean area 170 microm(2)). Patches of high NADPH-d activity and tyrosine hydroxylase (TH) immunoreactivity were also examined in the developing striatum. The distributions of NADPH-d patches overlapped with those of TH-immunoreactive patches by P10. The spatiotemporal appearance of nNOS and overlapping of nNOS patchy distribution with TH point to an important role of NO and to an interaction between nNOS and DA fibers during development of the striatum.

Pharyngeal branch of the vagus nerve carries intraepithelial afferent fibers in the cat pharynx: An elucidation of the origin and central and peripheral distribution of these components ☆ ☆☆

The presence of a sensory component in the pharyngeal branch of the vagus nerve (PhB), including its peripheral distribution and central projection, was studied by denervation and tracer experiments in the cat. The distribution of nerve fibers immunore-active to protein gene product 9.5, a sensitive neuronal marker; calcitonin gene-related peptide; and substance P in the pharyngeal epithelium was analyzed in both intact animals and animals subjected to partial denervation by means of sectioning two of the three nerve trunks, the glossopharyngeal nerve, the superior laryngeal nerve, and the PhB, while leaving one intact. The results of this study show that the glossopharyngeal nerve and superior laryngeal nerve carry nerve fibers to the pharyngeal epithelium rostral and caudal to the middle level of the epiglottis, respectively, whereas the PhB carries nerve fibers to the mesopharyngeal epithelium. Tracer experiments, by applying wheat germ agglutinin-horseradish peroxidase to the PhB, demonstrated retrogradely labeled primary sensory neurons in the jugular ganglion and transganglionic labeling of terminals in the interstitial subnucleus of the nucleus of the tractus solitarius. These results indicate that the PhB contains a sensory component that originates from the jugular ganglion, innervates the mesopharyngeal epithelium, and projects to the nucleus of the tractus solitarius.