Biomedical subjects

S Masuko

Publications and source records attributed to S Masuko.

At least 37 records · Page 2Linked to original sources

PubMed1996

Association of dopaminergic terminals and neurons releasing nitric oxide in the rat striatum: an electron microscopic study using NADPH-diaphorase histochemistry and tyrosine hydroxylase immunohistochemistry.

To examine synaptic input and association of terminals containing dopamine and other transmitters to rat striatal nitric oxide synthase-expressing neurons, an electron microscopic study using tyrosine hydroxylase (TH) immunohistochemistry combined with histochemistry for NADPH-diaphorase (NADPHd) was performed. NADPHd-positive neurons had medium-sized cell bodies containing a highly invaginated nucleus and received relatively sparse synaptic input; 3.6% of boutons apposed to the NADPHd-positive neurons were TH-immunoreactive. Of these TH-immunoreactive boutons, two synaptic contacts showing symmetrical synaptic specializations were found on a cell body and a proximal dendrite of a NADPHd-positive neuron. Other nonsynaptic TH-immunoreactive boutons were occasionally associated with unlabeled terminals adjacent to the NADPHd-positive dendrites and also forming asymmetric synaptic contacts with unlabeled spinous or dendritic profiles. These results suggest that activity of the striatal neurons that release nitric oxide may be regulated by direct synaptic input from dopaminergic neurons and also suggest that the TH-immunoreactive terminals associated with the dendrites of nitric oxide synthase-expressing neurons provide the sites where nitric oxide influences dopamine release from neighboring terminals.

Animals↗

PubMed1996

A technique for culturing brain nuclei from postnatal rats.

The technique for culturing brain nuclei from postnatal rats is described in detail. Key features of this simple method of culturing brain nuclei are: (a) to make brain slices of a particular brain region and to isolate the brain nucleus under direct visualization using a dissecting microscope; (b) to use papain for dissociation; (c) to grow neuron cultures over a glial feeder layer; and (d) to use rat serum (prepared in the laboratory) in the culture medium. We have developed neuronal cultures from several types of brain nuclei (such as the locus coeruleus) and identified the type of neurons immunocytochemically and histochemically. With this culture method, we can obtain high purity cultures of specific types of brain neurons. Our brain nucleus cultures are excellent materials for cellular and molecular physiological studies. Long-term changes of a single neuron belonging to a particular neuron type can be observed. Experiments using the patch clamp technique and intracellular injection of antibodies and antisense oligonucleotides are feasible.

Animals↗

PubMed1996

Distribution and origins of nitric oxide-producing nerve fibers in the dog tongue: correlated NADPH-diaphorase histochemistry and immunohistochemistry for calcitonin gene-related peptide using light and electron microscopy.

The distribution and origins of nitric oxide (NO)-producing nerves in the dog tongue with reference to calcitonin gene-related peptide (CGRP)-containing sensory fibers were investigated using NADPH-diaphorase (NADPH-d) histochemistry and immunohistochemistry for CGRP and NO synthase combined with retrograde axonal tracing and denervation experiments. The ultrastructural relationships between NADPH-d-positive and CGRP-immunoreactive neuronal elements were also examined electron microscopically. NADPH-d-positive and CGRP-immunoreactive varicose fibers were found within the taste buds and surrounding the epithelia of the fungiform papillae, and they disappeared completely after severance of the lingual nerve. Following injection of fast blue into the subepithelial layer of the anterior two thirds of the tongue, retrogradely labeled neurons possessing NO synthase and/or CGRP immunoreactivities were mainly detected in the trigeminal ganglion. Some of the retrogradely labeled trigeminal cells showed the coexistence of NADPH-d reactivity and CGRP immunoreactivity, but in the geniculate ganglion neither NADPH-d reactivity nor NO synthase immunoreactivity was found instead of retrogradely labeled CGRP-immunoreactive neurons. The lingual artery and its branches, including the arteriovenous anastomoses, showed dense distributions of NADPH-d-positive fibers, most of which were unaffected by the denervation experiments. There were many small ganglia in the tongue, and virtually all ganglionic neurons were NADPH-d reactive. CGRP-immuno-reactive varicose fibers were also found around the vascular walls and within the intralingual ganglia. Ultrastructural analysis revealed a close distribution of NADPH-d-positive and CGRP-immunoreactive varicose fibers within the arterial walls, and synaptic contacts between CGRP-immunoreactive terminals and NADPH-d-positive intralingual ganglionic neurons. These results indicated that the taste buds of epithelia of fungiform papillae in the anterior two thirds of the dog tongue receive NADPH-d-positive and CGRP-immunoreactive sensory fibers from the trigeminal ganglion, and that perivascular NADPH-d-positive fibers mainly originate from intrinsic ganglia in the tongue. The ultrastructural findings suggest an intrinsic peripheral nerve-reflex mechanism in the regulation of the lingual vascular function by NO-producing postganglionic parasympathetic neurons and CGRP-containing sensory fibers.

Animals↗

PubMed1996

Specific innervation of the rat thalamus by grafted noradrenergic locus coeruleus neurons.

Growth and distribution of noradrenaline (NA) fibres from the implant into the thalamus of host rats were examined at 5-13 months after the implantation by immunohistochemistry using NA or tyrosine hydroxylase antisera. Cell suspension dissociated from the locus coeruleus (LC) region of 14-day-old rat fetuses was implanted into the center of the unilateral thalamus in adult rats from which the noradrenergic afferents to the thalamus had been eliminated with 6-hydroxydopamine treatment. A dense network of varicose NA-immunoreactive (NA-IR) fibres extended laterally into the posterior thalamic nuclear group and the ventral posterolateral thalamic nucleus from the implant in a pattern similar to that the intrinsic noradrenergic fibres form in the normal thalamus, i.e. laterally rich and medially poor NA fibres. Electron microscopic observations revealed that varicosities of NA-IR fibres formed symmetrical as well as asymmetrical axodendritic synapses and axo-axonic synapses with the host neurons as seen in the normal thalamus. labelled dendrite-like fibres of graft origin penetrated deep into the host brain and received afferents from non-labelled axon terminals. Varicosities of NA-IR fibres in the LC implanted animal formed axo-dendritic synapses at the higher ratio than those in the normal animal did. These results show that implanted fetal noradrenergic neurons innervate target regions of the thalamus specifically as the noradrenergic fibres in the normal thalamus do and maintain the innervation for a long time in the noradrenergically denervated rats.

Animals↗

PubMed1995

Substance P innervation of neurons projecting to the paraventricular hypothalamic nucleus in the rat nucleus tractus solitarius.

After injection of WGA-HRP-colloidal gold in the rat paraventricular nucleus (PVN), retrogradely labeled neurons were found mainly in the medial and commissural subnuclei of the nucleus tractus solitarius (NTS) around 0.5 mm caudal to the obex which is closely related to cardiovascular function. Electron microscopic immunohistochemistry in these areas demonstrated synaptic contacts between retrogradely labeled neurons and substance P-immunoreactive terminals. Innervation of NTS-PVN projection systems by substance P is suggested.

Amino Acid Sequence↗

PubMed1995

Vascularization and innervation of the canine wrist joint synovial membrane.

The correlation between vascular distribution and synovial structures was investigated using corrosion cast of the synovial fold of the dog antebrachiocarpal joint (wrist joint) under scanning electron microscopy. Arterial branches arose from a main artery deep in the subsynovial layer, ascended toward the free margin of the fold, dividing into several branches, and finally formed a dense capillary network beneath the lining layer. Immunohistochemical study and a retrograde axonal tracing experiment revealed that almost all nerve fibers were associated with the arterial tree and that: (1) proximal parts of the arterial tree were innervated with neuropeptide Y (NPY)-containing noradrenergic sympathetic fibers, vasoactive intestinal peptide-containing non-catecholaminergic sympathetic fibers from the stellate ganglion and substance P (SP)- and calcitonin gene-related peptide (CGRP)-containing sensory fibers from the C7-T1 segments of the dorsal root ganglia; (2) more distal parts of the arterial branches were associated with NPY-immunoreactive sympathetic fibers and SP- and CGRP-immunoreactive sensory fibers; and (3) the most peripheral precapillary arterioles were accompanied by only SP- and CGRP-containing sensory fibers. These results indicate that synovial blood flow is regulated by at least three different nerve systems which possess regional differences.

Animals↗

PubMed1993

Synaptic inputs of neuropeptide Y-immunoreactive noradrenergic nerve terminals to neurons in the nucleus preopticus medianus which project to the paraventricular nucleus of the hypothalamus of the rat: a combined immunohistochemical and retrograde tracing method.

The nucleus preopticus medianus (POMe) is known to serve as a relay site in the neural pathway, from the subfornical organ to the paraventricular nucleus of the hypothalamus (PVN), and to play an important role in the regulation of fluid balance and cardiovascular control. A neural connection of noradrenergic nerve terminals in the POMe was examined using electron microscopic immunohistochemistry with the retrograde tract tracing method. Double immunofluorescent labeling revealed nerve terminals immunoreactive to both tyrosine hydroxylase (TH) and neuropeptide Y (NPY) and those immunoreactive to both TH and noradrenaline in the POMe. This indicates that there is an NPY-immunoreactive noradrenergic innervation in the POMe. At the electron microscopic level, nerve terminals immunoreactive to TH or NPY in the POMe formed synapses with dendrites or cell bodies of neurons which were retrogradely labeled after injection of the retrograde tracer, WGA-HRP-colloidal gold, in the PVN. These observations suggest that neurons in the POMe with projections to the PVN may be directly affected by NPY-immunoreactive noradrenergic afferent fibers which presumably originate in the brainstem.

Animals↗

PubMed1993

Membrane properties and dendritic arborization of the intermediolateral nucleus neurons in the guinea-pig thoracic spinal cord in vitro.

The morphological and electrophysiological properties of neurons in the intermediolateral nucleus (IML) were studied in the transverse and longitudinal slice of guinea-pig thoracic spinal cord (T2-T3) using intracellular staining and recording techniques. Two morophologically different types of neurons were observed: fusiform cells with craniocaudally oriented dendrites, and multipolar cells with dendrites diffusely extending in the IML. The ratio of fusiform to multipolar cells was 4:1. The fusiform cells were identified as sympathetic preganglionic neurons (SPNs) by their antidromic responses to stimulation of the ventral root exit zone, while the multipolar cells were not antidromically activated by stimulation of this site. Both cell types showed similar resting membrane potential and input resistance. The tonic responses of these neurons to hyperpolarizing current pulses were characteristically different: the SPNs had a marked hyperpolarizing sag at the break of the pulse, caused by an A current, while the unidentified neurons showed no A current. In addition, the SPNs had much longer duration of spike and afterhyperpolarization, as well as lower frequency of spontaneous or current-evoked firing, than the unidentified neurons. These observations suggest that, in the absence of the criterion of antidromic activation by stimulation of the axon, it is still possible to differentiate SPNs from other IML neurons on the basis of morphological and electrophysiological properties of the neuron.

Animals↗

PubMed1993

An immunohistochemical study of sensory and autonomic innervation of the dog tongue with special reference to substance P- and calcitonin gene-related peptide-containing fibers in blood vessels and the intralingual ganglia.

The distribution, pathways and origins of peptide-containing nerve fibers in the anterior two thirds of the dog tongue were investigated using immunohistochemistry combined with retrograde axonal tracing and denervation experiments. Within the epithelium of the fungiform papillae, varicose nerve fibers immunoreactive to substance P (SP) and calcitonin gene-related peptide (CGRP) were present. These disappeared completely after severance of the lingual nerve (LN) alone. Dense CGRP-immunoreactive varicose fibers surrounded cell bodies in the intralingual ganglia (ILG), which consisted of neurons immunoreactive to vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and SP. These CGRP-immunoreactive fibers disappeared following severance of the chorda tympani (CT) alone. SP-, CGRP-, VIP-, NPY- and tyrosine hydroxylase (TH)-immunoreactive nerve fibers were distributed around the walls of blood vessels, especially arteriovenous anastomoses (AVAs). None of these immunoreactive fibers completely disappeared after severance of the LN or CT alone, but SP- and CGRP-immunoreactive fibers disappeared following severance of both the LN and CT. TH-immunoreactive fibers disappeared after ganglionectomy of the superior cervical ganglion (SCG) or severance of the hypoglossal nerve (HGN). VIP- and NPY-immunoreactive fibers invariably remained after various denervation experiments. In tracing experiments, CGRP-immunoreactive as well as SP and CGRP-immunoreactive cells in the trigeminal ganglion were labelled from the LN, and those in the geniculate ganglion and jugular ganglion were labelled from the CT. A large number of neurons in the SCG were labelled from the HGN, with some of these being SP and CGRP-immunoreactive. These results demonstrate that SP- and CGRP-immunoreactive fibers from the trigeminal ganglion are distributed to the lingual epithelium; vascular walls receive SP- and CGRP-immunoreactive sensory fibers from the LN as well as CT, some SP and CGRP-immunoreactive fibers from the SCG in addition to catecholaminergic sympathetic fibers, and VIP- and NPY-immunoreactive parasympathetic fibers from the ILG. The ILG is also considered to be innervated by CGRP-immunoreactive sensory fibers from the CT.

Animals↗

PubMed1993

Immunoelectron microscopic studies of synaptic organization in the intralaryngeal ganglia of the cat.

The synaptic organization of nerve terminals containing calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), substance P (SP) and enkephalin (ENK) in the intralaryngeal local ganglia of the cat was investigated by immunoelectron microscopy. CGRP-immunoreactive (IR) and VIP-IR varicose fibers formed mainly axo-dendritic synapses, whereas SP-IR and ENK-IR varicose fibers made axo-somatic synapses to the principal neurons of the local ganglion. The synaptic specializations of the CGRP-IR varicosities were asymmetrical, or Gray's type I, whereas the other peptide-IR varicosities showed symmetrical, or Gray's type II, synaptic specializations. After denervation of the extrinsic nerves, CGRP-IR varicose fibers disappeared from the ganglion, but VIP-IR, SP-IR and ENK-IR varicose fibers and synapses remained. These results suggest that local ganglia act as an integration center of laryngeal function rather than as a unidirectional parasympathetic relay center.

Animals↗

PubMed1992

Met-enkephalin-Arg6-Gly7-Leu8- and substance P-containing projections from the nucleus preopticus medianus to the paraventricular hypothalamic nucleus.

The nucleus preopticus medianus (POMe) is known to be important for the regulation of fluid balance and cardiovascular control. Direct projections from the POMe to the paraventricular hypothalamic nucleus (PVN), where vasopressin-containing neurons exist, were examined in the rat using immunohistochemistry combined with a retrograde tract tracing method. After injection of WGA-HRP-colloidal gold into the PVN, many neurons were retrogradely labeled in the POMe; some of them were immunoreactive to Met-enkephalin-Arg6-Gly7-Leu8 (mE8) or substance P (SP). The results indicate that mE8- and SP-immunoreactive neurons in the POMe send their axons to the PVN.

Animals↗

PubMed1992

Neurite regeneration in the cat recurrent laryngeal nerve: an immunohistochemical study.

The recurrent laryngeal nerve (RLN) consists of various motor, sensory and autonomic nerve fibers, although it has not been established whether different neuronal types exhibit a similar ability to regenerate. To address this question, freezing was used to injure the cat RLN fibers and the presence or absence of immunoreactivity for neuropeptides or transmitter-synthesizing enzymes was then examined as a marker to classify the fibers. In the control RLN, calcitonin gene-related peptide-immunoreactive (CGRP-IR) fibers were the highest in number and were distributed throughout the nerve fascicles. The number of substance P-immunoreactive (SP-IR) fibers was about 40% that of CGRP-IR fibers, while a portion of CGRP-IR fibers was found to contain SP immunoreactivity. Relatively low numbers of tyrosine hydroxylase-immunoreactive (TH-IR) and neuropeptide Y (NPY-IR) nerve fibers were seen which tended to form clusters. The distribution pattern of NPY-IR fibers was very similar to that of TH-IR fibers. In the regenerating RLN 1 week after the freezing injury, the fastest growing axons were CGRP-IR, while the regenerating rates of SP-IR, TH-IR and NPY-IR fibers were slower than that of CGRP-IR fibers. These results suggest that the ability for neurite regeneration varies among neuron types and that CGRP-IR fibers possess the most rapid ability to regenerate.

Animals↗

PubMed1992

Dissociated high-purity dopaminergic neuron cultures from the substantia nigra and the ventral tegmental area of the postnatal rat.

We have developed dissociated primary neuronal cultures obtained from the substantia nigra and from the ventral tegmental area of postnatal rats (two to three days old). After making brain slices, the regions of the substantia nigra and the ventral tegmental area were separately dissected. The removed fragments of brain tissue were dissociated and cultured on a glial feeder layer. Double immunocytochemical labeling for tyrosine hydroxylase and GABA on cultures grown for two to three weeks showed the presence of 42% dopaminergic and 39% GABAergic neurons in substantia nigra cultures, whereas in ventral tegmental area cultures there were 65% dopaminergic and 21% GABAergic neurons. The dopaminergic neurons were characterized by thick and straight primary processes dividing into several branches. Varicosities were found mainly on distal parts of the processes. In contrast, GABAergic neurons possessed highly branched thick and thin primary processes with intensive arborization and numerous varicosities. Co-existence of dopamine and cholecystokinin was found in about 70% of dopaminergic neurons from the substantia nigra and in about 35% of dopaminergic neurons from the ventral tegmental area. Physiological properties of these cultured dopaminergic neurons were investigated with the whole-cell version of the patch-clamp method. After each physiological experiment, immunocytochemical labeling confirmed that the cell was dopaminergic. Properties of single action potentials, with an action potential height of 92 mV and duration of 1.6 ms, were similar to those reported for dopaminergic neurons in brain slices. The neurons showed a high resting potential, and no spontaneous firing of action potentials. Constant current depolarizations elicited trains of action potentials. In the majority of cells, the train stopped firing within a few seconds, while in some cells it lasted indefinitely. When the cell was hyperpolarized, the voltage response started to decline slowly (sag), indicating the presence of hyperpolarization-activated currents (time-dependent inward rectification). These results show that by using our culture method it is possible to obtain separate dissociated cultures of the substantia nigra and the ventral tegmental area from newborn rats. Because they are rich in functional dopaminergic neurons, these cultures will be a useful tool for studying various properties of dopaminergic neurons.

Action Potentials↗

PubMed1992

Fine structures around the orifice of the intercostal artery of the rabbit thoracic aorta.

In hypercholesterolemic rabbits, atherosclerotic lesions easily occur in the thoracic aorta, especially at the distal and lateral sides of the walls around the orifices of the dorsal intercostal arteries. In order to examine whether some special structures that lead to atherosclerotic lesions are present even in normal conditions, the authors investigated the morphologic features around the orifice of the intercostal artery of 20 normal rabbit aortae under electron microscopy. The endothelial cells were generally fusiform but tended to be round and have a cobblestone-like appearance at the lateral side. There was intimal protrusion at the distal and lateral sides of the orifice, where the distribution and arrangement of elastic fibers and smooth muscle cells were different from those at the proximal side. At the proximal edge of the orifice, elastic fibers formed a thick plate-like internal elastic lamina beneath the endothelial cells. On the other hand, at the distal and lateral sides, elastic fibers formed close-meshed structures over the proper plate-like internal elastic lamina. These results indicate that the aortic walls at the distal and lateral sides of the orifice are structurally different from those at other regions even in normal conditions and suggest the involvement of special structures at the distal and lateral sides of the orifice in atherogenesis.

Animals↗

PubMed1992

Heterogeneity of rabbit aortic endothelial cells, with special reference to phagocytosis.

Heterogeneity of aortic endothelial cells with regard to phagocytotic ability was examined by injecting India ink into normal rabbits. Light and electron microscopic analyses revealed that particles of India ink were phagocytosed in the endothelial cells, which in turn were localized at the distal side of the orifice of aortic branches, especially those of brachiocephalic, left clavicular, and dorsal intercostal arteries. No remarkable differences were found ultrastructurally between phagocytosing and nonphagocytosing endothelial cells. Ingested India ink particles were present within phagosomes of the endothelial cells for several hours after injection; the particles eventually accumulated in the subendothelial space twenty-four hours after injection. These results indicate that an active transport system of large molecules via the phagocytotic processes is present in endothelial cells located at the distal sides of the orifice of aortic branches. These regions are known to develop initial atherosclerotic lesions in hypercholesterolemic animals. Thus, a possible correlation between phagocytotic ability of endothelial cells and development of atherosclerosis is suggested.

Animals↗

PubMed1992

Immunohistochemical study of intralaryngeal ganglia in the cat.

To study the mechanism of autonomic regulation in the larynx, intralaryngeal local ganglia of the cat were investigated using immunohistochemical techniques. Small intralaryngeal ganglia were found in the peripheral portions of internal branches of the superior laryngeal nerve. Ninety-one percent of the ganglionic neurons were immunoreactive (IR) to vasoactive intestinal polypeptide (VIP), and 10% of the VIP-IR cells were also immunoreactive to enkephalin (ENK) and/or substance P (SP). The immunoreactivity of neuronal cell bodies remained unchanged even after denervation of the bilateral superior and recurrent laryngeal nerves. A dense distribution of calcitonin gene-related peptide (CGRP)-IR nerve fibers was found around almost all neuronal cells in the intralaryngeal ganglia. A few VIP-IR, ENK-IR, and SP-IR nerve fibers were also observed. Only the CGRP-IR fibers disappeared after the denervation experiments. In the laryngeal glands and mucosal arterioles, VIP-IR nerve terminals were found that were also immunoreactive to ENK and/or SP. However, these immunoreactive nerve endings in the glands and arterioles remained after the denervation experiments. The results of our study indicate that laryngeal exocrine secretion and blood flow are regulated by postganglionic autonomic parasympathetic fibers from intralaryngeal ganglia that contain VIP alone or VIP with ENK and/or SP, and that these ganglionic neurons may be innervated by CGRP-IR extrinsic nerve fibers.

Animals↗

PubMed1992

Immunohistochemical study of the sympathetic and sensory innervation to the blood vessels of the dog forepaw.

Immunohistochemical staining of arteries supplying the dog forepaw showed a dense distribution of nerve fibers which were immunoreactive to tyrosine hydroxylase (TH), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), substance P (SP), and calcitonin gene-related peptide (CGRP) around the vascular walls. The density of each immunoreactive fiber tended to increase in the peripheral branch of the vascular tree. Retrograde axonal tracing with Fast Blue from the artery revealed that these immunoreactive fibers originated from NPY-containing catecholaminergic as well as VIP/SP/CGRP-containing non-catecholaminergic neurons in the stellate ganglion and SP/CGRP-containing neurons in the dorsal root ganglia of segments C7 to Th1. After stellate ganglionectomy, TH-, NPY-, and, VIP-immunoreactive fibers disappeared completely from the arterial walls while approximately 40% of SP- and CGRP-immunoreactive fibers remained. The present results indicate that the artery of the dog forepaw receive triple innervation of adrenergic sympathetic, non-adrenergic sympathetic, and sensory fibers, and suggest that about 40% of SP- and CGRP-immunoreactive fibers are of sensory origin.

Animals↗

PubMed1992

Postnatal changes in development of serotonin-, neuropeptide Y-, Leu-enkephalin- and substance P- terminals in the rat locus coeruleus; a quantitative immunohistochemical study.

Postnatal developmental changes were investigated in afferent terminals immunoreactive to serotonin (5-HT), neuropeptide Y (NPY), Leu-Enkephalin (ENK) and substance P (SP) within the locus coeruleus from postnatal rat from day 1 (1 D) to 9 week (9 W) adult using immunohistochemical techniques. Quantitative study using a light microscopic image analyzing system revealed that the number of immunoreactive terminals increased after birth to reach a peak at 5 W, then decreased by 26% of this value and stabilized at 7 W; terminals immunoreactive to 5-HT or NPY increased gradually after birth, while those immunoreactive to ENK or SP increased suddenly at 3 W. Electron microscopic analysis revealed similar changes in the total terminal number during development. Synaptic terminals, on the other hand, increased sharply from 1 D to 3 W, then gradually until 5 W, and remained stable thereafter. These results suggest that surplus afferent terminals are eliminated prior to the establishment of afferent innervation and that 5 W postnatal is the critical time for maturation of the afferent system. Electron microscopy also demonstrated morphological characteristics of terminals immunoreactive to each peptide or 5-HT and developmental changes in their characteristics.

Aging↗