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Biomedical subjects

R Nishi

Publications and source records attributed to R Nishi.

At least 55 records · Page 3Linked to original sources
PubMed1992

Tyrosine hydroxylase expression in non-catecholaminergic cells in cerebellar cultures.

Organotypic cerebellar cultures, some with incorporated portions of brainstem, were immunostained after 12-19 days in vitro with three different antibodies to tyrosine hydroxylase. Similar cultures were reacted with glyoxylic acid and examined for catecholamine histofluorescence. Locus coeruleus and other subcortical neurons were positive for tyrosine hydroxylase, as were Purkinje cells and outgrowth zone astrocytes. By contrast, only locus coeruleus neurons and their axons exhibited catecholamine histofluorescence after reaction with glyoxylic acid. These results confirm previously reported in vivo developmental studies indicating that tyrosine hydroxylase can be expressed in the absence of its normal biosynthetic products, and suggest that tyrosine hydroxylase cannot be considered to be a specific marker for catecholaminergic neurons in vitro, as well as during development in vivo.

Animals↗
PubMed1992

Cloning, expression during development, and evidence for release of a trophic factor for ciliary ganglion neurons.

Ciliary ganglion (CG) neurons undergo a period of cell death during development that may be regulated by the limited availability of trophic factor produced by their target tissues. We have previously reported the purification of a ciliary neurotrophic factor from adult chick sciatic nerve that we called growth promoting activity (GPA). Here we demonstrate that GPA can be purified and cloned from embryonic day 15 (E15) chick eyes, which contain all the target tissues of the CG. Our studies show the following: GPA mRNA is induced in embryonic chick eyes during the period of CG neuron cell death; GPA mRNA is expressed specifically in the layer of the eye that contains the targets of the CG and in primary cultures of smooth muscle cells isolated from the choroid layer of the eye; and biologically active GPA is released from cells transfected with a GPA cDNA.

Amino Acid Sequence↗
PubMed1992

Localization of acidic fibroblast growth factor in specific subcortical neuronal populations.

The effects of fibroblast growth factors (FGFs) in vitro include the stimulation of mitogenesis in a variety of non-neuronal cell types and the promotion of the survival of various central and peripheral neuronal populations. The precise physiological role of FGFs in vivo is currently not known. As a step toward understanding the role of FGFs in the nervous system, the present study determined the distribution of acidic FGF (aFGF) in the rat CNS. The levels of aFGF in dissected areas of the nervous system were quantified using a biological assay method, and the cellular distribution of aFGF was determined in tissue sections using immunohistochemical methods. aFGF was found to be localized within specific neuronal populations in the CNS and was absent from non-neuronal cells. Neurons containing aFGF immunoreactivity included magnocellular neurons in the septal area and nucleus basalis; some additional defined neuronal groups in the subcortical telencephalon; specific neuronal populations in the hypothalamus, the thalamus, the substantia nigra, the reticular formation, and the pons; and motor and sensory neurons. Cerebral cortex and hippocampus contained only a very limited number of aFGF-immunoreactive neurons. A significant overlap of neuronal populations known to express the low-affinity NGF receptor (LNGFR) with populations containing aFGF immunoreactivity was also observed. These neuronal populations are known to be affected by neurodegenerative diseases, and the possible functional implications of the presence of aFGF and the LNGFR in these cells are discussed.

Animals↗
PubMed1992

Nuclear and cytoplasmic localization of basic fibroblast growth factor in astrocytes and CA2 hippocampal neurons.

Fibroblast growth factors (FGFs) are known to stimulate mitogenesis in a variety of non-neuronal cell types and to support the survival in vitro of many neuronal cell types. The physiological role of FGFs in the CNS is currently not known. The present study determined the distribution in the rat CNS of a prominent member of the FGF family, basic FGF (bFGF). Immunohistochemical analysis showed that bFGF immunoreactivity was found predominantly in astrocytes throughout all regions of the CNS. In contrast, only a few neuronal populations were found to contain bFGF immunoreactivity, most prominent among them, neurons in the CA2 area of the hippocampus. This predominant localization of bFGF to astrocytes was confirmed by two other observations: (1) highly enriched cultures of astrocytes contained bFGF immunoreactivity and bioactivity, whereas highly enriched cultures of cerebral cortical neurons contained no detectable bFGF, and (2) neonatal rat cerebral cortex, which contains only a few differentiated astrocytes, also contained no detectable bFGF immunoreactivity and only low amounts of bFGF bioactivity. Immunocytochemical analysis also suggested that bFGF immunoreactivity was present in the nucleus as well as the cytoplasm of astrocytes and CA2 neurons. This nuclear localization was confirmed by EM analysis of the intracellular distribution of the immunoperoxidase reaction product. In addition, preparations of both nuclear and soluble fractions of brain extracts contained bFGF immunoreactivity and bioactivity. These data suggest that bFGF might be involved in mediating astrocytic influences on the late postnatal maturation and plasticity in the CNS, and that the nuclear localization of bFGF within astrocytes may play an important role in the differentiation of these cells. In addition, bFGF may play a similar role in a few specific neuronal populations, such as CA2 hippocampal neurons.

Animals↗
PubMed1991

Differential localization and possible functions of aFGF and bFGF in the central and peripheral nervous systems.

We investigated the relative distribution of acidic and basic FGF (aFGF and bFGF) in the nervous system of the rat, using a combination of biological, biochemical, immunochemical, and immunohistochemical methods that can differentiate unambiguously between aFGF and bFGF. We found that different regions of the nervous system contained varying levels of aFGF and bFGF. In the central nervous system, bFGF was present nearly exclusively in astrocytes. Most neurons did not contain detectable amounts of bFGF immunoreactivity, with the notable exception of pyramidal cells in hippocampal area CA2. Interestingly, bFGF immunoreactivity was localized to the nucleus of both CA2 neurons and astrocytes. Astrocytes in vitro were also found to express bFGF, whereas cortical neurons in culture did not contain detectable amounts of bFGF. Transection of the optic nerve led to an approximately twofold increase of bFGF in the distal stump, which is consistent with the observation that bFGF is expressed by astrocytes. Transection of rat and chicken sciatic nerve resulted in a rapid and complete disappearance of aFGF from the distal nerve stump, suggesting that aFGF is present in axons projecting through the sciatic nerve. We observed, in agreement with this notion, that cultured sensory neurons contain reasonably high levels of FGF-like bioactivity. Similar levels of activity were found in developing sciatic nerve, suggesting that neuronal aFGF might be involved in regulating the development of the peripheral nervous system.

Animals↗
PubMed1991

Stimulation of somatostatin expression in developing ciliary ganglion neurons by cells of the choroid layer.

An important component of neuronal development is the matching of neurotransmitter expression with the appropriate target cell. We have examined how peptide transmitter expression is controlled in a simple model system, the avian ciliary ganglion (CG). This parasympathetic ganglion contains 2 distinct types of neurons: choroid neurons, which project to vasculature in the eye's choroid layer and use somatostatin as a co-transmitter with ACh, and ciliary neurons, which innervate the ciliary body and iris and use ACh but no known peptide co-transmitter. We have found that the earliest developmental stage in which neurons with somatostatinlike immunoreactivity (SOM-IR) are consistently found in vivo is stage 30 (embryonic day 6.5), a time shortly after the extension of neurites to targets in the eye's choroid layer. In cell culture, CG neurons expressed SOM-IR in co-culture with choroid cells, but not when cultured with striated muscle myotubes or with ganglion non-neuronal cells. No significant differences in neuronal survival or in ChAT activity were observed under these different co-culture conditions, which suggests that somatostatin expression is independently regulated. The stimulation of somatostatin expression was also specific in that other neuropeptides commonly found in autonomic neurons [neuropeptide Y (NPY), substance P (SP), vasoactive intestinal polypeptide (VIP)] were not induced in the presence of choroid cells. The ability to stimulate SOM-IR was not contact dependent because a macromolecule of greater than or equal to 10 kDa in choroid-conditioned medium (ChCM) was found to stimulate somatostatin expression in a dosage-dependent fashion. The somatostatin-stimulating activity induced SOM-IR in more than 90% of CG neurons, as well as in retrogradely labeled ciliary neurons, which would not normally express SOM-IR. Thus, the expression of somatostatin in cultured CG neurons is regulated by a macromolecule produced by cells in the choroid layer, a target normally innervated in vivo by CG neurons expressing somatostatin.

Actins↗
PubMed1991

Acidic and basic fibroblast growth factors in the nervous system: distribution and differential alteration of levels after injury of central versus peripheral nerve.

Acidic and basic fibroblast growth factors (aFGF and bFGF) are known to stimulate mitogenesis in a variety of non-neuronal cell types. Recent work has also established that FGFs can act as neurotrophic factors that promote the survival and regeneration in vitro of a variety of neurons. The present study investigates the distribution of aFGF and bFGF in vivo by using a mitogenic bioassay on AKR-2B cells coupled with Western-blot analysis to estimate the levels of aFGF and bFGF in different areas of the rat nervous system. Acidic FGF and bFGF from extracts of nervous tissue were found to differ considerably in their relative dependencies upon heparin to potentiate their mitogenic activities: the effect of aFGF was strongly dependent upon heparin, whereas the effect of bFGF was only slightly potentiated by heparin. Heparin was also found to stimulate differentially the mitogenic activity of extracts prepared from different areas of the nervous system, indicating that spinal cord, cortex, pituitary, and optic nerve contained different ratios of aFGF to bFGF, whereas sciatic nerve contained extremely high levels of only aFGF. These results were confirmed in Western-blot experiments, using antibodies specific for either aFGF or bFGF. Transection of nerves had opposing effects in sciatic and optic nerves: aFGF rapidly declined in the sciatic nerve distal to the cut, whereas bFGF increased slightly in the distal portion of the cut optic nerve. This differential effect of injury on FGF levels in central versus peripheral nerves may reflect the differential regenerative potential of these two types of nerves.

Acids↗
PubMed1990

Purification and characterization of a trophic factor for embryonic peripheral neurons: comparison with fibroblast growth factors.

The validation of NGF as a physiologically important neurotrophic factor has led to intense efforts to identify novel polypeptide growth factors for neurons. We report here the details of a greater than 80,000-fold purification of a neurotrophic molecule, referred to as growth-promoting activity (GPA), from chicken sciatic nerves. The final product of the purification migrated as a protein band of 21.5 kd, its apparent pI was approximately 4.8, and the ED50 of the most active preparation was approximately 10 pg/ml. Amino acid sequence of a proteolytic digestion fragment of GPA revealed homology with the recently published sequences for rabbit and rat sciatic nerve CNTF. Thus this molecule may be the chicken form of CNTF. Analysis of the specificity of action of GPA showed that, in addition to E8 ciliary ganglion neurons, the factor was able to support short-term survival of E8 dorsal root ganglion and E12 sympathetic neurons. This range of specificities of biological action was also seen with both acidic and basic FGF in the presence of heparin. The biological activity of GPA differed from that of FGF in that it was not potentiated by heparin and did not stimulate mitogenesis in chick fibroblasts.

Amino Acid Sequence↗
PubMed1988

Conditioned medium alters electrophysiological and transmitter-related properties expressed by rat enteric neurons in cell culture.

We have previously shown that rat enteric neurons display many of their in vivo phenotypes when they are dissociated and grown in long-term cell culture. To assess the degree of plasticity of these phenotypes we have examined the effect of medium conditioned by rat heart cells because this treatment strongly affects transmitter properties in rat sympathetic neurons in culture. Growth of enteric neurons for 3-4 weeks in conditioned medium caused several changes that are similar to previously described effects of conditioned medium on other neuronal cell types in culture. When compared to cultures grown in control medium, cultures grown in conditioned medium: (i) contained three times as many large (greater than 25 micron) neurons; (ii) synthesized and stored 3-4 times as much acetylcholine; (iii) contained 4-5 times as many neurons with detectable 5-hydroxytryptamine immunoreactivity; and (iv) contained 10 times as many neurons that fired repetitively during sustained depolarization. Several other changes, which have not been reported in other systems, were also observed. Conditioned medium cultures: (i) contained many fewer neuronal processes with immunohistochemically detectable vasoactive intestinal polypeptide, substance P, somatostatin, and [Met]enkephalin; (ii) contained 70% fewer neuronal cell bodies with vasoactive intestinal polypeptide-like immunoreactivity; and (iii) contained four times as many neurons that had muscarinic responses to acetylcholine. None of the changes in properties described above uniformly affected all enteric neurons, even after 6 weeks of growth in conditioned medium. We conclude that the heterogeneity of enteric neuron phenotypes is established prior to birth and limits the capacity of certain subsets of neurons to respond to exogenous factors in the environment. Nevertheless, the phenotypes of other subsets of neurons displayed considerable plasticity when exposed to conditioned medium.

Acetylcholine↗
PubMed1987

Neuropeptides mark functionally distinguishable cholinergic enteric neurons.

Subpopulations of physiologically identified cholinergic enteric neurons in cell culture contain somatostatin (SOM)- or vasoactive intestinal peptide (VIP)- like immunoreactivity (LIR). These subpopulations differ in their synaptic effects on other neurons: cholinergic neurons that contain SOM-LIR cause fast nicotinic excitatory postsynaptic potentials (EPSPs) that have significantly larger amplitudes than do EPSPs caused by cholinergic neurons that lack SOM-LIR. Cholinergic neurons containing VIP-LIR cause slow non-cholinergic depolarizations in addition to fast nicotinic EPSPs. These findings are the first correlation between neuropeptide content and functional differences in the synaptic effects of subpopulations of cholinergic enteric neurons.

Action Potentials↗
PubMed1986

Activation of alveolar macrophages in pulmonary sarcoidosis: lack of correlation with serum angiotensin-converting enzyme activity.

To identify the activation of alveolar macrophages as a direct measure of disease activity in pulmonary sarcoidosis and to evaluate the possible relationship with serum angiotensin-converting enzyme (ACE) levels, we studied the morphology and function of alveolar macrophages obtained by bronchoalveolar lavage from patients with active disease (n = 12) and inactive disease (n = 5), and from normal controls (n = 11). At the time of lavage, 6 of 12 patients with active disease showed an elevation of serum ACE levels. When alveolar macrophages were cultured, more cells from active disease adhered to glass, spread out, and were positive for NBT reduction (p less than 0.001). Lysosomal enzyme (beta-galactosidase) activity was higher only in patients with active disease (p less than 0.001). In active disease, there were no significant differences in morphology or function of alveolar macrophages between higher and normal serum ACE levels. Thus, alveolar macrophages were activated in active pulmonary sarcoidosis and no correlation was found with serum ACE levels. These results suggest that serum ACE levels do not accurately reflect the activity of disease in the lung.

Adrenal Cortex Hormones↗
PubMed1985

Neurons dissociated from rat myenteric plexus retain differentiated properties when grown in cell culture. I. Morphological properties and immunocytochemical localization of transmitter candidates.

We have developed procedures for dissociating neurons from the myenteric plexus of the small intestine of newborn rats and for growing those neurons in cell cultures for up to 3 months. Neurons in these cultures retain many of the differentiated properties of myenteric neurons in vivo. This is the first of a series of 3 papers describing those properties. In this paper, we describe the morphology of cultured neurons that we have observed with light and electron microscopy; we also describe the patterns of straining observed when immunocytochemical techniques were used to localize neurotransmitter candidates in the cultured neurons. Intracellular injections of a fluorescent dye, Lucifer yellow, revealed that many of the cultured neurons had morphologies similar to those of myenteric neurons in vivo. When thin sections of cultures were viewed in an electron microscope, many neurons were observed to have numerous small (40-60 nm), clear synaptic vesicles and/or large (80-150 nm), opaque-cored (p-type) vesicles. Synaptic profiles were most often observed on neuronal somata. Neurons containing immunoreactive serotonin, substance P, somatostatin, enkephalin, bombesin and gastrin/cholecystokinin were observed in about the same proportions as they occur in the intact myenteric plexus. Neurons containing immunoreactive vasoactive intestinal polypeptide were found in higher numbers than reported in vivo. Neurons containing immunoreactive neurotensin, secretin and glutamate decarboxylase were not observed. An antiserum directed against choline acetyltransferase stained 40-50% of the neurons. We conclude that myenteric neurons continue to express much of their normal differentiated properties even when they are removed from the gut, dissociated into a suspension of single cells and grown in culture. Such cultures will be useful for correlating the morphological, biophysical, pharmacological and synaptic properties of individual myenteric neurons and for testing the ability of altered environmental conditions to change those properties.

Animals↗
PubMed1985

Neurons dissociated from rat myenteric plexus retain differentiated properties when grown in cell culture. II. Electrophysiological properties and responses to neurotransmitter candidates.

We have used intracellular recordings to study the electrophysiological and pharmacological properties of neurons that have been grown in cell cultures after having been dissociated from the myenteric plexus of the small intestine of newborn rats. Studies of action potential mechanisms revealed that all of the neurons could generate Na+-dependent action potentials in the presence of Ca2+-channel blockers and that about 70% could generate Ca2+-dependent action potentials when Na+ channels were blocked with tetrodotoxin. No neurons generated long afterhyperpolarizations after single action potentials but about 50% of neurons did so following trains of action potentials. Over 95% of the neurons tested accommodated rapidly to sustained depolarization. The effects of several enteric neurotransmitter candidates were studied by superfusing or pressure-ejecting test solutions while recording neuronal responses. All of the cultured neurons tested had nicotinic responses to acetylcholine. Subsets of neurons responded to muscarinic cholinergic agonists (slow depolarization and increased excitability), serotonin (fast depolarization or slow depolarization and increased excitability), gamma-aminobutyrate (fast depolarization), substance P (slow depolarization, biphasic fast and slow depolarization or increased excitability without a change in membrane potential), vasoactive intestinal peptide (slow depolarization and increased excitability), or [Met]enkephalin (slow hyperpolarization and/or decreased action potential duration). We conclude that myenteric neurons grown in cell culture retain many of the physiological and pharmacological properties that they have in situ. Such cultures will permit detailed biophysical and pharmacological studies of the mechanisms of action of enteric neurotransmitter candidates.

Acetylcholine↗
PubMed1985

Neurons dissociated from rat myenteric plexus retain differentiated properties when grown in cell culture. III. Synaptic interactions and modulatory effects of neurotransmitter candidates.

We have used intracellular recordings to study synaptic interactions between myenteric neurons grown in dissociated cell culture. Intracellular stimulation of individual myenteric neurons caused several types of synaptic effects in nearby neurons: fast excitatory synaptic potentials mediated by nicotinic acetylcholine receptors; slow, non-cholinergic synaptic potentials; dual transmission having both fast cholinergic and slow non-cholinergic components and inhibition of spontaneously occurring fast nicotinic synaptic potentials. Fast nicotinic synaptic potentials were elicited by about 40% of neurons tested and often occurred spontaneously. The fast synaptic potentials were similar to those that have been studied in other autonomic neurons with respect to their estimated reversal potential and their sensitivity to cholinergic antagonists. The amplitudes of the fast synaptic potentials declined if evoked at frequencies greater than 0.5 Hz. Potentiation of the fast synaptic potentials was observed following high-frequency stimulation of presynaptic neurons. Several transmitter candidates modulated fast cholinergic transmission. Substance P and vasoactive intestinal peptide promoted nicotinic transmission by causing increased amplitudes of evoked and spontaneous fast synaptic potentials and an increased frequency of spontaneous synaptic potentials. gamma-Aminobutyrate and [Met]enkephalin both caused decreased amplitudes and frequency of nicotinic synaptic potentials. Serotonin depressed synaptic potentials in some neurons while enhancing them or having no effect in others. Slow, non-cholinergic, synaptic potentials were elicited by about 10% of neurons tested. These synaptic effects lasted 15-300s, caused depolarizations of 3-15 mv and were accompanied by increased neuronal input resistance. The transmitter(s) causing these slow synaptic potentials has not yet been identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Surface morphology and function of human pulmonary alveolar macrophages from smokers and non-smokers.

Pulmonary alveolar macrophages were obtained by saline lavage from 23 healthy male volunteers--10 non-smokers and 13 cigarette smokers. Lavage produced three times as many alveolar macrophages in smokers than in non-smokers. When macrophages from smokers and from non-smokers were incubated in vitro, more cells from smokers adhered to glass, spread out, and showed enhanced nitroblue tetrazolium (NBT) reduction. The surface morphology of alveolar macrophages from smokers showed more with a plate like appearance and ridge like membrane surface, while the macrophages from non-smokers were predominantly spherical with ruffles. The proportions of cells which stained highly for beta galactosidase were 55% in smokers and 11% in non-smokers. Thus, in a resting state in vitro, alveolar macrophages from smokers were more active than those from non-smokers. When, however, macrophages from smokers and non-smokers were incubated with immunobeads and with opsonised or non-opsonised BCG, the phagocytic activity and stimulated NBT reduction of alveolar macrophages from smokers were similar to or somewhat less than those of non-smokers.

Adult↗