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At least 19 recordsLinked to original sources
PubMed1976

Preparation of pure neuronal and non-neuronal cultures from embryonic chick sympathetic ganglia: a new method based on both differential cell adhesiveness and the formation of homotypic neuronal aggregates.

A new method has been developed for the preparation of essentially pure primary cultures of neurons and non-neuronal cells from 11-day embryonic chick sympathetic ganglia. This method utilizes (1) differences in cell-to-substrate adhesiveness between neurons and non-neuronal cells and (2) the capacity of neurons to form homotypic aggragates. The maximum difference in adhesiveness between neuronal and non-neuronal cells occurred when the ganglia were dissociated with trypsin following collection in a salt solution lacking divalent cations. This difference allowed the preparation of highly purified non-neuronal cultures and 85-90% pure neuronal cultures. Intermittent agitation during the period of cell separation markedly increased the purity of the neuronal cultures by (1) inhibiting neuronal but not non-neuronal cell attachment and (2) facilitating the formation of homotypic neuronal aggregates in the supernatant. Neuronal and non-neuronal cultures prepared under these conditions were more than 99% pure on the basis of both morphological and biochemical analyses. Both cell types exhibited attachment efficiencies greater than 95% and have been maintained for several weeks in vitro. Thus, completely isolated neuronal and non-neuronal cultures can be prepared and maintained for prolonged periods in the absence of cells of the other type.

Animals↗
PubMed1992

Quantitative analysis of neurons and glial cells in the rat somatosensory cortex, with special reference to GABAergic neurons and parvalbumin-containing neurons.

The number of neuronal and glial cells in the rat somatosensory cortex (barrel area) has been estimated by a stereological method, the disector, using pairs of toluidine blue-stained, plastic-embedded 0.5-microns-thick sections, 1.5 microns distant from each other. Chemical properties of those disector-counted cells were further analyzed by postembedding immunocytochemical methods on adjacent semithin sections. Thus we were able to analyze quantitatively number, distribution, and proportion of five cell types: (1) gamma-aminobutyric acid-(GABA)-negative neurons; (2) GABA-like immunoreactive (GABA-LIR) neurons; (3) a specific calcium-binding protein parvalbumin-immunoreactive (PV-IR) neurons, a subpopulation of GABA-LIR neurons; (4) S-100 beta-LIR glial cells (astrocytes); and (5) S-100 beta-negative glial cells (oligodendrocytes and microglia). The densities of total cells, glial cells, and neurons in the rat somatosensory cortex were 85.4 +/- 10(3)/mm3, 30.5 x 10(3)/mm3, and 54.9 x 10(3)/mm3, respectively. Of all neurons 25% and 14% were GABA-LIR and PV-IR, respectively; all PV-IR neurons are GABA-LIR, and thus about 54% of GABA-LIR neurons are PV-positive. The number of total cells under a unit surface area of 1 mm2 through the thickness of the somatosensory cortex was 171.6 x 10(3); the number of neurons and glial cells were 110.2 x 10(3) and 61.4 x 10(3), respectively. There were 27.7 x 10(3) GABA-LIR neurons and 15.0 x 10(3) and 12.7 x 10(3) PV-IR neurons and PV-negative GABA-LIR neurons, respectively. The laminar distribution of each group of cells shows prominent differences, indicating that the cellular composition was different from layer to layer. The density of GABA-LIR neurons was highest in layer IV. The numerical density of PV-IR neurons was 2-4 times higher in layer IV than in layers II/III, V, and VI, whereas that of PV-negative GABA-LIR neurons was almost constant throughout the layers.

Animals↗
PubMed1992

Organization of the serotonergic innervation of spinal neurons in rats--I. Neuropeptide coexistence in varicosities innervating some spinothalamic tract neurons but not in those innervating postsynaptic dorsal column neurons.

Previous studies have suggested that peptides such as substance P and thyrotropin-releasing hormone coexist with serotonin in the same varicosities in the ventral horn and intermediate gray of the spinal cord in rat. However, coexistence of these peptides with serotonin is rare in fibers in the superficial dorsal horn. Since it has been proposed that serotonergic fibers in the superficial dorsal horn act to modulate nociception, it was hypothesized that the serotonergic neurons that contain neither substance P nor thyrotropin-releasing hormone might constitute a specifically antinociceptive subset of serotonergic neurons. This being the case, it would be expected that different types of serotonergic neurons innervate nociceptive and non-nociceptive spinal neurons. In order to test this hypothesis, a group of cells that include nociceptive neurons (spinothalamic tract neurons) and a group of predominantly non-nociceptive neurons (postsynaptic dorsal column neurons) in the spinal cord of rat were retrogradely labeled. Sections of the spinal cord containing retrogradely labeled spinothalamic tract or postsynaptic dorsal column neurons were stained for serotonin and either substance P or thyrotropin-releasing hormone using two-color immunohistochemistry. A retrogradely labeled cell was classified as "apposed" if there was no discernible distance between an immunohistochemically labeled varicosity and the cell. Eighty per cent of spinothalamic tract and 83% of postsynaptic dorsal column profiles were apposed by serotonin-immunoreactive varicosities in the spinal cord. Thirty-one per cent of the spinothalamic tract profiles that were apposed by serotonergic varicosities were apposed by serotonergic varicosities that were also stained for thyrotropin-releasing hormone. The distribution of the latter spinothalamic neurons was similar to that reported for spinothalamic tract neurons responsive to joint movement. In addition, at least 63% of the spinothalamic tract profiles which were apposed by serotonergic varicosities were apposed by "serotonin-only" varicosities, including most spinothalamic tract neurons in the marginal zone, suggesting that at least some "serotonin-only" neurons are antinociceptive. However, contrary to the hypothesis, at least 94% of the postsynaptic dorsal column profiles apposed by serotonergic varicosities were apposed by "serotonin-only" varicosities. These findings suggest that there may be a relationship between the sensory modality to which a spinal neuron responds and the type of serotonergic innervation it receives. However, it appears that "serotonin-only" neurons may not constitute a specifically antinociceptive category of serotonergic neurons.

Animals↗
PubMed1990

Neuron-glia interactions of rat hippocampal cells in vitro: glial-guided neuronal migration and neuronal regulation of glial differentiation.

To examine neuron-glia interactions of hippocampal cells, including glial-guided neuronal migration, glial organization of neuronal positioning and neuronal regulation of astroglial differentiation, rat hippocampal tissue, harvested between embryonic day 16 (E16) and postnatal day 3 (P3), was dissociated into a single cell suspension and plated in glass coverslip microcultures (Hatten and Liem, 1981; Hatten et al., 1984). Immunostaining the cells with antibodies against the glial filament protein (AbGFP) revealed developmental stage-specific changes in the number and extent of morphological differentiation of hippocampal astroglial cells. At E16-E18, fewer than 5% of the cells were AbGFP-positive; stained cells were immature, bearing very short processes. By E19-E20, the number of stained cells increased to 15% of the total cell population. Three forms of differentiated glial cells predominated, a bipolar form bearing processes 30-50 microns, an elongated form which resembled the radial glia of hippocampus, bearing processes 120 microns in length, and a stellate form with 3 or more processes 30-50 microns in length. At P0-P3, glial morphological differentiation varied with the culture substratum; differentiated forms resembling those seen at E20 occurred on Matrigel, but not on polylysine. Quantitation of the distribution of neurons relative to AbGFP-stained glial processes revealed developmental stage-specific changes in glial organization of neuronal positioning in the cultures. In cultures of E16-E18 hippocampal cells, the neurons did not preferentially associate with astroglial cells. By E19-E20, extensive neuron-glia interactions occurred, with 80-90% of the neurons being located within 5-10 microns of a glial process. In addition to their organization of neuronal positioning, E20 hippocampal astroglial cells supported extensive neuronal migration. Migrating hippocampal neurons displayed a cytology and neuron-glia cell apposition identical to that described for migrating cerebellar granule cells in vitro (Edmondson and Hatten, 1987), closely apposing their cell soma against the hippocampal glial process and moving along the glial arm by extending a thickened, leading process. Migration was seen only along highly elongated glial profiles resembling radial glial seen in vivo. The morphological differentiation of hippocampal glial cells in vitro was dependent on cell-cell interactions with neurons. In the absence of neurons, purified hippocampal astroglia had flat, undifferentiated profiles and proliferated rapidly. The addition of hippocampal neurons rapidly arrested glial growth and induced glial process extension.

Animals↗
PubMed1992

Aminergic neurons in the brain of blowflies and Drosophila: dopamine- and tyrosine hydroxylase-immunoreactive neurons and their relationship with putative histaminergic neurons.

The distribution and morphology of neurons reacting with antisera against dopamine (DA), tyrosine hydroxylase (TH) and histamine (HA) were analyzed in the blowflies Calliphora erythrocephala and Phormia terraenovae. TH-immunoreactive (THIR) and HA-immunoreactive (HAIR) neurons were also mapped in the fruitfly Drosophila melanogaster. The antisera against DA and TH specifically labeled the same neurons in the blowflies. About 300 neurons displayed DA immunoreactivity (DAIR) and THIR in the brain and subesophageal ganglion of the blowflies. Most of these neurons were located in bilateral clusters; some were distributed as bilateral pairs, and two ventral unpaired median (VUM) neurons were seen in the subesophageal ganglion. Immunoreactive processes were found in all compartments of the mushroom bodies except the calyces, in all divisions of the central body complex, in the medulla, lobula and lobula plate of the optic lobe, and in non-glomerular neuropil of protocerebrum, tritocerebrum and the subesophageal ganglion. No DA or TH immunoreactivity was seen in the antennal lobes. In Drosophila, neurons homologous to the blowfly neurons were detected with the TH antiserum. In Phormia and Drosophila, 18 HA-immunoreactive neurons were located in the protocerebrum and 2 in the subesophageal ganglion. The HAIR neurons arborized extensively, but except for processes in the lobula, all HAIR processes were seen in non-glomerular neuropil. The deuto- and tritocerebrum was devoid of HAIR processes. Double labeling experiments demonstrated that TH and HA immunoreactivity was not colocalized in any neuron. In some regions there was, however, substantial superposition between the two systems. The morphology of the extensively arborizing aminergic neurons described suggests that they have modulatory functions in the brain and subesophageal ganglion.

Animals↗
PubMedDate not supplied

Differential inhibition of neurone-neurone, neurone-astrocyte and astrocyte-astrocyte adhesion by L1, L2 and N-CAM antibodies.

The cell adhesion molecules L1, N-CAM and Ng-CAM have been implicated in cell-cell interactions among developing neural cells. L1 and N-CAM are structurally and functionally distinct molecular entities and act synergistically in mediating Ca2+-independent adhesion between re-aggregating early postnatal cerebellar cells. N-CAM has been reported to be neurone-specific in the chicken and to mediate fasciculation of neurites and of nerve-muscle interactions. L1, which in the central nervous system has been found only on post-mitotic neurones, mediates migration of granule cell neurones in the mouse cerebellar cortex. In view of the molecules' distinct effects on cell interactions, we wondered whether different neural cell types are involved in the actions of each molecule. Here we report that L1 antigen promotes neurone-neurone adhesion. N-CAM, which is expressed on both neurones and glia, mediates neurone-neurone, neurone-astrocyte and astrocyte-astrocyte adhesion. The L2 carbohydrate epitope shared between the two adhesion molecules seems to be involved in neurone-astrocyte and astrocyte-astrocyte adhesion and acts in a more than additive manner in N-CAM-mediated neurone-neurone adhesion.

Animals↗
PubMed1990

Distinct and different effects of the oncogenes v-myc and v-src on avian sympathetic neurons: retroviral transfer of v-myc stimulates neuronal proliferation whereas v-src transfer enhances neuronal differentiation.

Immature avian sympathetic neurons are able to proliferate in culture for a limited number of divisions albeit expressing several neuron-specific properties. The effect of avian retroviral transfer of oncogenes on proliferation and differentiation of sympathetic neurons was investigated. Primary cultures of 6-d-old quail sympathetic ganglia, consisting of 90% neuronal cells, were infected by Myelocytomatosis virus (MC29), which contains the oncogene v-myc, and by the v-src-containing Rous sarcoma virus (RSV). RSV infection, in contrast to findings in other cellular systems, resulted in a reduction of neuronal proliferation as determined by 3H-thymidine incorporation (50% of control 4 d after infection) and in increased morphological differentiation. This is reflected by increased neurite production, cell size, and expression of neurofilament protein. In addition, RSV-infected neurons, unlike uninfected cells, are able to survive in culture for time periods up to 14 d in the absence of added neurotrophic factors. In contrast, retroviral transfer of v-myc stimulated the proliferation of immature sympathetic neurons preserving many properties of uninfected cells. The neuron-specific cell surface antigen Q211 and the adrenergic marker enzyme tyrosine hydroxylase were maintained in MC29-infected cells and in the presence of chick embryo extract the cells could be propagated over several weeks and five passages. Within 7 d after infection, the number of Q211-positive neurons increased approximately 100-fold. These data demonstrate distinct and different effects of v-src and v-myc-containing retroviruses on proliferation and differentiation of sympathetic neurons: v-src transfer results in increased differentiation, whereas v-myc transfer maintains an immature status reflected by proliferation, immature morphology, and complex growth requirements. The possibility of expanding immature neuronal populations by transfer of v-myc will be of considerable importance for the molecular analysis of neuronal proliferation and differentiation.

Animals↗
PubMed1988

Neuronal maintenance and neurite extension of adult mouse neurones in non-neuronal cell-reduced cultures is dependent on substratum coating.

Adult mouse DRG neurones have been maintained for 14 days in cultures where non-neuronal cell proliferation was inhibited by the inclusion of 5 x 10(-6) microM-cytosine arabinoside (AraC) in the medium from the onset of culture. On uncoated plastic neurone numbers significantly declined in the absence of non-neuronal cell outgrowth compared with uninhibited co-cultures. However, when neurones were maintained in the presence of AraC on certain coated surfaces this decrease in neurone numbers was not observed. Combinations of fibronectin (FN) and laminin (LAM) proved most effective for 7 and 14 days in vitro, although either was beneficial if used separately. Microexudates produced by the fibroblast line, 3T6, also significantly improved neuronal counts for 14 days in vitro. However, a microexudate derived from primary cultures of mouse hepatocytes, although advantageous for 7 days in vitro, was not effective in maintaining neurones over the 14-day culture period, reminiscent of previous observations when synthetic cationic agents were used. Electrophoretic analysis of the fibroblast exudate indicated that fibronectin was present in the substrate-attached material generated by this cell line. The reduction in non-neuronal cell growth facilitated the monitoring of neuronal structural detail by scanning electron microscopy. Examination of neurite extension, indicative of neurone differentiation, was particularly improved. FN/LAM and the fibroblast-derived exudate increased nerve fibre growth, whilst the hepatocyte exudate had little effect on neurite regeneration, and polylysine had a detrimental effect. The data demonstrate that substrata can have a significant effect on maintenance and differentiation of adult neurones in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Structure, expression, and function of Ng-CAM, a member of the immunoglobulin superfamily involved in neuron-neuron and neuron-glia adhesion.

The neuron-glia cell adhesion molecule (Ng-CAM) mediates neuron-neuron adhesion by a homophilic mechanism and neuron-astrocyte adhesion by a heterophilic mechanism. The protein is expressed on neurons and Schwann cells but not on astrocytes. It is most prevalent during development on cell bodies of migrating neurons and on axons during formation of nerves. Ng-CAM expression is greatly increased following nerve injury. Anti-Ng-CAM antibodies inhibited migration of granule cells along Bergmann glia in cerebellar explants and fasciculation of neurites in outgrowths from explants of dorsal root ganglia. The combined results indicate that Ng-CAM on neurons binds to Ng-CAM on adjacent neurons and to as yet unidentified ligands on astrocytes. Ng-CAM is synthesized in chicken neurons from a 6 kb mRNA as Mr approximately 200,000 forms which are cleaved to yield two components of Mr 135,000 and 80,000. It is glycosylated and can be phosphorylated. Amino acid sequence analysis indicates that it contains six immunoglobulin domains, five fibronectin type III repeats, a transmembrane domain and a cytoplasmic region. Structural analyses indicate that Ng-CAM is most closely related to the mammalian glycoprotein L1 but significant differences between them strongly suggest that they are not equivalent molecules. The recent identification of another structurally related molecule in the chicken called Nr-CAM underscores the notion that these molecules are members of a subfamily of neural cell adhesion molecules within the immunoglobulin superfamily that have related or complementary functions in the nervous system.

Animals↗
PubMed1986

Influence of the extracellular potassium environment on neurite growth in sensory neurons, spinal cord neurons and sympathetic neurons.

The influence of the extracellular potassium concentration ([K+]o) on neurite growth in rat sensory neurons, spinal cord neurons and sympathetic neurons was investigated. Experiments carried out in 3-compartment culture dishes showed that although neurites from sensory and spinal cord neurons were capable of growing in both 5 mM [K+]o and 20 mM [K+]o, they were virtually unable to grow from a region of 5 mM [K+]o into a region of 20 mM [K+]o. Neurites from sympathetic neurons behaved similarly although [K+]o exceeding 20 mM was required to exclude sympathetic neurites. We suggest the possibility of a negative chemotaxis to [K+]o by growth cones in these neurons. Neurite regeneration following axotomy in sensory neurons was partially inhibited distal to a proximo-distal increase in [K+]o. The nature of this inhibition was somewhat different from that described previously in sympathetic neurons. The possibility is raised that [K+]o plays a role in the development of the nervous system.

Animals↗
PubMed1986

Specific asparagine-linked oligosaccharides are not required for certain neuron-neuron and neuron-Schwann cell interactions.

To determine whether specific asparagine-linked (N-linked) oligosaccharides present in cell surface glycoproteins are required for cell-cell interactions within the peripheral nervous system, we have used castanospermine to inhibit maturation of N-linked sugars in cell cultures of neurons or neurons plus Schwann cells. Maximally 10-15% of the N-linked oligosaccharides on neuronal proteins have normal structure when cells are cultured in the presence of 250 micrograms/ml castanospermine; the remaining oligosaccharides are present as immature carbohydrate chains not normally found in these glycoproteins. Although cultures were treated for 2 wk with castanospermine, cells always remained viable and appeared healthy. We have analyzed several biological responses of embryonic dorsal root ganglion neurons, with or without added purified populations of Schwann cells, in the presence of castanospermine. We have observed that a normal complement of mature, N-linked sugars are not required for neurite outgrowth, neuron-Schwann cell adhesion, neuron-induced Schwann cell proliferation, or ensheathment of neurites by Schwann cells. Treatment of neuronal cultures with castanospermine increases the propensity of neurites to fasciculate. Extracellular matrix deposition by Schwann cells and myelination of neurons by Schwann cells are greatly diminished in the presence of castanospermine as assayed by electron microscopy and immunocytochemistry, suggesting that specific N-linked oligosaccharides are required for the expression of these cellular functions.

Alkaloids↗
PubMed1985

Factors involved in expression of neuron-specific and non-neuronal enolase activity in developing chick brain and in primary cultures of chick neurons.

The effect of various factors affecting non-neuronal enolase (NNE) and neuron-specific enolase (NSE) was investigated in developing brain of two different chick strains, in primary cultures of pure neurons and of mixed cultures of neuronal and glial cells. NNE and NSE activities reached their maximum at an earlier stage of brain development in the fast growing Hybro strain than in the Leghorn strain. In pure neurons cultured during 6 days, NNE was stimulated by hydrocortisone in presence or in absence of serum. Dibutyryl cyclic AMP (diBcAMP) stimulated NNE only in serum-free medium. NSE activity was increased by glial cell-conditioned medium in presence of serum and by removal of serum from the medium. Hydrocortisone and diBcAMP had no effect on NSE. In mixed cultures of neurons and glial cells both enolase activities were raised in absence of serum. Hydrocortisone and diBcAMP had no effect. Steroid hormones, insulin and serum albumin also modify both enolase activities in pure neurons and in mixed cultures of neurons and glial cells. Our results suggest that NNE and NSE are regulated separately by various factors involved in nerve cell maturation.

Animals↗
PubMed1988

Reciprocal age-dependent pattern of two neuronal markers, tetanus toxin and neuron-specific enolase, in postnatal rat sensory and sympathetic neurons.

Tetanus toxin (TT) and antibodies to neuron-specific enolase (NSE) were used to study the developmentally regulated expression of two neuronal markers in isolated and short-term cultured rat sensory and sympathetic neurons at different postnatal ages. The results show a reciprocal pattern of TT binding and NSE-immunoreactive neurons during postnatal maturation: there is a gradual transition from TT-positive, NSE-negative to TT-negative, NSE-positive phenotypic expressions. We conclude that TT may serve as a neuronal marker for newborn, anti-NSE antibodies as markers for adult neurons, and a combination of both for adolescent neurons.

Adrenergic Fibers↗
PubMed1989

Differentiation of neuron-like cells in cultured rat optic nerves: a neuron or common neuron-glia progenitor?

The optic nerve consists of axons, glia, and undifferentiated cells; neuronal cell bodies are absent. To study the developmental potential of glia and precursor cells in vitro, we devised an original, long-term culture system of optic nerve explants, called minisegments, of newborn rats; at this stage the nerves are composed of naked axons, astrocytes, and undifferentiated cells. After about 4-5 weeks in culture, neuron-like cells appeared, which showed morphological, fine structural, and immunocytochemical properties ascribed to neurons. These neuron-like cells may be derived from a common neuron-glia progenitor or from a small population of precursors, which never produce glia in situ.

Animals↗
PubMed1979

Projections of intestinal neurons showing immunoreactivity for vasoactive intestinal polypeptide are consistent with these neurons being the enteric inhibitory neurons.

Experiments were performed to determine if the distribution of vasoactive intestinal peptide(VIP)-like immunoreactivity in nerve cell bodies and axons of the myenteric plexus and circular muscle of the small intestine is consistent with VIP being the transmitter of enteric inhibitory neurons. Immunoreactivity for VIP was found in nerve cell bodies of the myenteric plexus and in axons within the myenteric plexus and circular muscle. When the axons in the myenteric plexus were interrupted, there was accumulation of material showing reactivity for VIP on the oral side, indicating that the neurons project in an anal direction. The VIP-like immunoreactivity in axons which supply the circular muscle disappeared after a myectomy in which the overlying myenteric plexus was removed, but remained intact when extrinsic nerves were served. The projections of VIP neurons from the myenteric plexus to the circular muscle correspond to the expected projections of enteric inhibitory neurons determined by functional studies.

Animals↗
PubMed1991

Inhibition of caudal medullary expiratory neurones by retrofacial inspiratory neurones in the cat.

1. Comparisons between the spike discharge of inspiratory neurons within the retrofacial area (RFN), and the membrane potential of expiratory neurones within the caudal medulla were made in pentobarbitone-anaesthetized, vagotomized, artificially ventilated cats. Spike-triggered averaging (STA) of synaptic potentials, triggered by the discharge of inspiratory RFN neurones, was utilized to test for synaptic connectivity. 2. Eighty-nine neurons with respiratory-phased discharge patterns were recorded in the vicinity of the RFN. Fifty-four neurones discharged at or slightly before the onset of the inspiratory burst activity of the phrenic nerve and continued firing throughout inspiration. Two continued to fire during post-inspiration. Forty-five of fifty-four inspiratory RFN neurones exhibited incrementing discharge patterns, six discharged with a plateau pattern, while only three neurones had a decrementing discharge pattern. 3. The membrane potential trajectories of caudal expiratory neurones revealed a typical wave of early inspiratory hyperpolarization. Occasionally, a second wave of hyperpolarization occurred during late inspiration, in conjunction with increased phrenic nerve activity. 4. Spike-triggered averaging revealed averaged inhibitory postsynaptic potentials (IPSPs), indicative of inhibitory synaptic connections, between eight and sixty-three pairs of RFN inspiratory and caudal expiratory neurones. 5. Inhibitory postsynaptic potentials detected by STA exhibited a relatively long latency and a slow time course. The IPSPs began, on average, 3.8 ms after an RFN action potential. The rise times, half-widths and durations of IPSPs were longer than expected for a monosynaptic somal input from myelinated axons of inspiratory RFN neurones. It is suggested that an inhibitory relay neurone in the immediate vicinity of the expiratory neurones is activated by a collateral of the RFN inspiratory neurone. 6. Retrofacial inspiratory neurones were antidromically activated only when high-intensity electrical stimulation was applied in the vicinity of caudal expiratory neurones. 7. The averaged IPSPs were preceded by diphasic and triphasic 'spike potentials'. The averaged spike potentials were highly entrained to the action potentials of RFN inspiratory neurones which triggered IPSPs. The spike potentials may be terminal potentials recorded from axons of RFN inspiratory neurones. 8. Evidence for convergence of synaptic inputs was obtained from STA tests in a caudal expiratory neurone receiving IPSPs from four RFN neurones. 9. The functional significance of this observation is discussed. We conclude that RFN inspiratory neurones exert a moderate inhibitory influence and act conjointly with other types of medullary inspiratory neurones.

Animals↗
PubMed1976

Neuronal stimulation of (3H)thymidine incorporation by primary cultures of highly purified non-neuronal cells.

A specific intercellular interaction has been demonstrated between neuronal and non-neuronal cells that appears to increase the rate of non-neuronal cell proliferation. Isolated and recombined primary cultures of both cell types were prepared from 11-day embryonic chick sympathetic ganglia by a method recently developed in this laboratory. When non-dividing neurons were added to an equal number of proliferating non-neuronal cells, the amount of [methyl-3H]thymidine incorporated by these mixed cultures was 230% greater than that incorporated by 99% pure non-neuronal cultures. Removal of all neurons from such non-neuronal cultures by a 48-h preincubation without nerve growth factor resulted in an even greater increase in [3H]thymidine incorporation upon addition of neurons (370%). When increasing numbers of isolated neurons were added to non-neuronal cell cultures, the amount of [3H]thymidine incorporation initially increased in a dose-dependent fashion until it reached a plateau. In contrast, the addition of increasing numbers of non-neuronal cells to a constant number of neurons resulted in a linear increase in [3H]thymidine incorporation. In some cases neurons and non-neuronal cells were not grown in direct physical contact but were only allowed to communicate with one another through the culture medium. Such indirect communication never resulted in a stimulation of [3H]thymidine incorporation. When neurons were added to cultures of embryonic chick fibroblasts, the neurons grew well but did not stimulate [3H]thymidine incorporation by the fibroblasts. These results suggest that embryonic sympathetic neurons selectively stimulate the proliferation of non-neuronal cells derived from the same source.

Animals↗
PubMed1988

Neuron-glia cell adhesion molecule interacts with neurons and astroglia via different binding mechanisms.

The neuron-glia cell adhesion molecule (Ng-CAM) is present in the central nervous system on postmitotic neurons and in the periphery on neurons and Schwann cells. It has been implicated in binding between neurons and between neurons and glia. To understand the molecular mechanisms of Ng-CAM binding, we analyzed the aggregation of chick Ng-CAM either immobilized on 0.5-micron beads (Covaspheres) or reconstituted into liposomes. The results were correlated with the binding of these particles to different types of cells as well as with cell-cell binding itself. Both Ng-CAM-Covaspheres and Ng-CAM liposomes individually self-aggregated, and antibodies against Ng-CAM strongly inhibited their aggregation; the rate of aggregation increased approximately with the square of the concentration of the beads or the liposomes. Much higher rates of aggregation were observed when the ratio of Ng-CAM to lipid in the liposome was increased. Radioiodinated Ng-CAM on Covaspheres and in liposomes bound both to neurons and to glial cells and in each case antibodies against Ng-CAM inhibited 50-90% of the binding. Control preparations of fibroblasts and meningeal cells did not exhibit significant binding. Adhesion between neurons and glia within and across species (chick and mouse) was explored in cellular assays after defining markers for each cell type, and optimal conditions of shear, temperature, and cell density. As previously noted using chick cells (Grumet, M., S. Hoffman, C.-M. Chuong, and G. M. Edelman. 1984 Proc. Natl. Acad. Sci. USA. 81:7989-7993), anti-Ng-CAM antibodies inhibited neuron-neuron and neuron-glia binding. In cross-species adhesion assays, binding of chick neurons to mouse astroglia and binding of mouse neurons to chick astroglia were both inhibited by anti-Ng-CAM antibodies. To identify whether the cellular ligands for Ng-CAM differed for neuron-neuron and neuron-glia binding, cells were preincubated with specific antibodies, the antibodies were removed by washing, and Ng-CAM-Covasphere binding was measured. Preincubation of neurons with anti-Ng-CAM antibodies inhibited Ng-CAM-Covasphere binding but similar preincubation of astroglial cells did not inhibit binding. In contrast, preincubation of astroglia with anti-astroglial cell antibodies inhibited binding to these cells but preincubation of neurons with these antibodies had no effect. Together with the data on Covaspheres and liposome aggregation, these findings suggested that Ng-CAM-Covaspheres bound to Ng-CAM on neurons but bound to different molecules on astroglia.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗