Root Ganglia Of Chick Embryos Research Articles

Effects of male and female sex steroids on the development of normal and the transient Froriep's dorsal root ganglia of the chick embryo

Sex steroids can influence developmental processes and support the survival of neurons in the embryonic central nervous system. Recent studies have shown that estrogen receptors are also expressed in the peripheral nervous system, in the dorsal root ganglia (DRG) of chick embryos. However, no studies have examined the effects of sex steroids on development of embryonic DRG. In the present study, 0.2 μg, 1.0 μg, 5.0 μg 10 μg, 20 μg, 25 μg, and 40 μg doses of testosterone or estradiol were delivered to chick embryos at Hamburger and Hamilton stage 18 (E3). The actions of these doses of sex steroids on the development of the C5DRG (fifth cervical ganglion, a “normal” DRG) and C2DRG (a transient ganglion known as a “Froriep's DRG”) were then evaluated by quantifying ganglionic volumes, cell number, proliferation, and apoptosis after 1 day of growth to stage 23. We found that both testosterone and estradiol promoted proliferation of cells in both normal DRG and the Froriep's ganglia. By contrast, estradiol significantly increased the number of apoptotic cells, while testosterone strongly inhibited apoptosis. These actions of sex steroids on DRG development were dose-dependent, and C5DRG and C2DRG showed different sensitivities to the applied sex steroids. In addition, the present results demonstrated that specific ER and AR inhibitors (tamoxifen and flutamide) did not influence the effects of 5 μg E2 and 5 μg T on C2 and C5DRG significantly. These results demonstrate that male and female sex steroids can modulate DRG development through an epigenetic mechanism, as had been shown for the central nervous system.

Expression, purification, and characterization of recombinant human neurturin secreted from the yeast Pichia pastoris

Neurturin (NTN), a potent neurotrophic factor acting specifically on dopaminergic neurons, is comprised of 102 amino acids as a mature protein. We artificially synthesized a gene for mature human NTN (hNTN) using codons preferred by the yeast Pichia pastoris. This synthesized gene, fused in frame with sequences encoding the α-factor signal peptide gene from Saccharomyces cerevisiae was cloned into P. pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-α-hNTN was then transformed into the yeast and stable multicopy recombinant P. pastoris strains were selected by G418 resistance. SDS–PAGE and Western blot assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hNTN, a 16 kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using CM–Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Bioactivity of the recombinant hNTN was confirmed by the ability of the protein to stimulate growth of nerve fibers from the dorsal root ganglia of chick embryos in vitro.

Muscarinic cholinergic receptors in dorsal root ganglia of chick embryo: a radioligand binding and immunocytochemical study

The presence and micro-anatomical localization of muscarinic cholinergic receptors were assessed in dorsal root ganglia of chick embryo during development using radioligand binding and immunocytochemical techniques, respectively. The non-selective muscarinic cholinergic receptor radioligand [ 3H]quinuclidinyl benzilate was specifically bound to sections of chick dorsal root ganglia with a dissociation constant value ( K d) of 0.75 ± 0.02 nM and a maximum density of binding sites ( B max) of 7.2 ± 0.5 fmol/mg tissue. [ 3H]Quinuclidinyl benzilate binding was partially sensitive to pirenzepine displacement. This suggests that muscarinic cholinergic receptors expressed by dorsal root ganglia of chick embryo at least in part belong to the M 1 muscarinic receptor subtype. Immunocytochemical analysis confirmed the presence of muscarinic receptors in the ganglia. These findings suggest that neurons of dorsal root ganglia, which are known to express cholinergic markers such as choline acetyltransferase, acetylcholinesterase and high affinity choline uptake, are also cholinoceptive.

Staurosporine induces the outgrowth of neurites from the dorsal root ganglion of the chick embryo and PC12D cells

Staurosporine, a potent inhibitor of protein kinases, caused the rapid outgrowth of neurites from cultured dorsal root ganglia of chick embryos and from PC12D cells, a subline of PC12 cells. Treatment of dorsal root ganglia with 1 to 20 nM staurosporine resulted in the extensive outgrowth of neurites that were indistinguishable from those induced by NGF, as assessed by phase-contrast microscopy, electron microscopy and cytochemical staining of actin and tubulin. However, neurites generated from the ganglia in response to the higher concentrations of staurosporine (40–100 nM) seemed to have different characteristics, possible as a result of the inhibition of cell migration from ganglia. The sequential changes in morphology of PC12D cells in response to staurosporine and to NGF were revealed by staining of actin. Ruffling membranes emerged at the margins of PC12D cells within 4 min after the addition of staurosporine or of NGF. From 10 min to 24 h after the addition of either compound, the ruffles were transformed into several projections that became growing neurites. The formation of ruffles and the outgrowth of neurites were both apparent at a concentration of staurosporine of 10 nM. The neurites that emerged from PC12D cells in response to staurosporine and in response to NGF were indistinguishable under the phase-contract microscope and after staining of actin and tubulin. However, staurosporine never promoted survival of PC12D cells in serum-free conditions as that promoted by NGF. The observations indicate that staurosporine at nanomolar concentrations may reproduce the neurogenic changes that induced by NGF in primed neuronal cells, although it can not mimic the action of NGF that supports survival of neurons.

An analysis of dorsal root ganglia differentiation using three tissue culture systems.

The histogenesis of the dorsal root ganglia of chick embryos (ages 3 to 9 days) was followed in three different tissue culture systems. Organotypic explants included dorsal root ganglia connected to the lumbosacral segment of the spinal cord or isolated explants of the contralateral ganglia. Additionally, dissociated monolayer cultures of ganglia tissue were established. The gradual differentiation of progenitor neuroblasts into distinct populations of large ventrolateral and small dorsomedial neurons was observed in vivo and in vitro. Neurites developed after 3 days in the presence or absence of nerve growth factor in the medium. In contrast, autoradiographic analysis indicates that [3H]thymidine incorporation in neuronal cultures differed significantly from intact embryos. In vivo, the number of neuronal progenitor cells labeled with [3H]thymidine decreased in older embryos; in vitro, uptake of [3H]thymidine label was not observed in ganglionic progenitor cells regardless of the age of the donor embryo or the type of culture system. Lack of proliferation in ganglionic progenitor cells was not due to degeneration because vital staining and uptake of [3H]deoxyglucose indicated that neurons were metabolically active. Furthermore, the block in mitotic activity in vitro was limited to presumptive ganglionic neuronal cells. In the ependyma of the spinal cord segment connected to the dorsal root ganglia, neuronal progenitor cells were heavily labeled as were non-neuronal cells within both spinal cord and ganglia. Our results suggest that in vitro conditions can promote the differentiation of sensory neurons from early embryos (E3.5-4.5) without proliferation of progenitor cells.

Influence of peripheral and central targets on subpopulations of sensory neurons expressing calbindin immunoreactivity in the dorsal root ganglion of the chick embryo

The influence of central and peripheral target tissues on the expression of calbindin D-28k by sensory neurons of the chick dorsal root ganglia was tested under various experimental conditions. Firstly, dorsal root ganglia of chick embryos were transplanted at two stages of development onto the chorioallantoic membrane of a host embryo for a period of 4 or 8 days. In dorsal root ganglia grafted at E12, 20% of the ganglion cell bodies were immunoreactive to calbindin 4 and 8 days later; the percentage of calbindin-immunostained neurons in grafted dorsal root ganglia was similar to that observed in control dorsal root ganglia of the same embryonic age (E16 or 20). In contrast, when grafted dorsal root ganglia were taken from a donor embryo at E8, no calbindin-immunoreactive neuron was found 4 or 8 days later. However, when dorsal root ganglia at E8 were cotransplanted with musculature cells, 14% of the grafted ganglion cell bodies were again immunoreactive to calbindin 4 or 8 days later. Secondly, peripheral targets of sensory neurons were suppressed by excision of one hindlimb. After excision at E6, virtually all the ipsilateral dorsal root ganglia cells were free of calbindin immunoreaction after 6 days of reincubation. In contrast, when the excision was performed at E11, calbindin was expressed in about 9% of the nerve cell bodies. Thirdly, central connections were destroyed by cauterization of the lumbosacral spinal cord at E6 or E11. Six days after deprivation of central connections, the percentage of calbindin-immunoreactive ganglion cells was the same as in control dorsal root ganglia of the same age. These results indicate that initiation of calbindin expression by sensory neurons requires interaction with skeletal muscle but not with spinal cord. Once expressed, calbindin immunoreaction may be maintained in sensory neurons deprived of musculature connections, at least for 8 days.

Inhibition by K-252a, a new inhibitor of protein kinase, of nerve growth factor-induced neurite outgrowth of chick embryo dorsal root ganglion cells

The effect of K-252a, a new alkaloid-like kinase inhibitor from the culture broth of Nocardiopsis sp., on the action of nerve growth factor on normal nerve cells was examined. Nerve cells were isolated from the dorsal root ganglia of chick embryo by trituration without using digesting enzymes, and were seeded on collagen- or poly- l-lysine-coated plastic dishes containing serum-free medium. K-252a at 50–100 nM reversibly inhibited the formation of neurites by dorsal root ganglion cells elicited by the treatment with nerve growth factor.

A proteolytic 140-kDa fragment of neurite outgrowth factor and its cell-binding activity

A neurite outgrowth factor (NOF) from chicken gizzard was treated with elastase, and a protease-resistant fragment of 140 kDa (F-140) with heparin-binding activity, was isolated from the digested mixture. Although F-140 showed no neurite outgrowth activity, it could bind dissociated cells from the dorsal root ganglia of chick embryos. The antiserum to F-140 inhibited the cell binding activity of native NOF in a dose-dependent manner, but not the neurite outgrowth activity. When membrane proteins from whole chick embryos were incubated with NOF or F-140, a protein band of 41 kDa was detected as a major NOF-binding component.

Sprouting inducing activity of human sera: low capacity in sera from patients with cranial polyneuropathy.

Cranial idiopathic acute peripheral facial nerve paralysis (Bell palsy) has been used as a model disease in the present study mainly to investigate trophic capacity of serum components. The ability to induce sprouting from chick embryo dorsal root ganglia was used as the bioassay for trophic activity. Sera from normal patients induce neuritic outgrowth from dorsal root ganglia of chick embryo in the absence of any exogenous factor. Sprouting, to a very limited extent or none, has been induced by 80% of the sera derived from the Bell palsy patients. Further analysis of the biochemical nature of the reduced activity has revealed that in sera of Bell palsy patients the substances that are fractionated within the Ig fraction are responsible for the reduced capacity to induce sprouting without loss of supportive molecules.

Chemotactic response of nerve fiber elongation to nerve growth factor

These studies seek to determine whether the locomotory behavior of the extending tip of growing nerve fibers exhibits chemotaxis to nerve growth factor (NGF). Sensory neurons from dissociated dorsal root ganglia of chick embryos were cultured within semisolid agar matrices containing concentration gradients of NGF which diffused from an adjacent source. A preferential orientation of approximately 60% of the tips of nerve fibers and an enhanced extension of fibers up NGF gradients were observed. The initiation sites of nerve fibers on cell bodies were not consistently oriented by these NGF gradients. Orientation of nerve fiber extension toward the NGF source was observed in chambers with NGF sources ranging from 25 to 1000 ng/ml of β-NGF and from 400 to 5000 ng/ml of 7 S NGF, but no orientation was observed when the concentration of the NGF source was 15000 ng/ml of β-NGF. This oriented response apparently is not a concentration-dependent trophic response to NGF. The evidence supports the notion that chemotaxis is a regulatory factor in neuronal morphogenesis.

Changes in glia-neuron relationships in cell cultures of spinal ganglia caused by puromycin.

Dorsal root ganglia of chick embryos were cultured for one to four weeks on Maximow-slides. Puromycin was added to cultures for a pulse of 30' in a dose of 100 mug/ml medium. Particular interest was given to the ultrastructural features of the glia-neuron relations. Puromycin caused a shrinkage of the glial processes and consequently the continous glial envelope of the spinal ganglion neurons disappeared. These changes of glia-neuron contacts were reversible after a few days when puromycin was withdrawn from the medium. The newly formed glial sheaths around the neurons were comparable to those in untreated cultures and to those in vivo. The effects of an inhibition of protein synthesis caused by puromycin are discussed in relation to the effects on the cell motility, on the renewal of cell membranes and on the formation of cell contacts in nervous tissue.

Neurotoxicity of organophosphates: Effects of tri- o-tolyl phosphate in chick ganglia cell cultures

A mixed cell nerve tissue culture system from dorsal root ganglia of chick embryos was used to test the effects of tri- o-tolyl phosphate (TOTP), its metabolites and di-isopropylphosphorofluoridate (DFP). Cytotoxic responses consisted of reduction in number and length of nerve fibres and increased vacuolization of neuroglial cells. Unmetabolized TOTP was shown to produce degenerative changes in both nerve fibres and neuroglial cells. Metabolized TOTP and DFP were slightly more potent than TOTP itself. The nerve fibres were generally more sensitive to all treatments than the neuroglial cells. A well defined, sensitive, tissue culture system was shown to yield results after only 3 to 5 days of culture.