Cultured Chick Ciliary Ganglion Neurons Research Articles

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The role of protein tyrosine kinase (PTK) activity in the development of the retinal projection was examined in vivo by applying inhibitors of cytoplasmic PTKs, herbimycin A and lavendustin A,...

Bridging the Resolution Gap: Correlated 3D Light and Electron Microscopic Analysis of Large Biological Structures

Abstract One class of biological structures that has always presented special difficulties to scientists interested in quantitative analysis is comprised of extended structures that possess fine structural features. Examples of these structures include neuronal spiny dendrites and organelles such as the Golgi apparatus and endoplasmic reticulum. Such structures may extend 10's or even 100's of microns, a size range best visualized with the light microscope, yet possess fine structural detail on the order of nanometers that require the electron microscope to resolve. Quantitative information, such as surface area, volume and the micro-distribution of cellular constituents, is often required for the development of accurate structural models of cells and organelle systems and for assessing and characterizing changes due to experimental manipulation. Performing estimates of such quantities from light microscopic data can result in gross inaccuracies because the contribution to total morphometries of delicate features such as membrane undulations and excrescences can be quite significant. For example, in a recent study by Shoop et al, electron microscopic analysis of cultured chick ciliary ganglion neurons showed that spiny projections from the plasmalemma that were not well resolved in the light microscope effectively doubled the surface area of these neurons. While the resolution provided by the electron microscope has yet to be matched or replaced by light microscopic methods, one drawback of electron microscopic analysis has always been the relatively small sample size and limited 3D information that can be obtained from samples prepared for conventional transmission electron microscopy. Reconstruction from serial electron micrographs has provided one way to circumvent this latter problem, but remains one of the most technically demanding skills in electron microscopy. Another approach to 3D electron microscopic imaging is high voltage electron microscopy (HVEM). The greater accelerating voltages of HVEM's allows for the use of much thicker specimens than conventional transmission electron microscopes.

TGF-? regulates the survival of ciliary ganglionic neurons synergistically with ciliary neurotrophic factor and neurotrophins

We investigated putative roles of transforming growth factor (TGF)-beta expressed in peripheral ganglia in the regulation of neuronal cell survival during the period of ontogenetic neuron death (OD). The chick ciliary ganglion (CG), where OD occurs between embryonic days (E) 6 and 10, was employed as a model system. We show that CG neurons (E8) are immunoreactive (ir) for TGF-beta2 and -beta3 as well as the TGF-beta receptor TbetaR-II, but are not ir for TGF-beta1. Ciliary neurotrophic factor (CNTF) and fibroblast growth factor (FGF)-2, established neurotrophic molecules for CG neurons, up-regulate TGF-beta3 mRNA and TGF-beta biological activity in cultures of E8 CG neurons. None of the TGF-beta isoforms--beta1, beta2, or beta3--has a trophic, survival-promoting effect on cultured CG neurons. However, all isoforms enhance CG neuron survival mediated by CNTF or FGF-2, significantly and over a wide range of concentrations. In combination with the neurotrophins (NT) nerve growth factor (NGF) and NT-3, which are not neurotrophic for CG neurons, TGF-beta significantly promotes CG neuron survival. However, TGF-beta does not act synergistically with the neuropoietic cytokines oncostatin M, leukemia inhibiting factor, or interleukin-6. Immunoneutralization of endogenous TGF-beta released from CG neurons using an antibody to TGF-beta1/-beta2/-beta3 significantly reduces the potency of CNTF or FGF-2 to promote CG neuron survival. The blocking effect of the anti-pan-TGF-beta antibody could be rescued by adding exogenous TGF-beta. Together, these data suggest that para-/autocrine TGF-beta signaling has an important effect on the regulation of neuron survival in a model system of peripheral neurons.

TGF‐β regulates the survival of ciliary ganglionic neurons synergistically with ciliary neurotrophic factor and neurotrophins

We investigated putative roles of transforming growth factor (TGF)-β expressed in peripheral ganglia in the regulation of neuronal cell survival during the period of ontogenetic neuron death (OD). The chick ciliary ganglion (CG), where OD occurs between embryonic days (E) 6 and 10, was employed as a model system. We show that CG neurons (E8) are immunoreactive (ir) for TGF-β2 and -β3 as well as the TGF-β receptor TβR-II, but are not ir for TGF-β1. Ciliary neurotrophic factor (CNTF) and fibroblast growth factor (FGF)-2, established neurotrophic molecules for CG neurons, up-regulate TGF-β3 mRNA and TGF-β biological activity in cultures of E8 CG neurons. None of the TGF-β isoforms—β1, β2, or β3—has a trophic, survival-promoting effect on cultured CG neurons. However, all isoforms enhance CG neuron survival mediated by CNTF or FGF-2, significantly and over a wide range of concentrations. In combination with the neurotrophins (NT) nerve growth factor (NGF) and NT-3, which are not neurotrophic for CG neurons, TGF-β significantly promotes CG neuron survival. However, TGF-β does not act synergistically with the neuropoietic cytokines oncostatin M, leukemia inhibiting factor, or interleukin-6. Immunoneutralization of endogenous TGF-β released from CG neurons using an antibody to TGF-β1/-β2/-β3 significantly reduces the potency of CNTF or FGF-2 to promote CG neuron survival. The blocking effect of the anti–pan-TGF-β antibody could be rescued by adding exogenous TGF-β. Together, these data suggest that para-/autocrine TGF-β signaling has an important effect on the regulation of neuron survival in a model system of peripheral neurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 563–572, 1998

Developmental Regulation of Neuronal K+ Channels by Target-Derived TGFβ In Vivo and In Vitro

The functional expression of Ca2+-activated K+ channels (KCa) in developing chick ciliary ganglion (CG) neurons requires interactions with target tissues and preganglionic innervation. Here, we show that the stimulatory effects of target tissues are mediated by an isoform of TGFβ. Exposure of cultured CG neurons to TGFβ1, but not TGFβ2 or TGFβ3, caused robust stimulation of KCa. The KCa stimulatory effects of target tissue extracts were blocked by a neutralizing pan-TGFβ antiserum but not by specific TGFβ2 or TGFβ3 antisera. Intraocular injection of TGFβ1 caused robust stimulation of KCa, whereas intraocular injection of pan-TGFβ antiserum inhibited expression of KCa in CG neurons developing in vivo. The effects of TGFβ1 were potentiated by β-neuregulin-1, a differentiation factor expressed in preganglionic neurons.

Ciliary Neurotrophic Factor Stimulates the Phosphorylation of Two Forms of STAT3 in Chick Ciliary Ganglion Neurons

Ciliary neurotrophic factor (CNTF) is a neuropoietic cytokine that was identified, purified, and cloned based on its neurotrophic activity on cultured chick ciliary ganglion neurons. The molecular mechanisms by which CNTF elicits its effects on these neurons are unknown. We have previously identified functional receptors for CNTF on ciliary ganglion neurons and demonstrated the CNTF-specific tyrosine phosphorylation of an approximately 90-kDa protein. Here we show that CNTF induced the rapid tyrosine phosphorylation and nuclear accumulation of this protein and identify it as an avian form of the transcription factor, STAT3. Identification was confirmed by its recognition with two distinct anti-STAT3 antibodies and the lack of binding to antibodies against STAT1, -2, -4, -5, or -6. The phosphorylation was stable for up to 2 h but required the continued presence of CNTF. CNTF also induced the tyrosine phosphorylation of a similar protein in cultured chick dorsal root ganglion and retinal neurons. In addition, we identify a second, 100-kDa form of STAT3 that appears in response to CNTF. Unlike previous reports, utilizing mammalian cell lines that detected a slower migrating form of STAT3 resulting from H7-sensitive protein phosphorylation, H7 did not prevent the appearance of the 100-kDa form in ciliary neurons. Thus, the 100-kDa avian protein may represent a novel form of CNTF-inducible STAT3.

Inhibition of protein tyrosine kinases impairs axon extension in the embryonic optic tract

The role of protein tyrosine kinase (PTK) activity in the development of the retinal projection was examined in vivo by applying inhibitors of cytoplasmic PTKs, herbimycin A and lavendustin A, to intact brain preparations of Xenopus embryos. The inhibitors were present during the period when retinal ganglion cell axons first navigate through the optic tract to reach their target, the optic tectum. A majority of inhibitor-treated retinal axons stalled at the beginning of the optic tract, leading to an 80% reduction in projection length at the highest doses. All inhibitor-treated axons that did extend into the optic tract exhibited normal pathfinding behavior. Tyrosine kinase assays of inhibitor-treated brains demonstrated that at doses at which retinal axon extension was severely impaired, PTK activity, including that of src family proteins, was reduced by 50-60%. Consistent with the in vivo findings, PTK inhibitors reduced neurite outgrowth from cultured retinal neurons by 70-80%. This contrasts with the strong enhancement of outgrowth induced by the same inhibitors in cultured chick ciliary ganglion neurons and suggests that the mediation of outgrowth by PTK activity may vary in different neuronal types. Inhibitor-treated growth cones cultured on laminin were larger than normal, suggesting that tyrosine phosphorylation can modulate growth cone-substrate adhesive interactions. Our results in vivo and in vitro provide complementary evidence that retinal axon outgrowth is inhibited by pharmacological blockers of PTK activity and indicate that inhibitor-sensitive PTKs normally play a role in promoting retinal neurite extension.

Differential distribution of purine metabolizing enzymes between glia and neurons.

Previous studies showed that in cultured chick ciliary ganglion neurons and CNS glia, adenosine can be synthesized by hydrolysis of 5'-AMP and that the accumulation of the adenosine degradative products inosine and hypoxanthine was significantly greater in glial than in neuronal cultures. Furthermore, previous immunochemical and histochemical studies in brain showed that adenosine deaminase and nucleoside phosphorylase are localized in endothelial and glial cells but are absent in neurons; however, adenosine deaminase may be found in a few neurons in discrete brain regions. These results suggested that adenosine degradative pathways may be more active in glia. Thus, we have determined if there is a differential distribution of adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase enzyme fluxes in glia, comparing primary cultures of central and ciliary ganglion neurons and glial cells from chick embryos. Hypoxanthine-guanine phosphoribosyltransferase and production of adenosine by S-adenosylhomocysteine hydrolase activity were also examined. Our results show that there is a distinct profile of purine metabolizing enzymes for glia and neurons in culture. Both cell types have an S-adenosylhomocysteine hydrolase, but it was more active in neurons than in glia. In contrast, in glia the enzymatic activities of xanthine oxidase (443 +/- 61 pmol/min/10(7) cells), nucleoside phosphorylase (187 +/- 8 pmol/min/10(7) cells), and adenosine deaminase (233 +/- 32 pmol/min/10(7) cells) were more active at least 100, 20, and five times, respectively, than in ciliary ganglion neurons and 100, 100, and nine times, respectively, than in central neurons.

Fibroblast Growth Factors, Depolarization, and Substratum Interact in a Combinatorial Way to Promote Neuronal Survival

Neuronal survival in vivo may be determined by the combined effects of multiple agents rather than simply by the effect of an individual agent. This idea is supported by experimental evidence showing that neuronal survival can independently be influenced by target-derived factors as well as afferent inputs. To test this idea directly, cultured chick ciliary ganglion neurons were used to study the potentially interactive and combinatorial effects of trophic factors (acidic and basic fibroblast growth factor; FGF), depolarization (as would be expected from afferent activity), and substrate (laminin and collagen IV). Our results were consistent with the idea that combinatorial interactions between multiple agents may be critical in the regulation of cell survival. Exposure to either basic FGF (bFGF) or depolarization on a laminin substrate promoted neuron survival. However, bFGF did not promote survival and depolarization-mediated survival was significantly reduced when neurons were plated on collagen. The simultaneous addition of FGF and depolarization affected survival synergistically when plated on both laminin and collagen. Surprisingly, while survival by FGF or depolarization alone was greatly dependent on substrate, the simultaneous addition of FGF and depolarization appeared to greatly reduce this dependency on substrate. Taken together, these data demonstrate the potential importance of synergistic interactions between trophic factors and depolarizing stimuli.

Muscle membrane preparation restores sensitivity to acetylcholine in cultured chick ciliary ganglion neurons

Ciliary ganglion (CG) neurons grown in culture in the absence of muscle cells rapidly lose sensitivity to acetylcholine (ACh), while neurons grown in the presence of muscle or muscle cell membranes maintain sensitivity to ACh for extended periods of time. The present study examined whether exposure to muscle membrane preparation or stimulation of cAMP-dependent processes could restore sensitivity to ACh in cultured neurons which had lost responsiveness to ACh. CG neurons from 11- to 14-day-old chick embryos were grown on collagen substrate in the absence of muscle cells. Sensitivity to ACh was assessed by measuring peak current responses following application of ACh (IACh) to neurons under whole-cell voltage clamp. In control cultures IACh decreased from an average of 837 pA the day of plating to 145 pA following 4 days in culture. Stimulation of cAMP-dependent processes with forskolin and 3-isobutyl-1-methylxanthine (IBMX) or 8'Br-cAMP and IBMX had variable effects on IACh. These treatments increased peak IACh in some neurons maintained in culture for less than 48 h. Treatment with these agents decreased peak IACh in cultures which were more than 48 h old. Exposure of neurons, which had lost sensitivity to ACh in culture, to muscle membranes increased IACh 2- to 3-fold over 24 to 48 h. This membrane-induced restoration of sensitivity to ACh was blocked by exposure to the protein synthesis inhibitor cycloheximide. Stimulation of cAMP-dependent processes in neurons exposed to muscle membrane decreased IACh. In conclusion, these results indicate that some element associated with the membranes of muscle cells has the ability to restore ACh responsiveness to CG neurons which have become insensitive to ACh in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Single-channel recordings of three K+-selective currents in cultured chick ciliary ganglion neurons

Multiple distinct K+-selective channels may contribute to action potential repolarization and afterpotential generation in chick ciliary neurons. The channel types are difficult to distinguish by traditional voltage-clamp methods, primarily because of coactivation during depolarization. I have used the extracellular patch-clamp technique to resolve single-channel K+ currents in cultured chick ciliary ganglion (CG) neurons. Three unit currents selective for K+ ions were observed. The channels varied with respect to unit conductance, sensitivity to Ca2+ ions and voltage, and steady-state gating parameters. The first channel, GK1, was characterized by a unit conductance of 14 pico-Siemens (pS) under physiological recording conditions, gating that was relatively independent of membrane potential and intracellular Ca2+ ions, and single-component open-time distributions with time constants of approximately 9 msec. The second channel, GK2, was characterized by a unit conductance of 64 pS under physiological recording conditions and gating that was affected by membrane potential but was not dependent on the activity of intracellular Ca2+ ions. Open-time distributions indicated 2 open states, with open-time constants of 0.09 (61%) and 0.35 (39%) msec, at +40 mV membrane potential. The third channel, GKCa2+, was identified in isolated patch recordings in which the concentration of internal Ca2+ was 10(-7) M or greater, which was an absolute prerequisite for channel opening. GKCa2+ was characterized by a unit conductance of 193 pS in symmetrical 0.15 M KCl solutions, an open-state probability that was a function not only of [Ca2+]i, but also of membrane potential, and single-component open-time distribution with a time constant of 1.11 msec at -10 mV patch potential. These results suggest the presence of at least 3 distinct K+ channel populations in the membrane of cultured chick CG neurons.

Extra-synaptic localization of α-bungarotoxin receptors in cultured chick ciliary ganglion neurons

Surface [ 125I]α-bungarotoxin receptors were localized on dissociated cultures of embryonic chick ciliary ganglion neurons by electron microscopic autoradiography. Grain density was 4–5-fold higher on cell body membrane versus neuritic process membrane, and there was no apparent concentration of grains at morphologically identifieble inter-neuronal synapses.

Developments of α-bungarotoxin receptors in cultured chick ciliary ganglion neurons

We have maintained embryonic chick ciliary ganglion neurons in dissociated cell culture and studied the progressive appearance of surface receptors for [ 125I]α-bungarotoxin. Cultures were established from 8-day-old embryos and fed a medium supplemented with 180 μg/ml of a soluble protein extract prepared from the eye, the target organ for the ciliary ganglion. Approximately 8064 neurons survived per ganglion and there was no evident loss of neurons through two weeks in culture. Binding of [ 125I]α-bungarotoxin was determined at room temperature on intact cells still attached to their coverslips. Non-specific binding was less than 2% of the total. Specific binding of [ 125I]α-bungarotoxin was saturable with respect to both time of incubation (20–30 min) and concentration of toxin (5–10 nM), with an apparent K d = 1.0 nM. Binding sites for [ 125I]α-bungarotoxin increased during the first week in culture from 1.8 fmol per 10 4 neurons at 1 day in vitro (DIV) to 8.6 fmol per 10 4 neurons at 7 DIV, after which the number of sites seemed to plateau. Light microscopic autoradiography was performed on cultures at 4 DIV and showed most of the grains associated with the surfaces of neuronal cell bodies, while scattered grains occurred over neuronal processes. When compared with previous reports on the in vivo development of α-bungarotoxin receptors in chick ciliary ganglia, the appearance of receptors in these cultured neurons followed a time course similar to, but at lower levels than, their in vivo counterparts. Nevertheless, this culture system should prove useful for the study of questions concerning the regulation, surface distribution and intracellular pathways of neuronal α-bungarotoxin receptors.

Binding properties of a neurotoxin from the venom of the green mamba, Dendroaspis viridis.

A toxin, alpha-mambatoxin, was purified from the venom of the green mamba Dendroaspis viridis using the procedures of Shipolini et al. (Shipolini, R. A., Bailey, G. S., Edwardson, J. A., and Banks, E. C. (1973) Eur. J. Biochem. 40, 337-344). The purified toxin blocks agonist-induced activation of acetylcholine receptors on muscle cells but, like alpha-bungarotoxin, it does not affect agonist-induced activation of receptors on a clonal sympathetic nerve cell line (PC12), an endothelial cell line, cultured chick ciliary ganglion neurons, or frog cardiac ganglion neurons. The toxin does block binding of alpha-bungarotoxin to cultures of muscle and nerve, and iodinated alpha-mambatoxin binds to cultures of muscle and nerve. The alpha-mambatoxin binding component on muscle was identified as acetylcholine receptor on the basis of sedimentation, immunoprecipitation, and rate of degradation. The alpha-mambatoxin binding component on PC12 cells, like the alpha-bungarotoxin binding component on these cells, is not recognized by anti-acetylcholine receptor antisera which do recognize acetylcholine receptor on these cells. The number of alpha-mambatoxin binding sites on both nerve and muscle when assayed in situ is twice that of alpha-bungarotoxin binding sites. However, when muscle cells are solubilized in nonionic detergents and then labeled with toxins, the number of alpha-mambatoxin binding sites is reduced and the two toxins bind in equal molar amounts. Finally, unlike alpha-bungarotoxin, which dissociates from complexes formed with nerve, alpha-mambatoxin forms complexes with nerve which dissociate only very slowly if at all.