Chick Dorsal Root Ganglion Neurons Research Articles

Temperature dependencies of Ca 2+ current, Ca 2+-activated CI- current and Ca 2+ transients in sensory neurones

We recorded Ca 2+ current (I ca) and Ca 2+-activated Cl- current (I cl(ca)) in isolated chick dorsal root ganglion neurons. At room temperature, I Cl(Ca) is activated by Ca 2+ influx (e.g. I ca) or by caffeine-stimulated release of Ca 2+ via ryanodine receptors. Warming from room temperature to 37°C increased the amplitude of Ica as well as the amplitude and rate of deactivation of I Cl(Ca) activated by Ca 2+ influx. In contrast, the activation of I CI(Ca) by caffeine-stimulated release of Ca 2+ from intracellular stores abruptly failed between 19 and 28°C. Warming from 22 to 37°C reduced the amplitude of [Ca 2+] i transients (measured with Indo-1) in chick neurons by more than 50% and reduced [Ca 2+] i transients in mouse neurons by more than 40%. We investigated the role of mitochondria in these phenomena using carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) to inhibit mitochondrial Ca 2+ uptake. 1–4 μM FCCP slowed the deactivation of I ca activated I CI(Ca) at 20°C and at 36°C, having a greater effect at the higher temperature. In the presence of FCCP, the rate of deactivation of I Cl(Ca) was relatively insensitive to temperature in this protocol. In contrast, FCCP had little effect on I CI(Ca) activated by caffeine at warmer temperatures (> 22°C) but prolonged I Cl(Ca) at cooler temperatures (< 22°C). Thus, we find that warming reduces the ability of Ca 2+ release to raise [Ca 2+] i, increases the effect of mitochondria on the deactivation of I Cl(Ca) if I CI(Ca) is activated by Ca 2+ influx, and reduces the effect of mitochondria if I Cl(Ca) is activated by caffeine-stimulated Ca 2+ release.

Structure of the mouse gene for the serine protease inhibitor neuroserpin (PI12).

Neuroserpin (PI12), initially identified as an axonally secreted protein in cultured chicken dorsal root ganglion neurons, belongs to the serpin family of the serine protease inhibitors and is mainly expressed by neurons of both the developing and the adult nervous system. Here we report on the cloning and structural characterization of the neuroserpin gene of the mouse. The murine neuroserpin gene spans over more than 55kb and consists of nine exons. The positions and phases of the exonīntron borders are completely conserved between neuroserpin and its nearest homologues, protease nexin-1 and plasminogen activator inhibitor-1. A single transcription initiation site, which is colocalized with a potential initiation (Inr) sequence, has been determined by primer extension and RNase protection. Sequence analysis revealed a TATA-less promoter with a CAAT box and several sites for the general transcription factor Sp1 and the neuron-specific transcription factor AP-2.

2′-Deoxyadenosine selectively kills nonneuronal cells without affecting survival and growth of chick dorsal root ganglion neurons

Recently, we have demonstrated that adenosine and 2′-deoxyadenosine are toxic to embryonic sympathetic neurons and proposed that purine and pyrimidine metabolism may play a critical role in the growth and development of sympathetic neurons. To extend this hypothesis further, we examined the effects of these nucleosides on two other neuronal populations in the chick embryo, sensory dorsal root ganglion neurons and parasympathetic ciliary ganglion neurons. Now, we show that 2′-deoxyadenosine and adenosine have no visible adverse effect on the viability of either sensory or parasympathetic neurons. Instead, 2′-deoxyadenosine proved to be highly toxic to the nonneuronal cells. The toxic effects of 2′-deoxyadenosine were markedly enhanced by inhibition of adenosine deaminase. In contrast, adenosine was much less toxic to nonneuronal cells than 2′-deoxyadenosine and its effect was not potentiated by inhibition of adenosine deaminase. Priming of pyrimidine pools by exogenous uridine and the specific inhibitor of the nucleoside transporter, nitrobenzylthioinosine, did not protect nonneuronal cells from 2′-deoxyadenosine toxicity. Since phosphorylation of internalized nucleosides was a key step in the initiation of toxicity in sympathetic neurons, adenosine kinase activity was compared in sensory and sympathetic neuronal cultures. The adenosine kinase activity in dorsal root ganglion cultures was only 20% of that in sympathetic ganglion cultures. Furthermore, inhibition of phosphorylation by blocking 2′-deoxyadenosine kinase with iodotubercidin and 5′-amino-5′-deoxyadenosine had no protective effect against 2′-deoxyadenosine toxicity. [ 3H]-thymidine incorporation was inhibited over 90% by 2′-deoxyadenosine as early as 6 h following its addition and for up to 4 days, suggesting inhibition of proliferation of nonneuronal cells by 2′-deoxyadenosine. The nucleoside was also able to wipe out already well established nonneuronal cells, leaving behind an enriched population of sensory neurons. The selective vulnerability of nonneuronal cells to 2′-deoxyadenosine offers a convenient and effective tool for removing nonneuronal cells from neuronal cultures as well as providing a new model for studying the mechanisms of nucleoside toxicity.

Functional involvement of synaptotagmin I/II C2A domain in neurite outgrowth of chick dorsal root ganglion neuron

Abstract Synaptotagmin I or II (Syt I/II) is involved in Ca 2+ -regulated exocytosis of secretory vesicles, probably serving as a Ca 2+ -sensor via its C2A domain. Synaptotagmin is also known to be expressed in neuronal growth cone vesicles, but its functional involvement in neurite outgrowth remains largely unknown. In this study, we examined the function of Syt I/II in neurite outgrowth in cultured chick dorsal root ganglion neurons using an anti-synaptotagmin I and II C2A domain (anti-STI/II-C2A) antibody that inhibits Ca 2+ -regulated exocytosis. Immunoblots confirmed the high specificity of the anti-STI/II-C2A antibody and showed the expression of synaptotagmin I or II in chick dorsal root ganglion neurons. Immunocytochemistry revealed that synaptotagmin I or II is enriched at the growth cone region of chick dorsal root ganglion neurons, in both lamellipodia and filopodia. Whole or Fab-fragment of the anti-STI/II-C2A antibody loaded into dorsal root ganglion neurons by trituration significantly inhibited neurite outgrowth, whereas preimmune IgG had no effect. These results showed that the C2A domain of synaptotagmin I or II plays a crucial role in neurite outgrowth.

Laminin Directs Growth Cone Navigation via Two Temporally and Functionally Distinct Calcium Signals

During development, growth cones navigate to their targets via numerous interactions with molecular guidance cues, yet the mechanisms of how growth cones translate guidance information into navigational decisions are poorly understood. We have examined the role of intracellular Ca2+ in laminin (LN)-mediated growth cone navigation in vitro, using chick dorsal root ganglion neurons. Subsequent to contacting LN-coated beads with filopodia, growth cones displayed a series of stereotypic changes in behavior, including turning toward LN-coated beads and a phase of increased rates of outgrowth after a pause at LN-coated beads. A pharmacological approach indicated that LN-mediated growth cone turning required an influx of extracellular Ca2+, likely in filopodia with LN contact, and activation of calmodulin (CaM). Surprisingly, fluorescent Ca2+ imaging revealed no LN-induced rise in intracellular Ca2+ in filopodia attached to their parent growth cone. However, isolation of filopodia by laser-assisted transection unmasked a rapid, LN-specific rise in intracellular Ca2+ (+73 +/- 11 nM). Additionally, a second, sustained rise in intracellular Ca2+ (+62 +/- 8 nM) occurred in growth cones, with a distinct delay 28 +/- 3 min after growth cone filopodia contacted LN-coated beads. This delayed, sustained Ca2+ signal paralleled the phase of increased rates of outgrowth, and both events were sensitive to the inhibition of Ca2+/CaM-dependent protein kinase II (CaM-kinase II) with 2 microM KN-62. We propose that LN-mediated growth cone guidance can be attributed, in part, to two temporally and functionally distinct Ca2+ signals linked by a signaling cascade composed of CaM and CaM-kinase II.

Verapamil block of the delayed rectifier K current in chick embryo dorsal root ganglion neurons.

We have used the patch-clamp method in the whole-cell configuration to investigate the mechanism of block of the delayed rectifier K current (IDRK) by verapamil in embryonic chick dorsal root ganglion (DRG) neurons. Verapamil induced a dose-dependent decay of the current, without altering its activation kinetics. This observation, together with the good description of IDRK time course at various blocker concentrations with the computer simulation of a three-state chain model (closed left and right arrow open left and right arrow open-blocked), indicates that verapamil acts as a state-dependent, open-channel blocker. To account for the double-exponential time course of recovery from block, this minimal kinetics scheme was expanded to include a closed-blocked state resulting from channel closure (at hyperpolarized voltages) with verapamil still bound to it. The apparent block and unblock rate constants assessed from verapamil-induced current decay in the presence of external Na were 0.95 +/- 0.05 ms-1mM-1 and 0.0037 +/- 0.0016 ms-1, respectively. When external Na was replaced by K, only the unblock rate constant changed, to 0.02 +/- 0.009 ms-1. Under these ionic conditions it was also observed that the recovery from block was modified from the double-exponential time course in the presence of external Na (tau1 = 160 ms; tau2 = 1600 ms), to a faster single-exponential recovery (tau = 100 ms). We tested the voltage dependence of block by applying stimulation protocols aimed at eliminating bias easily introduced by the shift of the gating equilibrium and by the coupling of channel activation and block. Under these experimental conditions the resulting block rate constant was not measurably voltage dependent.

Ca2+ and Na+ permeability of high-threshold Ca2+ channels and their voltage-dependent block by Mg2+ ions in chick sensory neurones.

1. The Mg2+ block of Na+ and Ca2+ currents through high-voltage activated (HVA; L- and N-type) Ca2+ channels was studied in chick dorsal root ganglion neurones. 2. In low extracellular [Ca2+] (< 10(-8) M) and with Na+o and Cs+i as the main charge carriers (120 mM), HVA Na+ currents started to activate at -40 mV, reached inward peak values near 0 mV and reversed at about +40 mV. 3. Addition of 30-500 microM Mg2+ to the bath caused a strong depression of inward Na+ currents that was voltage and dose dependent (KD = 39 microM in 120 mM Na+ at -10 mV). The block was maximal at negative potentials (< -70 mV) and decreased with increasing positive potentials, suggesting that Mg2+ cannot escape to the cell interior. 4. Block of Ca2+ currents by Mg2+ was also voltage dependent, but by three orders of magnitude less potent than with Na+ currents (KD = 24 mM in 2 mM Ca2+ at -30 mV). The high concentration of Mg2+ caused a prominent voltage shift of channel gating kinetics induced by surface charge screening effects. To compensate for this, Mg2+ block of inward Ca2+ currents was estimated from the instantaneous I-V relationships on return from very positive potentials (+100 mV). 5. Inward Na+ and Ca2+ tail currents following depolarization to +90 mV were markedly depressed, suggesting that channels cleared of Mg2+ ions during strong depolarization are quickly re-blocked on return to negative potentials. The kinetics of re-block by Mg2+ was too fast (< 100 microseconds) to be resolved by our recording apparatus. This implies a rate of entry for Mg2+ > 1.45 x 10(8) M-1 S-1 when Na+ is the permeating ion and a rate approximately 3 orders of magnitude smaller for Ca2+. 6. Mg2+ unblock of HVA Na+ currents at +100 mV was independent of the size of outward currents, whether Na+, Cs+ or NMG+ were the main internal cations. 7. Consistent with the idea of a high-affinity binding site for Ca2+ inside the channel, micromolar amounts of Ca2+ caused a strong depression of Na+ currents between -40 and 0 mV, which was effectively relieved with more positive as well as with negative potentials (KD = 0.7 microM in 120 mM Na+ at -20 mV). In this case, the kinetics of re-block could be resolved and gave rates of entry and exit for Ca2+ of 1.4 x 10(8) M-1 S-1 and 2.95 x 10(2) s-1, respectively. 8. The strong voltage dependence and weak current dependence of HVA channel block by divalent cations and the markedly different KD values of Na+ and Ca2+ current block by Mg2+ can be well described by a previously proposed model for Ca2+ channel permeation based on interactions between the permeating ion and the negative charges forming the high-affinity binding site for Ca2+ inside the pore (Lux, Carbone & Zucker, 1990).

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.

A family of glycoproteins (GP55), which inhibit neurite outgrowth, are members of the Ig superfamily and are related to OBCAM, neurotrimin, LAMP and CEPU-1.

We have previously identified a glycosylphosphatidylinositol-linked glycoprotein of 55 kDa (GP55) which inhibits neurite outgrowth. We now provide evidence that GP55, isolated from adult chick brain, consists of at least two bands, both of which are active, i.e., block outgrowth of neurites from chick dorsal root ganglion neurons. An antiserum raised against the adult proteins reverses the inhibition and preliminary experiments suggest that GP55 is restricted to the nervous system, increases during development from very low levels at embryonic day 10 and is most abundant after hatching. Immunofluorescence reveals that GP55 is expressed on neurons cultured from an embryonic day 14 chick brain but is barely detectable on embryonic day 10 dorsal root ganglion neurons or embryonic day 8 forebrain neurons; the neurons which respond to substrate-bound GP55. Peptide sequencing revealed considerable homology with OBCAM, a protein previously identified on the basis of binding opiates. Nested polymerase chain reaction using primers to the OBCAM sequence and internal primers to GP55 peptides produced two different polymerase chain reaction fragments with homology to OBCAM. A full length clone (E19S) corresponding to one polymerase chain reaction product and a partial length clone (E14S) corresponding to the second have been isolated from an embryonic chick brain library. Both are members of the immunoglobulin superfamily and have (or are expected to have) three C2 domains. E19S has 90% homology with LAMP at the amino acid level. This sequence only partially matches the peptides from the adult protein and hence is probably not a major component of the adult proteins. E14S (GP55-A) has 83% homology to OBCAM at the amino acid level over the region sequenced. The sequence matches several of the peptides from the adult protein and is hence likely to correspond to a major component of the adult proteins. Thus members of the GP55 family are related to OBCAM, neurotrimin, LAMP and a recently discovered chick protein CEPU-1. Our results suggest molecules within this family are capable of acting as cell adhesion molecules and inhibitors of neurite outgrowth.

Characterization of a neuronal delayed rectifier K current permeant to Cs and blocked by verapamil.

We have used the patch-clamp method in the whole-cell configuration to characterize the delayed rectifier K current (IDRK) in embryonic chick dorsal root ganglion (DRG) neurons. The IDRK is activated by depolarizing pulses positive to -40 mV, and its V1/2 is near -20 mV. The slope factor of 10.4 mV for an e-fold change in conductance indicates an equivalent gating charge of 2.4e. Inactivation during sustained depolarizing pulses displays two distinct time constants of 200-300 msec and 6-9 sec, respectively. Outward current through the delayed rectifier K (DRK) channels could also be carried by internal Cs, which however exerts mild block when in mixtures with K, as evidenced by the anomalous mole fraction effect. The relative permeability of Cs vs. K, PCs/PK, as calculated from reversal potential measurements, is 0.25. Rb likewise permeates the DRK channel (PRb/PK = 0.67). The IDRK was effectively suppressed by external application of the Ca channel blocker Verapamil, with apparent dissociation constant of ca. 4 microM. The time course of Verapamil block, its good description by equations derived from open-channel block kinetic scheme, and the frequency-dependent effect of the blocker indicate that Verapamil can bind to the channel only when it is in the open state.

Function of myosin-V in filopodial extension of neuronal growth cones.

The molecular mechanisms underlying directed motility of growth cones have not been determined. The role of myosin-V, an unconventional myosin, in growth cone dynamics was examined by chromophore-assisted laser inactivation (CALI). CALI of purified chick brain myosin-V absorbed onto nitrocellulose-coated cover slips inhibited the ability of myosin-V to translocate actin filaments. CALI of myosin-V in growth cones of chick dorsal root ganglion neurons resulted in rapid filopodial retraction. The rate of filopodial extension was significantly decreased, whereas the rate of filopodial retraction was not affected, which suggests a specific role for myosin-V in filopodial extension.

Developmentally Regulated Neurite Outgrowth Response from Dorsal Root Ganglion Neurons to Heparin‐binding Growth‐associated Molecule (HB‐GAM) and the Expression of HB‐GAM in the Targets of the Developing Dorsal Root Ganglion Neurites

Heparin-binding growth-associated molecule (HB-GAM) is a highly conserved cell surface- and extracellular matrix-associated protein that enhances neurite outgrowth in brain neurons in vitro. To study the possible response of peripheral neurons, we cultured chicken dorsal root ganglion neurons from different developmental stages from embryonic day 4.5 (E4.5; St 25) to E9 (St 35) on recombinant HB-GAM. We discovered that the neurite outgrowth response to HB-GAM is maximal at E5.5-6.5 (St 28-30). In order to correlate this in vitro phenomenon with in vivo phenomena, immunohistochemical staining and in situ hybridization were performed on cryosections. The protein expression of HB-GAM peaked at E6 (St 29) and was most extensive on the dorsal spinal cord and dorsal roots. Using Dil labelling, we confirmed that at the time when sensory afferents travel longitudinally in the bundle of His of the spinal cord, HB-GAM protein expression there is at its peak. Though HB-GAM is a secreted protein, at the RNA level the timing of HB-GAM appearance and existence in the spinal cord and sensory ganglia is in accordance with its protein expression. Our results demonstrate that peripheral neurons are responsive to substrate-bound HB-GAM in a developmentally regulated manner, and that the expression of both HB-GAM mRNA and protein in vivo is spatially and temporally matched to this in vitro phenomenon. HB-GAM is therefore a putative cue for the growth of sensory afferents to and within the dorsal spinal cord.

Neurite outgrowth on non-permissive substrates in vitro is enhanced by ectopic expression of the neural adhesion molecule L1 by mouse astrocytes.

Axonal regrowth in the lesioned central nervous system (CNS) of adult mammals is, in part, prevented by non-permissive properties of glial cells and myelin. To test if ectopic expression of the neurite outgrowth promoting recognition molecule L1 will overcome these non-permissive influences and promote neurite outgrowth, L1 was expressed in astrocytes of transgenic mice using regulatory sequences of the glial fibrillary acidic protein (GFAP) gene. Northern blot analysis of different transgenic lines revealed different levels of transgenically expressed L1. Cultured astrocytes derived from transgenic animals displayed L1 immunoreactivity at the cell surface and in situ hybridization and immunocytochemical analysis of optic nerves from adult transgenic mice localized L1 expression to astrocytes. Expression of L1 protein by transgenic astrocytes was significantly upregulated in lesioned optic nerves. When mouse small cerebellar neurons or chick dorsal root ganglion neurons were cultured on cryosections of lesioned optic nerves or astrocyte monolayers from transgenic mice, respectively, neurite outgrowth was increased up to 400% on tissue sections and 50% on astrocytes compared with similar preparations from non-transgenic mice. The increase in neurite outgrowth on tissue sections or astrocyte monolayers from different transgenic lines was proportional to the different levels of L1 expression. Moreover, increased neurite outgrowth on these substrates was specifically inhibited by polyclonal L1 antibodies. In vivo, rescue of severed axons was enhanced in transgenic versus wild type animals, while regrowth of axons was slightly, but not significantly, increased. Together, our observations demonstrate that L1 promotes neurite outgrowth when expressed ectopically by astrocytes and that L1 is able to overcome, at least partially, the non-permissive substrate properties of differentiated CNS glial cells in vitro.

GROWTH CONE BEHAVIOR IN THE PRESENCE OF SOLUBLE CHONDROITIN SULFATE PROTEOGLYCAN (CSPG), COMPARED TO BEHAVIOR ON CSPG BOUND TO LAMININ OR FIBRONECTIN

Proteoglycans (PGs) are complex macromolecules of the extracellular matrix (ECM) that have a wide variety of effects on developing and regenerating neurons in vivo and in vitro. One hypothesis regarding the mechanisms of PG regulation of neuronal behavior states that the conformation of PGs may be critical, and thus that ECM- or cell surface-bound PGs may operate differently than secreted (soluble) PGs. Therefore, this study examined differences between the effects of soluble chondroitin sulfate proteoglycan (CSPG) and substratum-bound CSPG on neuronal growth cone behavior. Dissociated chicken dorsal root ganglion (DRG) neurons were cultured on either laminin (LN) or fibronectin (FN), both sensory neurite outgrowth-promoting glycoproteins. CSPG (or chondroitin sulfate alone) was either bound to FN or LN, or was added to the culture media. Subsequently, using time lapse video microscopy and image analysis, this study measured: (1) neuronal attachment, (2) neurite outgrowth, (3) rate of neurite elongation, and (4) filopodial length and lifespan. To determine the site of CSPG action, DRG neurons were grown on either: CS-1, a FN peptide [Humphries M. J. et al. (1987) J. biol. Chem. 262, 6886–6892], or a recombinant FN protein, RFNIIICS (Maejne, submitted), both of which permit DRG attachment and outgrowth but do not have recognized CSPG binding sites, and the resulting neuronal behavior was compared to that of DRG neurons grown on intact FN.The results of these studies confirm that the effect of CSPG on DRG neurons is concentration-, conformation- and substratum-dependent. On LN, soluble CSPG had little to no effect on neurite initiation or outgrowth, while substratum-bound CSPG inhibited neurite outgrowth. In contrast, on FN, soluble CSPG inhibited neurite outgrowth and decreased the rate of neurite elongation. Soluble CSPG did not affect the length of sensory growth cone filopodia or filopodial lifespan on either LN or FN. From the FN fragment experiments, we found that: (1) soluble CSPG reduces neurite outgrowth on FN or FN fragments, but not on LN, up to 80%, and reduces elongation rate on FN up to 50%, and (2) soluble CSPG regulates neuronal behavior by binding directly to growth cones elongating on FN.Given that substratum-bound CSPG from a variety of sources is inhibitory to neurite outgrowth and to the rate of neurite elongation, while soluble CSPG often has different effects on growth cone behavior, the regulation of growth cone behavior by CSPGs may be dependent upon CSPG conformation. Further, CSPG may affect growth cone behavior by either binding to the substratum or by binding directly to growth cones. Copyright © 1996 ISDN. Published by Elsevier Science Ltd.

Cisplatin‐induced changes in the number of argyrophilic nucleolar granules in cultured chick dorsal root ganglion neurons

Cisplatin‐induced changes in the number of argyrophilic nucleolar granules in cultured chick dorsal root ganglion neurons

Cisplatin-induced changes in the number of argyrophilic nucleolar granules in cultured chick dorsal root ganglion neurons

Changes in the number of argyrophilic nucleolar granules (ANGs) caused by cisplatin treatment were studied in sensory neurons isolated from dorsal root ganglia of 12-day-old chick embryos. The number of ANGs changed during culture in untreated cultures. The mean number of ANGs per cell was 10.6 + 4.6 at 24 h. A dramatic increase of ANGs (mean number 32.8 + 6.7) was observed after 48 h in vitro, followed by a slight decrease to 26.8 + 0.3 at 72 h. Cisplatin treatment caused a significant decrease of the number of ANGs per neuron depending on the concentration of the drug in culture medium as well as on the exposure time. The profile of changes of ANGs within the time was at low concentrations of cisplatin (1 and 2.5 μg/ml) identical with untreated cultures. The mean number of ANGs was unchanged in the cells exposed for 24 h only and reduced in cultures treated for 48 and 72 h. At higher concentrations of cisplatin (from 5 to 20 μg/ml), a prominent decrease of the mean number of ANGs was observed in all cultures in all time intervals. These findings provide evidence that cisplatin influences the nucleolar organizer regions of dorsal root ganglia neurons. Thus, nucleolar RNA and protein synthesis can be reduced. This alteration of cell metabolism probably contributes to the development of peripheral sensory neuropathy.

Proteins from chromaffin granules promote survival of dorsal root ganglionic neurons: comparison with neurotrophins

Neurotrophins are established survival and differentiation factors for sensory dorsal root ganglionic (DRG) neurons. We have previously shown that proteins from the secretory granules of adrenal chromaffin cells have a capacity to promote the survival of cultured chick DRG neurons. Using DRG neurons from embryonic day (E) 8 chick embryos we show now that this material is (i) as effective as nerve growth factor (NGF), (ii) additive to NGF, neurotrophin-3, or -4, (iii) unlikely to be a neurotrophin, since the survival promoting effect can not be blocked by K252b, a specific inhibitor of the signal transduction pathways of neurotrophin high affinity receptors, (iv) partially blockable by antibodies to transforming growth factor-β (TGF-β) 1/2/3, and (v) more potent than any other out of 30 cytokines tested individually, including fibroblast growth factor (FGF)-5, epidermal growth factor (EGF), TGF-α, platelet-derived growth factor (PDGF)-AB, insulin-like growth factor, (IGF)-I and -II, leukemia inhibitory factor (LIF), TGF-β, glial cell line-derived neurotrophic factor (GDNF), stem cell factor, granulocyte-colony stimulating factor (G-CSF), oncostatin M, tumor necrosis factor (TNF)-α, and interleukins (IL)-1 through-12. We conclude that chromaffin cells, which are known to receive a sensory innervation, can provide (a) trophic factor(s), which, in addition to neurotrophins, may be relebant for the maintenance of DRG neurons.

Synthesis and calcium antagonistic activity of 8-[N-[2-(3,4-dimethoxy- phenyl)ethyl]-beta-alanyl]-5,6,7,8-tetrahydrothieno[3,2-b][1,4]thiazepi ne fumarate.

KT-362 is an antiarrhythmic and antihypertensive agent with vasodilating activity. Since it carries a homoveratryl group in the side chain, an obvious relation exists to the verapamil-type calcium antagonists. Replacement of the fused aromatic moiety in KT-362 with thiophene provided 8-[N-[2-(3,4-dimethoxyphenyl) ethyl]-beta-alanyl]-5,6,7,8-tetrahydrothieno[3,2-b][1,4] thiazepine (1). Compound 1 shows a negative chronotropic activity in spontaneously beating right atria (IC50 = 23 microM, n = 7), and a negative inotropic effect in papillary muscles (IC50 = 2.7 microM, n = 7) and left atria (IC50 = 4 microM, n = 6) of the guinea-pig heart. The decrease of contractility in papillary muscles could be antagonized by increasing the extracellular calcium concentration. Compound 1 was found to affect high (IC50: 70 +/- 5 microM) and low (IC50: 129 +/- 34 microM) voltage-activated calcium channel currents as well as voltage-activated sodium channel currents (IC50: 80 +/- 13 microM) in chick dorsal root ganglion neurons. In addition nicotine-induced currents were potently inhibited (IC50: 6 +/- 0.7 microM) in bovine chromaffin cells.

Human CNTF and related cytokines: effects on DRG neurone survival

Ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), oncostatin M (OSM), interleukin-6 (IL-6), and interleukin-11 (IL-11) are structurally and functionally related cytokines. We compared their survival-promoting activities on embryonic chick and newborn rat dorsal root ganglion (DRG) neurones. Human CNTF showed the well known trophic effect on both chick and rat DRG neurones. Human and murine LIF and, at unphysiologically high doses, human OSM were trophic for rat neurones, but failed to promote chick DRG cell survival. Human IL-11, murine IL-6 and human IL-6 did not improve chick or rat DRG neurone survival; soluble human IL-6 receptor α did not increase sensitivity to human IL-6. Thus, human CNTF as well as murine and human LIF had special neurotrophic properties compared with other related cytokines.

Human CNTF and related cytokines

Ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), oncostatin M (OSM), interleukin-6 (IL-6), and interleukin-11 (IL-11) are structurally and functionally related cytokines. We compared their survival-promoting activities on embryonic chick and newborn rat dorsal root ganglion (DRG) neurones. Human CNTF showed the well known trophic effect on both chick and rat DRG neurones. Human and murine LIF and, at unphysiologically high doses, human OSM were trophic for rat neurones, but failed to promote chick DRG cell survival. Human IL-11, murine IL-6 and human IL-6 did not improve chick or rat DRG neurone survival; soluble human IL-6 receptor alpha did not increase sensitivity to human IL-6. Thus, human CNTF as well as murine and human LIF had special neurotrophic properties compared with other related cytokines.