Schwann cells promote neurite outgrowth of dorsal root ganglion neurons through secretion of nerve growth factor

The transplantation of Schwann cells (SCs) could successfully promote axonal regeneration. This is likely to attribute to the adhesion molecules expression and growth factors secretion of SCs. But which factor(s) play a key role has not been precisely studied. In this study, an outgrowth assay using dorsal root ganglia (DRG) neuron-SC co-culture system in vitro was performed. Co-culture of SCs or application of SC-conditioned medium (CM) substantially and significantly increased DRG neurite outgrowth. Further, nerve growth factor and NGF receptor (TrkA) mRNA were highly expressed in Schwann cells and DRG neuron, respectively. The high concentration of NGF protein was detected in SC-CM. When K-252a, a specific inhibitor of NGF receptor was added, DRG neurite outgrowth was significantly decreased in a concentration-dependent manner. These data strongly suggest that SCs play important roles in neurite outgrowth of DRG neurons by secreted NGF.     

Ionic behaviors and nerve growth factor dependence in developing chick ganglia. II. Studies with neurons of dorsal root ganglia

Using intact dorsal root ganglia (DRG) from embryonic (E) chick and measuring 22Na+ accumulation, the authors have recently shown that (i) ionic control by the ganglia has a complete requirement for exogenous NGF between E6 and E10, and (ii) control of ion pump mechanisms independent of exogenous NGF is progressively acquired by these ganglia from E10 to E16. Similar experiments have now been carried out using enriched suspensions of ganglionic neurons to test whether the acquisition of endogenous control by older ganglia was (1) due to the close association between neurons and nonneurons, and (2) correlated with a decreasing need by these neurons for exogenous NGF for survival in culture. In this enriched neuronal population, Na+ accumulation in the absence of NGF increases from E7 to E10, paralleling the increase in Na+ accessible space under ouabain, but then decreases conspicuously between E10 and E16, despite little change in the ouabain-sensitive Na+ space. NGF prevents Na+ accumulation during the early period, and becomes increasingly irrelevant for this behavior in later (after E10) development. K+ movements (traced with 86Rb+) behaved similarly. Active K+ influx (Na+, K+-pump mediated) also increases severalfold between E7 and E10. This K+ influx is sensitive to NGF at E7 and E10 but not at E14, paralleling the observed Na+ and K+ behaviors. These data suggest that the control of Na+, K+-pump performances acquired by these neurons between E10 and E16 represents the development of a neuronal self-sufficiency. This increase in ionic control is not due to an increase in pump molecules or pumping efficiency. No increases in the binding of [3H]ouabain or active K+ influx occur between E10 and E16, when ionic control is developing. The ionic dependence on NGF by the DRG neurons changes with their developmental age along the same temporal pattern displayed by their survival response to NGF in culture.     

Okadaic acid reversibly inhibits neurite outgrowth in embryonic dorsal root ganglion neurons

The aim of this study was to assess the effects of low concentrations of okadaic acid (OA) on neurite outgrowth and cellular integrity in cultures of dissociated dorsal root ganglion (DRG) neurons. The complete and fully reversible arrest of neurite outgrowth was achieved at 1 nM OA, thus ruling out the involvement of protein phosphatase 1 in the observed inhibitory effect. OA at 0.5 nM did not completely block neurite outgrowth, although it reduced the rate of growth by about one third. Protein phosphorylation and the integrity of microtubules and neurofilaments in neuron-enriched cultures were unaffected by 1 nM OA. The rate of synthesis of the low-molecular-weight neurofilament subunit (NFL) was also unchanged by OA treatment. Antimitotic agents used to eliminate proliferating cells did not alter the rate of neurite elongation. Since 1 nM OA does not suffice to inhibit neuronal protein phosphatase 2A fully, owing to the high concentration of this enzyme in neurons, we propose that the inhibitor is affecting a neuronal compartment that contains low levels of the phosphatase. This putative compartment is likely to be located in neurites, which were shown to contain levels of protein phosphatase 2A that were two- to threefold lower than in neuronal perikarya. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 193–201, 1997.     

Neutrophic influence of denervated sciatic nerve of on adult dorsal root ganglion neurons

Isolated adult frog dorsal root ganglion neurons survive in vitro in a defined medium for more than 4 weeks and extend processes. When co-cultured with a 1-mm piece of peripheral nerve the average tottal process lenght per neuron was 10 times longer than that of control neurons by 8 days, and the processes had a significantly different morphology from that of control neurons. This influence on process length increased with increasing time of nerve denervation length increased with increasing time of nerve denervation prior to co-culturing. These results suggest the release of a neurotrophic factor/s from the cells of the peripheral nerve. The neurotropic influence was completely blocked by antibodies against mouse nerve growth factor (NGF). Although NGF increased the average process length by twofold over control neurons, its influence never reached that of the nerve-released factor, and the NGF-induced processes had a distinctly different morphology. The frog nerve-released factor promoted process outgrowth from E11 chick sympathetic ganglia, although the process number, length, and their fasciculation differed greatly from those induced by NGF. These results suggest that the nerve-released factor/s are immunologically and functionally related to NGF but have not estabished whether a single factor or an aggregate of several secreted molecules are responsible. This article presents a new preparation in which the varied influences of different neurotrophic factors can be studied in great detail on large populations of isolated adult vertebrte neurons and sets the stage for the characterization and isolation of the frog peripheral nerve neurotrophic factor, as well as examining the influence of this facor on neuronal morphology and its ability to direct process outgrowth. 1994 John Wiley & Sons, Inc.     

Influence of factors released from sciatic nerve on adult dorsal root ganglion neurons

Previous experiments have shown that medium conditioned (CM) by denervated peripheral nerve contains a process outgrowth promoting factor (s) for cultured adult frog dorsal root ganglion (DRG) neurons. The present experiments further characterize the influences of these factors on DRG neurons. The growth factors increases average process length by threefold, restricts the number of processes extended from four to two while simultaneously altering the morphology of those processes. Neurons with preexisting processes respond to the factors by significantly increasing the length of 35% of these processes. Only the newly elongated portions of preexisting processes have morphology typical of factor-induced processes, while the previously extended portions retain their original morphology. The number of processes of these neurons remains unchanged. Although composed of two population according to size, neurons in both populations are similarly influenced, suggesting that the factors influence neurons of all sensory modalities. To look at other possible influences of the nerve-released factors, a novel simple culture system has been developed in which concentration gradients of these factors can be established and maintained. The front of the outgrowth-promoting influence in these cultures could be followed over time (up to 9 days) as it affected the process length and morphology of neurons at increasing distances (up to 8 mm) from the source of the factors. The trophic factors may play important roles during regeneration in vivo by influencing the cytoskeletal organization in the cell body and growth cones to bring about a stabilization and consolidation of growth cone membrane of only a limited number of processes resulting in increasing the rate of process elongation. The factors may also serve to direct process outgrowth, which can be examined using the new culture system. 1994 John Wiley & Sons, Inc.     

Effects of ion channel activity on development of dorsal root ganglion neurons

Studies of mouse dorsal root ganglion neurons in vitro demonstrate that ion channel function and regulation can influence a wide range of developmental processes. The work suggests that much as exposure to different trophic factors, the pattern of impulse activity a neuron experiences can have significant structural and functional effects during development. Studies concerning effects of ion channel activity on growth cone motility, axon fasciculation, synaptic plasticity, myelination, and intracellular signaling pathways regulating gene expression are presented in the context of changes in endogenous firing patterns during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 158–170, 1998     

Radiation-induced apoptosis in dorsal root ganglion neurons

Ionizing radiation (IR) results in apoptosis in a number of actively proliferating or immature cell types. The effect of IR on rat dorsal root ganglion (DRG) neurons was examined in dissociated cell cultures. After exposure to IR, embryonic DRG neurons, established in cell culture for six days, underwent cell death in a manner that was dose-dependent, requiring a minimum of 8 to 16 Gy. Twenty-five per cent cell loss occurred in embryonic day 15 (E-15) neurons, grown in cell culture for 6 days (“immature”), and then treated with 24 Gy IR. In contrast, only 2% cell loss occurred in E-15 neurons maintained in culture for 21 days ("mature") and then treated with 24 Gy IR. Staining with a fluorescent DNA-binding dye demonstrated clumping of the nuclear chromatin typical of apoptosis. Initiation of the apoptosis occurred within 24 h after exposure to IR. Apoptosis was prevented by inhibition of protein synthesis with cycloheximide. Apoptosis induced by IR occurred more frequently in immature than in mature neurons. Immature DRG neurons have a lower concentration of intracellular calcium ([Ca2+]i) than mature neurons. Elevation of [Ca2+]i by exposure to a high extracellular potassium ion concentration (35 μM) depolarizes the cell membrane with a resultant influx of calcium ions. The activation of programmed cell death after nerve growth factor (NGF) withdrawal is inversely correlated with [Ca2+]i in immature DRG neurons. When treated with high extracellular potassium, these immature neurons were resistant to IR exposure in a manner similar to that observed in mature neurons. These data suggest that [Ca2+]i modulates the apoptotic response of neurons after exposure to IR in a similar manner to that proposed by the “Ca2+ setpoint hypothesis” for control of NGF withdrawal-induced apoptosis.     

Inositol trisphosphate-induced hyperpolarization in rat dorsal root ganglion neurons.

Inositol 1,4,5-trisphosphate (1,4,5-InsP3) was perfused into rat dorsal root ganglion (DRG) neurons by whole-cell patch-clamp electrodes, while measuring the membrane potential. This operation evoked a transient (2-3 min) membrane hyperpolarization of about -15 mV (from -42 mV) followed by a depolarization. The membrane hyperpolarization was abolished when 30 mM EGTA was perfused together with 1,4,5-InsP3 or when 0.2 mM quinine was added to the bath solution. The hyperpolarizing response was enhanced when a low-Ca2+ EGTA-free intracellular solution was used. Two InsP2 isomers induced a different response. Our results suggest that the hyperpolarization is due to 1,4,5-InsP3-induced Ca2+ release which may trigger Ca-sensitive K+ channels to open. Present results show that cultured DRG neurons are able to respond to 1,4,5-InsP3 perfusion in the whole-cell configuration.     

MicroRNA-143 expression in dorsal root ganglion neurons

The unpleasant sensory and emotional experience of pain is initiated by excitation of primary afferent nociceptive neurons. Nerve damage or inflammation induces changes in nociceptive DRG neurons which contribute to both peripheral and central sensitization of pain-sensitive pathways. Recently, blockade of microRNA synthesis has been found to modulate the response of nociceptive neurons to inflammatory stimuli. However, little is known about the contributions of individual miRNAs to painful conditions. We compared miRNA expression in mouse sensory neurons and focussed on the localisation and control of miR-143. Using miRNA-arrays we compared the microRNA expression profile of intact lumbar DRG with one-day-old DRG cultures and found that nine miRNAs including miR-143 showed lower expression levels in cultures. Subsequent RT-qPCR confirmed array data and in-situ hybridisation localised miR-143 in the cytosol of sensory DRG neurons in situ and in vitro. Analysis of microbead-enriched neuron cultures showed significantly higher expression levels of miR-143 in isolectin B4 (I-B4) binding sensory neurons compared with neurons in the I-B4 negative flow-through fraction. In animal models of peripheral inflammation (injection of Complete Freund's Adjuvant, CFA) and nerve damage (transection of the sciatic nerve), we found that expression levels of miR-143 were significantly lower in DRGs ipsilateral to CFA injection or after nerve damage. Taken together, our data demonstrate for the first time miR-143 expression in nociceptive neurons. Since expression levels of miR-143 were higher in I-B4 positive neurons and declined in response to inflammation but not axotomy, miR-143 could selectively contribute to mRNA regulation in specific populations of nociceptors.     

Neurotoxicity caused by didanosine on cultured dorsal root ganglion neurons

The purpose of the present study was to investigate whether didanosine (ddI) directly causes morphological and ultrastructural abnormalities of dorsal root ganglion (DRG) neurons in vitro. Dissociated DRG cells and organotypic DRG explants from embryonic 15-day-old Wistar rats were cultured for 3 days and then exposed to ddI (1 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) for another 3 days and 6 days, respectively. Neurons cultured continuously in medium served as normal controls. The diameter of the neuronal cell body and neurite length were measured in dissociated DRG cell cultures. Neuronal ultrastructural changes were observed in both culture models. ddI induced dose-dependent decreases in neurite number, length of the longest neurite in each neuron, and total neurite length per neuron in dissociated DRG cell cultures with 3 days treatment. There were no morphological changes seen in organotypic DRG cultures even with longer exposure time (6 days). But ddI induced ultrastructural changes in both culture models. Ultrastructural abnormalities included loss of cristae in mitochondria, clustering of microtubules and neurofilaments, accumulation of glycogen-like granules, and emergence of large dense particles between neurites or microtubules. Lysosome-like large particles emerged inconstantly in neurites. ddI induced a neurite retraction or neurite loss in a dose-dependent manner in dissociated DRG neurons, suggesting that ddI may partially contribute to developing peripheral neuropathy. Cytoskeletal rearrangement and ultrastructural abnormalities caused by ddI in both culture models may have a key role in neurite degeneration.     

Neuroprotective properties of ciliary neurotrophic factor for cultured adult rat dorsal root ganglion neurons

We observed that recombinant ciliary neurotrophic factor (CNTF) enhanced survival and neurite outgrowth of cultured adult rat dorsal root ganglion (DRG) neurons. Among other neurotrophic factors (NGF and GDNF) and interleukin (IL)-6 cytokine members [IL-6, LIF, cardiotrophin-1, and oncostatin M (OSM)] at the same concentration (50 ng/ml), CNTF, as well as LIF and OSM, displayed high efficacy for the promotion of the number of viable neurons and neurite-bearing cells. CNTF enhanced the number of neurite-bearing cells in both small neurons (soma diameter <30 mum) and large neurons (soma diameter >/=30 mum), whereas NGF and GDNF promoted that in only small neurons. Western blot analysis revealed that CNTF induced phosphorylation of STAT3, Akt, and ERK1/2 in the neurons. Furthermore, the neurite outgrowth-promoting activity of CNTF was diminished by co-treatment with Janus kinase (JAK) 2 inhibitor, AG490; STAT3 inhibitor, STA-21; phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor, LY294002; and mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, in a concentration-dependent manner. Its survival-promoting activity was also affected by AG490, STA-21, and LY294002 at higher concentrations, but not by PD98059. These findings suggest the involvement of JAK2/STAT3, PI3K/Akt, and MEK/ERK signaling pathways in CNTF-induced neurite outgrowth, where the former two pathways are thought to play major roles in mediating the survival response of neurons to CNTF.     

Calcium- and dynamin-independent endocytosis in dorsal root ganglion neurons

Synaptic vesicle endocytosis is believed to require calcium and the GTPase dynamin. We now report a form of rapid endocytosis (RE) in dorsal root ganglion (DRG) neurons that, unlike previously described forms of endocytosis, is independent of calcium and dynamin. The RE is tightly coupled to calcium-independent but voltage-dependent secretion (CIVDS). Using FM dye and capacitance measurements, we show that membrane depolarization induces RE in the absence of calcium. Inhibition of dynamin function does not affect RE. The magnitude of RE is proportional to that of preceding CIVDS and stimulation frequency. Inhibitors of protein kinase A (PKA) suppress RE induced by high-frequency depolarization, while PKA activators enhance RE induced by low-frequency depolarization. Biochemical experiments demonstrate that depolarization directly upregulates PKA activity in calcium-free medium. These results reveal a calcium- and dynamin-independent form of endocytosis, which is controlled by neuronal activity and PKA-dependent phosphorylation, in DRG neurons.     

Sodium channels in cultured chick dorsal root ganglion neurons

Isolated Na currents were studied in cultured chick sensory neurons using the patch clamp technique. On membrane depolarization, whole cell currents showed the typical transient and voltage-dependent time course as in nerve fibres. Na currents appeared at about-40 mV and reached maximum amplitude at around-10 mV. At low voltages (-30 to 0 mV), their turning-on was sigmoidal and inactivation developed exponentially. The ratio of inactivation time constants was found to be smaller than in squid axons and comparable to that of mammalian nodes of Ranvier. Peak conductance and steady-state inactivation were strongly voltage-dependent, with maximum slopes at-17 and-40 mV, respectively. The reversal potential was close to the Nernst equilibrium potential, indicating a high degree of ion-selectivity for the channel. Addition of 3M TTX, or replacement of Na by Choline in the external bath, abolished these currents. Internal pronase (1 mg/ml) and N-bromoacetamide (0.4 mM) made inactivation incomplete, with little effect on its rate of decay.Single Na channel currents were studied in outside-out membrane patches, at potentials between-50 and-20 mV. Their activation required large negative holding potentials (-90 mV). They were fully blocked by addition of TTX (3 M) to the external bath. At-40 mV their mean open time was about 2ms and the amplitude distribution could be fitted by a single Gaussian curve, indicating the presence of a homogeneous population of channels with a conductance of 11±2 pS. Probability of opening increased and latency to first opening decreased with increasing depolarization. Inactivation of the channel became faster with stronger depolarizations, as measured from the inactivation time course of sample averages. Internal pronase (0.1 mg/ml) produced effects on inactivation comparable to those on whole cell currents. Openings of the channel had a tendency to occur in bursts and showed little inactivation during pulses of 250 ms duration. The open lifetime of the channel at low potentials (-50,-40 mV) was only three times larger than in control patches, suggesting that Na channels in chick sensory neurons can close several times before entering an inactivating absorbing state.