Cultured Rat Sympathetic Neurons Research Articles

Target tissue influence on somatostatin expression in the avian ciliary ganglion.

Activin as a neurodifferentiation factor. Our studies of neurotransmitter expression have focused on the expression of neuropeptide transmitters in the avian ciliary ganglion (CG) and have examined the influence of choroidal vascular smooth muscle cells in regulating the differential expression of somatostatin in the CG. In these activities we have identified activin A as a potential target-derived neurodifferentiation factor that can stimulate somatostatin expression in cultured CG neurons. In cultured CG neurons, activin can stimulate the expression of somatostatin in choroid neurons, the pattern of neurotransmitter expression found in vivo, and in the ciliary neurons that would normally not express somatostatin. In vivo, mRNA transcripts of the cActR-IIA appear to be expressed by both choroid and ciliary CG neurons. This suggests that activin might serve as an instructive factor in controlling neuropeptide phenotype. For activin to serve as an instructive factor requires that activin be produced by choroid smooth-muscle target cells. Indeed, activin mRNA and activin-like immunoreactivity are found in choroid cells, in vitro. However, the lack of somatostatin expression by ciliary neurons suggests that activin is not produced by their targets, the iris and ciliary body. This simple view is countered by the observation that activin A mRNA is also present in the iris and activin-like immunoreactivity is detectable in the iris and ciliary body. Instead, the production of the specific activin inhibitor follistatin in the iris and ciliary body is likely to limit the availability of activin to only those neurites innervating the choroid layer, thus accounting for the differential expression of somatostatin in only the choroid CG neurons. This somewhat more complicated arrangement is similar to the mechanism thought to be employed for primary induction during frog embryogenesis. The observations reviewed here are all consistent with the hypothesized role for activin as a molecule whose availability to neurites in the target regulates neurotransmitter expression. Additional in vivo perturbation experiments are needed to further examine this hypothesis; nevertheless, activin appears as a strong candidate for a target-derived neurotransmitter differentiation factor. Activin's potential roles in differentiation: A wide variety of biological effects have been ascribed to activin. Initially identified and purified as a gonadal hormone stimulating the production and release of FSH from the pituitary, activin is also implicated in the stimulation of erythroid differentiation, as a modulator of follicular granulosa cell differentiation, as a mesodermalizing factor in both amphibian and avian early development, and as a component in establishing left-right axial patterning in the chicken embryo. Activin has also been found to be a survival factor for several neuronal cell lines and for rat embryonic neural retina cells in culture. However, activin is not a survival factor for chicken CG neurons in culture. Our observation that activin may play a function in target-derived control of neuropeptide expression adds yet another aspect to the list of its potential biological functions. In addition, activin shares regions of amino acid sequence identity with members of the TGF-beta superfamily, which includes the TGF-betas, Mullerian inhibitory substance, Drosophila decapentaplegic gene product, dorsalin, bone morphogenetic proteins, inhibin, and glial-derived neurotrophic factor. Interestingly, these are all factors that have effects upon cellular differentiation. Effects of activin on other neurons. Activin A--as well as two other TGF-beta superfamily members, BMP-2 and BMP-6--has been shown to induce expression of mRNAs for several neuropeptides in cultured rat sympathetic neurons. In addition, activin A induces ChAT mRNA in cultured sympathetic neurons. (ABSTRACT TRUNCATED)

Retrograde Transport and Steady-State Distribution of125I-Nerve Growth Factor in Rat Sympathetic Neurons in Compartmented Cultures

We have used compartmented cultures of rat sympathetic neurons to quantitatively examine the retrograde transport of 125I-nerve growth factor (NGF) supplied to distal axons and to characterize the cellular events that maintain steady-state levels of NGF in cell bodies. In cultures allowed to reach steady-state 125I-NGF transport, cell bodies contained only 5-30% of the total neuron-associated 125I-NGF, whereas 70-95% remained associated with the distal axons. This was true over an 8 pM to 1.5 nM 125I-NGF concentration range, indicating that saturation of high affinity receptors could not account for the large fraction of 125I-NGF remaining in axons. Dissociation assays indicated that 85% of 125I-NGF associated with distal axons was surface-bound. At steady-state, only 2-25% of the distal axon-associated 125I-NGF was retrogradely transported each hour, with higher transport rates associated with younger cultures and lower 125I-NGF concentrations. The velocity of 125I-NGF retrograde transport was estimated at 10-20 mm/hr. However, as in a previous report, almost no 125I-NGF transport was observed during the first hour after 125I-NGF administration, indicating a significant lag between receptor binding and loading onto the retrograde transport system. During 125I-NGF transport through axons spanning an intermediate compartment in five-compartment cultures, little or no 125I-NGF was degraded or released from the axons. After transport, 125I-NGF was degraded with a half-life of 3 hr. In summary, although some cellular events promoted NGF accumulation in cell bodies, distal axons represented by far the principal site of NGF-receptor interaction at steady-state as a result of a low retrograde transport rate.

Elevation of Ceramide within Distal Neurites Inhibits Neurite Growth in Cultured Rat Sympathetic Neurons

Sphingolipids are abundant constituents of neuronal membranes and have been implicated in intracellular signaling. We show that two analogs of glycosphingolipid biosynthetic intermediates, fumonisin B1 (which inhibits dihydroceramide synthesis) and DL-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) (which inhibits glucosylceramide synthesis) decrease glycosphingolipid synthesis in rat sympathetic neurons. Although both fumonisin and PPMP inhibit glycosphingolipid synthesis, these inhibitors have differential effects on ceramide metabolism in axons. threo-PPMP, but not erythro-PPMP or fumonisin, induces an accumulation of [3H]palmitate-labeled ceramide and impairs axonal growth. Moreover, exogenously added, cell-permeable C6-ceramide, but not C6-dihydroceramide, mimicks the effect of PPMP. Our studies suggest that the lipid second messenger ceramide acts in distal axons, but not cell bodies, as a negative regulator of neurite growth.

Genetic and metabolic status of NGF-deprived sympathetic neurons saved by an inhibitor of ICE family proteases.

Sympathetic neurons undergo programmed cell death (PCD) when deprived of NGF. We used an inhibitor to examine the function of interleukin-1 beta-converting enzyme (ICE) family proteases during sympathetic neuronal death and to assess the metabolic and genetic status of neurons saved by such inhibition. Bocaspartyl(OMe)-fluoromethylketone (BAF), a cell-permeable inhibitor of the ICE family of cysteine proteases, inhibited ICE and CPP32 (IC50 approximately 4 microM) in vitro and blocked Fas-mediated apoptosis in thymocytes (EC50 approximately 10 microM). At similar concentrations, BAF also blocked the NGF deprivation-induced death of rat sympathetic neurons in culture. Compared to NGF-maintained neurons, BAF-saved neurons had markedly smaller somas and maintained only basal levels of protein synthesis; readdition of NGF restored growth and metabolism. Although BAF blocked apoptosis in sympathetic neurons, it did not prevent the fall in protein synthesis or the increase in the expression of c-jun, c-fos, and other mRNAs that occur during neuronal PCD, implying that the ICE-family proteases function downstream of these events during PCD.NGF and BAF rescued sympathetic neurons with an identical time course, suggesting that NGF, in addition to inhibiting metabolic and genetic events associated with neuronal PCD, can act posttranslationally to abort apoptosis at a time point indistinguishable from the activation of cysteine proteases. Both poly-(ADP ribose) polymerase and pro-ICE and Ced-3 homolog-1 (ICH-1) appear to be cleaved in a BAF-inhibitable manner, although the majority of pro-CPP32 appears unchanged, suggesting that ICH-1 is activated during neuronal PCD. Potential implications of these findings for anti-apoptotic therapies are discussed.

Histone hyperacetylating agents stimulate promoter activity of human choline acetyltransferase gene in transfection experiment

Butyrate (5 mM), Trichostatin A (1 microM) or Trapoxin A (30 nM) increased choline acetyltransferase (ChAT) activity in cultured rat sympathetic neurons 3- to 8-fold in 2 days. On the contrary, the three drugs decreased ChAT activity in human CHP126 cells. Butyrate had little effect on ChAT mRNA level in these cells, suggesting post-transcriptional mechanisms for the decrease in ChAT activity. However, transient transfection experiments using CHP126 cells revealed that the M promoter, but not the R promoter, of human ChAT gene was activated 20- to 130-fold by the three hyperacetylating agents. A butyrate-responsive element was localized in the 1 kbp region upstream of exon M. Constructs containing in addition the genomic segment between exons M and 1 displayed maximal basal activity and inducibility by butyrate, suggesting the presence of butyrate-activated promoter/enhancer elements in this region. The stimulatory effects of butyrate and Trichostatin A were also observed in stably transfected CHP126 clones, suggesting that the chromatin environment was not preventing the induction of the endogenous ChAT gene by butyrate. Rather, the data suggest different chromatin organizations for the stable transgene and the endogenous ChAT gene.

THE EFFECTS OF EXTRACELLULAR MATRIX AND OSTEOGENIC PROTEIN-1 ON THE MORPHOLOGICAL DIFFERENTIATION OF RAT SYMPATHETIC NEURONS

The growth patterns of axons and dendrites differ with respect to their number, length, branching, and spatial orientation; therefore, it is likely that these processes differ in their growth requirements. To examine this hypothesis, we have been analyzing the responses of cultured rat sympathetic neurons to three types of stimuli: large structural proteins of the extracellular matrix, matrix-associated growth factors, and neurotrophins. Purified structural proteins such as laminin and collagen IV have been found to promote only axonal growth; whereas the matrix associated growth factor, osteogenic protein-1, selectively stimulates dendritic growth. In contrast, nerve growth factor modulates the growth of both types of processes. These data suggest that process-specific interactions with the extracellular environment may be critical determinants of cell shape in neurons. Perinatal rat sympathetic neurons grown in culture in the absence of serum or glial cells extend a single process which is axonal in nature. Exposure to osteogenic protein-1 causes the formation of additional processes which express the morphological, cytoskeletal, and ultrastructural characteristics of dendrites. Consistent with observations on the regulation of dendritic growth in sympathetic neurons in situ, the dendrite-promoting activity of osteogenic protein-1 is independent of synaptic or electrical activity, but is modulated by nerve growth factor. In the presence of optimal concentrations of osteogenic protein-1 and nerve growth factor, the size of the dendritic arbor extended by cultured sympathetic neurons approximates that seen in situ at comparable developmental stages. Osteogenic protein-1 does not promote dendritic growth in cultured neurons obtained from embryonic ciliary, dorsal root, trigeminal or nodose ganglia, suggesting that its morphogenetic effects are cell selective. Since mRNA for osteogenic protein-1 is expressed in mature as well as embryonic target tissues of the sympathetic nervous system, we also examined the effects of osteogenic protein-1 on cultures of sympathetic neurons derived from adult rats. Consistent with results obtained with perinatal neurons, osteogenic protein-1 selectively promoted dendritic growth in adult neurons. These data suggest that this matrix-associated growth factor could play a role not only in the morphogenesis of the developing nervous system, but also in the maintenance and remodeling of dendritic structures in the mature animal. Copyright © 1996 ISDN. Published by Elsevier Science Ltd.

Proton sensitivity of the GABA(A) receptor is associated with the receptor subunit composition.

1. Modulation of GABA(A) receptors by external H(+) was examined in cultured rat sympathetic neurones, and in Xenopus laevis oocytes and human embryonic kidney (HEK) cells expressing recombinant GABA(A) receptors composed of combinations of alpha 1, beta 1, beta 2, gamma 2S and delta subunits. 2. Changing the external pH from 7.4 reduced GABA-activated currents in sympathetic neurones. pH titration of the GABA-induced current was fitted with a pH model which predicted that H(+) interact with two sites (PK(a) values of 6.4 and 7.2). 3. For alpha 1 beta 1 GABA(A) receptors, low external pH (< 7.4) enhanced responses to GABA. pH titration predicted the existence of two sites with PK(a) values of 6.6 and 7.5. The GABA concentration-response curve was shifted to the left by low pH and non-competitively inhibited at high pH (> 7.4). 4. alpha 1 beta 1 gamma 2S receptor constructs were not affected by external pH, whereas exchanging the beta 1 subunit for beta 2 conferred a sensitivity to pH, with predicted PK(a) values of 5.16 and 9.44. 5. Low pH enhanced the responses to GABA on alpha 1 beta 1 delta subunits, whilst high pH caused an inhibition (PK(a) values of 6.6 and 9.9). The GABA concentration-response curves were enhanced (pH 5.4) or reduced (pH 9.4) with no changes in the GABA EC(50). 6. Immunoprecipitation with subunit and epitope-specific antisera to alpha 1, beta 1 and delta subunits demonstrated that these subunits could co-assemble in cell membranes. 7. Expression of alpha 1 beta 1 gamma 2S delta constructs resulted in a 'bell-shaped' pH titration relationship. Increasing or decreasing external pH inhibited the responses to GABA. 8. The pH sensitivity of recombinant GABA(A) receptors expressed in HEK cells was generally in accordance with data accrued from Xenopus oocytes. However, rapid application of GABA to alpha 1 beta 1 constructs at high pH (> 7.4) caused an increased peak and reduced steady-state current, with a correspondingly increased rate of desensitization. 9. Modulation of GABA(A) receptor function was apparently unaffected by the internal pH. Moreover, pH values between 5 and 9.5 did not significantly affect the charge distribution on the zwitterionic GABA molecules. 10. In conclusion, this study demonstrates that external pH can either enhance, have little effect, or reduce GABA-activated responses, and this is apparently dependent on the receptor subunit composition. The potential importance of H(+) sensitivity of GABA(A) receptors is discussed.

Antisense oligodeoxynucleotides to bax mRNA promote survival of rat sympathetic neurons in culture.

Previous in vitro studies have shown that the presence of high levels of Bax protein accelerated the rate of cell death following growth factor deprivation and that the ratio of cell death repressor Bcl-2 to cell death effector Bax may determine the susceptibility to apoptosis. Both Bcl-2 and Bax protein expression has been detected in sympathetic neurons in vivo, and overexpression of bcl-2 in cultured sympathetic neurons prevented apoptosis after deprivation of nerve growth factor (NGF). In the present study, we investigated the expression of bax and bcl-2 in primary cultures of sympathetic neurons from rat superior cervical ganglia. Furthermore, we tested the effects of a partially phosphorothioated bax antisense oligodeoxynucleotide (ODN) on the survival of sympathetic neurons in cultures supplied with suboptimal concentrations of NGF (0.5 ng/ml). A constitutive expression of bax mRNA at high levels was detected by reverse transcription and polymerase chain reaction which did not change significantly following NGF reduction or treatment with bax antisense ODN. A decrease in Bcl-2 immunoreactivity was observed by immunocytochemistry in tyrosine hydroxylase-positive neurons when cultured under suboptimal NGF concentrations, whereas Bcl-2 immunolabeled non-neuronal cells were not affected. Maximal number of neurons was obtained in control cultures containing 50 ng/ml of NGF. Few neurons survived in cultures grown in 0.5 ng/ml of NGF for 2 days (12.0 +/- 1.5% of controls, mean +/- SEM). Addition of two control ODNs at 1 microM had no effect on neuronal survival (10.1 +/- 1.2% and 11.0 +/- 1.3%, respectively), while the number of neurons was significantly increased in NGF-reduced cultures treated with a bax antisense ODNs (1 microM) (31.5 +/- 1.9%). Administration of fluorescein-labeled ODNs demonstrated intracellular uptake into cultured neurons. Treatment with bax antisense ODNs caused a significant reduction of Bax protein levels in SCG neurons by 46 +/- 2.6% as assessed by immuno-cytochemistry and digital image analysis. Taken together, our data demonstrate a constitutive expression of bax mRNA in sympathetic neurons suggesting that activation of bax expression may not be required for neuronal cell death after NGF withdrawal. After changing to suboptimal NGF concentrations, the cell-specific reduction in Bcl-2 immunoreactivity preceded morphological signs of degeneration indicating that growth factor starvation may down-regulate neuronal bcl-2 expression. Treatment with bax antisense ODNs indicated that suppression of Bax protein synthesis may promote neuronal survival in the threshold situation of insufficient trophic support.

Antisense oligodeoxynucleotides to bax mRNA promote survival of rat sympathetic neurons in culture

Previous in vitro studies have shown that the presence of high levels of Bax protein accelerated the rate of cell death following growth factor deprivation and that the ratio of cell death repressor Bcl-2 to cell death effector Bax may determine the susceptibility to apoptosis. Both Bcl-2 and Bax protein expression has been detected in sympathetic neurons in vivo, and overexpression of bcl-2 in cultured sympathetic neurons prevented apoptosis after deprivation of nerve growth factor (NGF). In the present study, we investigated the expression of bax and bcl-2 in primary cultures of sympathetic neurons from rat superior cervical ganglia. Furthermore, we tested the effects of a partially phosphorothioated bax antisense oligodeoxynucleotide (ODN) on the survival of sympathetic neurons in cultures supplied with suboptimal concentrations of NGF (0.5 ng/ml). A constitutive expression of bax mRNA at high levels was detected by reverse transcription and polymerase chain reaction which did not change significantly following NGF reduction or treatment with bax antisense ODN. A decrease in Bcl-2 immunoreactivity was observed by immunocytochemistry in tyrosine hydroxylase-positive neurons when cultured under suboptimal NGF concentrations, whereas Bcl-2 immunolabeled non-neuronal cells were not affected. Maximal number of neurons was obtained in control cultures containing 50 ng/ml of NGF. Few neurons survived in cultures grown in 0.5 ng/ml of NGF for 2 days (12.0 ± 1.5% of controls, mean ± SEM). Addition of two control ODNs at 1 μM had no effect on neuronal survival (10.1 ± 1.2% and 11.0 ± 1.3%, respectively), while the number of neurons was significantly increased in NGF-reduced cultures treated with a bax antisense ODNs (1 μM) (31.5 ± 1.9%). Administration of fluorescein-labeled ODNs demonstrated intracellular uptake into cultured neurons. Treatment with bax antisense ODNs caused a significant reduction of Bax protein levels in SCG neurons by 46 ± 2.6% as assessed by immunocytochemistry and digital image analysis. Taken together, our data demonstrate a constitutive expression of bax mRNA in sympathetic neurons suggesting that activation of bax expression may not be required for neuronal cell death after NGF withdrawal. After changing to suboptimal NGF concentrations, the cell-specific reduction in Bcl-2 immunoreactivity preceded morphological signs of degeneration indicating that growth factor starvation may down-regulate neuronal bcl-2 expression. Treatment with bax antisense ODNs indicated that suppression of Bax protein synthesis may promote neuronal survival in the threshold situation of insufficient trophic support. © 1996 Wiley-Liss, Inc.

Role of axons in membrane phospholipid synthesis in rat sympathetic neurons

The axonal synthesis of phospholipids has been demonstrated in compartmented cultures of rat sympathetic neurons. In this model of neuron culture, metabolic events occurring in distal axons were studied independently of those occurring in cell bodies. Using radiolabeled tracers the axonal biosynthesis of the major membrane phospholipids and fatty acids but not cholesterol was detected. The capacity of axons for synthesis of phosphatidycholine (PC), the major membrane lipid, was confirmed by the demonstration that key enzymes of PC biosynthesis were present in distal axons. A double-labeling experiment showed that at least 50% of axonal PC was synthesized locally in axons, with the remainder being made in cell bodies and transported into axons. The requirement of axonal PC synthesis for axonal elongation was investigated. When PC biosynthesis in distal axons alone was inhibited by two independent approaches (deprivation of choline or addition of the inhibitor hexadecylphosphocholine) axonal growth was markedly retarded. Our experiments demonstrated that PC synthesis in cell bodies was neither necessary nor sufficient for growth of distal axons, whereas local synthesis of PC in distal axons was required for normal axonal elongation.

Impairment of syntaxin by botulinum neurotoxin C1 or antibodies inhibits acetylcholine release but not Ca2+ channel activity.

The involvement of syntaxin, an omega-conotoxin-sensitive Ca2+ channel-associated protein, in acetylcholine release was studied at synapses formed between rat sympathetic neurons in culture. Transmission at these synapses involved omega-conotoxin-sensitive Ca2+ channels because a dose-dependent inhibition was observed when omega-conotoxin was bath-applied. Confocal microscope examination of immunofluorescent staining showed that syntaxin had a similar distribution to synaptic vesicle-associated membrane proteins, synaptophysin and vesicle-associated membrane protein/synaptobrevin-2, indicating that syntaxin molecules are concentrated in the presynaptic terminals. Botulinum neurotoxin C1 applied extracellularly or intracellularly into presynaptic neurons blocked synaptic transmission. Introduction of a monoclonal antibody, or polyclonal antibodies, to syntaxin into the presynaptic neuron depressed the evoked release of acetylcholine without affecting Ca2+ influx through Ca2+ channels. These results suggest that syntaxin plays an important role in release of neurotransmitter by a nerve impulse and that this mechanism is downstream of Ca2+ influx.

Addition of Purified Basal Lamina Molecules Enables Schwann Cell Ensheathment of Sympathetic Neurites in Culture

Our aim was to determine the effect of purified basal lamina components on the differentiation of non-myelin-forming Schwann cells accompanying rat sympathetic neurons in culture. Previous work has demonstrated that Schwann cells contacting superior cervical ganglion neurons cultured in the presence of ascorbate and serum fall to effectively deposit matrix and ensheathe neurites unless fibroblasts are also present. We questioned if an increase in the amount of available basal lamina components could mimic fibroblast-induced Schwann cell differentiation. Superior cervical ganglion neuron plus Schwann cell cultures were supplemented with purified basal lamina molecules for up to 7 weeks. The addition of laminin, type IV collagen, heparan sulfate proteoglycan, or a combination of all three components, led to increased basal lamina deposition and increased neurite ensheathment compared with cultures with no additions. In long-term cultures receiving component additions, however, ensheathment was exaggerated, and the Schwann cells were hypertrophic. The ensheathing conformations adopted by these Schwann cells surpassed normal-appearing ensheathment and resembled more unusual unmyelinated nerve morphology found in vivo by others in normal senescence and in several peripheral neuropathies. These experiments show that increased availability of exogenous basal lamina components leads to increased basal lamina deposition and that basal lamina elaboration can have dramatic effects on the morpholgy of nonmyelinating Schwann cells. These findings suggest that influences of extracellular matrix should be considered when unusual Schwann cell/axon conformations are seen in vivo.

Myosin II is involved in transmitter release at synapses formed between rat sympathetic neurons in culture

The presynaptic function of myosin II was studied at cholinergic synapses formed between rat superior cervical ganglion neurons in culture. Immunofluorescent staining showed that myosin 11 was colocalized with synaptophysin at the presynaptic nerve terminals. Antimyosin II antibody introduced into presynaptic neurons inhibited synaptic transmission. Transmission was also inhibited in a dose-dependent manner by two inhibitors of myosin light chain kinase: a peptide, SM-1, and an organic inhibitor, wortmannin. The inhibition produced by these agents was dependent on presynaptic activity. Extracellularly applied wortmannin also blocked synaptic transmission, but its effects were slower in onset. Wortmannin also decreased postsynaptic potentials and post-tetanic potentiation in intact superior cervical ganglia. These results suggest a model in which myosin light chain kinase phosphorylates myosin, and the resultant change in actin-myosin interactions is involved in neurotransmitter release.

Analysis of the mechanism for acetylcholine release at the synapse formed between rat sympathetic neurons in culture.

Superior cervical ganglion neurons (SCGNs) were isolated from 7-day-old rat SCG and cultured in MEM containing horse serum, fetal calf serum, and nerve growth factor. In this culture condition, it is well known that the SCGNs form cholinergic synapse. In 3-4 weeks cultured neurons, immunofluorescent staining for synaptophysin, a small synaptic vesicle associated protein, showed the presence of synaptophysin as small dots on the surface of the soma. Postsynaptic potentials could be recorded in 50-80% of the neurons responding to evoked action potentials elicited in neighboring neurons. Because of its relatively large cell size and the short distance to the terminal, this synapse is a useful model for studying the mechanisms of acetylcholine (ACh) release by introducing substances such as antibodies or selective inhibitors into the presynaptic neuron by means of the whole-cell clamp technique. In this model synapse we tested the possible role of myosin in ACh release. The distribution of myosin was studied by the immunofluorescent staining technique. Myosin was recognized by the anti-myosin II IgG at the same synaptic terminals that showed the presence of synaptophysin with its antibody. The functional blockade of myosin by the antibody itself, and that of myosin light chain kinase (MLCK) by a pseudosubstrate inhibitor of MLCK, SM-1, or by a selective inhibitor of MLCK, wortmannin, induced depression of synaptic transmission in a dose-dependent manner. These indicate that phosphorylation of myosin by MLCK may be necessary for ACh release mechanisms.

NGF and the local control of nerve terminal growth

It is generally believed that the mechanism of action of neurotrophic factors involves uptake of neurotrophic factor by nerve terminals and retrograde transport through the axon and back to the cell body where the factor exerts its neurotrophic effect. This view originated with the observation almost 20 years ago that nerve growth factor (NGF) is retrogradely transported by sympathetic axons, arriving intact at the neuronal cell bodies in sympathetic ganglia. However, experiments using compartmented cultures of rat sympathetic neurons have shown that neurite growth is a local response of neurites to NGF locally applied to them which does not directly involve mechanisms in the cell body. Recently, several NGF-related neurotrophins have been identified, and several unrelated molecules have been shown to act as neurotrophic or differentiation factors for a variety of types of neurons in the peripheral and central nervous systems. It has become clear that knowledge of the mechanisms of action of these factors will be crucial to understanding neurodegenerative diseases and the development of treatments as well as the means to repair or minimize neuronal damage after spinal injury. The concepts derived from work with NGF suggest that the site of exposure of a neuron to a neurotrophic factor is important in determining its response.

IL-6 increases choline acetyltransferase but not neuropeptide transcripts in sympathetic neurons.

The effect of interleukin (IL)-6 on cell survival, and the expression of choline acetyltransferase (ChAT) and neuropeptide mRNAs was examined in cultured rat sympathetic neurons. Reverse transcription-polymerase chain reaction analysis indicated that IL-6 treatment led to six-fold increase in ChAT mRNA without a concomitant increase in ChAT immunoreactivity. In contrast, treatment with ciliary neurotrophic factor or leukemia inhibitory factor/cholinergic differentiation factor increased ChAT mRNA levels 25-fold and resulted in intense ChAT immunoreactivity. Moreover, the latter factors increased somatostatin and preprotachykinin mRNA levels 10-fold whereas IL-6 had no effect. Maximal doses of IL-6 had no effect on cell survival. These findings provide a basis for the overlapping and yet divergent actions of these three factors which share some components of their signal transduction machinery.

Leukemia Inhibitory Factor and Nerve Growth Factor are Retrogradely Transported and Processed by Cultured Rat Sympathetic Neurons

How neurons convert the presence of factors at their axon terminals into signals that affect mechanisms in their cell bodies is unknown, but retrograde axonal transport of the factors themselves may be involved. Nerve growth factor (NGF) and leukemia inhibitory factor (LIF) have previously been shown to produce changes in cell bodies of sympathetic neurons when applied to their peripheral neurites, and it is well established that NGF is retrogradely transported along sympathetic axons. In this study we show that 125 I-LIF applied to terminal neurites of rat sympathetic neurons in compartmented cultures is retrogradely transported, but at a much lower level compared to the retrograde transport of 125I-NGF. Transport of 125 I-LIF was competed by cotreatment with unlabeled LIF and was blocked by cotreatment with dinitrophenol. The rate of 125I-LIF transport was independent of NGF concentration. However, both 125 I-LIF and 125I-NGF transport was reduced by pretreating neurons with LIF. SDS-PAGE analysis showed that retrogradely transported radiolabel which accumulated in cell body-containing extracts following transport of both 125I-LIF and 125I-NGF consisted of intact as well as partially processed species. Radiolabel also accumulated in the medium bathing the cell bodies and migrated near the dye front on SDS-PAGE, implying that both factors were extensively degraded and released by the neurons. These results are consistent with the suggestion that the retrograde transport of LIF, as thought for NGF, may be important for retrograde signaling mechanisms.

Bradykinin augments a PKC-regulated choride conductance in cultured rat sympathetic neurones

Bradykinin augments a PKC-regulated choride conductance in cultured rat sympathetic neurones

Characterization of apoptosis in cultured rat sympathetic neurons after nerve growth factor withdrawal.

Sympathetic neurons depend on nerve growth factor (NGF) for their survival both in vivo and in vitro. In culture, the neurons die after NGF withdrawal by an autonomous cell death program but whether these neurons die by apoptosis is under debate. Using vital DNA stains and in situ nick translation, we show here that extensive chromatin condensation and DNA fragmentation occur before plasma membrane breakdown during the death of NGF-deprived rat sympathetic neurons in culture. Furthermore, kinetic analysis of chromatin condensation events within the cell population is consistent with a model which postulates that after NGF deprivation nearly all of the neurons die in this manner. Although the dying neurons display membrane blebbing, cell fragmentation into apoptotic bodies does not occur. Apoptotic events proceed rapidly at around the time neurons become committed to die, regardless of neuronal culture age. However the duration of NGF deprivation required to commit neurons to die, and the rate at which apoptosis occurs, increase with culture age. Thus, within the first week of culture, apoptosis is the predominant form of cell death in sympathetic neurons.

Dequalinium, a selective blocker of the slow afterhyperpolarization in rat sympathetic neurones in culture

The actions of dequalinium have been investigated in cultured rat sympathetic neurones. It produced a rapid and reversible inhibition of the slow apamin-sensitive component of the afterhyperpolarization (AHP) which follows a single action potential in these cells. The IC 50 for this effect was 1.1 μM and in voltage clamp experiments, 1 μM dequalinium produced 45% inhibition of the underlying current I AHP. When the small conductance Ca 2+-activated K + channels were blocked by 20 nM apamin the slow component of the AHP was abolished, and dequalinium (10 μM) produced no further change in the residual AHP. Dequalinium (10 μM) had no effect on the voltage-activated Ca 2+ current in these cells, suggesting that the inhibition of the AHP was the result of a direct interaction with the K + channels. The A-current as well as a composite current made up of I K and I C were all unaffected by 10 μM dequalinium. However, at this concentration it did produce 18% inhibition of the M-current. These results show dequalinium to be a potent and selective non-peptide blocker of the apamin-sensitive small conductance Ca 2+-activated K + channel in rat sympathetic neurones.