Cultured Rat Sympathetic Neurons Research Articles

Activation and inhibition of neuronal G protein-gated inwardly rectifying K(+) channels by P2Y nucleotide receptors.

Neuronal signaling by G protein-coupled P2Y nucleotide receptors is not well characterized. We studied here the coupling of different molecularly defined P2Y receptors to neuronal G protein-gated inward rectifier K(+) (GIRK) channels. Individual P2Y receptors were coexpressed with GIRK1+GIRK2 (Kir3.1 + 3.2) channels by intranuclear plasmid injections into cultured rat sympathetic neurons. Currents were recorded using perforated-patch or whole-cell (disrupted patch) techniques, with similar results. P2Y(1) receptor stimulation with 2-methylthio ADP (2-MeSADP) induced activation of GIRK current (I(GIRK)) followed by inhibition. In contrast, stimulation of endogenous alpha(2)-adrenoceptors by norepinephrine produced stable activation without inhibition. P2Y(1)-mediated inhibition was also seen when 2-MeSADP was applied after I(GIRK) preactivation by norepinephrine or by expression of Gbeta(1)gamma(2) subunits. In contrast, stimulation of P2Y(4) receptors with UTP or P2Y(6) receptors with UDP produced very little I(GIRK) activation but significantly inhibited preactivated currents. Current activation was prevented by pertussis toxin (PTX) or after coexpression of the betagamma-scavenger transducin-Galpha.I(GIRK) inhibition by all three nucleotide receptors was insensitive to PTX and was significantly reduced after coexpression of RGS2 protein, known to inhibit G(q)alpha signaling. Inhibition was not affected 1) after coexpression of RGS11, which interferes with G(q)betagamma action; 2) after coexpression of phospholipase C (PLC) delta-Pleckstrin homology domain, which sequesters the membrane phospholipid phosphatidylinositol 4,5-bisphosphate; (3) after buffering intracellular Ca(2+) with 1,2-bis(2-aminiphenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM); and (4) after pretreatment with the protein kinase C inhibitor 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF 109203X). We conclude that activation of I(GIRK) by P2Y receptors is mediated by G(i/o)betagamma, whereas I(GIRK) inhibition is mediated by G(q)alpha. These effects may provide a mechanism for P2Y-modulation of neuronal excitability.

Block of slow axonal transport and axonal growth by brefeldin A in compartmented cultures of rat sympathetic neurons

Disruption of the Golgi by brefeldin A (BFA) has been reported to block fast axonal transport and axonal growth. We used compartmented cultures of rat sympathetic neurons to investigate its effects on slow axonal transport. BFA (1 μg/ml) applied to cell bodies/proximal axons for 6–20 h disrupted the Golgi, reversibly blocked axonal growth, and reversibly blocked anterograde transport of all proteins, including tubulin. The retrograde transport of nerve growth factor (NGF) was also blocked. The phosphorylation of Erk1 and Erk2 in response to NGF was unaffected after 6 h of treatment with BFA, suggesting that the block of axonal transport was specific and direct. Consistent with its principal site of action at the Golgi, no effects were observed when BFA was applied only to the distal axons. Block of fast anterograde and retrograde axonal transport is consistent with the role of the Golgi in supplying transport vesicles. Block of slow axonal transport was surprising, and further results indicated that transport of tubulin en route along the axon was arrested by application of BFA to the cell bodies, suggesting that a continuous supply of anterograde transport vesicles from the Golgi is required to maintain slow axonal transport of cytoskeletal proteins.

Growing and Working with Peripheral Neurons

Cultures of vertebrate peripheral neurons have been used to address a variety of issues related to the cell biology of the neuron. They are particularly amenable to experimental manipulations, such as microinjection, and can be cultured under a variety of different conditions designed to meet the needs of the particular experiment. This chapter focuses on cultures of rat sympathetic neurons from the superior cervical ganglia and on cultures of chick sensory neurons from the lumbosacral dorsal root ganglia. Information is provided on methods for dissection, preparation of culture dishes and substrates, composition of media, relevant growth factors, reduction of nonneuronal contamination, and maintenance of the cultures.

Ionic basis of the resting membrane potential in cultured rat sympathetic neurons.

The conductances which determine the resting membrane potential of rat superior cervical ganglia (SCG) neurons were investigated using perforated voltage- and current-clamp whole-cell techniques. The resting potential of SCG cells varied from -47 to -80 mV (-58.3 +/- 0.8 mV, n = 55). Blockade of M and h currents induced a depolarisation (7.4 +/- 0.7 mV, n = 22) and a hyperpolarisation (7.2 +/- 0.7 mV, n = 20) respectively; however, no correlation between the amplitude of these currents and the resting potential was found. The inhibition of the Na/K pump also induced membrane depolarisation (3.2 +/- 0.2 mV, n = 8). Inhibition of voltage-gated currents unmasked a voltage-independent resting conductance reversing at -50 mV. The reversal potential of the voltage-independent conductance, which included the electrogenic contribution of the Na/K pump, was strongly correlated with the resting potential (R = 0.87, p < 0.0001, n = 30). Ionic substitution experiments confirmed the existence of a voltage-independent conductance (leakage) with four components, a main potassium conductance, two minor sodium and chloride conductances and a small contribution of the Na/K pump. It is concluded that the resting potential of SCG cells strongly depends on the reversal potential of the voltage-independent conductance, with voltage-activated M and h currents playing a prominent stabilising role.

Retrograde support of neuronal survival without retrograde transport of nerve growth factor.

Application of nerve growth factor (NGF) covalently cross-linked to beads increased the phosphorylation of TrkA and Akt, but not of mitogen-activated protein kinase, in cultured rat sympathetic neurons. NGF beads or iodine-125-labeled NGF beads supplied to distal axons resulted in the survival of over 80% of the neurons for 30 hours, with little or no retrograde transport of iodine-125-labeled NGF; whereas application of free iodine-125-labeled NGF (0.5 nanograms per milliliter) produced 20-fold more retrograde transport, but only 29% of the neurons survived. Thus, in contrast to widely accepted theory, a neuronal survival signal can reach the cell bodies unaccompanied by the NGF that initiated it.

Ceramide Inhibits Axonal Growth and Nerve Growth Factor Uptake without Compromising the Viability of Sympathetic Neurons

Ceramide inhibits axonal growth of cultured rat sympathetic neurons when the ceramide content of distal axons, but not cell bodies, is increased (Posse de Chaves, E. I., Bussiere, M. Vance, D. E., Campenot, R. B., and Vance, J.E. (1997) J. Biol. Chem. 272, 3028-3035). We now report that inhibition of growth does not result from cell death since although ceramide is a known apoptotic agent, C(6)-ceramide given to the neurons for 24 h did not cause cell death but instead protected the neurons from death induced by deprivation of nerve growth factor (NGF). We also find that a pool of ceramide generated from sphingomyelin in distal axons, but not cell bodies, inhibits axonal growth. Analysis of endogenous sphingomyelinase activities demonstrated that distal axons are rich in neutral sphingomyelinase activity but contain almost no acidic sphingomyelinase, which is concentrated in cell bodies/proximal axons. Together, these observations are consistent with the idea that generation of ceramide from sphingomyelin by a neutral sphingomyelinase in axons inhibits axonal growth. Furthermore, we demonstrate that treatment of distal axons with ceramide inhibits the uptake of NGF and low density lipoproteins by distal axons by approximately 70 and 40%, respectively, suggesting that the inhibition of axonal growth by ceramide might be due, at least in part, to impaired endocytosis of NGF. However, inhibition of endocytosis of NGF by ceramide could not be ascribed to decreased phosphorylation of TrkA.

A conservedα-herpesvirus protein necessary for axonal localization of viral membrane proteins

Pseudorabies virus, an α-herpesvirus, is capable of infecting the nervous system and spreading between synaptically connected neurons in diverse hosts. At least three viral membrane proteins (gE, gI, and Us9) are necessary for the spread of infection from presynaptic to postsynaptic neurons (anterograde spread) in infected rodents. To understand how these proteins effect anterograde spread between neurons, we analyzed the subcellular localization of viral proteins after infection of cultured rat sympathetic neurons with wild-type or mutant viruses. After Us9-null mutant infections but not gE-null mutant infections, only a subset of the viral structural proteins had entered axons. Surprisingly, capsid and tegument proteins but not viral membrane proteins were detected in axons. The spread of Us9 missense mutants in the rodent nervous system correlated with the amount of viral membrane proteins localized to axons. We conclude that the Us9 membrane protein controls axonal localization of diverse viral membrane proteins but not that of capsid or tegument proteins. The data support a model where virion subassemblies but not complete virions are transported in the axon. Our results provide new insight into the process of virion assembly and exit from neurons that leads to directional spread of herpesviruses in the nervous system.

A ryanodine fluorescent derivative reveals the presence of high-affinity ryanodine binding sites in the Golgi complex of rat sympathetic neurons, with possible functional roles in intracellular Ca 2+ signaling

The plant alkaloid ryanodine (Ry) is a high-affinity modulator of ryanodine receptor (RyR) Ca 2+ release channels. Although these channels are present in a variety of cell types, their functional role in nerve cells is still puzzling. Here, a monosubstituted fluorescent Ry analogue, B-FL-X Ry, was used to reveal the distribution of RyRs in cultured rat sympathetic neurons. B-FL-X Ry competitively inhibited the binding of [ 3H]Ry to rabbit skeletal muscle SR membranes, with an IC 50 of 150 nM, compared to 7 nM of unlabeled Ry. Binding of B-FL-X Ry to the cytoplasm of sympathetic neurons is saturable, reversible and of high affinity. The pharmacology of B-FL-X Ry showed marked differences with unlabeled Ry, which are partially explained by its lower affinity: (1) use-dependent reversible inhibition of caffeine-induced intracellular Ca 2+ release; (2) diminished voltage-gated Ca 2+ influx, due to a positive shift in the activation of voltage gated Ca 2+ currents. B-FL-X Ry-stained sympathetic neurons, viewed under confocal microscopy, showed conspicuous labeling of crescent-shaped structures pertaining to the Golgi complex, a conclusion supported by experiments showing co-localization with Golgi-specific fluorescent probes and the breaking up of crescent-shaped staining after treatment with drugs that disassemble Golgi complex. The presence of RyRs to the Golgi could be confirmed with specific anti-RyR 2 antibodies, but evidence of caffeine-induced Ca 2+ release from this organelle could not be obtained using fast confocal microscopy. Rather, an apparent decrease of the cytosolic Ca 2+ signal was detected close to this organelle. In spite of that, short-term incubation with brefeldin A (BFA) suppressed the fast component of caffeine-induced Ca 2+ release, and the Ca 2+ release process lasted longer and appeared less organized. These observations, which suggest a possible role of the Golgi complex in Ca 2+ homeostasis and signaling in nerve cells, could be relevant to reports involving derangement of the Golgi complex as a probable cause of some forms of progressive neuronal degeneration, such as Alzheimer's disease and amyotrophic lateral sclerosis.

Evidence for Redox Regulation of Cytochrome c Release during Programmed Neuronal Death: Antioxidant Effects of Protein Synthesis and Caspase Inhibition

Sympathetic neurons die by apoptosis when they are deprived of nerve growth factor (NGF). Activation of caspases by cytochrome c released from mitochondria is central to this death. In this report we present evidence that cellular redox state regulates cytochrome c redistribution in these neurons. An increase of mitochondrial-produced reactive oxygen species (ROS) occurred in rat sympathetic neurons in cell culture within 3 hr of NGF withdrawal. Caspase inhibitors blocked this ROS burst. By 6 hr after NGF deprivation, glutathione (GSH) levels had increased, neutralizing elevated hydrogen peroxide levels and returning cellular redox state to basal levels. By 12 hr after deprivation, ROS levels had again increased and remained elevated during the rest of the apoptotic process. The later ROS burst appeared to have both caspase-dependent and caspase-independent components and was coincident with the period of cytochrome c release. Inhibition of protein synthesis with cycloheximide (CHX) and treatment with the antioxidant compound, N-acetyl-l-cysteine (l-NAC), blocked both the early and late ROS bursts by increasing cellular GSH levels (Ratan et al., 1994; Tan et al., 1998). Both compounds, and a membrane-permeant form of GSH, also inhibited cytochrome c release and death. Treatment of NGF, CHX-, l-NAC-, and GSH-saved cells with hydrogen peroxide caused rapid cytochrome c release. These data suggest a role for cellular redox state in regulating cytochrome c release during apoptosis induced by NGF withdrawal.

Mechanisms of blockade by the novel migraine prophylactic agent, dotarizine, of various brain and peripheral vessel contractility

The novel antimigraineur, dotarizine, inhibited 5-HT (5 hydroxytryptamine)-evoked contractions of rabbit vertebral, aorta, femoral and mesenteric arteries, with IC 50s of 1.35, 1.40, 0.52 and 1.09 μM, respectively. Flunarizine had little effect on these contractions, while ketanserin was more potent (IC 50s of 0.17 μM for vertebral, 0.22 μM for aorta, 0.05 μM for femoral and 0.03 μM for mesenteric arteries). At 10 μM, dotarizine caused 40% blockade of K +-evoked contractions of rabbit aorta, and 70% inhibition of 5-HT-evoked responses; these values were 30% and 20% for 10 μM flunarizine. Contractions of rabbit aorta elicited by noradrenaline, angiotensin II or prostaglandin F 2α were not affected by 10 μM dotarizine or flunarizine. Ketanserin shifted to the right, in parallel, the concentration–response curves for 5-HT in rabbit aorta; however, dotarizine caused a non-competitive type of blockade, increasing the maximum 5-HT contraction at 30 nM and decreasing it at 3 and 30 μM. K +-evoked contractions of rabbit aorta were halved by 3 μM dotarizine in a voltage-independent manner; flunarizine caused a delayed-type, non-reversible post-drug blockade, and exhibited some voltage-dependence. Blockade by nifedipine was voltage-dependent and fully reversible. Ca 2+-evoked contractions of depolarised bovine middle cerebral arteries were blocked by 1–3 μM dotarizine in a non-surmountable manner. Contraction of these vessels evoked by electrical stimulation was blocked 50% and 70% by 1 and 3 μM dotarizine, respectively. Dotarizine (1–3 μM) also inhibited to a similar extent the K +-evoked [ 3H]noradrenaline release from cultured rat sympathetic neurones. These data suggest that the mechanism of blockade by dotarizine of cerebral vessels contractility has three components: (i) presynaptic inhibition of noradrenaline release; (ii) blockade of postsynaptic vascular 5-HT receptors; (iii) blockade of Ca 2+entry into the vascular smooth muscle cell cytosol. The compound does not affect the vascular receptors for noradrenaline, angiotensin II or prostaglandin F 2α.

Differential gene expression of dopamine D-2 receptor subtypes in rat chromaffin cells and sympathetic neurons in culture.

Chromaffin cells and sympathetic neurons arise from a common bipotential progenitor which, if exposed to nerve growth factor (NGF), matures into a sympathetic neuron, but if exposed to glucocorticoids (GCs), differentiates into a mature chromaffin cell. Pharmacological evidence indicates that, in adrenal medulla and sympathetic neurons, dopamine (DA) receptors belonging to the D-2 family inhibit catecholamine secretion. The molecular characterization of these receptors, however, is not been yet described. Our data suggest that bipotential cells obtained from newborn rat adrenal medulla express both isoforms of the D-2 receptor, while D-3 receptor and D-4 receptor messenger RNAs (mRNAs) are not present. GC-mediated maturation induces the expression of D-4 receptors, without modification of D-2 isoforms. Sympathetic neurons differentiated in vitro selectively express the D-2short mRNA. Taken together, present results suggest that NGF and GCs play a role in regulating D-2 family receptor expression in neural crest-derived cells.

Uptake of Lipoproteins for Axonal Growth of Sympathetic Neurons

Lipoproteins originating from axon and myelin breakdown in injured peripheral nerves are believed to supply cholesterol to regenerating axons. We have used compartmented cultures of rat sympathetic neurons to investigate the utilization of lipids from lipoproteins for axon elongation. Lipids and proteins from human low density lipoproteins (LDL) and high density lipoproteins (HDL) were taken up by distal axons and transported to cell bodies, whereas cell bodies/proximal axons internalized these components from only LDL, not HDL. Consistent with these observations, the impairment of axonal growth, induced by inhibition of cholesterol synthesis, was reversed when LDL or HDL were added to distal axons or when LDL, but not HDL, were added to cell bodies. LDL receptors (LDLRs) and LR7/8B (apoER2) were present in cell bodies/proximal axons and distal axons, with LDLRs being more abundant in the former. Inhibition of cholesterol biosynthesis increased LDLR expression in cell bodies/proximal axons but not distal axons. LR11 (SorLA) was restricted to cell bodies/proximal axons and was undetectable in distal axons. Neither the LDL receptor-related protein nor the HDL receptor, SR-B1, was detected in sympathetic neurons. These studies demonstrate for the first time that lipids are taken up from lipoproteins by sympathetic neurons for use in axonal regeneration.

Activity-dependent enhancement of miniature excitatory postsynaptic current amplitude and its modulation by protein kinase C in cultured rat sympathetic neurons

A high K + solution increased the frequency of miniature excitatory postsynaptic currents (MEPSCs) and intracellular Ca 2+ concentration ([Ca 2+] i) in cultured rat sympathetic neurons. Repetition or continuation of high K + treatment increased MEPSC amplitude, acetylcholine-induced currents and the averaged rise in [Ca 2+] i per single MEPSC. The enhancement of MEPSCs lasted over 30 min and was inhibited by intracellular BAPTA and phorbol ester, but not by atropine. The results suggest that repeated Ca 2+ entry through the channel pore of nicotinic acetylcholine receptor enhances the efficacy of its opening and the activation of protein kinase C inhibits the enhancement.

CEP‐1347 (KT7515), an Inhibitor of JNK Activation, Rescues Sympathetic Neurons and Neuronally Differentiated PC12 Cells from Death Evoked by three Distinct Insults

The c-Jun N-terminal kinase signaling cascade appears to play a role in some cases of cell death, including neuronal apoptosis. CEP-1347 (KT7515), an indolocarbazole of the K252a family, blocks this stress signaling cascade and promotes survival. Here, we used CEP-1347 to probe whether neuronal death pathways activated by distinct insults also possess elements in common. Cultured rat sympathetic neurons and neuronally differentiated PC12 cells were induced to die by withdrawal of nerve growth factor, exposure to ultraviolet irradiation, or subjection to oxidative stress. In each case, death was prevented by 100-200 nM CEP-1347. Moreover, in each of these death paradigms, c-Jun N-terminal kinase 1 activity in neuronally differentiated PC12 cells was elevated by two- or threefold, and this increase was totally blocked by CEP-1347 at concentrations that promoted survival. In contrast, 200 nM CEP-1347 did not block death due to serum withdrawal from undifferentiated PC12 cells or to activation of Fas in Jurkat T cell cultures, even though in each case c-Jun N-terminal kinase 1 activation occurred and was inhibited by CEP-1347. These observations suggest that some but not all death pathways triggered by different insults can include a common mechanistic component, a likely candidate for which is activation of the c-Jun N-terminal kinase signaling cascade.

Somatic and prejunctional nicotinic receptors in cultured rat sympathetic neurones show different agonist profiles.

1. The release of [3H]-noradrenaline ([3H]-NA) in response to nicotinic acetylcholine receptor (nAChR) agonists was compared with agonist-induced currents in cultured rat superior cervical ganglion (SCG) neurones. 2. [3H]-NA release in response to high concentrations of nicotinic agonists was reduced, but not fully inhibited, by the presence of either tetrodotoxin (TTX) or Cd2+ to block voltage-gated Na+ or Ca2+ channels, respectively. We used the component of transmitter release that remained in the presence of these substances (named TTX- or Cd2+-insensitive release) to pharmacologically characterize nAChRs in proximity to the sites of vesicular exocytosis (prejunctional receptors). Prejunctional nAChRs were activated by nicotinic agonists with a rank order of potency of dimethylphenylpiperazinium iodide (DMPP) > nicotine > cytisine > ACh, and with EC50 values ranging from 22 microM (DMPP) to 110 microM (ACh). 3. [3H]-NA release in response to low concentrations of nAChR agonists was fully inhibited by the presence of either TTX or Cd2+ (named TTX- or Cd2+-sensitive release). TTX-sensitive release was triggered by nicotinic agonists with a rank order of potency of DMPP > cytisine approximately nicotine approximately ACh, which due to its similarity to TTX-insensitive release indicates that it might also be triggered by prejunctional-type nAChRs. The EC50 values for TTX (Cd2+)-sensitive release were less than 10 microM for all four agonists. 4. By contrast to transmitter release, somatic nAChRs as seen by patch clamp recordings were most potently activated by cytisine, with a rank order of potency of cytisine > nicotine approximately DMPP > ACh. EC50 values for the induction of currents exceeded 20 microM for all four agonists. 5. The nicotinic antagonist mecamylamine potently inhibited all transmitter release in response to nicotine. alpha-Bungarotoxin (alpha-BuTX) was, on the other hand, without significant effect on nicotine-induced TTX-insensitive release. The competitive antagonist dihydro-beta-erythroidine (DHbetaE) caused rightward shifts of the dose-response curves for both TTX-sensitive and TTX-insensitive transmitter release as well as for currents in response to nicotine, with pA2 values ranging from 4.03 to 4.58. 6. Due to clear differences in the pharmacology of agonists we propose that nAChRs of distinct subunit composition are differentially targeted to somatic or axonal domains.

Neurotoxicity of 25-OH-Cholesterol on Sympathetic Neurons

Cultured rat sympathetic neurons derived from postnatal rat superior cervical ganglia (SCG) were used to compare the neurotoxicity of several cholesterol oxides. The cholesterol oxides tested included: 7-beta-OH-, 7-keto-, 19-OH-, 22(R)-OH-, 22(S)-OH-, and 25-OH-cholesterol. These agents caused an acute as well as a delayed toxicity in sympathetic neurons with 25-OH-cholesterol appearing to be the most toxic. A time-dependent experiment indicated that 25-OH-cholesterol at 4 μg/ml (10 μM) was able to kill 50% of the cells in 36 h. Morphological studies indicate that most of the cells do not exhibit a structural change similar to that observed in neuronal programmed cell death. Whole-cell patch clamp recording of untreated controls and 25-OH-cholesterol (2 μg/ml)-treated cells indicated that this toxicity was not accompanied by significant changes in voltage-dependent calcium channel activity. A number of pharmacological agents including ethylene glycolbis (beta-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), cycloheximide, KCl, vitamin E, and methyl-beta-cyclodextrin were able to prevent the 25-OH-cholesterol-induced cell death to various degrees. These results suggest that, in addition to causing pathological changes in cells directly involved in atherosclerosis, cholesterol oxides may induce neurotoxicity in sympathetic neurons.

Sympathetic neurons can produce and respond to interleukin 6.

Neuronal expression of cytokines is an area of active investigation in the contexts of development, disease, and normal neural function. Although cultured rat sympathetic neurons respond very weakly to exogenous interleukin 6 (IL-6), we find that addition of soluble IL-6 receptor (sIL-6R) and IL-6 enhances neuronal survival in the absence of nerve growth factor. Neutralizing monoclonal antibodies against IL-6 block these effects. Addition of IL-6 and sIL-6R also induces a subset of neuropeptide and transmitter synthetic enzyme mRNAs identical to that demonstrated for leukemia inhibitory factor, ciliary neurotrophic factor, and oncostatin M. Both of these effects are duplicated by addition of a highly active fusion protein of sIL-6R and IL-6, covalently linked by a flexible peptide chain, which is designated H-IL-6. In addition, we show that sympathetic neurons produce IL-6. In situ hybridization indicates a neuronal localization of IL-6 mRNA in superior cervical ganglia, and bioactive IL-6 protein is detected in ganglion culture supernatants. Interestingly, the IL-6 produced by sympathetic neurons does not lead to survival of these cells in culture unless sIL-6R is added. Thus, sympathetic neurons can produce IL-6 and may respond to it in an autocrine/paracrine manner if sIL-6R is present. Moreover, the prior findings of sIL-6R in serum and inflammatory fluids now have added interest in the context of neuro-immune interactions.

Leukemia inhibitory factor and ciliary neurotrophic factor regulate dendritic growth in cultures of rat sympathetic neurons

Cytokines such as leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have previously been shown to regulate neurotransmitter and neuropeptide synthesis in sympathetic neurons [P.H. Patterson, Leukemia inhibitory factor, a cytokine at the interface between neurobiology and immunology, Proc. Natl. Acad. Sci. USA 91 (1994) 7833–7835]. We considered the possibility that these agents may also affect the development of neuronal cell shape. Intracellular dye injection and immunocytochemistry were used to assess dendritic growth in cultures of perinatal rat sympathetic neurons and the effects of LIF and CNTF were compared to those of osteogenic protein-1 (OP-1), a growth factor that induces profuse dendritic growth in these neurons [P. Lein, M. Johnson, X. Guo, D. Rueger, D. Higgins, Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons, Neuron 15 (1995) 597–605]. Under control conditions, sympathetic neurons formed only axons. Exposure to either LIF or OP-1 stimulated dendritic growth, but the magnitude of the response to LIF was much less than that obtained with OP-1 with respect to both dendritic number and length. Simultaneous exposure to LIF and OP-1 resulted in dendritic growth equivalent to that observed in the presence of LIF alone, suggesting that LIF inhibits the response of neurons to OP-1. Both the stimulatory and inhibitory effects of LIF were mimicked by CNTF, but not by other growth factors. These data suggest that LIF and CNTF regulate dendritic development in a complex manner that is dependent on both the morphological state of the neuron and the presence of other growth factors. However, the net effect of exposure to these cytokines appears to be the production of a population of neurons with rudimentary arbors consisting of only one or two short dendrites.

Role of Lipoproteins in the Delivery of Lipids to Axons during Axonal Regeneration

Nerve fiber elongation involves the input of lipids to the growing axons. Since cell bodies are often a great distance from the regenerating tips, alternative sources of lipids have been proposed. We previously demonstrated that axonal synthesis of phosphatidylcholine is required for axonal growth (Posse de Chaves, E., Vance, D. E., Campenot, R. B. and Vance, J. E. (1995) J. Cell Biol. 128, 913-918; Posse de Chaves, E., Vance, D. E., Campenot, R. B. and Vance, J. E. (1995) Biochem. J. 312, 411-417). In contrast, cholesterol is not made in axons. We now show that when compartmented cultures of rat sympathetic neurons are incubated with pravastatin, in the absence of exogenously supplied lipids, cholesterol synthesis is inhibited and axonal growth is impaired. The addition of cholesterol to the axons or cell bodies of neurons treated with this inhibitor restores normal axonal elongation. Similarly, a supply of cholesterol via lipoproteins restores normal axonal growth. In contrast, lipoproteins do not provide axons with sufficient phosphatidylcholine for normal elongation when axonal phosphatidylcholine synthesis is inhibited. Thus, our studies support the idea that during axonal regeneration lipoproteins can be taken up by axons from the microenvironment and supply sufficient cholesterol, but not phosphatidylcholine, for growth. We also show that neither apoE nor apoA-I within the lipoproteins is essential for axonal growth.

Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na+-dependent mechanism in cultured rat sympathetic neurons

Superior-cervical ganglion (SCG) cells dissociated from newborn rats depend on nerve growth factor (NGF) for survival. Membrane depolarization with elevated K+ is known to prevent neuronal death following NGF deprivation and/or to promote survival via a Ca2+-dependent mechanism. Here we have exploited the possibility of whether or not a Na+-dependent pathway for neuronal survival is present in these cells. Veratridine (ec50=40 nM), a voltage-dependent Na+ channel activator, significantly delayed the onset of apoptotic cell death in NGF-deprived SCG neurons that had been cultured for 7 days in the presence of NGF. This effect was blocked completely by Na+ channel blockers including tetrodotoxin (TTX, 1 μM), benzamil (25 μM) and flunarizine (1 μM), but was not attenuated by nimodipine (1 μM), an L-type Ca2+ channel blocker. The saving effect of veratridine on cultured neurons was observed even in low Ca2+ media (0–1.0 mM), but was completely abolished in a low Na+ medium (38 mM). Sodium-binding benzofuran isophthalate was employed as a fluorescent probe for monitoring the level of cytoplasmic free Na+, which revealed a sustained increase in its level (12.9 mM, 307% of that of control) in response to veratridine (0.75 μM). The TTX or flunarizine completely blocked veratridine-induced Na+ influx in these cultured neurons. Moreover, no appreciable increase in intracellular Ca2+ was detected under these conditions. Though Na+ channels were effectual in SCG neurons which were freshly isolated from newborn rats, the Na+-dependent saving effect of veratridine was not observed in these young neurons. These lines of evidence suggest that the death-suppressing effect of veratridine on cultured SCG neurons depends on the Na+ influx via voltage-dependent Na+ channels, and suggests the presence of Na+-dependent regulatory mechanism(s) in neuronal survival.