Cultured Rat Sympathetic Neurons Research Articles

An efficient deletion mutant packaging system for defective herpes simplex virus vectors: potential applications to human gene therapy and neuronal physiology.

We have previously described a defective herpes simplex virus (HSV-1) vector system that permits the introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli beta-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; ts mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. We now report an efficient packaging system for HSV-1 vectors using a deletion mutant, D30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses beta-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system.

Interferon Suppresses Sympathetic Neuronal Cell Death Caused by Nerve Growth Factor Deprivation

Cultured rat sympathetic neurons die within 48 h after being deprived of nerve growth factor. Addition of interferons (IFN-alpha/beta or IFN-gamma) prevented the cell death in a dose-dependent manner. Upon longer periods of nerve growth factor deprivation, IFNs failed to maintain survival. Thus, IFNs retarded neuronal death, but did not prevent it. Ligand binding, autoradiography, and cross-linking experiments demonstrated the presence of specific IFN-gamma receptors on sympathetic neurons similar to those seen on other cell types. The possible relationships of the death-suppressing actions of IFNs are compared to the mechanisms of the antiviral or antiproliferative actions of IFNs.

Ca2+ transients are not required as signals for long-term neurite outgrowth from cultured sympathetic neurons.

A method for clamping cytosolic free Ca2+ ([Ca2+]i) in cultures of rat sympathetic neurons at or below resting levels for several days was devised to determine whether Ca2+ signals are required for neurite outgrowth from neurons that depend on Nerve Growth Factor (NGF) for their growth and survival. To control [Ca2+]i, normal Ca2+ influx was eliminated by titration of extracellular Ca2+ with EGTA and reinstated through voltage-sensitive Ca2+ channels. The rate of neurite outgrowth and the number of neurites thus became dependent on the extent of depolarization by KCl, and withdrawal of KCl caused an immediate cessation of growth. Neurite outgrowth was completely blocked by the L type Ca2+ channel antagonists nifedipine, nitrendipine, D600, or diltiazem at sub- or micromolar concentrations. Measurement of [Ca2+]i in cell bodies using the fluorescent Ca2+ indicator fura-2 established that optimal growth, similar to that seen in normal medium, was obtained when [Ca2+]i was clamped at resting levels. These levels of [Ca2+]i were set by serum, which elevated [Ca2+]i by integral of 30 nM, whereas the addition of NGF had no effect on [Ca2+]i. The reduction of [Ca2+]o prevented neurite fasciculation but this had no effect on the rate of neurite elongation or on the number of extending neurites. These results show that neurite outgrowth from NGF-dependent neurons occurs over long periods in the complete absence of Ca2+ signals, suggesting that Ca2+ signals are not necessary for operating the basic machinery of neurite outgrowth.

Nerve growth factor-dependence of herpes simplex virus latency in peripheral sympathetic and sensory neurons in vitro

Previously, we reported that nerve growth factor (NGF) is required to maintain herpes simplex virus (HSV) latency in cultures of rat sympathetic neurons (Wilcox and Johnson, 1987, 1988). Here, we extend these results by showing that NGF was also required to maintain HSV latency in cultures of sensory neurons obtained from dorsal root ganglia of rats, monkeys, and humans. The interruption of the neuronal supply of NGF for 1 hr reactivated HSV, indicating that the latent virus was exquisitely sensitive to perturbations in the concentration or binding of NGF. A species-specific monoclonal antibody directed against the human NGF-receptor, which blocks NGF binding, reactivated latent HSV in human, but not rat, sensory neurons. In contrast, a monoclonal antibody against the rat NGF-receptor, which binds the receptor without blocking NGF action, did not produce reactivation. These results indicate that the effects of NGF on HSV latency are mediated via NGF binding to the NGF receptor. In addition, treatments that interfere with specific steps in the transduction of the NGF signal, including treatment with 6-hydroxydopamine and colchicine, reactivated latent HSV. Further, in neurons harboring latent virus, interruption of protein synthesis or RNA transcription for 1 hr resulted in viral reactivation, suggesting that a short-lived factor may be present in neurons which represses viral reactivation.

Induction of the vesicular monoamine transporter by elevated potassium concentration in cultures of rat sympathetic neurons

The expression of the vesicular monoamine transporter was studied in newborn rat sympathetic neurons and compared to that of the catecholamine biosynthesis enzymes tyrosine hydroxylase and dopamine-β-hydroxylase. The vesicular monoamine transporter was assayed using the specific ligand [ 3H]dihydrotetrabenazine. In cultures grown for 10 days in the presence of 35 mM K +, tyrosine hydroxylase activity and the density of [ 3H]dihydrotetrabenazine binding sites were increased by a similar 2–3-fold factor, while dopamine-β-hydroxylase activity and protein level were unchanged. Under these conditions, choline acetyltransferase activity was depressed by 90%. The induction of the vesicular monoamine transporter by high K + was dependent upon Ca 2+ entry through slow calcium channels since it was inhibited by the diphenylbutylpiperidine antagonist fluspirilene and by 20 mM Mg 2+, and was enhanced by the dihydropyridine agonist, Bay K8644. The induction of the vesicular monoamine transporter by neuronal depolarization indicates the existence of a Ca 2+-independent mechanism of coregulation for this intrinsic component of monoaminergic synaptic vesicles and tyrosine hydroxylase. On the other hand, the apparent absence of dopamine-β-hydroxylase induction is probably due to the continuous secretion of this intravesicular enzyme by the depolarized sympathetic neurons, an effect already observed in trans-synaptically stimulated adult sympathetic ganglion and adrenal medulla.

Different biological activities in conditioned media control the expression of a variety of neuropeptides in cultured sympathetic neurons

An intriguing question regarding neuronal development is how neurons choose which neurotransmitter and/or peptide to express among over 40 candidates. We find that heart cell conditioned medium (CM) induces a number of neuropeptides and/or their precursor mRNAs, as well as acetylcholine, in cultured rat sympathetic neurons: substance P, somatostatin, vasoactive intestinal polypeptide, enkephalin derivatives, and cholecystokinin, but not neuropeptide Y. Different patterns of peptide induction were observed for CMs from primary cultures of heart, gut, and skin. Acetylcholine and substance P were induced most effectively by serum-free heart cell CM; enkephalin derivatives were induced most effectively by skin cell CM; and somatostatin and vasoactive intestinal polypeptide were induced equally well by all of the CMs. These observations suggest the possibility that many distinct, diffusible factors can influence the choice of transmitter and/or peptide phenotype in developing neurons.

The Cholinergic Neuronal Differentiation Factor from Heart Cells Is Identical to Leukemia Inhibitory Factor

A protein secreted by cultured rat heart cells can direct the choice of neurotransmitter phenotype made by cultured rat sympathetic neurons. Structural analysis and biological assays demonstrated that this protein is identical to a protein that regulates the growth and differentiation of embryonic stem cells and myeloid cells, and that stimulates bone remodeling and acute-phase protein synthesis in hepatocytes. This protein has been termed D factor, DIA, DIF, DRF, HSFIII, and LIF. Thus, this cytokine, like IL-6 and TGF beta, regulates growth and differentiation in the embryo and in the adult in many tissues, now including the nervous system.

The role of Ca 2+ channels of the L-type in neurotransmitter plasticity of cultured sympathetic neurons

We have studied the effects of Ca 2+ antagonists and agonists on the development of choline acetyltransferase (ChAT), tyrosine hydroxylase (TOH) and acetylcholinesterase (AChE) in cultures of rat sympathetic neurons maintained for 6–9 days in low K + (5 mM) or high K + (35 mM) medium. Previous experiments have shown that high K + medium increases TOH activity and TOH-mRNA level up to 3.5-fold and depresses the development of AChE, in particular of its asymmetric A 12 form. Moreover, high K + medium inhibits ChAT induction by 90% in muscle-conditioned medium (Raynaud et al., Dev. Biol., 119 (1987) 305–312; 121 (1987) 548–558). None of the Ca 2+ antagonists tested affected the development of ChAT, TOH or AChE in low K + medium. In high K + medium, nitrendipine (3 μM) or fluspirilene (1 μM) fully restored ChAT induction by conditioned medium to the level observed in low K + medium. Other drugs (1 μM) gave partial reversion: flunarizine > (+)-PN 200-110 > (−)-D-888 > cinnarizine = lidoflazine. On the other hand, ChAT induction was not restored by a calmodulin inhibitor, calmidazolium (1 μM). Fluspirilene, PN 200-110, and nitrendipine also totally abolished TOH induction by high K + medium; fluspirilene (1 μM) suppressed the inhibitory effect of high K + medium on AChE development and restored the development of A 12 AChE. Conditioned medium also depresses AChE and blocks the development of A 12 AChE (Swerts et al., Dev. Biol., 103 (1984) 230–234), but these effects were insensitive to fluspirilene. The Ca 2+ agonist Bay K 8644 (1 μM) potentiated the effects of elevated K + on both ChAT and TOH. The data suggest that the effects of long-term depolarization on ChAT, TOH and AChE are mediated by Ca 2+ entry specifically through voltage-sensitive channels of the L-type. Our results on cultured sympathetic neurons raise the possibility that Ca 2+ antagonists, which are widely used clinically, may affect the expression of neurotransmitter phenotypic traits in vivo and interfere with trans-synaptic induction of enzymes.

Growth cone-growth cone interactions in cultures of rat sympathetic neurons

Growth cones of sympathetic neurons from the superior cervical ganglia of neonatal rats were studied using videomicroscopy to determine events following contact between growth cones and other cell surfaces, including other growth cones and neurites. A variety of behaviors were observed to occur upon contact between growth cones. Most commonly, one growth cone would collapse and subsequently retract upon establishing filopodial contact with the growth cone of another sympathetic neuron. Contacts resulting in collapse and retraction were often accompanied by a rapid and transient burst of lamellipodial activity along the neurite 30–50 μm proximal to the retracting growth cone. In no instances did interactions between growth cones and either fibroblasts or red blood cells result in the growth cone collapsing, suggesting that a specific recognition event was involved. On several occasions, growth cones were seen to track other growth cones, although fasciculation was rare. In some cases, there was no obvious response between contacting growth cones. Growth cone-growth cone contact was almost four times more likely to result in collapse and retraction than was growth cone-neurite contact (45% vs 12%, respectively). These observations suggest that the superior cervical ganglion may be composed of neurons with different cell surface determinants and that these determinants are more concentrated on the surface of growth cones than on neurites. These results further suggest that contact-mediated inhibition of growth cone locomotion may play an important role in growth cone guidance.

On the transduction mechanism for muscarine‐induced inhibition of M‐current in cultured rat sympathetic neurones.

1. Dissociated adult or fetal rat superior cervical ganglion cells were voltage-clamped through a single patch pipette. The voltage-dependent K+ current, IM (M-current), was maintained by including MgATP in the pipette solution and by buffering the solution pH to 6.7. 2. Bath-applied muscarine (0.4 microM) produced a reversible inhibition of IM. 3. Addition of Gpp(NH)p (200 microM) or GTP-gamma-S (500 microM) to the pipette solution induced a slowly developing inhibition of IM and prevented recovery from subsequent muscarine-induced inhibition. 4. Addition of GDP-beta-S (500 microM) to the pipette solution reduced the amount of IM inhibition produced by 0.4 microM-muscarine by 42% and reduced the associated inward shift of the holding current by 56%. 5. Cells responded normally to muscarine after pre-treatment for 4-27 h with 500 ng ml-1 pertussis toxin (PTx). 6. IM was not diminished by extracellular addition of 1 mM-dibutyryl cyclic AMP, 8-bromo-cyclic AMP or dibutyryl cyclic GMP, or of 10 microM-forskolin. 7. IM was not reduced by inclusion of Li+ (2 mM) or inositol 1,4,5-trisphosphate (IP3, 100 microM) in the patch pipette, nor by ionophoretic injection of IP3 from an inserted micropipette. 8. Addition of 4-beta-phorbol 12,13-dibutyrate (PDBu, 0.5-2 microM) to the extracellular medium partly inhibited IM and reduced an additional component of resting membrane current. This effect was not replicated by 4-alpha-phorbol 12,13-didecanoate. 9. It is concluded that the inhibition of IM by muscarine is mediated through activation of a PTx-insensitive GTP-binding protein. The effect of muscarine appears not to be mediated by cyclic nucleotides or IP3 but may possibly involve the generation of diacylglycerols and activation of protein kinase C.

Dynamics of the endoplasmic reticulum and other membranous organelles in growth cones of cultured neurons

The fluorescent lipophilic dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] was used to examine the distribution of membrane-bound organelles in growth cones of cultured rat sympathetic neurons. Within chemically fixed growth cones, intense DiOC6(3) fluorescence was localized predominately to the base or central region of growth cones. However, in most growth cones several thin DiOC6(3)-fluorescent processes radiated from the base into the periphery, and double fluorescence imaging of single growth cones indicated that these processes were highly colocalized (approximately 79%) with microtubules. The distribution of DiOC6(3) fluorescence in living growth cones was examined using low light-level fluorescence video microscopy. We observed thin fluorescent processes within the periphery of growth cones to undergo length excursions (extension/retraction) and to change orientation (move laterally). During growth cone advance, processes became progressively thicker and were gradually engulfed by the advancing fluorescent mass. When growth cones were viewed with video-enhanced differential interference contrast microscopy, the position of the fluorescent processes correlated with thickened extensions of central-type cytoplasm through which vesiclelike organelle transport often occurred. These observations indicate several features concerning the organization and movement of membranous organelles (MOs) in growth cones: (1) MOs are highly compartmentalized, the majority being localized to the growth cone base; (2) MOs advance into the periphery along distinct pathways probably associated with microtubules; (3) one or more thin continuous MOs, which most likely represent a thin tubular component of the endoplasmic reticulum, generally precedes advance of vesiclelike MOs along individual transport pathways; and (4) transport pathways with their associated MOs are spatially and temporally dynamic.

Ciliary neurotrophic factor induces cholinergic differentiation of rat sympathetic neurons in culture.

Ciliary neurotrophic factor (CNTF) influences the levels of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) in cultures of dissociated sympathetic neurons from newborn rats. In the presence of CNTF both the total and specific activity of ChAT was increased 7 d after culture by 15- and 18-fold, respectively, as compared to cultures kept in the absence of CNTF. Between 3 and 21 d in culture in the presence of CNTF the total ChAT activity increased by a factor of greater than 100. Immunotitration demonstrated that the elevated ChAT levels were due to an increased number of enzyme molecules. In contrast to the increase in ChAT levels, the total and specific activity levels of TH were decreased by 42 and 36%, respectively, after 7 d in culture. Half-maximal effects for both ChAT increase and TH decrease were obtained at CNTF concentrations of approximately 0.6 ng and maximal levels were reached at 1 ng of CNTF per milliliter of medium. The effect of CNTF on TH and ChAT levels were seen in serum-containing medium as well as in serum-free medium. CNTF was shown to have only a small effect on the long-term survival of rat sympathetic neurons. We therefore concluded that the effects of CNTF on ChAT and TH are not due to selective survival of cells that acquire cholinergic traits in vitro, but are rather due to the induction of cholinergic differentiation of noradrenergic sympathetic neurons.

Morphological differentiation of embryonic rat sympathetic neurons in tissue culture: I. Conditions under which neurons form axons but not dendrites

We have examined the morphology of fetal rat sympathetic neurons grown in serum-free medium in the absence of nonneuronal cells. Because cell density can affect phenotypic expression in vitro, the morphological analysis was subdivided into the study of isolated neurons (neurons whose somata were at least 150 μm from their nearest neighbor) and of more highly aggregated neurons. When isolated neurons were injected with intracellular markers, it was found that most (79%) had a single process emanating from their somata and that this unipolar state persisted for at least 8 weeks in vitro. The processes of unipolar sympathetic neurons had the appearance of axons in that they were thin and long, had a constant diameter, and were relatively unbranched. Cytochemical methods revealed that such processes had other axonal characteristics: (1) they were more reactive with a monoclonal antibody against phosphorylated forms of the M and H neurofilament subunits than with an antibody to nonphosphorylated forms of these proteins; (2) they also reacted with antibodies to the τ microtubule-associated protein and to the phosphorylated forms of the H neurofilament subunit; and (3) they contained only small amounts of RNA as determined by [ 3H]uridine autoradiography. These data indicate that neurons which normally form dendrites in vivo need not express this capacity in vitro and that axonal and dendritic growth can be dissociated under some conditions in culture. While most isolated neurons were unipolar, neurons in regions of high neuronal cell density were usually multipolar. In addition to axons, multipolar neurons had processes with some of the characteristics expected of rudimentary dendrites: they ended locally (usually within 100 μm), were often highly branched, and reacted with an antibody to nonphosphorylated forms of the M and H neurofilament subunits. The effects of density were most prominent when neurons were within aggregates in which the somata were in close apposition. Density-dependent changes in morphology were less frequently observed when neuronal somata were separated by greater distances (30–100 μm). These data indicate that the morphology of sympathetic neurons is subject to environmental regulation and that neuron-neuron interactions can promote the extension of rudimentary dendrites in vitro.

Morphological differentiation of embryonic rat sympathetic neurons in tissue culture: II. Serum promotes dendritic growth

In the preceding paper, we reported that embryonic rat sympathetic neurons formed axons, but not dendrites, when they were maintained in the absence of serum and nonneuronal cells. To assess the effects of serum-derived factors on cellular morphology, cultures were initially maintained in serum-free medium while nonneuronal cells were eliminated. Subsequently some cultures were chronically exposed either to fetal calf serum (10%) or to a high-molecular-weight ammonium sulfate fraction of serum (P40 material, 500 μg/ml). Phase-contrast microscopy revealed that serum and P40 material did not alter neuronal survival, but did cause flattening of the somata and fasciculation of processes. When neurons exposed to serum or P40 material were injected with Lucifer Yellow, it was found that the majority (⩾90%) had local, tapered processes that could be identified as dendrites by light microscopic criteria. These local processes also exhibited other dendritic characteristics in that (1) they reacted with monoclonal antibodies to nonphosphorylated forms of the M and H neurofilament subunits and to microtubule-associated protein 2; and (2) they had substantial amounts of RNA as determined by [ 3H]uridine autoradiography. Quantitative measurements of the effects of serum and P40 material on dendritic morphology revealed that (1) an 8-day exposure caused most neurons (>80%) to form dendrites; (2) neurons typically had more than one dendrite (mean of 4.1 ± 0.2 dendrites/cell after a 28-day exposure); and (3) the dendrites were relatively short with the maximum extent of the dendritic arbor being 110 ± 13 μm after 4 weeks. Serum and P40 material did not routinely cause the formation of supernumerary axons, did not alter radial axonal outgrowth from ganglion explants, and did not significantly increase [ 3H]leucine incorporation. Thus, serum contains a factor (or factors) which selectively stimulates the extension of dendrites, but not axons. If such a factor were operative in situ, it could play an important role in determining the morphology of sympathetic neurons. In examining the mechanism of serum-induced dendritic growth, we found that even high concentrations (5 μg/ml) of nerve growth factor failed to promote dendritic growth in the absence of serum; thus, nerve growth factor by itself is not a sufficient condition for the extension of dendrites. It was also observed that the percentage of cells forming dendrites in either serum-free or serum-containing media was unaffected by (1) agonists and antagonists known to bind to various cholinergic and adrenergic receptors; or (2) agents (25 m M K +, tetrodotoxin) known to affect electrical activity in vitro. Thus, serum-induced dendritic growth does not require normal electrical activity or afferent input.

Conditioned medium alters electrophysiological and transmitter-related properties expressed by rat enteric neurons in cell culture

We have previously shown that rat enteric neurons display many of their in vivo phenotypes when they are dissociated and grown in long-term cell culture. To assess the degree of plasticity of these phenotypes we have examined the effect of medium conditioned by rat heart cells because this treatment strongly affects transmitter properties in rat sympathetic neurons in culture. Growth of enteric neurons for 3–4 weeks in conditioned medium caused several changes that are similar to previously described effects of conditioned medium on other neuronal cell types in culture. When compared to cultures grown in control medium, cultures grown in conditioned medium: (i) contained three times as many large (> 25 μm) neurons; (ii) synthesized and stored 3–4 times as much acetylcholine; (iii) contained 4–5 times as many neurons with detectable 5-hydroxytryptamine immunoreactivity; and (iv) contained 10 times as many neurons that fired repetitively during sustained depolarization. Several other changes, which have not been reported in other systems, were also observed. Conditioned medium cultures: (i) contained many fewer neuronal processes with immunohistochemically detectable vasoactive intestinal polypeptide, substance P, somatostatin, and [Met]enkephalin; (ii) contained 70% fewer neuronal cell bodies with vasoactive intestinal polypeptide-like immunoreactivity; and (iii) contained four times as many neurons that had muscarinic responses to acetylcholine. None of the changes in properties described above uniformly affected all enteric neurons, even after 6 weeks of growth in conditioned medium. We conclude that the heterogeneity of enteric neuron phenotypes is established prior to birth and limits the capacity of certain subsets of neurons to respond to exogenous factors in the environment. Nevertheless, the phenotypes of other subsets of neurons displayed considerable plasticity when exposed to conditioned medium.

A muscle-derived substrate-bound factor that promotes neurite outgrowth from neurons of the central and peripheral nervous systems

The development and survival of spinal motor neurons depends upon muscle-derived trophic factors. Some circumstantial evidence suggested to us that the regulatory subunit of cyclic adenosine 3′:5′-monophosphate-dependent protein kinase (cAMP-dPK)-type II might be involved in neuritic outgrowth from spinal neurons. In the present study, we tested a commercial preparation of cAMP-dPK for neurite-promoting activity. Commercial cAMP-dPK-type II from skeletal and cardiac muscles elicited a significant neurite outgrowth from cultured embryonic chicken neurons when the enzyme preparation was bound to polylysine-coated substrata; type I cAMP-dPK from skeletal muscle was ineffective. Neither cAMP-dPK-type I nor -type II had a significant effect on the survival of spinal neurons in culture. Type II cAMP-dPK also stimulated neurite outgrowth from chicken cerebral hemisphere neurons, dorsal root ganglionic neurons, ciliary ganglionic neurons, and rat sympathetic ganglionic neurons in culture. The neurite-promoting activity appears to reside in a contaminant of the preparation since neither the purified regulatory nor catalytic subunits of cAMP-dPK-type II had an effect on neurite outgrowth per se from cultured neurons and since neurite-promoting activity did not correlate with [ 3H]cAMP binding or cAMP-dependent kinase activity. The neurite-promoting protein was then partially purified from commercial cAMP-dPK-type II by gel filtration on Sephadex G-200 followed by ion-exchange chromatography on DE-52 cellulose. Sodium dodecyl sulfate gel electrophoresis of the active protein peak revealed a major protein band (MW 50 kDa) and several minor bands (e.g., MW 200 kDa, 52 kDa, 45 kDa). Also, immunoblot analysis and immunoprecipitation revealed that the partially purified neurite-promoting protein was distinct from laminin, heparan sulfate proteoglycan, nerve growth factor, neural cell adhesion molecule, and fibronectin. Furthermore, the neurite-promoting activity was not diminished by treatment with heparinase nor was it bound to heparin conjugated to Sepharose. Our results demonstrate that a protein unrelated to laminin or its associated macromolecules and which copurifies with the type II cAMP-dPK of striated muscle stimulates neurite outgrowth from neurons of the central and peripheral nervous systems.

Rapid regulation of neuronal growth cone shape and surface morphology by nerve growth factor.

Scanning electron microscopy was used to study regulation of growth cone shape and surface morphology by nerve growth factor (NGF). The growth cones of cultured rat sympathetic neurons and neuronally-differentiated PC12 cells were observed under conditions of continuous NGF exposure, NGF withdrawal, and NGF readdition. Growth cones of cells cultured in the continuous presence of NGF were mostly spread in shape and about 60% possessed surface ruffles. Ruffles appeared to be largely restricted to growth cones in that few were observed on cell bodies and neurites. Withdrawal of NGF for 4-5 hr caused most of the growth cones to take on a non-spread or contracted appearance and to lose their ruffles. Readdition of NGF promoted rapid changes in growth cone properties. Within 30 sec, ruffling was again evident on the growth cones and remained prominent there throughout the course of treatment (up to 5 hr). This was in contrast to cell bodies on which, as previously reported, ruffling also occurred following NGF readdition, but only transiently (for less than 15 min). Respreading of growth cones also occurred under these conditions. This was evident within 1 min of NGF readdition and reached the levels observed in continuously-treated cultures within 1-2 hr. Neurites were also examined. Ruffles were only rarely present in the continuous presence of NGF and were absent after NGF withdrawal. NGF readdition elicited ruffling along neurites within 30 sec; the prevalence of such ruffles diminished to that seen in continuously-treated cultures within about an hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of an inhibitor of neuronal plasminogen activator released by heart cells

A basic understanding of growth cone dynamics and developmental events involving growth cones requires an understanding of the function and regulation of molecules associated with and released by growth cones. Rat sympathetic neurons in culture release a urokinase-like plasminogen activator from their distal processes and/or growth cones (Pittman, 1985a). When sympathetic neurons are grown in cocultures with heart cells, however, plasminogen activator activity is not detected. The absence of plasminogen activator activity in cocultures of sympathetic neurons and heart cells appears to be due to the release of an inhibitor of plasminogen activator by heart cells. This inhibitor has a molecular weight of approximately 50 kDa in the presence of SDS and apparent molecular weights of approximately 50 and greater than 2000 kDa under native conditions. A significant fraction of the large-molecular-weight form of the inhibitor is converted to the smaller form following treatment with heparinase. Extremely stable complexes of 68 and 80 kDa are formed between the heart inhibitor and the plasminogen activator, urokinase, such that the complexes withstand boiling in SDS/mercaptoethanol. The data are consistent with the formation of an 80 kDa urokinase-inhibitor complex in the presence of heparan sulfate proteoglycan and a 68 kDa complex in the absence of heparan sulfate proteoglycan. A highly purified preparation of the heart inhibitor produces a 2- to 3-fold increase in neurite outgrowth from sympathetic neurons. These data indicate that the activity of the plasminogen activator released by sympathetic neurons can be regulated by a normal target tissue and that this regulation may result in increased neurite outgrowth from the neurons.

Single calcium‐activated potassium channels recorded from cultured rat sympathetic neurones.

1. The properties of single Ca2+-activated K+ channels in cultured rat superior cervical ganglionic neurones were studied in cell-attached and excised patches using the patch-clamp technique. 2. In cell-attached patches using an external K+ concentration ([K+]o) of 150 mM, approximately equal to the internal [K+], the channel slope conductance was approximately 200 pS and independent of membrane voltage between -50 and +50 mV. Using [K+]o of 4.7 mM (providing a near physiological K+ gradient), the I-V relationship was non-linear with a slope conductance of approximately 120 pS at 0 mV. 3. The channel was selective for K+ over Cs+ and Na+ which were impermeant from either side of the membrane. Both Na+ and Cs+ also blocked the movement of K+ through the channel. Cs+ was active on either side of the membrane, whereas Na+ apparently blocked the channel only when applied to the cytoplasmic side. 4. The channel was activated by increasing the Ca2+ concentration on the inside of the membrane ([Ca2+]i). The channel was virtually inactive when [Ca2+]i = 0.01 microM. Depolarizing the patch at a constant [Ca2+]i usually further increased the opening probability. 5. The gating properties of the channel were studied using cell-attached patches. At potentials more negative than the resting membrane potential, the open-time distribution was described by a single exponential. On depolarization, two exponentials were required. The closed-time distribution was fitted by three exponentials. 6. Depolarization of the patch caused the long mean open lifetime to increase whilst the short mean open and closed lifetimes were unaffected. Both the intermediate and long mean closed lifetimes decreased with depolarization from -60 to +60 mV. 7. In cell-attached patches, the long mean open lifetimes were usually smaller than those observed in excised patches at depolarized potentials (greater than 0 mV). 8. A fourth closed state, possibly representing an inactivated form of the channel, was infrequently observed. A 50% substate of the full single-channel current was also observed occasionally. This substate was always associated with openings to the full current state. 9. The channel was blockable by external tetraethylammonium (25 microM-1 mM), Ba2+ (1-10 mM), and quinine (10-200 microM). External d-tubocurarine (25-100 microM) also blocked this IC channel. However it was insensitive to apamin (100-300 nM), muscarine (10 microM) and 4-aminopyridine (1-3 mM). The channel was also blocked by internal tetraethylammonium (5-10 mM) or Ba2+ (0.3-1 mM).(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of the effects of elevated K + ions and muscle-conditioned medium on the neurotransmitter phenotype of cultured sympathetic neurons

Neuronal depolarization and culture media conditioned by certain nonneuronal cells (CM) are known to exert opposite effects on the expression of cholinergic and noradrenergic traits in cultured rat sympathetic neurons. We have compared their effects on the developments of choline acetyltransferase (CAT), tyrosine hydroxylase (TOH), dopa decarboxylase (AADC) and acetylcholinesterase (AcChE) in these cultures. A macromolecular factor which was partially purified from CM increased CAT development in a dose-dependent manner and depressed the development of TOH and AADC by 5- to 10-fold. In the presence of intermediate concentrations of this partially purified factor, both CAT and catecholamine synthesizing enzymes developed to high levels, whereas high concentrations caused a long-lasting, but not total, impairment of TOH development. The effects of CM on both CAT and AADC activities resulted from variations in the number of immunotitratable enzyme molecules. Conversely, K + ions (30–40 m M) depressed the development of CAT by 90% and stimulated TOH development 2.5-fold. Cultures grown with CM in high K + medium had similar CAT and TOH activities as compared to those cultures grown without CM in low K + medium, suggesting that CM and K + ions had antagonistic effects on the expression of these enzymes. However, K + ions did not affect the development of AADC in these cultures. CM suppressed in a reversible manner the development of the 16 S form of AcChE. In the presence of 40 m M K +, the rate of development of AcChE was reduced. In particular, the development of 16 S AcChE was strikingly impaired, although not totally suppressed. The effect of elevated K + ions on the percentage of 16 S AcChE was rapidly reversible. It is concluded that CM and elevated K + ions have antagonistic effects on CAT and TOH, but not on AADC development; AcChE, in particular its asymmetric 16 S form, is regulated independently of the cholinergic/noradrenergic status of sympathetic neurons.