Tetrahydrobiopterin Synthesis Rate and Turnover Time in Neuronal Cultures from Embryonic Rat Mesencephalon and Hypothalamus

6-(R)-(L-erythro-1',2'-Dihydroxypropyl)-2-amino- 4-hydroxy-5,6,7,8-tetrahydropteridine (tetrahydrobiopterin, BH4) synthesis rate and turnover time were estimated in cultures derived from the embryonic rat mesencephalon (MES) and hypothalamus (HYP) by following the decline in BH4 levels after blockade of BH4 biosynthesis by N-acetylserotonin (NAS) or 2,4-diamino-6-hydroxypyrimidine (DAHP). BH4 content of both culture systems decreased by 75% following an 8-h incubation with maximally effective concentrations of NAS (200 microM) or DAHP (10 mM). Parameters describing BH4 metabolism were calculated from steady-state levels of BH4 and first-order rate constants determined by a nonlinear regression analysis of the exponential BH4 decline. These parameters were confirmed using an alternative procedure that examined the first-order rate of recovery of BH4 following termination of BH4 synthesis inhibition. Steady-state levels of BH4 in HYP cultures (70.3 +/- 9.4 pg/culture) were significantly greater than that for MES (46.5 +/- 2.8 pg/culture). The average fractional rate constants of BH4 loss for MES (0.153 +/- 0.015/h) and HYP (0.159 +/- 0.014/h) were equivalent. The calculated rate of BH4 synthesis was significantly greater for HYP (11.29 +/- 2.13 pg/culture/h) than for MES (7.11 +/- 0.85 pg/culture/h), owing to the greater steady-state concentration of BH4. BH4 turnover time for MES (6.68 +/- 0.67 h) and HYP (6.40 +/- 0.62 h) and half-life for MES (4.63 +/- 0.46 h) and HYP (4.44 +/- 0.43 h) did not differ. The turnover of the cofactor is thus rapid enough that alterations in its synthesis or degradation could acutely modify the rate of monoamine biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Wild-Type and Mutated Copper/Zinc Superoxide Dismutase on Neuronal Survival and l-DOPA-Induced Toxicity in Postnatal Midbrain Culture

Abstract: Mutations in the free radical-scavenging enzyme copper/zinc superoxide dismutase (Cu/Zn-SOD) are associated with neuronal death in humans and mice. Here, we examine the effects of human wild-type (WT SOD) and mutant (Gly⁹³→ Ala; G93A) Cu/Zn-SOD enzyme on the fate of postnatal midbrain neurons. One-week-old cultures from transgenic mice expressing WT SOD enzyme had significantly more midbrain neurons and fewer necrotic and apoptotic neurons than non-transgenic cultures. In contrast, 1-week-old cultures from transgenic G93A mice expressing mutant SOD enzyme had significantly fewer midbrain neurons and more necrotic and apoptotic neurons than nontransgenic cultures. To subject postnatal midbrain neurons to oxidative stress, cultures were incubated with l -DOPA. l -DOPA at 200 µ M caused ∼50% loss of tyrosine hydroxylase (TH)-positive neurons in nontransgenic cultures and even greater loss in transgenic G93A cultures; no alterations were noted in GABA neuron numbers. In contrast, 200 µ M l -DOPA did not cause any significant reductions in TH-positive or GABA neuron numbers in transgenic WT SOD cultures. l -DOPA at 50 µ M had opposite effects, in that it significantly increased TH-positive, but not GABA neuron numbers in transgenic WT SOD and G93A and in nontransgenic cultures. These results indicate that increased amounts of WT SOD enzyme promote cell survival and protect against l -DOPA-induced dopaminergic neurotoxicity, whereas increased amounts of mutated Cu/Zn-SOD enzyme have inverse effects. As the spontaneous loss and l -DOPA-induced loss of postnatal dopaminergic midbrain neurons appear to be mediated by free radicals, our study supports the view that mutated Cu/Zn-SOD enzyme kills cells by oxidative stress.     

Conditions increasing the adrenergic properties of dissociated chick superior cervical ganglion neurons grown in long-term culture

Neurons dissociated from the embryonic chick superior cervical ganglion (SCG) were separated from ganglionic nonneuronal cells using a density gradient formed with Percoll. The sympathetic neurons were then grown for 3-4 weeks in serum containing medium on a polyornithine substrate precoated with heart-conditioned medium. Both catecholamine (CA) and acetylcholine (ACh) are synthesized and accumulated by these neurons, but the amount of CA is higher and increases much more over time in culture than the amount of ACh. The cultures become therefore more adrenergic with time. We report here that the adrenergic properties of these cells can be enhanced. A 3-fold increase in CA synthesis, as expressed on a per neuron basis, is obtained by increasing neuron cell density 3- to 4-fold. ACh synthesis, however, is decreased at high neuronal density. Optimal CA production is obtained at densities of 120-150,000 neurons/cm2. This effect is due to direct cell contact since it cannot be transferred to low density cultures by medium conditioned by high density cultures. Nerve growth factor concentrations 5-10-fold higher than the amount necessary for optimal neuronal survival (1 microgram/ml 7S NGF) increases CA production but do not affect ACh synthesis. This effect is highest at low plating densities (20-30,000 neurons/cm2, 10-fold increase) and progressively decreases with increasing neuronal density. No increase is obtained in high density cultures where CA production is maximal. In addition, we made the novel observation that medium conditioned by chick liver cells in culture (LCM) increases CA production approximately 4-fold, whereas it does not increase ACh production by the SCG neurons. Work is in progress to biochemically characterize the active component(s) present in the LCM and to determine whether they favor the survival of a subpopulation of adrenergic neurons possible present in these ganglia. Alternatively, the adrenergic differentiation of neurons initially capable of synthesizing both CA and ACh could be selectively increased by LCM.     

Biophysical properties of hypoglossal neurons in vitro: intracellular studies in adult and neonatal rats

A brain stem slice preparation from adult and neonatal (less than or equal to 12 days old) rats and intracellular recordings were used to examine the cellular properties of neurons within the hypoglossal (HYP) nucleus. Resting membrane potential (Vm) for adult hypoglossal neurons was -80 +/- 2 (SE) mV. Rheobase was 2.1 +/- 0.4 nA, and input resistance (RN) was 20.8 +/- 1.5 M omega and decreased during the hyperpolarizing period ("sag"). Compared with adult HYP cells, newborn HYP neurons had significantly lower resting potentials (Vm = -73 +/- 2 mV), lower rheobase (0.7 +/- 0.2 nA), and higher RN (27.6 +/- 3.9 M omega). Single action potentials, elicited by short depolarizing-current pulses, were followed by a slow afterhyperpolarization in adult [6.4 +/- 0.3 mV, time constant (tc) 31.0 +/- 1.2 ms] and newborn cells (7.4 +/- 0.2 mV, tc 37.2 +/- 8.2 ms). Prolonged outward current (2 s) produced little spike frequency adaptation in either adult or newborn neurons. Onset of spike activity was not delayed by hyperpolarizing pulses preceding depolarizations. In addition, pharmacological experiments showed that HYP neurons have a tetrodotoxin-sensitive Na+ current and a delayed and an inward rectifier current but no major Ca2+ current. We conclude the following. 1) Electrophysiological membrane properties mature postnatally in HYP neurons; some of these developmental changes can be ascribed to an increase in soma size and dendritic outgrowth but others cannot. 2) Adult HYP neurons, compared with other brain stem neurons (i.e., vagal cells or cells in the nucleus tractus solitarius), are not endowed with major Ca2+ currents or K+ currents such as the A current and the Ca2(+)-activated K+ current.     

Effect of chronic ethanol exposure on the electric membrane properties of DRG neurons in cell culture

Neural cell cultures of adult mouse dorsal root ganglia were utilized to investigate the effects of chronic ethanol exposure on neuronal electric membrane properties (EMP). After 12 days of exposure to various ethanol concentrations, the EMP of the neurons were determined in ethanol-free medium. Significant changes in a number of EMP were observed. Of particular physiological significance were decreased specific membrane resistance, increased specific membrane capacitance, relatively little change in membrane time constant, and increased electrical excitability. Various features of the action potential were also affected, e.g., reduced overshoot, afterhyperpolarization, and rate of rise. In preliminary experiments, EMP were determined at varying periods after the cultures had been withdrawn from ethanol medium and maintained in ethanol-free medium. These results indicated that the altered EMP persisted as long as one (C_m) to two (R_m) weeks after ethanol withdrawal. A possible mechanism for these ethanol-induced changes in EMP was suggested, utilizing the membrane expansion theory of anesthesia. Because of few previous reports demonstrating significant electrophysiological effects of ethanol at pharmacological concentrations, the neural cell culture system provides a useful new experimental model for studying the action of chronic ethanol exposure on neuronal EMP and the physical basis of the tolerance and withdrawal phenomena found in alcoholism and addiction in general. After being maintained for 12 days in culture media containing various concentrations of ethanol, non-neuronal cell survival was observed to have decreased in an approximately linear manner with increasing ethanol levels. By contrast, neuron survival was not affected until ethanol concentrations greater than 0.34 g % were used. This decreased cell survival due to chronic exposure to physiological levels of ethanol has not been reported previously. Neural cell cultures may therefore be useful for investigating the cellular pathology of chronic alcoholism and fetal alcohol syndrome.     

False transmitter, 5,6-dihydroxytryptamine as a tool in selecting serotonergic Helix neurons for studies with isolated,dialysed cells

1. Dialysed serotonergic neurons were identified, isolated from the ganglia of 5,6-dihydroxytryptamine (5,6-DHT) treated snail, Helix pomatia L. Twenty-four to 40 days after injection of 5,6-DHT into the animal, serotonergic neurons show a specific brown pigmentation, which stays there for several weeks. After protease digestion (0.5–1.0 mg/ml for 10–12 min) the labelled neurons can be easily separated. This method ensures the reliable identification of serotonergic neurons for intracellular dialysis.2. We showed that isolated serotonergic neurons maintain their membrane characteristics, and ion-currents can be registered under voltage clamp, just as from neurons of untreated animals. The threshold concentration of serotonin (10 ⁻⁷ M) and the survival time of pigment labelled dissociated cells were the same as for the control cells.3. Following 5-HT application, the voltage activated Ca-currents were either increased or decreased, depending on the neuron used.4. The different responses are probably caused by different receptors on the cell membrane or by the presence of different types of Ca-channels.5. The deactivation time constant of the Ca-current, calculated from the tail current, was also altered in the pigment labelled neuron following serotonin treatment.     

Back Cover: J. Biophotonics 10/2012

The cover art shows simultaneous multi‐site two‐photon photostimulation along the three‐dimensional (3D) dendritic arbor of a layer II neocortical neuron. Three foci positioned at different planes are shown at which glutamate (Glu) is locally released from its chemical cage (MNI) within each focus. Binding of this neurotransmitter to ionotropic ligand‐gated ion‐channels causes an excitatory post‐synaptic current flow of Na⁺/Ca⁺⁺ into the neuron. Photostimulating synapses at multiple 3D sites could elucidate how neurons process information. (Picture: M. A. Go et al., pp. 101–118 in this issue)     

Effect of the weaver (wv) mutation on cerebellar neuron differentiation. I. Qualitative observations of neuron behavior in culture

A mutant gene dose-dependent inhibition of cerebellar granule cell neuron survival and neurite growth in dissociated cultures of cerebellum from 7-day-old heterozygous (+/wv) and homozygous (wv/wv) weaver mutant mice (M. Willinger, D. M. Margolis, and R. L. Sidman. (1981), J. Supramol. Struc. 17, 79-86) has previously been observed. In the present phase-contrast study time-lapse microcinematography was performed between 10 and 80 hr in culture to determine which properties of neurite growth and neuron migration are affected by weaver gene expression. Neurite growth in +/+ cultures is rapid and discontinuous. Neurites are thin and cylindrical. Membrane movement occurs only at the growth cone. Growth cone contact with cell aggregates or glial somas results in the cessation of cone advancement and the induction of translocation of the neuronal soma toward the astrocyte. In cultures of +/wv and wv/wv cerebellar cells, abnormal neurite growth is characterized by frequent neurite retractions and reinitiations. Neuronal somas and neurite shafts are motile during elongation. Homozygous neurites and cones are pleomorphic. Normal, +/wv, and wv/wv neurons undergo nuclear translocation. Like +/+ neurons, +/wv neurons migrate in response to growth cone-cell soma contact. In contrast, homozygous soma frequently reverse direction and migrate independently of cell contact. Granule cell death occurs with increasing frequency with increasing gene dosage. Neurons are unusually active prior to the rapid onset of cell death. In summary, the weaver mutation impairs granule cell differentiation by affecting neurite maintenance, membrane motility, and neuron morphology. The loss of viability appears to be independent of, or secondary to, these targets of gene action.     

Defective interfering particles modulate VSV infection of dissociated neuron cultures.

Infection of dissociated neuron cultures of mice with VSV and its defective particle DI-T was studied using fluorescent light microscopy as well as transmission and scanning electron microscopy. When cultures are infected with wild virus, VSV replicates selectively in neurons, producing cell death within 24-48 hr. Sensory and immature neurons express viral antigen most rapidly. Viral antigen and viral budding sites are detected along the neuron soma and dendrites. When large amounts of DI-T particles are added to the wild virus inoculum, viral growth is completely suppressed in mature neurons, the cell killing effects of VSV are considerably delayed and co-infected cultures survive for 5-16 days. Viral antigen accumulates in cytoplasmic inclusions and on the membrane of neuron cell somas and dendrites in the virtual absence of viral assembly. Identical modulation of VSV infection in mature neuron cultures is obtained when DI-T particles are added before or after the wild virus, but ultraviolet inactivation of DIs completely abolishes their protective effect. Immature neurons or Vero cells cannot be protected from acute cytopathic changes by an equivalent amount of DI particles. Thus DIs interfere with replication and assembly of the wild virus and attenuate cell killing effects in mature neurons in vitro.     

Regulation of cholinergic expression in cultured spinal cord neurons

Factors regulating development of cholinergic spinal neurons were examined in cultures of dissociated embryonic rat spinal cord. Levels of choline acetyltransferase (CAT) activity in freshly dissociated cells decreased rapidly, remained low for the first week in culture, and then increased. The decrease in enzyme activity was partially prevented by increased cell density or by treatment with spinal cord membranes. CAT activity was also stimulated by treatment with MANS, a molecule solubilized from spinal cord membranes. The effects of MANS were greatest in low-density cultures and in freshly plated cells, suggesting that the molecule may substitute for the effects of elevated density and cell-cell contact. CAT activity in ventral (motor neuron-enriched) spinal cord cultures was similarly regulated by elevated density or treatment with MANS, whereas enzyme activity was largely unchanged in mediodorsal (autonomic neuron-enriched) cultures under these conditions. These observations suggest that development of cholinergic motor neurons and autonomic neurons are not regulated by the same factors. Treatment of ventral spinal cord cultures with MANS did not increase the number of cholinergic neurons detected by immunocytochemistry with a monoclonal CAT antibody, suggesting that MANS did not increase motor neuron survival but rather stimulated levels of CAT activity per neuron. These observations indicate that development of motor neurons can be regulated by cell-cell contact and that the MANS factor may mediate the stimulatory effects of cell-cell contact on cholinergic expression.     

Primary Cultures From Cerebral Cortex and Hippocampus Enriched in Glutamatergic and GABAergic Neurons

The aim was to define a primary culture system enriched in neurons using a defined culture medium, and characterize the model system as to cellular morphology and neuronal phenotypes. We found that these primary neuron enriched cultures from either newborn rat cerebral cortex or hippocampus contain small GABAergic and large glutamatergic neurons as well as astrocytes and microglia. Astrocytes in these cultures are morphologically differentiated with long, slender processes and interact with soluble factors responsible for induction and expression of the glutamate transporter GLT-1. The cultures achieve the highest expression of the vesicular glutamate transporter 1 (VGLUT1) and GLT-1 after 20 days in vitro. Conditioned media from these neuron enriched cultures also induced GLT-1 expression in primary astrocytic cultures, which were free from neurons. The amount of glutamatergic neurons guides the morphological maturation of astrocytes and GLT-1 expression both in the neuron enriched cultures and in the conditioned media supplemented astrocytic cultures. Interestingly, these cultures were not influenced or activated by the inflammatory stimulus lipopolysaccharide. This suggests that soluble factors from neurons protect microglia and astrocytes to become inflammatory reactive. In conclusion we have developed a well characterized culture model system enriched in neurons, taken from newborn rats and cultured in defined media. The neurons express different neuronal phenotypes. Such a model system is valuable when studying interactions between neurons and glial cells.     

Electric membrane properties of adult mouse DRG neurons and the effect of culture duration

The electrical membrane properties (EMP) of adult mouse dorsal root ganglion (DRG) neurons were characterized by an extensive electrophysiological investigation of 450 cells. The neurons were divided into two types: an M-type having an action potential with monophasic falling phase and a B-type with a more complex biphasic or triphasic falling phase. Compared to M-type, B-type were “slow” neurons with a higher specific membrane resistance (R_m), and a longer time constant (τ), duration of action potential (Δt), and absolute refractory period (ARP). B-type also had a larger amplitude action potential, afterhyperpolarization and positive overshoot. The action potential of the M-type neuron had only a Na⁺ component while that of the B-type had both a Na⁺ and a Ca²⁺ component. After two days in culture, M-type neurons exhibited phase bright cytoplasmic granules, which were seldom observed for B-type neurons. Although neuron survival remained constant during the first six days in culture (DIV), the relative frequency of occurrence of the M-type decreased from 82 to 50%. Thereafter, it decreased more gradually to a final value of approximately 20% after 40 DIV. It was concluded that at least during the first 6 DIV and possibly through to 40 DIV, M-type neurons transformed into B-type. Both M- and B-type neurons showed significant and similar changes in their EMP with increasing DIV (up to 40 DIV). For M- and B-types combined, R_m increased approximately 142%, τ by 204%, and no significant change in specific membrane capacitance was observed. Rheobasic threshold depolarization decreased 58%, while the resting membrane potential decreased by only 19%. These changes in the EMP of adult neurons are strikingly similar to changes in EMP observed in adult denervated muscle and in cultures of either embryonic nerve or muscle. This similarity suggested that the adult DRG neurons in cell culture undergo progressive dedifferentiation because of isolation from their usual trophic interactions. Determination of neuronal membrane electrical characteristics provides a new method for evaluating the effects of various possible trophic agents, e.g., hormones and tissue extracts, on the state of differentiation of neurons in cell culture.     

Haematological data for Matsumoto Eosinophilic Shinshu rats as determined by an automated haematology analyser

The Matsumoto Eosinophilic Shinshu (MES) rat originated from an inbred mutant colony of rats with spontaneous eosinophilia. As part of an investigation of the pathogenesis of the MES rat, we examined the haematology data for 106 males and 88 females and age-associated changes using an automated haematology analyser, flow cytometric analysis and morphological examination. The data at 10 weeks of age showed the MES rats had higher counts for eosinophils and neutrophils, slightly higher counts for lymphocytes, monocytes, basophils, and large unstained cells (LUCs), and slightly lower values for the erythrocytic parameters when compared with Sprague-Dawley (SD) rats. In data for MES rats aged 8 to 20 weeks, eosinophil counts increased with age up to 20 weeks together with some increased neutrophil counts. After 11 weeks of age, counts for lymphocytes, monocytes, basophils, and LUCs in the MES rats were also slightly increased. In female MES rats, flow cytometric analysis showed increased counts for pan-T+ cells, but blasts, abnormal granulocytes and lymphocytes were not detected morphologically. The MES rat characterized by the haematological findings could be a useful animal model for studies of hypereosinophilia.     

Transgenic Murine Dopaminergic Neurons Expressing Human Cu/Zn Superoxide Dismutase Exhibit Increased Density in Culture, but No Resistance to Methylphenylpyridinium-Induced Degeneration

Abstract: Primary dopaminergic neuronal cultures with increased superoxide dismutase (SOD) activity were established for studying the role of superoxide anion (O₂^?) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced degeneration of dopamine (DA) neurons. Mean SOD activity in cultures prepared from transgenic (human) Cu/Zn SOD (hSOD1) mice was 2.46–2.60 times greater than in cultures prepared from nontransgenic control mice. After 1 and 2 weeks in culture, the mean density of DA neurons [number of tyrosine hydroxylase-immunoreactive (TH-ir) cells per visual field] was significantly higher in cultures prepared from transgenic mice compared with those prepared from nontransgenic control mice (4.55–5.63 TH-ir neurons per field in hSOD1 cultures vs. 2.66–2.8 TH-ir neurons per field in control cultures). However, uptake of [³H]DA relative to uptake of [³H]GABA was only slightly greater in hSOD1 cultures than in normal cultures (14.1 nmol of DA/100 nmol of GABA vs. 12.1 nmol of DA/100 nmol of GABA). Resistance to MPP⁺ toxicity was not significantly different from that in normal cultures when based on density of surviving TH-ir cell bodies (EC₅₀ = 0.54 µM in hSOD1 and EC₅₀ = 0.37 µM in normal cultures). A more sensitive measure of DA neuron integrity and function ([³H]DA uptake) also failed to demonstrate increased resistance of hSOD1 cultures to the toxin (EC₅₀ = 73.7 nM in hSOD1 and EC₅₀ = 86.2 nM in controls). These results do not support the hypothesis that neurotoxicity of the active metabolite of MPTP, MPP⁺, is mediated by generation of O₂^? in the cytoplasm. Nevertheless, mesencephalic cultures with increased hSOD1 activity appear to survive better than normal control cultures in the oxidatively stressful environment of cell culture incubators, and such mesencephalic cells may be useful for cell grafting studies in animal models of Parkinson's disease.     

Mitochondria-Targeted Catalase Reverts the Neurotoxicity of hSOD1G93A Astrocytes without Extending the Survival of ALS-Linked Mutant hSOD1 Mice

Dominant mutations in the Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. The molecular mechanism underlying the toxic gain-of-function of mutant hSOD1s remains uncertain. Several lines of evidence suggest that toxicity to motor neurons requires damage to non-neuronal cells. In line with this observation, primary astrocytes isolated from mutant hSOD1 over-expressing rodents induce motor neuron death in co-culture. Mitochondrial alterations have been documented in both neuronal and glial cells from ALS patients as well as in ALS-animal models. In addition, mitochondrial dysfunction and increased oxidative stress have been linked to the toxicity of mutant hSOD1 in astrocytes and neurons. In mutant SOD1-linked ALS, mitochondrial alterations may be partially due to the increased association of mutant SOD1 with the outer membrane and intermembrane space of the mitochondria, where it can affect several critical aspects of mitochondrial function. We have previously shown that decreasing glutathione levels, which is crucial for peroxide detoxification in the mitochondria, significantly accelerates motor neuron death in hSOD1^G93A mice. Here we employed a catalase targeted to the mitochondria to investigate the effect of increased mitochondrial peroxide detoxification capacity in models of mutant hSOD1-mediated motor neuron death. The over-expression of mitochondria-targeted catalase improved mitochondrial antioxidant defenses and mitochondrial function in hSOD1^G93A astrocyte cultures. It also reverted the toxicity of hSOD1^G93A-expressing astrocytes towards co-cultured motor neurons, however ALS-animals did not develop the disease later or survive longer. Hence, while increased oxidative stress and mitochondrial dysfunction have been extensively documented in ALS, these results suggest that preventing peroxide-mediated mitochondrial damage alone is not sufficient to delay the disease.     

Acceleratory nervous regulation of juvenile myogenic hearts in the isopod crustacean Ligia exotica

We examined regulation of the myogenic heart by two identified cardioacceleratory neurons (CA1, CA2) in early juveniles of the isopod Ligia exotica. Repetitive stimulation of either the CA1 or CA2 axon increased the frequency and plateau amplitude of the action potential and decreased the maximum hyperpolarization of the cardiac muscle. These effects were larger with increasing stimulus frequency. The rate of increase in the frequency caused by CA1 stimulation was significantly larger than that by CA2. No impulse activity of the cardiac ganglion was induced by acceleratory nerve stimulation. The frequency of the muscle activity was decreased by injection of a hyperpolarizing current into the muscle during stimulation of the acceleratory nerve. In a quiescent heart, acceleratory nerve stimulation caused an overall depolarization in the muscle membrane and the amplitude of the depolarization induced by CA1 stimulation was significantly larger than that by CA2. These results suggest that CA1 and CA2 neurons regulate the myogenic heart affecting directly the cardiac muscle; the CA1 neuron produces more potent effects than does the CA2 neuron.     

Role of nerve growth factor in the development of rat sympathetic neurons in vitro. I. Survival, growth, and differentiation of catecholamine production

To study the effect of nerve growth factor (NGF) on neuronal survival, growth, and differentiation, cultures of dissociated neonatal rat sympathetic neurons virtually free of other cell types were maintained for 3-4 wk. In the absence of NGF, the neurons did not survive for more than a day. Increased levels of NGF increased neuronal survival and growth (total protein and total lipid phosphate); saturation occurred at 0.5 microgram/ml 7S NGF. Neuronal differentiation examined by measuring catecholamine (CA) production from tyrosine also depended on the level of NGF in the culture medium. As the NGF concentration was raised, CA production per neuron, per nanogram protein, or per picomole lipid phosphate increased until saturation was achieved between 1 and 5 microgram/ml 7S NGF. Thus, NGF induces neuronal survival, growth, and differentiation of CA production in a dose-dependent fashion. Neuronal growth and differentiation were quantitatively compared in the presence of the high and low molecular weight forms of NGF; no significant functional differences were found.     

Induction of cholinergic function in cultured sympathetic neurons by periosteal cells: cellular mechanisms.

Periosteum, the connective tissue surrounding bone, alters the transmitter properties of its sympathetic innervation during development in vivo and after transplantation. Initial noradrenergic properties are downregulated and the innervation acquires cholinergic and peptidergic properties. To elucidate the cellular mechanisms responsible, sympathetic neurons were cultured with primary periosteal cells or osteoblast cell lines. Both primary cells and an immature osteoblast cell line, MC3T3-E1, induced choline acetyltransferase (ChAT) activity. In contrast, lines representing marrow stromal cells or mature osteoblasts did not increase ChAT. Growth of periosteal cells with sympathetic neurons in transwell cultures that prevent direct contact between the neurons and periosteal cells or addition of periosteal cell-conditioned medium to neuron cultures induced ChAT, indicating that periosteal cells release a soluble cholinergic inducing factor. Antibodies against LIFRbeta, a receptor subunit shared by neuropoietic cytokines, prevented ChAT induction in periosteal cell/neuron cocultures, suggesting that a member of this family is responsible. ChAT activity was increased in neurons grown with periosteal cells or conditioned medium from mice lacking either leukemia inhibitory factor (LIF) or LIF and ciliary neurotrophic factor (CNTF). These results provide evidence that periosteal cells influence sympathetic neuron phenotype by releasing a soluble cholinergic factor that is neither LIF nor CNTF but signals via LIFRbeta.