Denervation-induced decreases in enzyme activity of rat superior cervical ganglia differ in vivo and in vitro

The decrease in tyrosine hydroxylase activity following denervation in vivo and subsequent culture in vitro was examined to determine the role of the preganglionic nerve in this decrease. Ganglia denervated in vivo prior to culture showed a greater decrease than ganglia maintained for the same total period in vitro, and hence denervated for the same time, suggesting factors in addition to the loss of the preganglionic nerve are involved in vivo.     

Tissue culture analysis of neurite outgrowth in the presence and absence of serum: possible relevance for central nervous system regeneration

A tissue culture model has been developed to examine the hypothesis that axons can only regenerate when their growing tips are surrounded by extracellular fluid containing proteins derived from the blood. Fetal rat cerebral explants were cultured in serum medium for 10 days, followed by serum-free (SF) medium (from which serum had been removed) until 18 days in vitro (DIV). All explants cultured in serum medium for 0-10 DIV exhibited greater than 77% neurite viability (neurite viability ratio, NVR, 3.10). This degree of neurite viability was maintained for those explants exposed to serum until 18 DIV (NVR 2.82 at 18 DIV). By contrast, explants maintained in SF medium from 10-18 DIV had a much lower NVR, which, by 18 DIV, had declined to 0.30 (7.5% viability). Transmission electron microscopic analysis of explants fixed at 18 DIV confirmed these phase-contrast results and also showed a predominance of axonal profiles within the neurite population. In the center of explants, tissue viability was in excess of 75% in both the serum and SF media, suggesting that serum is of primary importance for axonal extension rather than neuronal survival. These data strengthen the hypothesis that blood-derived proteins may be needed for prolonged regeneration.     

Quantitation of the growth-associated protein B-50/GAP-43 and neurite outgrowth in PC12 cells

A combined assay to measure neurite outgrowth and B-50/GAP-43 levels in PC12 cells is reported. During NGF-induced neuritogenesis, B-50/GAP-43 expression was monitored by enzyme-linked immunosorbent assay (ELISA). Neurite outgrowth was quantified at the same time by the use of video image analysis. Sensitivity and reliability of the methods are shown with a dose-response and time curve of beta-NGF-induced neuritogenesis. A linear increase in total length of neurites was induced by concentrations of beta-NGF greater than or equal to 5 ng/ml and was accompanied by a linear increase in the amount of B-50/GAP-43. The combined methods presented here can conveniently and reliably establish subtle changes in neurite outgrowth and intracellular protein contents.     

Second-generation antipsychotic drugs, olanzapine, quetiapine, and clozapine enhance neurite outgrowth in PC12 cells via P13K/AKT, ERK, and pertussis toxin-sensitive pathways

Second-generation antipsychotic drugs, olanzapine, quetiapine, and clozapine, were found to enhance neurite outgrowth induced by nerve growth factor (NGF) in PC12 cells. These drugs increased the number of cells bearing neurites, the length of primary neurites, and the size of the cell body of NGF-differentiated PC12 cells. In addition, the drugs induced sprouting of neurite-like processes in PC12 cells in the absence of NGF. Olanzapine, quetiapine, and clozapine enhanced the phosphorylation of Akt and ERK in combination with NGF, and specific inhibitors of these pathways attenuated these effects. Pretreatment of cells overnight with pertussis toxin had no effect on NGF-induced differentiation but significantly decreased the effects of the antipsychotic drugs on neurite outgrowth, suggesting that G_i/G_o-coupled receptors are involved in the response to drug. A better understanding of the mechanisms underlying the effects of the second-generation drugs might suggest new therapeutic targets for enhancement of neurite outgrowth.     

Variation in number of small intensely fluorescent cells (SIF cells) in rat superior cervical ganglia

Changes in the relative synthesis rates of a number of specific proteins from rat dorsal root ganglia (DRG) were observed in response to transection of the sciatic nerve. The pattern of these changes was compared to that in rat sympathetic ganglia after transection of the postganglionic nerves. Although the overall pattern of proteins synthesized, and the pattern of changes seen after nerve transection, were quite similar in both ganglia, some specific differences were observed.     

The alpha-bungarotoxin site and its relation to the cholinergic and nerve growth factor mediated increases in tyrosine hydroxylase activity in cultures of sympathetic ganglia and chromaffin cells

alpha-Bungarotoxin has been proposed to interact with a membrane site in neuronal tissue which has the characteristics of a nicotinic acetylcholine receptor and also a trophic receptor. A nerve cell function which is affected by both nicotinic stimulation and nerve growth factor is the induction of tyrosine hydroxylase. For this reason, alpha-bungarotoxin was tested on the carbachol-induced increase and nerve growth factor-mediated increase in tyrosine hydroxylase activity in two preparations of neuronal origin, organ cultures of rat superior cervical ganglia and cultured bovine adrenal medullary cells. The results demonstrate that the alpha-bungarotoxin site is not involved in tyrosine hydroxylase induction.     

The intercellular communication via nitric oxide and its regulation in coupling of cyclic GMP synthesis upon stimulation of muscarinic cholinergic receptors in rat superior cervical sympathetic ganglia

Cyclic GMP (cGMP) production in rat superior cervical sympathetic ganglia (SCG) was markedly increased (ca. 7–9-fold) by the addition of either acetylcholine (ACh; 0.1 mM) or a muscarinic agonist, carbachol (Carb; 0.1 mM), in the presence of an inhibitor (3-isobutyl-1-methylxanthine) for cGMP hydrolytic enzyme during in vitro aerobic incubation at 37°C for 5 min. The ACh-induced accumulation of cGMP in SCG was effectively blocked (−73%) by the further addition of atropine (10 μM), a muscarinic antagonist, whereas a nicotinic blocker, hexamethonium (10 μM) partially antagonized (−41%) this ACh stimulation. The inhibitory effect of hexamethonium on ACh-evoked ganglionic cGMP production was effectively augmented (−83%) by addition ofN^G-monomethyl-l-arginine, (l-NMMA, 50 μM), a compound that inhibits nitric oxide (NO) synthesis froml-arginine. Comparable inhibition of cGMP formation was observed following application ofl-NMMA to the SCG upon stimulation of Carb. In contrast,l-NMMA had no effect on the decreased level of ACh-evoked cGMP production caused by the muscarinic antagonist. The Carb-induced elevation of ganglionic cGMP synthesis was significantly reduced within 1 min of incubation in the medium containing hemoglobin (Hb; 20 μM), an agent that scavenges only the extracellular fraction of NO. Thereafter, the tissue cGMP formation attenuated to the control level by subsequent incubation for several minutes. Addition of protein kinase C (PKC) activator,12-O-tetradecanoylphorbol 13-acetate (TPA; 1 μM) to the medium significantly decreased Carb-evoked cGMP synthesis (−61%) in SCG, whereas superoxide dismutase (SOD; 30 U/ml) only slightly suppressed the Carb stimulation. This finding supports the idea that PKC might play a role in dampening the muscarinic receptor-mediated increase in NO release within ganglionic tissue. In axotomized SCG one week prior to examination, where sympathetic neurons were degenerated and reactive proliferation of glial cells was in progress, no stimulatory effect of Carb-induced cGMP production via NO release was seen. These results provide evidence that a large fraction of NO generated upon stimulation of muscarinic receptors in sympathetic neuronal cells can possibly freely diffuse in extracellular space, and then be taken back into the same group of surrounding cells to stimulate cGMP production.     

The α-bungarotoxin site and its relation to the cholinergic and nerve growth factor mediated increases in tyrosine hydroxylase activity in cultures of sympathetic ganglia and chromaffin cells

α-Bungarotoxin has been proposed to interact with a membrane site in neuronal tissue which has the characteristics of a nicotinic acetylcholine receptor and also a trophic receptor. A nerve cell function which is affected by both nicotinic stimulation and nerve growth factor is the induction of tyrosine hydroxylase. For this reason, α-bungarotoxin was tested on the carbachol-induced increase and nerve growth factor-mediated increase in tyrosine hydroxylase activity in two preparations of neuronal origin, organ cultures of rat superior cervical ganglia and cultured bovine adrenal medullary cells. The results demonstrate that the α-bungarotoxin site is not involved in tyrosine hydroxylase induction.