Cultured Rat Superior Cervical Ganglion Research Articles

Cyclic AMP modulates but does not mediate the inhibition of [ 3H]norepinephrine release by activation of alpha-2 adrenergic receptors in cultured rat ganglion cells

The purpose of this study was to determine whether a decrease in cyclic AMP accumulation mediates the inhibition of norepinephrine release in response to alpha-2 adrenergic receptor activation in cultured rat superior cervical ganglion cells. Superior cervical ganglia from neonatal rats were dissociated and cultured on collagen-coated plastic strips. Neurotransmitter release was assessed by measuring the fractional overflow of tritium in superfused cells prelabeled with [ 3H]norepinephrine. Intracellular cyclic AMP accumulation was measured using radioimmunoassay. Electrical field stimulation at 1 Hz, 30 pulses, 1 ms duration at 20 min intervals produced an increase in the fractional overflow of tritium that was composed predominantly of intact [ 3H]norepinephrine. The alpha-2 adrenergic receptor agonist UK-14,304 dose-dependently attenuated the increase in fractional tritium overflow elicited by electrical field stimulation. The adenylyl cyclase activator, forskolin, increased cyclic AMP accumulation in superior cervical ganglion cells and UK-14,304 dose-dependently inhibited forskolin-stimulated cyclic AMP accumulation. UK-14,304 had no effect on basal cyclic AMP accumulation or cyclic AMP accumulation during electrical field stimulation. Forskolin (1–10 μM) or the non-hydrolysable cAMP analog, 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (1–100 μM), slightly increased basal and dose-dependently potentiated the increase in fractional tritium overflow in response to electrical stimulation. Despite enhancement by forskolin and 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate of fractional tritium overflow caused by electrical field stimulation, UK-14304 (1–10 μM) reduced release to a similar degree as that observed in the absence of forskolin or 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate. These data suggest that in cultured rat superior cervical ganglion cells, stimulation of alpha-2 adrenergic receptors decreases cyclic AMP accumulation when adenyl cyclase is activated. A decrease in cyclic AMP does not mediate the inhibition of [ 3H]norepinephrine release in response to alpha-2 adrenergic receptor activation, although elevations in cyclic AMP can enhance electrically-stimulated release of [ 3H]norepinephrine.

Electrical activity increases growth cone calcium but fails to inhibit neurite outgrowth from rat sympathetic neurons

Previous studies have shown that the growth of axons from both mouse dorsal root ganglion neurons and Helisoma neurons is arrested when the cells are electrically stimulated (Cohan and Kater, 1986; Fields et al., 1990a). Furthermore, in the case of Helisoma neurons, this arrest has been attributed to a rise in the calcium concentration in the growth cones (Cohan et al., 1987). To test the generality of these results, we examined the response of cultured rat superior cervical ganglion (SCG) neurons to electrical stimulation and changes in cytoplasmic calcium. Suprathreshold electrical stimulation of SCG neurons at 10 Hz by extracellular patch electrodes for periods of up to 1 hr had no measurable effect on their rate of growth. In agreement with previous studies, electrical stimulation was accompanied by a rise in the internal calcium concentration: when measured by the fluorescence of fura-2, growth cone calcium levels rose from about 100 nM to greater than 500 nM and then settled to a plateau value of about 350 nM. Despite this increase, however, growth of SCG neurons' processes continued. Our results show that electrical activity is not a universal signal for neurons to stop growing and that a rise in internal calcium does not always arrest the migration of growth cones.

Mechanism of Neurotransmitter Release Elicited by the Preferential ?1-Adrenergic Receptor Agonist Phenylephrine in Superfused Superior Cervical Ganglion Cells in Culture

Phenylephrine increased [3H]norepinephrine efflux and accumulation of cyclic AMP in cultured rat superior cervical ganglion cells superfused with Tyrode's solution. The purpose of this study was to determine the mechanism and relationship between these two events. Electrical stimulation (1-2 Hz), potassium chloride (50 mM), and the preferential alpha 1-adrenergic receptor agonist phenylephrine (1-100 microM) increased fractional tritium efflux, whereas methoxamine, cirazoline, and amidephrine were relatively ineffective. Phenylephrine, but not methoxamine and cirazoline, also increased cyclic AMP accumulation. Phenylephrine-induced tritium efflux was not altered by alpha- and beta-adrenergic receptor antagonists or by removal of extracellular calcium. Phenylephrine-induced cyclic AMP accumulation was blocked by the beta-adrenergic receptor antagonists propranolol and atenolol. Forskolin (10 microM) and the nonhydrolyzable cyclic AMP analogue 8-(4-chlorophenylthio)cyclic AMP (100 microM) had minimal effect on tritium efflux. However, phenylephrine-evoked increase in tritium efflux was dose dependently attenuated by the neuronal uptake blocker cocaine, and phenylephrine dose-dependently inhibited the incorporation of [3H]norepinephrine into neuronal stores. We conclude that the increase in tritium efflux induced by phenylephrine is independent of cyclic AMP accumulation and appears to be mediated by uptake of phenylephrine via the neuronal carrier-mediated amine transport process, which in turn promotes efflux of the adrenergic transmitter from its storage sites.

Mediators of nerve growth factor action in cultured rat superior cervical ganglion neurones

Mediators of nerve growth factor action in cultured rat superior cervical ganglion neurones

Autoradiographic localization of muscarinic receptors on cultured, peptide-containing neurones from newborn rat superior cervical ganglion

In order to identify subpopulations of cultured rat superior cervical ganglion (SCG) neurones which express muscarinic receptors, a combination of immunocytochemistry and autoradiography was performed on these cultures. Antibodies to vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) were used to immunostain cultures that had previously been labelled with the irreversible muscarinic antagonist, [ 3H]propylbenzylylcholine mustard (PrBCM). Binding sites for [ 3H]PrBCM were observed on a large subpopulation of 65–85% of the ganglionic neuronal cell bodies. Specific labelling was not associated with non-neuronal cells found in these cultures. Approximately 60% of the SCG neurones were NPY-like immunoreactive (NPY-LI), a high proportion of which expressed muscarinic receptors. Five to 10% of the SCG neurones were VIP-LI, a small subpopulation of which displayed [ 3H]PrBCM binding sites. Receptor distribution on cell bodies was usually uniform, but occasionally, regions of high receptor density were seen. Dense networks of both varicose and non-varicose NPY-LI fibres were seen throughout the culture, a subpopulation of which expressed muscarinic receptors. Occasional VIP-LI fibres were also labelled with silver grains for [ 3H]PrBCM, but in less abundance than those for NPY-LI fibres. Conversely, neurones expressing muscarinic receptors were often immunonegative for either VIP or NPY: therefore, the identity of some of the neurones which express muscarinic receptors remains to be determined.

The organization of myosin and actin in rapid frozen nerve growth cones.

Rapid freezing and freeze substitution were used in conjunction with immunofluorescence, whole mount EM, and immunoelectron microscopy to study the organization of myosin and actin in growth cones of cultured rat superior cervical ganglion neurons. The general cytoplasmic organization was determined by whole mount EM; tight microfilament bundles formed the core of filopodia while a dense meshwork formed the underlying structure of lamellipodia. Although the central microtubule and organelle-rich region of the growth cone had fewer microfilaments, dense foci and bundles of microfilaments were usually observed. Anti-actin immunofluorescence and rhodamine phalloidin staining of f-actin both showed intense staining of filopodia and lamellipodia. In addition, staining of bundles and foci were observed in central regions suggesting that the majority of the microfilaments seen by whole mount EM are actin filaments. Anti-myosin immunofluorescence was brightest in the central region and usually had a punctate pattern. Although less intense, anti-myosin staining was also seen in peripheral regions; it was most prominent at the border with the central region, in portions of lamellipodia undergoing ruffling, and in spots along the shaft and at the base of filopodia. Immunoelectron microscopy of myosin using postembedment labeling with colloidal gold showed a similar distribution to that seen by immunofluorescence. Label was scattered throughout the growth cone, but present as distinct aggregates in the peripheral region mainly along the border with the central region. Less frequently, aggregates were also seen centrally and along the shaft and at the base of filopodia. This distribution is consistent with myosins involvement in the production of tension and movements of growth cone filopodia and lamellipodia that occur during active neurite elongation.

Autoradiographic localization of binding sites for 125I-substance P on neurones from cultured rat superior cervical ganglion

Using an autoradiographic receptor binding technique, the distribution of substance P (SP) receptors on cells cultured from the superior cervical ganglia (SCG) of newborn rats was investigated. Binding sites for 125I-Bolton-Hunter-SP were observed on a subpopulation of 35–50% of the ganglion neurones. The percentage of labelled neurones remained constant whether the cultures were seeded densely or sparsely. Variation in the density of labelling was observed on different neuronal clusters. Neuronal cell bodies were often densely labelled, but neuronal processes were rarely labelled. In contrast with the neuronal cells, specific labelling was not associated with other cell types found in this culture preparation, including fibroblasts, glial cells and other non-neuronal supporting cells. These results are interpreted to suggest that there is a subpopulation of SCG neurones which, by virtue of their expressing SP receptors, are responsive to SP and have a physiological role within the ganglion.

Selective destruction of nerve growth factor receptor-bearing cells in vitro using a hybrid toxin composed of ricin A chain and a monoclonal antibody against the nerve growth factor receptor.

A hybrid toxin composed of ricin A chain and a monoclonal antibody directed against the rat nerve growth factor (NGF) receptor (192-IgG) was prepared using the heterobifunctional cross-linking agent N-succinimidyl-3-(2-pyridyldithio)-propionate and purified by affinity chromatography. Characterization studies showed that the hybrid, 192-s-s-A, displaced bound 125I-labeled 192-IgG from rat superior cervical ganglion (SCG) membranes with an IC50 3-5 times lower than that of unconjugated 192-IgG. When incubated with cultured rat SCG neurons, 192-s-s-A inhibited protein synthesis in a concentration-dependent fashion. The effect of 192-s-s-A on these neurons was reversed by coincubation with an excess of 192-IgG. The IC50 of 192-s-s-A on protein synthesis in rat SCG neurons was 4 nM. Intact ricin and ricin A chain inhibited protein synthesis in these neurons with IC50 values of 5 pM and 500 nM, respectively. The 192-s-s-A hybrid had no effect on mouse SCG neurons or a human melanoma cell line known to have NGF receptors. This is consistent with the finding that 192-IgG recognizes only the rat NGF receptor. Also, 192-s-s-A did not inhibit protein synthesis in primary cultures of rat skeletal muscle or Vero cells, which do not have cell surface receptors for NGF. 192-s-s-A was able to inhibit protein synthesis in PC12 cells but the potency was 10-100 times less in these cells compared to rat SCG neurons. Ricin and A chain were also 10-100 times less potent in PC12 cells than neurons. Rat SCG neurons exposed to 192-s-s-A lost their refractile appearance under phase-contrast optics, showed granular degeneration of neurites, and died. Thus the decreased protein synthesis caused by the hybrid toxin correlated with the morphological destruction of the neurons. 192-s-s-A represents a potentially powerful tool by which to selectively destroy NGF receptor-bearing cells in vitro. The hybrid toxin may prove useful as an in vivo toxin.

Tyrosine hydroxylase activity decreases with induction of cholinergic properties in cultured sympathetic neurons

Establishment of transmitter phenotype is an essential step in neuronal development. Studies on rat sympathetic neurons both in vivo and in vitro have provided evidence that mature cholinergic sympathetic neurons arise from previously noradrenergic neurons. Cultured rat superior cervical ganglion neurons can be influenced by their environment to remain noradrenergic, to acquire dual transmitter function, or to become predominantly cholinergic. Several other neuronal traits, such as a variety of surface molecules and released proteins, change simultaneously with levels of catecholamine and acetylcholine production, suggesting that various components of transmitter phenotype are regulated in concert. In this report, tyrosine hydroxylase levels are compared in neurons cultured under noradrenergic, dual function, or cholinergic conditions. Both enzyme activity in cell extracts and immunocytochemical staining were measured. These methods showed significantly less tyrosine hydroxylase enzyme activity and immunoreactive material in cholinergic cultures compared to noradrenergic and dual function cultures. These data support the interpretation that a switch in transmitter status from noradrenergic to cholinergic has occurred. This interpretation contrasts with that of Iacovitti et al. (Iacovitti, L., T. H. Joh, D. H. Park, and R. P. Bunge (1981) J. Neurosci. 1: 685-690), who conducted their experiments under critically different culture conditions.