On the Role of Cyclic Nucleotides in the Transmitter Choice Made by Cultured Sympathetic Neuron9

Previous investigations have established that electrical activity or chronic depolarization influences the development of neonatal rat sympathetic neurons in dissociated cell culture. Depolarization reduces their ability to respond to a cholinergic inducing factor produced by non-neuronal cells, allowing normal adrenergic differentiation to proceed (Walicke, P., R. Campenot, and P. Patterson (1977) Proc. Natl. Acad. Sci. U. S. A. 74: 5767-5771). The present study examines whether the developmental effects of depolarization are mediated through cyclic nucleotides. Addition of dibutyryl CAMP, dibutyryl cGMP, adenosine, prostaglandin El, and cholera toxin all raise neuronal cyclic nucleotide levels and qualitatively mimic the developmental effects of depolarization. However, the quantitative decrease in acetylcholine production caused by these cyclic nucleotide agents is much smaller than that caused by depolarization. Short (48-hr) exposures to the cyclic nucleotide derivatives do not alter transmitter synthesis, indicating that long term developmental changes are involved. Chronic depolarization with elevated K’ increases neuronal CAMP 2-fold but has little effect on cGMP. The increase in CAMP is maintained during several weeks of depolarization and is present as early as the 3rd day in vitro, preceding the significant alterations in adrenergic and cholinergic differentiation. Exposure to 2 mM theophylline also increases neuronal CAMP, but, in contrast to the other agents, it enhances cholinergic differentiation. In combination with elevated K’, theophylline further increases neuronal CAMP but still favors cholinergic differentiation. Thus, although CAMP satisfies some criteria for being the second messenger in the developmental effects of depolarization, several findings are inconsistent with the nucleotide playing a central role: (i) Depolarization has much larger effects on transmitter choice than the cyclic nucleotide agents and (ii) theophylline can uncouple cyclic nucleotide levels from the developmental events. Cultures of dissociated sympathetic neurons from superior cervical ganglia of newborn rats have been studied extensively in order to delineate signals influencing neuronal development. When grown alone in culture, these neurons can complete the course of adrenergic differentiation that they had begun in vivo. However, when grown in the presence of a variety of non-neuronal cell types, or in medium conditioned by such non-neuronal cells (CM), the sympathetic neurons can be induced to express cholinergic properties. Concomitant with the increase in cholinergic function is a decrease in the ability to synthesize catecholamine (CA) (Patterson, 1978; Bunge et al., 1978). Analysis of single neurons showed that the vast majority of these cells can be induced to ’ This work was supported by the National Institute of Neurological and Communicative Disorders and Stroke, the Dysautonomia Foundation, and the American and Massachusetts Heart Associations. Dr. Walicke was a United States Public Health Service predoctoral fellow. Preliminary reports of this work have appeared previously (Walicke, P. A., and P. H. Patterson (1978) Sot. Neurosci. Abstr. 4: 129; Walicke, P. A., and P. H. Patterson (1979) Sot. Neurosci. Abstr. 5: 183). ’ To whom correspondence should be addressed. become cholinergic (Reichardt and Patterson, 1977). This observation was surprising, since most sympathetic neurons in vivo become adrenergic. It seemed possible that there were factors missing from the culture system which, in vivo, might counteract the response of the neurons to the cholinergic factor. It was observed that chronic depolarization with 20 mM K’ or veratridine (an alkaloid which opens voltagedependent Na’ channels) directed the neurons to complete adrenergic differentiation, even in the presence of CM (Walicke et al., 1977). The effect of these agents was very striking; the acetylcholine to catecholamine (ACh/ CA) ratio (used as an index of the proportion of neurons undergoing cholinergic or adrenergic differentiation) w IS lowered as much as 500-fold. The change was almost entirely due to suppression of ACh synthesis, although 20 mM K+, in the absence of CM, appeared to enhance adrenergic differentiation somewhat, roughly doubling the content of CA/neuron and increasing the proportion of small granular (adrenergic) vesicles (Landis, 1980). Direct electrical stimulation at a frequency of 1 Hz also markedly decreased the response to CM; thus, the effects 334 Walicke and Patterson Vol. 1, No. 4, Apr. 1981 of chronic depolarization appear to be the same as those of normal neuronal activity. The effect of depolarization or activity on transmitter choice was a specific one in that depolarization did not alter neuronal survival or overall growth, as monitored by total protein content (Walicke et al., 1977). The alterations in transmitter choice were interpreted as indicating that electrical activity acts as a determination or maturation signal, fixing the neurons in their initial adrenergic mode. Little is known about how electrical activity may lead to long term alterations in cellular metabolism. It was of interest, therefore, to use this system to investigate the intracellular mechanism through which activity has its effect on development. In adult adrenergic tissues, increased synaptic stimulation leads to induction of tyrosine hydroxylase (TH) and dopamine ,f?-hydroxylase (Zigmond and Chalazonitis, 1979), an effect somewhat analogous to that under study here. Cyclic adenosine 3’:5’monophosphate (CAMP) has been suggested to serve as the second messenger in trans-synaptic induction in the adrenal medulla (Guidotti and Costa, 1977). Derivatives of CAMP increase the levels of tyrosine hydroxylase and dopamine /?-hydroxylase and CA content in neuroblastoma cell lines (Prasad, 1975; Waymire et al., 1978) and organ-cultured ganglia (Goodman et al., 1974; Keen and McLean, 1974; Mackay and Iversen, 1972). Cyclic AMP, therefore, seemed a reasonable candidate for the second messenger in the developmental effects of depolarization. If CAMP is playing such a role, then one would expect that (i) exogenous cyclic nucleotides and effecters which increase cellular cyclic nucleotide levels should be able to mimic the developmental effects of activity, (ii) increases in activity should lead to increases in CAMP, (iii) the increase in CAMP should precede detectable changes in transmitter content, (iu) phosphodiesterase inhibitors should increase the developmental effects of activity, and (u) agents which antagonize the effects of activity on transmitter choice should block its effects on CAMP. These criteria are examined in the experiments presented in this paper. Materials and Methods Superior cervical ganglia were dissected and cultured as previously described (Hawrot and Patterson, 1978). All cultures were plated initially into normal L15-CO2 medium; pharmacological agents were added with the first change of medium on day 2. All of the medium needed for an experiment was mixed with the appropriate drugs at the time of the first feeding, and aliquots of appropriate size for one feeding were made up and frozen until needed. Cholera toxin was purchased from Schwarz/Mann. RO 20-1724 was a gift of Roche Pharmaceuticals. All other chemicals were obtained from Sigma. Conditioned medium. CM was made by incubation on monolayer cultures of rat heart or skeletal muscle cells as previously described (Patterson and Chun, 1977a). Instead of CM, some neuronal cultures were incubated with a CM preparation purified by ammonium sulfate precipitation (M. Weber, unpublished technique). For this, 2.5 mM EGTA (ethylene glycol his@-aminoethyl ether)-2\r, N’-tetra-acetic acid) and 0.3 mM phenylmethylsulfonyl flouride were added to pooled batches of Methocel-free CM at 4’C. The pH was held at about 7.3 with NH40H, observing the color of the indicator dye in the medium. Ammonium sulfate was added to 50% saturation and the CM was centrifuged at 4000 x g for 30 min. The precipitate was discarded, ammonium sulfate was added to saturation, and the CM was centrifuged at 4000 x g for 60 min. The precipitate was resuspended in a minimal volume of Hz0 and placed in Spectropore dialysis tubing (12,000 to 14,000 daltons cutoff). Dialysis tubing was prepared by boiling twice for 15 min in glass-distilled HzO, followed by autoclaving. Dialysis was for 2 days at 4°C against three l-liter changes of distilled HZO. The retentate was stored at -20°C except for an aliquot which was incubated at 37°C to test for sterility. Sterile technique was employed whenever possible, and the resulting material was usually sterile. The dialyzed ammonium sulfate fraction was added to give medium containing the equivalent of 60 to 100% CM, and it never constituted more than 10% of the final volume of the medium. Assays. Neuronal transmitter synthesis was determined by incubation with [“Hltyrosine and [:‘H]choline for 4 hr and electrophoretic separation of the products as previously described (Patterson and Chun, 1977a). All incubations were performed in normal L15-CO2 medium (in the absence of phosphodiesterase inhibitors, cyclic nucleotide derivatives, depolarizing agents, and CM). Cyclic nucleotides were assayed with CAMP and cGMP (cyclic guanosine 3’:5’-monophosphate) radioimmunoassay kits purchased from New England Nuclear using the acetylated version with a sensitivity of 5 fmol. The day before assay, Methocel-free L15-CO2 medium with all other additives and pertinent pharmacological agents was placed in the same incubator as the cultures to reach the same temperature and pH. Before harvesting, cultures were rinsed with 1 ml of this medium and then incubated 10 min in another ml. For cultures grown in dibutyryl CAMP (dbcAMP) or dibutyryl cGMP (dbcGMP), the cyclic nucleotide was omitted from the rinse, and the cultures were rinsed five times with 1 ml to remove the added nucleotide. The medium was removed, the culture was drained carefully with a cottontipped applicator, and the neurons were carefully scraped free of the collagen film with a small surgical blade. Two cultures were combined for each sample. They were homogenized in 100 ~1 of 6% trichloroacetic acid in a microhomogenizer (Micro or Kontes) which then was rinsed twice with 100 ~1 each of acid. The combined samples were centrifuged for 30 min at high speed in a Sorvall GLC-1 centrifuge at 4°C. The supernatant fraction was collected for cyclic nucleotide assay. The protein precipitate was redissolved and assayed by the method of Lowry (Lowry et al., 1951) using bovine serum albumin as a standard. The supernatant fractions were washed four times with 1.5 ml of water-saturated ether. They were incubated briefly in a warm water bath under a stream of air to remove the last of the ether and then frozen and lyophilized. The samples were resuspended in 200 fl of acetate buffer (0.05 M, pH 6.2); 100 ~1 was used for the cGMP assay and 50 to 100 ~1 for the CAMP assay. Recoveries of the [3H]cAMP and [3H]cGMP added to the assays were 30 to 40%. External standard curves were run with each assay. The Journal of Neuroscience Cyclic Nucleotides and Transmitter Choice 335