Determination of synaptic phenotype: insulin and cAMP independently initiate development of electrotonic coupling between cultured sympathetic neurons.

Abstract

Electrotonic coupling between pairs of sympathetic neurons dissociated from superior cervical ganglia of neonatal rats is rare when cells are cultured for 2 weeks in a nutrient medium plus serum and is common when cells are cultured for the same period in serum-free defined medium. This defined medium is the same nutrient medium with five added factors (progesterone, transferrin, putrescine, insulin, and selenium). When added singly to serum-containing medium, insulin and, to a lesser extent, selenium promote the development of electrotonic and dye coupling. The insulin effect is obtained with doses as low as 0.01 jag/nm and is maximal after exposures from 3 to 5 days. The incidence of electrotonic coupling is also enhanced by exposure of cells to dibutyryl cAMP. This effect is obtained with doses as low as 0.1 mM, is faster (being maximal at =12 hr exposure), and is prolonged in the presence of the phosphodiesterase inhibitor caffeine. Butyrate itself promotes coupling to a small extent, but cAMP involvement is confirmed by similar effects of other membrane permeant analogues. Endogenous levels of cAMP are significantly elevated in cultures grown in the defined medium but not in those in serum-containing medium to which insulin or selenium are added. We conclude that the promotion of coupling by cAMP and by insulin or selenium are independent.The development of coupling in the defined medium thus seems to be a consequence of the addition of promoting substances (insulin, selenium) and the removal of an inhibitory effect of serum on cAMP levels.Neuronal differentiation and specialization are influenced by the environment, so that the phenotype of a neuron may depend on the extracellular milieu (1-4). For example, it is now well established that neurons may alter the transmitters they synthesize and secrete, and thus alter their phenotypes, under appropriate circumstances (5-7). Alteration of neurotransmitter expression in response to experimental alteration in the environment has been particularly well defined in neural crest-derived sympathetic and sensory neurons. For example, regions of the neural crest that normally give rise to cholinergic neurons can be induced to develop noradrenergic progeny by transplanting them into a region of the crest from which noradrenergic ganglia normally arise (8). Conversely, sympathetic neurons that in vivo express predominantly noradrenergic characteristics become cholinergic when cultured in the presence of certain non-neuronal cells (5).Phenotypic modifiability is not restricted to choice of transmitter; it also involves other aspects of neuronal differentiation. For example, Higgins and Burton (9) found that embryonic sympathetic neurons cultured in a chemically defined serum-free medium become electrotonically coupled, a mode of synaptic transmission not normally utilized by sympathetic gangliaJ This observation was confirmed by Wolinsky et al. (10). The cultured neurons continue to express noradrenergic characteristics, including tyrosine hydroxylase and dopamin...

Modulation of an electrical synapse between solitary pairs of catfish horizontal cells by dopamine and second messengers.

“…In contrast, some neurohumoral agents and growth factors change coupling over the course of hours or days. Examples of this slower type of regulation are an increase in coupling between cultured superior cervical ganglion cells produced by insulin (Kessler, Spray, Saez & Bennett, 1984) or between cultured hepatocytes produced by certain proteoglycans . These slower changes in coupling have been attributed to an increase in the expression of the gap junction protein .…”

Purification and partial characterization of a cholinergic neuronal differentiation factor.

“…Single postmitotic rat sympathetic neurons, for instance, can be made to form noradrenergic, cholinergic, or dual-function synapses with cardiac myocytes in microcultures (5)(6)(7). Depending on the developmental cues present in vitro, these neurons also have the capacity to produce several neuropeptides (8) and purines (9,10), and they can be induced to form electrical synapses (11)(12)(13). This plasticity in choice of transmitter is not simply a reflection of the developmental potential these neurons possess under artificial conditions, because at least some of these phenotypic transitions are actually expressed during normal embryogenesis.…”

“…In the case of the sympathoadrenal lineage, a number of such cues have been identified, including nerve growth factor (22)(23)(24), glucocorticoids (22)(23)(24)(25)(26)(27), epidermal growth factor (26), insulin (12,13), electrical activity and Ca2+ influx (28,29), rat serum (30), and nonneuronal cells and their conditioned medium (CM) (31)(32)(33)(34). Such signals have been termed "instructive," in order to distinguish them from growth factors that have been termed "permissive" (2).…”

“…For instance, nerve growth factor is permissive for the development of both noradrenergic and cholinergic sympathetic neurons (35), but it can also act instructively by converting chromaffin cells into neurons (22,23). Similarly, glucocorticoids and insulin are necessary for the survival of certain cells in the sympathoadrenal lineage, but these hormones can also influence the choice of phenotype as well (12,13,(22)(23)(24)(25)(26).…”

Superoxide dismutase ameliorates neuronal death from hypoxia in culture.

“…18 Cells were grown in a medium consisting of Ham's nutrient mixture F-12 (Gibco), penicillin (50 U/mL), streptomycin (50 fig/mh), nerve growth factor (100 ng/mL), and 10% fetal calf serum. Cultures were maintained at 37°C in 95% air/5% CO 2 at nearly 100% relative humidity.…”