Neural regulation of acetylcholine sensitivity in embryonic sympathetic neurons.

Abstract

The development of transmitter sensitivity is an important component of synaptic differentiation. Despite a wealth of information about the appearance of acetylcholine (AcCho) sensitivity at the neuromuscular junction, the onset and regulation of this critical aspect of synaptogenesis has not previously been examined for synapse formation between neurons. To determine whether there is a role of presynaptic input in the induction of AcCho sensitivity at interneuronal synapses, AcCho-induced currents were measured in embryonic sympathetic neurons before and after synapse formation in vitro. The total AcCho sensitivity of postsynaptic neurons was increased nearly 10-fold after innervation. The effects of innervation are mimicked by medium conditioned by preganglionic neurons, suggesting that presynaptic neurons regulate postsynaptic AcCho sensitivity by release of a soluble factor.These observations provide evidence that presynaptic input regulates neuronal sensitivity to an identified synaptic transmitter.Synaptogenesis between motoneurons and muscle is accompanied by both pre-and postsynaptic cell differentiation (see ref. 1 for review). A well-characterized aspect of muscle cell development with innervation is the enhanced sensitivity to transmitter at the site of nerve-muscle contact (refs. 2-4 and see refs. 5-7 for review). Arrival of the motoneuron is followed by a rapid increase in acetylcholine (AcCho) sensitivity colocalized with sites of presynaptic transmitter release (3). This is due, in part, to an increase in the number of AcCho receptors (AcChoR; see ref. 6 for review) and may depend upon a factor released from the motor nerve (see refs. 5-7 for review).In contrast to the wealth of information available on the development of AcCho sensitivity at the neuromuscular junction, this important aspect of synaptogenesis has not been studied at synapses formed between neurons. In vivo studies have been hampered by the difficulties of recording from neurons before and shortly after the arrival of presynaptic input and establishment of synapses. Although this is technically straightforward using an in vitro approach, there are few culture preparations for which synapses have been described between identified pre-and postsynaptic neurons (8-10).In the current study the developmental regulation of neuronal AcCho sensitivity with synaptogenesis was studied by using an in vitro preparation to innervate sympathetic neurons with their preganglionic partners and assay transmitter-evoked responses before and after synapse formation. This study indicates that innervation of sympathetic neurons is accompanied by a 10-fold increase in the total AcCho sensitivity. Since this enhancement of AcCho sensitivity is mimicked by medium conditioned by the preganglionic neurons, these findings suggest that neural regulation of transmitter sensitivity may play an important role in synapse formation between neurons. A preliminary account of some of these findings has been reported in abstract form (11). MATERIALS AND METHODS...

Regulatory mechanisms that govern nicotinic synapse formation in neurons

“…Instead, it is likely to be due to the simplified cell culture environment where positive regulatory signals have more dramatic effects. Interestingly, the effect is mimicked by addition of conditioned media from the appropriate preganglionic, as well as other, cholinergic neurons, suggesting that input-derived soluble factors are responsible for increasing nAChR expression (Role, 1988;Gardette et al, 1991;Yang et al, 1998). Taken together, the in vivo and in vitro data show that innervation is a strong inductive influence on nAChR expression in developing autonomic neurons.…”

“…A particular neuregulin isoform containing cysteine-rich domains, CRD-NRG, increases the ␣3-nAChR and ␣7-nAChR subunit mRNA levels, as well as whole-cell ACh responses, in cultured chick SG neurons (Yang et al, 1998). The changes in nAChR expression induced by CRD-NRG resemble the effects of presynaptic input in vitro (Role, 1988;Gardette et al, 1991). Interestingly, in vivo, CRD-NRG is expressed in the appropriate SG preganglionic inputs prior to synaptogenesis and is detected in presynaptic terminals (Yang et al, 1998).…”

“…The coculture of chick SG neurons with appropriate preganglionic tissue increases ␣3-nAChR and ␣7-nAChR subunit transcript levels two-to fivefold in comparison to neurons grown alone (Yang et al, 1998). In addition, in vitro innervation of SG neurons causes a 10-fold increase in whole-cell ACh responses, arising from an accelerated rate of appearance of new nAChRs on the cell surface (Role, 1988;Gardette et al, 1991). Although this result contrasts with the lack of change in the whole-cell ACh response on input-deprived CG neurons in vivo, it is not likely to represent a difference in the regulatory response to innervation in the two different autonomic neurons types.…”

“…In sharp contrast to the large increases in muscle-type nAChR levels and AChsupersensitivity caused by denervation of vertebrate skeletal muscle (Schuetze and Role, 1987), neuronal nAChR levels decrease after denervation. The surgical disruption of functional presynaptic inputs (preganglionic deafferentation) of newly hatched chick CG neurons in vivo causes two-to threefold decreases in ␣3, ␣5, and ␤4 subunit mRNA levels and a corresponding threefold decrease in the total number of ␣3-nAChRs (Jacob and Berg, 1987;1988;Levey and Jacob, 1996). ␣7-nAChR protein levels decline to a similar level as ␣3-nAChRs, but with a more rapid time course (Jacob and Berg, 1987).…”

Kinetic basis for the voltage-dependent inhibition of N-type calcium current by somatostatin and norepinephrine in chick sympathetic neurons

“…Sympathetic neurons were prepared according to Role (1988). Lumbar sympathetic ganglia from embryonic day 11 chicks were incubated for 30 min in a phosphate-buffered medium containing 0.01% trypsin.…”