Innervation and target tissue interactions differentially regulate acetylcholine receptor subunit mRNA levels in developing neurons in situ

Abstract

Neurons engage in two distinct types of cell-cell interactions: they receive innervation and establish synapses on target tissues. Regulatory events that influence synapse formation and function on developing neurons are largely undefined. We show here that nicotinic acetylcholine receptor (AChR) subunit transcript levels are differentially regulated by innervation and target tissue interactions in developing chick ciliary ganglion neurons in situ. Using ganglia that have developed in the absence of pre- or postganglionic tissues and quantitative RT-PCR, we demonstrate that alpha 3 and beta 4 transcript levels are increased by innervation and target tissue interactions. In contrast, alpha 5 transcript levels are increased by innervation, but target tissues have little effect. Whole-cell ACh-induced currents, used to estimate the number of functional AChRs, change in correlation with alpha 3 and beta 4, but not alpha 5, transcript levels. A model is proposed in which the changes in AChR subunit expression regulate levels of synaptic activity, which is a critical determinant of synapse stabilization and elimination, and neuronal cell death.

Regulatory mechanisms that govern nicotinic synapse formation in neurons

“…Prior to innervation of autonomic neurons, few nAChRs are present intracellularly and no receptors are detected on the cell surface (Jacob, 1991). nAChR subunit transcript and protein levels increase significantly during the period of pre-and postganglionic synapse formation (Jacob, 1991;Corriveau and Berg, 1993;Devay et al, 1994;Mandelzys et al, 1994;Levey et al, 1995). Functional nAChRs at the neuronal cell surface also change during this period, with developmental increases in ACh current density and maturational changes in the relative abundance and kinetics of nAChR conductance classes (Margiotta and Gurantz, 1989;Moss et al, 1989;Engisch and Fischbach, 1990;Moss and Role, 1993).…”

“…The in vivo role of innervation in controlling nAChR expression was defined by in ovo microsurgical removal of the sole source of presynaptic inputs to chick CG neurons, the accessory oculomotor nucleus, prior to synaptogenesis (Furber et al, 1987;Engisch and Fischbach, 1992;Arenella et al, 1993;Levey et al, 1995;Brumwell et al, 2002). This surgery prevents innervation without direct damage to CG neurons or transection of their processes.…”

“…This surgery prevents innervation without direct damage to CG neurons or transection of their processes. In the absence of inputs, these neurons are ultrastructurally and electrophysiologically normal (Engisch and Fischbach, 1992;Dourado et al, 1994;Levey et al, 1995). Importantly, input-deprived CG neurons still form synapses on target tissues (Furber et al, 1987), enabling characterization of the specific effects of presynaptic innervation on nAChR expression.…”

“…The ␣7-nAChRs have other Ca 2ϩ -dependent nicotinic signaling and plasticity functions (Pugh and Berg, 1994;Broide and Leslie, 1999;Pugh and Margiotta, 2000;Chang and Berg, 2001;Bunker and Nishi, 2002). In CG neurons that have developed in the absence of presynaptic inputs, mRNAs encoding all subunits of both ␣3-nAChRs and ␣7-nAChRs are decreased compared to control age-matched CG neurons (Levey et al, 1995;Brumwell et al, 2002). All the nAChR subunit transcript levels are reduced to a similar extent in inputdeprived embryonic CG neurons, and intracellular protein levels for both ␣3-nAChRs and ␣7-nAChRs drop (Arenella et al, 1993;Brumwell et al, 2002;Kaiser et al, 2002).…”

“…These data demonstrate that inductive effects of presynaptic inputs on ␣3-nAChR and ␣7-nAChR subunit expression are specific, and that innervation coordinately regulates the 2␣7 ---expression of both receptor classes despite the fact that ␣3-nAChRs are synaptic and ␣7-nAChRs are extrasynaptic. Although ␣3-nAChR and ␣7-nAChR total protein levels are reduced in input-deprived CG neurons, whole-cell ACh responses are unchanged relative to age-matched controls (Engisch and Fischbach, 1992;Levey et al, 1995). The absence of a change in functional nAChR surface levels, despite declines in nAChR mRNA and protein levels, can possibly be explained by the observation that only a very small proportion (approximately 5%) of the internal biosynthetic pool of nAChRs is destined for the surface in developing CG neurons (Jacob et al, 1986;Stollberg and Berg, 1987;Levey et al, 1995).…”