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The signals that determine whether axons are ensheathed or myelinated by Schwann cells have long been elusive. We now report that threshold levels of neuregulin-1 (NRG1) type III on axons determine their ensheathment fate. Ensheathed axons express low levels whereas myelinated fibers express high levels of NRG1 type III. Sensory neurons from NRG1 type III deficient mice are poorly ensheathed and fail to myelinate; lentiviral-mediated expression of NRG1 type III rescues these defects. Expression also converts the normally unmyelinated axons of sympathetic neurons to myelination. Nerve fibers of mice haploinsufficient for NRG1 type III are disproportionately unmyelinated, aberrantly ensheathed, and hypomyelinated, with reduced conduction velocities. Type III is the sole NRG1 isoform retained at the axon surface and activates PI 3-kinase, which is required for Schwann cell myelination. These results indicate that levels of NRG1 type III, independent of axon diameter, provide a key instructive signal that determines the ensheathment fate of axons.

Neuregulin-1 (NRG-1) signaling has been implicated in inductive interactions between pre- and postsynaptic partners during synaptogenesis. We used gene targeting to selectively disrupt cysteine-rich domain-(CRD-) containing NRG-1 isoforms. In CRD-NRG-1-/-mice, peripheral projections defasciculated and displayed aberrant branching patterns within their targets. Motor nerve terminals were transiently associated with broad bands of postsynaptic ACh receptor (AChR) clusters. Initially, Schwann cell precursors accompanied peripheral projections, but later, Schwann cells were absent from axons in the periphery. Following initial stages of synapse formation, sensory and motor nerves withdrew and degenerated. Our data demonstrate the essential role of CRD-NRG-1-mediated signaling for coordinating nerve, target, and Schwann cell interactions in the normal maintenance of peripheral synapses, and ultimately in the survival of CRD-NRG-1-expressing neurons.

Elapid snake venom neurotoxins exert their effects through high-affinity interactions with specific neurotransmitter receptors. A novel murine gene, lynx1, is highly expressed in the brain and contains the cysteine-rich motif characteristic of this class of neurotoxins. Primary sequence and gene structure analyses reveal an evolutionary relationship between lynx1 and the Ly-6/neurotoxin gene family. lynx1 is expressed in large projection neurons in the hippocampus, cortex, and cerebellum. In cerebellar neurons, lynx1 protein is localized to a specific subdomain including the soma and proximal dendrites. lynx1 binding to brain sections correlates with the distribution of nAChRs, and application of lynx1 to Xenopus oocytes expressing nAChRs results in an increase in acetylcholine-evoked macroscopic currents. These results identify lynx1 as a novel protein modulator for nAChRs in vitro, which could have important implications in the regulation of cholinergic function in vivo.

We report here that neuregulin (NRG) isoforms with a conserved cysteine-rich domain (CRD) in their N terminus regulate expression of nicotinic acetylcholine receptors (nAChRs) at developing interneuronal synapses and report the isolation of transmembrane NRG isoforms with this CRD within the N-terminal portion. CRD-NRG mRNA and immunoreactive protein are detected early in developing presynaptic (visceral motor) neurons. The levels of expression of CRD-NRG peak prior to the formation of synapses with their postsynaptic partners, the ganglionic sympathetic neurons. Recombinant CRD-NRG mimics the effects of presynaptic input on target neurons. Functional deletion of CRD-NRG from presynaptic neurons abolishes the upregulation of nAChR expression induced by input-derived soluble material. Thus, CRD-NRG appears to be both a necessary and a sufficient signal for the control of neuronal nAChR expression during synaptogenesis.

Substance P (SP) is present in avian sympathetic ganglia and accelerates the decay rate of acetylcholine (ACh)-evoked macroscopic currents in sympathetic neurons. We demonstrate here that SP modulates ACh-elicited single channels in a manner consistent with an enhancement of ACh receptor (AChR) desensitization. Furthermore, since AChR channel function was monitored in cell-attached patches with SP applied to the extra-patch membrane, the peptide must act via a second messenger mechanism. SP specifically decreases the net ACh-activated single-channel current across the patch membrane by decreasing both channel opening frequency and mean open time kinetics. These experiments demonstrate that a peptide can modulate neuronal AChR function by a second messenger mechanism.

Measurement of acetylcholine (ACh)-induced currents indicates that the sensitivity of embryonic sympathetic neurons increases following innervation in vivo and in vitro. We have used single-channel recording to assess the contribution of changes in ACh receptor properties to this increase. Early in development (before synaptogenesis), we detect three classes of ACh-activated channels that differ in their conductance and kinetics. Molecular studies indicating a variety of neuronal receptor subunit clones suggest a similar diversity. Later in development (after innervation), changes in functional properties include increases in conductance and apparent mean open time, the addition of a new conductance class, as well as apparent clustering and segregation of channel types. These changes in channel function are compatible with the developmental increase in ACh sensitivity.