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The alpha3 subunit of the neuronal nicotinic acetylcholine receptor is widely expressed in autonomic ganglia and in some parts of the brain. The alpha3 subunit can form heteromultimeric ion channels with other alpha subunits and with beta2 and beta4 subunits, but its function in vivo is poorly understood. We prepared a null mutation for the alpha3 gene by deletion of exon 5 and found that homozygous (-/-) mice lacked detectable mRNA on Northern blotting. The -/- mice survive to birth but have impaired growth and increased mortality before and after weaning. The -/- mice have extreme bladder enlargement, dribbling urination, bladder infection, urinary stones, and widely dilated ocular pupils that do not contract in response to light. Detailed histological studies of -/- mice revealed no significant abnormalities in brain or peripheral tissues except urinary bladder, where inflammation was prominent. Ganglion cells and axons were present in bladder and bowel. Bladder strips from -/- mice failed to contract in response to 0.1 mM nicotine, but did contract in response to electrical field stimulation or carbamoylcholine. The number of acetylcholine-activated single-channel currents was severely reduced in the neurons of superior cervical ganglia in -/- mice with five physiologically distinguishable nicotinic acetylcholine receptor subtypes with different conductance and kinetic properties in wild-type mice, all of which were reduced in -/- mice. The findings in the alpha3-null mice suggest that this subunit is an essential component of the nicotinic receptors mediating normal function of the autonomic nervous system. The phenotype in -/- mice may be similar to the rare human genetic disorder of megacystis-microcolon-intestinal hypoperistalsis syndrome.

Although presynaptic input can influence the number and distribution of ACh receptors (AChRs) on muscle, the role of cellular interactions in the development of transmitter sensitivity in neurons is less clear. To determine whether presynaptic input modifies neuronal AChR channel function and distribution, we must first ascertain the profile of changes in receptor properties relative to the timing of synapse formation. We have examined the temporal aspects of synaptogenesis in the lumbar sympathetic ganglia of the embryonic chick in anatomical experiments with anterograde 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate labeling of presynaptic inputs and cytochrome oxidase histochemistry. Biophysical studies of sympathetic neurons, within hours of removal from animals at different stages relative to synapse formation, show that both the properties and distribution of AChR channels are modified concurrent with a significant increase in presynaptic input to the neurons. The most striking change in AChR channel distribution is revealed by patching multiple sites on the surface of individual neurons. Following innervation in vivo, many neurons express only one of the four AChR channel subtypes and the AChRs are clustered in discrete, high-activity patches. Furthermore, when neurons at this stage express more than one AChR channel subtype, the different classes are often spatially segregated from one another on the cell surface. This contrasts with patches from neurons removed earlier on, which have lower overall activity, often comprised of multiple channel subtypes. Comparison of the AChR properties of acutely dispersed neurons to those of neurons maintained in vitro indicates that most features of AChR channels are conserved despite their removal from presynaptic and other in vivo influences. These findings are consistent with inductive interactions between pre- and postsynaptic neurons playing an important regulatory role in transmitter receptor expression.

Although multiple related genes encoding nicotinic acetylcholine receptor (AChR) subunits have been identified, how each of these subunits contributes to AChRs in neurons is not known. Sympathetic neurons express four classes of AChR channels and six AChR subunit genes (alpha 3, alpha 4, alpha 5, alpha 7, beta 2, and beta 4). The contribution of individual subunits to AChR channel subtypes in these neurons was examined by selective deletion with antisense oligonucleotides. An alpha 3 antisense oligonucleotide decreased the number and altered the properties of the normally expressed ACh-activated channels. The remaining AChR channels have distinct biophysical and pharmacological properties that indicate an important functional contribution of the alpha 7 subunit.

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.