The well-documented effects of nicotine on learning and memory and its addictive potential implicate nicotinic acetylcholine receptors (nAchRs) in mediating or modulating synaptic transmission in the CNS. Indeed, nAchRs have been detected electrophysiologically in numerous neuronal cell types of the central nervous system. The vertebrate genome provides a tremendous potential for generating heteromeric nAchRs diversity by encoding at least sixteen different subunits that can be used to assemble receptors. The number of subtypes that are actually made in vivo, however, is far less than what is mathematically possible. An important question currently under investigation is what are the mechanisms that control the composition and distribution of nAchR subtypes made within the organism? Clearly one important and early step is that which limits the distribution and abundance of individual subunit mRNAs to particular tissues and cell types. Thus particular cells must transcriptionally select and accumulate from the sixteen available subunit mRNAs only those that are required to make appropriate subunit combinations. In contrast to the growing understanding of the function of various neuronal nAchR subtypes, the transcriptional mechanisms that direct expression of subunit genes to subsets of neurons remain poorly understood.

The focus of our investigation is a cluster of phylogenetically conserved vertebrate nAchR genes ordered β4, α3, and α5.

The β4α3α5 gene cluster has a rich capacity for generating numerous central and peripheral nAchR subtypes. For instance, all three subunits are assembled together into a β4α3α5 nAchR subtype in all post-ganglionic autonomic neurons. Gene targeting of the α3 and β4 genes has shown that the β4α3α5 subtype is essential for fast synaptic transmission between pre and postganglionic neurons in the autonomic nervous system. In the retina the β4 and α3 genes are assembled into retinal ganglion and amacrine cell nAchR subtypes and α3 containing subtypes have been implicated in the propagation of retinal waves in mice. Less well-documented are the nAchR subtypes in various brain nuclei that contain β4, α3, and α5 subunits.

Our investigation has identified an enhancer, β43’, embedded in the cluster. β43’ is particularly attractive because in dissociated primary retinal cultures its activity is restricted to neuronal cell types that express the clustered nAchR genes. Its location within the cluster raises the possibility that it functions as a shared regulatory element necessary for coordinating the expression of the clustered genes. Interestingly, we find that enhancer activity in primary cultured retinal neurons depends on two different kinds of ETS domain binding sites. Thus, ETS domain transcription factors appear to be involved in controlling gene expression in both the serotonergic and cholinergic neurotransmitter pathways.

Our current work on the enhancer is aimed at determining its biological relevance for expression of nAchR subtypes encoded by the cluster and what role ETS factor interactions play. Specifically, we want to know whether the enhancer coordinates the spatial and temporal expression of the clustered subunit genes in various neuronal populations. We are investigating this question using transgenic methods and eventually we will use knock out approaches. This project will help to advance an understanding of how cholinergic neurotransmitter systems are built and will help to reveal the function of ETS factors in neurons.