Coexpression of multiple acetylcholine receptor genes in neurons: quantification of transcripts during development

Abstract

A large family of genes encoding subunits of nicotinic ACh receptors (AChRs) has been identified in vertebrates and shown to be expressed in the nervous system. The multiplicity of genes raises questions about which gene products coassemble to produce native receptor subtypes and how the expression of receptor genes is regulated in neurons. We report here that five neuronal AChR genes are expressed in the chick ciliary ganglion at both early and late times in development. Quantitative RNase protection experiments demonstrated that at embryonic day 18 (E18) the ganglion contains about 1800 copies of alpha 7 transcript per neuron, 900 copies of alpha 3 transcript per neuron, and 200-300 copies each of alpha 5, beta 2, and beta 4 transcripts per neuron. The same five genes are expressed at significantly lower levels at E8 but show the same rank order of abundance in transcripts per neuron. Few, if any, transcripts were found for the alpha 2, alpha 4, alpha 8, and beta 3 AChR genes in ciliary ganglion RNA at either E8 or E18. The 6- and 13-fold increases previously reported for two classes of AChRs on the neurons between E8 and E18 approximate the 4-14-fold increases observed here in AChR gene mRNA levels per neuron over the same time period. The alpha 3, alpha 5, alpha 7, and beta 4 genes have previously been correlated with subunits of ciliary ganglion AChRs, but the beta 2 gene has not. The abundance of beta 2 transcripts raises the possibility either that the known AChRs in the ganglion have a more complex subunit composition than previously described or that additional receptor subtypes remain to be discovered. Northern blot analysis revealed no changes in transcript pattern for the alpha 3, alpha 5, and beta 4 genes between E8 and E18; a small change may occur in the transcript pattern for the alpha 7 gene. In situ hybridizations demonstrated that alpha 5 and beta 4 transcripts are expressed in essentially all ciliary ganglion neurons as has been shown previously for the more abundant alpha 3 transcript and inferred for the alpha 7 transcript. The results indicate that neurons can stably coexpress multiple AChR genes, including three of the alpha type, and that transcript levels may be rate limiting for accumulation of AChRs during development.

N-Methyl-d-aspartate Receptors Regulate a Group of Transiently Expressed Genes in the Developing Brain

“…RNase Protection-RNase protection experiments were carried out as previously described (15). Briefly, total RNA (10 -20 g) was annealed to a molar excess of 32 P-labeled single-stranded antisense RNA probe for 5 min at 85°C in 80% formamide, 40 mM PIPES buffer (pH 6.7), 0.4 M NaCl, and 1 mM EDTA, and the hybridization was continued for 12-18 h at 45°C.…”

“…Developmental studies suggest that mRNA abundance is rate-limiting for the accumulation of functional neurotransmitter receptors on neurons during development in vivo (15). Changes in gene expression also accompany activity regulated synaptic plasticity events in the developing visual system (16), as well as at the neuromuscular junction (17).…”

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).…”

“…This cluster is organized with ␤4 being most 5Ј, followed by ␣3 and ␣5; ␣5 is transcribed in the opposite direction from ␤4 and ␣3. These three genes are often coexpressed in central and peripheral neurons and this, along with their tight linkage, suggests coordinate regulation Corriveau and Berg, 1993;Mandelzys et al, 1994). A neuralspecific enhancer element identified in the 3Ј untranslated exon of the ␤4 gene may be important for regulating neuronal expression of these clustered nAChR subunit genes Deneris, 1997, McDonough et al, 2000).…”

Clustering of Nicotinic Acetylcholine Receptors: From the Neuromuscular Junction to Interneuronal Synapses

“…Chick ciliary ganglion neurons express α3, α5, β2, β4 and α7 subunits and, thus far, two major subtypes of receptors differing in their subunit-compositions and also functional properties have been identified, that is, the α-BTX sensitive component comprised of α7 subunits and mAb35-immunoreactive receptors of which at least two classes exist depending on the presence of β2 subunits, e.g. α3α5β2β4 and α3α5β4 (139)(140)(141). In the chick ciliary ganglion, α7 and mAb35-AChRs show interesting differences in their localizations with respect to synaptic sites.…”