Research report
Linkage of strain-specific nicotinic receptor α7 subunit restriction fragment length polymorphisms with levels of α-bungarotoxin binding in brain

https://doi.org/10.1016/S0169-328X(96)00149-0Get rights and content

Abstract

Inbred mouse strains have been shown to differ in their levels of brain α-bungarotoxin binding. These differences in α-bungarotoxin receptors have been shown to correlate with an animal's sensitivity to nicotine-induced seizures. Recent studies have shown that the α7 nicotinic acetylcholine receptor subunit is the major α-bungarotoxin binding site in rodent brain. In this report, we examined whether mouse strains that differ in levels of α-bungarotoxin binding and sensitivity to nicotine-induced convulsions also differ for the α7 subunit. A full-length murine α7 cDNA was cloned and sequenced and found to be identical to that of a mouse α7 cDNA recently reported. Subsequently, a comparison of α7 cDNA sequences and RNA species was performed between two strains (C3H/2 and DBA/2) that differ in levels of brain α-bungarotoxin binding and sensitivity to nicotine-induced seizures. The only difference observed was a single nucleotide difference in the open reading frame of α7 that does not affect the primary amino acid sequence. Inbred strains were also surveyed for restriction fragment length polymorphisms at the α7 locus. Strain-specific polymorphisms were identified, and F2 and backcross animals from a classic genetic cross between C3H/2 and DBA/2 mice were compared for the inheritance of α7 genotype and α-bungarotoxin receptor levels. A significant association between genotype and receptor levels was observed in both the F2 and backcross generations. These results indicate that α7 genotype is an important determinant of α-bungarotoxin receptor levels.

Introduction

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that are expressed throughout the central and peripheral nervous systems. To date, ten cDNAs (α2, α3, α4, α5, α6, α7, α9 and β2, β3, and β4) that code for putative neuronal nAChR subunit proteins have been identified in rodents 2, 3, 8, 9, 10, 11, 18, 36, 46, 49, 51. An additional subunit, α8, has also been found in chickens [44]. The mRNAs for each of these putative nAChR subunits display unique but overlapping patterns of expression 8, 10, 11, 23, 44, 46, 49, 50.

Two major types of nAChRs can be measured by ligand-binding strategies. One type binds nicotine, acetylcholine and cytisine with high affinity 4, 26, 28, 37, 41, 45while the other binds α-bungarotoxin (α-BTX) with high affinity 6, 26, 28. Minor receptor subtypes have recently been identified with the advent of new ligands such as neuronal bungarotoxin [20]and epibatidine 16, 39. The subunit stoichiometry of nicotinic receptors in vivo is not well established. However, the receptor type that binds nicotine with high affinity is believed to be comprised of α4 and β2 subunits 13, 35, 51whereas the α-BTX-binding receptor is believed to contain α7 subunits 44, 46. These two receptor types exhibit different patterns of expression 6, 26, 38. In addition, levels of these receptor types vary significantly among the regions where they are expressed. Moreover, a variety of mouse strains differ in their levels of these two receptor types and these differences in receptor levels have been found to correlate with physiological and behavioral sensitivity to nicotine 24, 27, 30, 31, 32, 33. For example, animals with higher levels of α-BTX binding in hippocampus are, in general, more sensitive to the convulsant effects of high doses of nicotine 30, 31, 32, 33. Therefore, understanding the regulation of expression of these receptor types not only between mouse strains but also across brain regions may provide insight into individual differences in sensitivity to nicotine.

In this report, we establish that two mouse strains, C3H/2Ibg and DBA/2Ibg, that differ in levels of brain α-BTX binding are polymorphic for the nAChR gene, α7, that codes for the α-BTX-binding subunit. The strain-specific α7 loci also segregated with α-BTX receptor levels in a fashion similar to the parental inbred strains indicating that α7 genotype is important in the regulation of the levels of α-BTX binding receptors.

Section snippets

Animals

AKR/J, BUB/BnJ, DBA/1J, St/bJ, and SWR/J mice were purchased from Jackson Laboratories. A/J/Ibg, BALB/cByJ, C3H/2Ibg, C57BL/6J/Ibg, DBA/2J/Ibg, LS/Ibg and SS/Ibg mice were from the colony maintained at IBG. F2 and backcross progeny derived from a classic genetic cross between C3H/2Ibg and DBA/2Ibg were generated at IBG. Offspring of the matings were weaned at 25 days of age and housed with 1–4 same sex siblings. Female mice were used for all experiments.

Mice were maintained on a 12 h light/12 h

Isolation of a murine α7 cDNA clone

Previous work has demonstrated that there is a correlation between the number of hippocampal α-bungarotoxin (α-BTX) receptors and susceptibility to nicotine-induced seizures 30, 31, 32, 33. More recent findings have shown that α7 is the major neuronal nAChR subunit in rodents that binds, and is functionally blocked by α-BTX [46]. Therefore, it is possible that mouse strains that differ in their levels of α-BTX receptor levels also differ, in some fashion, for the α7 nAChR subunit. Such a

Discussion

In this study, rflp analysis demonstrated that the nAChR α7 locus is polymorphic between inbred strains of mice. Of the twelve strains examined, ten exhibited one genotype (C3H-like) whereas the two DBA strains surveyed (DBA/1J and DBA/2JIbg) had a separate genotype. When F2 animals derived from a classic genetic cross between C3H and DBA mice were compared for α7 genotype vs. α-BTX binding receptor levels, a significant relationship between these two measures was found. In fact, the

Acknowledgements

The authors wish to thank Dr. Mike Marks for his helpful comments during the preparation of this manuscript. The authors would also like to thank Jian-Zhe Cao and Grant Burton, Jr. for technical assistance. This work was supported by grants from the National Institute on Drug Abuse (DA-03194 and DA-10156). A.C.C. is supported, in part, by a Research Scientist Award from NIDA (DA-00197).

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