Experiment
Fluid licking in mice is a rhythmic behavior that is controlled by a central pattern generator (CPG) located in a complex of brainstem nuclei. C57BL/6J and DBA/2J strains differ significantly in water-restricted licking, with a highly heritable difference in rates (h2 >= 0.62) and a corresponding 20% difference in interlick interval (mean +/- SEM = 116.3 +/- 1 vs 95.4 +/- 1.1 ms).
The authors systematically quantified motor output in C57BL/6J (n = 13) and DBA/2J (n = 14), their F1 hybrids (n = 13), and a set of 64 BXD recombinant inbred strains (n = 423 individuals). The mean primary interlick interval (MPI) varied continuously among progeny strains. Of the 489 total mice tested, 256 were female and 233 were male. With the exception of 9 of the 64 BXD lines, for which only one sex was tested, males and females were approximately equally represented in each genotype.
The D2 parent is at the fast extreme (lowest MPI) whereas B6 has a higher MPI (corresponds to slower licking).
All QTL mapping for MPI, total licks, and volume per lick (VPL) (both for water and sucrose trials) was conducted using interval-mapping software and genotypes in GeneNetwork.org.
The authors detected two significant QTL for a CPG controlling lick rate:
Lick1 (lick rate 1, water) maps to Chr 1: 172.5 - 175.5 Mb with a peak LRS score of 25.2 (LOD 5.5). The peak LRS is located between markers at 174.7 and 175.2 Mb. High MPI values (slow lick rates) are associated with the C57BL/6J allele. The additive effect of the C57BL/6J allele at this locus (rs13476241) on MPI is 4.35 ms. The difference between BXD strains that inherited the D/D and B/B haplotypes in this interval is approximately 9 ms.
Lick2 (lick rate 2, sucralose) maps to Chr 1: 172.5 - 175.5 Mb with a peak LRS score of 26.8. The peak LRS is located between markers at 174.7 and 175.2 Mb. High MPI values (slow lick rates) are associated with the C57BL/6J allele. The additive effect of the C57BL/6J allele at this locus (rs13476241) on MPI is 4.35 ms. The difference between BXD strains that inherited the D/D and B/B haplotypes in this interval is approximately 9 ms.
The authors controlled for the effect of Lick1 and Lick2 using the marker rs13476241 (Chr 1 at 174.698878 Mb) and remapped both phenotypes using composite interval mapping. This procedure is essentially the partial regression of lick rate and MPI after eliminating any genetic variance associated with the distal Chr 1 region. The authors identified a third significant QTL:
Lick3 (lick rate 3) maps to Chr 10: 61.5 - 69.0 Mb with a peak LRS of 20.5 at 67 Mb (rs13480629). Lick3 is in a SNP desert in the BXD family (the region is essentially identical by descent), but includes a small island of B vs D SNPs that extends from 67.5 to 69.0 Mb. The effect size of this Lick3 locus has the same polarity as Lick1 and Lick2 and each C57BL/6J allele increases the MPI by 3.3 ms.
There may be a third locus on Chr 13 between rs13481782 and rs6196305 that interacts epistatically with Lick1 to generate especially long MPIs when both loci have the C57BL/6J haplotype. This interaction has an LRS of 30.9 and has a genome-wide p<0.1. The suggestive Chr 13 locus is also associated with a small additive effect of +23 ms per C57BL/6J allele.
Lick1 and Lick2 account for 33% of the total betweenstrain variance in MPI among the BXD strains, and Lick3 accounts for 19%. These three loci therefore account for approximately 50% of the genetic variation in lick rate. When BXD strains are grouped according to haplotype at Lick1-3, those possessing mixed haplotypes are intermediate in phenotype to those possessing either solely DBA/2J or C57BL/6J haplotypes.
Linkage was verified by testing of B6.D2-1D congenic stock in which a segment of Chr 1 of the DBA/2J strain was introgressed onto the C57BL/6J parent. B6.D2 1D (n = 14) and B6.D2 10M (n = 12). For B6.D2 1D, the introgressed fragment (from D2 onto the B6 background) spanned from proximal marker rs6267646 (154.395967 Mb) to distal marker rs29609526 (197.134686 Mb). For B6.D2 10M, the introgressed fragment spanned from proximal marker D10Mit299 (66.154402 Mb) to distal marker rs3706484 (114.541067 Mb; locations from NCBI37/mm9).
Mice from the congenic strain B6.D2.10M were phenotypically similar to C57BL/6J mice, although this result does not necessarily indicate failure to confirm the QTL. The introgressed region on these mice spans from approximately 66 MB to 115 MB on Chr 10. Although the peak LRS score for MPI on Chr 10 was in this region, the lack of phenotypic difference between B6.D2.10M and C57BL/6J nevertheless can in part be explained by a physical location for Lick3 proximal to this fragment (but still within the confidence interval).
A suggestive QTL was found for licks to either stimulus on chromosome 16. For VPL for water, suggestive QTL were found on Chr 1, 11, and 16, and for sucrose VPL suggestive QTL were found on Chr 8, 13, and 16. The suggestive QTL on Chr 1 for water VPL (peak at approximately 95 MB) did not overlap with the significant lick rate QTL (i.e. confidence intervals do not overlap).
The Lick1 and Lick2 intervals on distal Chr 1 contain several strong candidate genes. One of these is a sodium/potassium pump subunit (Atp1a2) with widespread expression in astrocytes, as well as in a restricted population of neurons. Both this subunit and the entire Na+/K+ -ATPase molecule have been implicated in rhythmogenesis for respiration and locomotion. Sequence variants in or near Apt1a2 strongly modulate expression of the cognate mRNA in multiple brain regions. This gene region has recently been sequenced exhaustively and we have cataloged over 300 non-coding and synonymous mutations segregating among BXD strains, one or more of which is likely to contribute to differences in central pattern generator tempo. Thus, Atp1a2 is given as a leading candidate gene for Lick1 and Lick2 in this study.
Analysis Tools