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* Department of Veterinary and Biomedical Sciences, and
Department of Animal Sciences Science, University of Nebraska-Lincoln, Lincoln, Nebraska, USA
1Correspondence: Department of Veterinary and Biomedical Science, University of Nebraska-Lincoln, Beadle Center, Rm. E117, 1901 Vine St., Lincoln, NE 68588-0665, USA. E-mail: yzhou2{at}unl.edu
We have recently reported differences between two mouse lines, which were generated by selection for high (SH) or low (SL) stress response, in distribution frequency of polymorphic forms of glucocorticoid receptors (GR), one with 8 (GRwt or GR8q) and the other with 16 (GRQn or GR16q) glutamines (1)
. The SH and SL lines of mice were developed, respectively, from two lines (Replicate #1), previously selected for high (MH) or low (ML) heat loss (2
, 3)
, because the difference in corticosterone response to restraint stress between the MH and ML lines was observed only in Replicate #1, but not in mice of Replicates #2 and #3 (unpublished data). The SH and SL lines of mice should still have the genetic bases of the MH and ML mice because we intentionally carried out our selection within the existing lines. After we identified the polymorphic form of GR in the 4th generation of SH and SL mice, we screened all three replicates of MH and ML lines to determine whether there is any linkage to or pleiotropy with heat loss. Although the GR polymorphism was also observed in MH and ML lines, the distribution frequencies of the two forms of GR were not different between the MH and ML mice in Replicates #2 and #3, which had no difference in corticosterone responses to stress as observed in our prescreening prior to our SH and SL line establishment and subsequent selection. However, the mice in Replicate #1, which showed the differences in corticosterone response to stress and were used as base population for SH and SL selection, had similar GR allelic distribution as that found after stress-response selection in SH and SL mice (Table 1
). This indicates that GR polymorphism is not linked to heat loss but may play a role in the mechanism of altered stress response.
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We agree with the comments made by Mormede et al. that the experiment yet to be done is to determine the stress responsiveness between mice with different GR genotypes within the same SH or SL lines. However, there were not enough mice in certain GR genotypes, as shown in our manuscript, and we did not have much success in generating sufficient numbers of mice with three GR allelic combinations within the same SH or SL line in next three generations of selections subsequent to our report. This was because most of the litters with "GR8q/8q" in SL line and those with "GR16q/16q" in the SH line died at a very young age or because some females produced no litter at all, which was probably due to their advanced age at mating. In two recent generations, we have used younger breeders based on GR genotypes without going through the previously described selection process and have been more successful. We are now able to evaluate all three GR genotypes within the SH and SL lines. Here we report data obtained from the 10th generation of SH and SL mice with all three GR allelic combinations within the same line. Based on genotyping using the GR primers as we previously described (1)
, a total number of 144 mice (72 per line) were used in this study and were divided into three groups (8 mice per GR genotype per group from each line): control, acute restraint (30 min restraint before the maze test); and repeated restraint was performed (two sessions of restraint before the maze test: 30 min restraint per day for consecutive 3 d and repeated once after 4 non-stress d). Each of the mice was tested in an elevated plus maze for 5 min and results were recorded using a Noldus EthoVision® automated video-tracking system as described previously (1)
. Due to the large number of mice, we performed the experiment in three days in order to test three genotypes of mice of different groups in a comparable time frame to minimize errors associated with the time of day (e.g., in sequence: 4 SH-GR8q/8q control mice, 4 SL-GR8q/8q control, 4 SH-GR8q/8q acute restraint, and 4 SL-GR8q/8q acute restraint, and so on). Percentage of time spent in defined zones (center, open arm, and closed arm), distance traveled, and velocity were analyzed and compared between SH and SL lines, between GR genotypes, and between control and stressed groups. Statistical analysis was performed using the mixed procedure of SAS (SAS Inst., Cary, NC, USA) as well as with paired t tests for comparisons of genotypes within the SL or SH lines.
Longer distance traveled (F=72.68, P<0.0001) and greater velocity (F=96.50, P<0.0001) were observed in SH mice, but not between the GR genotypes, and there was no line by genotype interaction (P>0.50). Both acute and repeated stress resulted in an increase in velocity (F=15.66, P<0.0001) and distance traveled (F=11.34, P=0.001). However, mice with GR8q/8q in both acute and repeated restraint groups of SL line had greater velocity (P=0.029) and traveled more distance (P=0.007) in response to acute or repeated restraint when compared to those with GR8q/8q in the same line. Such an effect was also seen between the GR16q/16q mice in SL line (velocity: P=0.031; distance traveled: P=0.037) but not in SH line. This suggests an additive effect of GR8q on stress response in the SL population while the GR16q has no effect in SH mice in terms of stress responses. The difference between the GR genotypes in the SL line was also observed in their behaviors in different zones of the plus maze (Fig. 1
).
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Similar to what we reported previously, the SL mice spent more time in the closed arm (F=121.7; P<0.0001) and much less time in the open arm (F=84.51; P<0.0001) in all treatment groups, when compared to the SH mice which traveled in all 4 arms during the given time of testing. As shown in Fig. 1
, the SL-GR16q/16q mice in the control group showed a high degree of fear to move and spent 
30% of time in the center zone before moving into the closed arm, However, all of the SL-GR8q/8q mice spent much less time in the center (P=0.019) before moving to the closed arm. They spent longer time in the closed arm (P=0.0076) and less time in the open arm (P=0.0386) than the SL-GR16q/16q mice. The SH-GR8q/8q showed significant decrease in time spent in the closed arm in response to either acute (P=0.005) and repeated (P=0.0007) restraints, as they traveled more to the open arm (P=0.003) in response to stress. Similar patterns of stress response (P=0.037) were also seen in the GR8q/8q mice exposed to repeated restraint in the SL line. Such stress-induced difference in time spent in the open or closed arm between control and restrained mice was not seen in the GR16q/16q mice in both SH and SL lines.
In addition, we recorded the body weight of all mice at 13 wk of age, before starting the stress experiment, and the average of two consecutive days of body weight was used for statistical analysis. As shown in Fig. 2
, the SL mice have overall greater body weight than SH mice. Body weight was significantly different between the three GR genotypes within the SL: GR16q/16q > GR16q/8q > GR8q/8q. No such difference in body weight was found between the GR genotypes in the SH line.
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We conclude that the mice with GR8q/8q have greater stress responsiveness than the GR16q/16q mice independent of line. The GR-16q allelic effect is much weaker in the SH line while both polymorphic forms of GRs exhibited a strong additive effect on behavior under either control or stressed conditions, as well as on body weight, in the SL line of mice. However, this GR polymorphism does not appear to be linked to heat loss. Additional experiments are underway, including creating SH-SL line-cross combination of GR genotypes, to further confirm this conclusion.
FOOTNOTES
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