Mu opioid receptor knockout mouse: Phenotypes with implications on restless legs syndrome

Abstract

Restless legs syndrome (RLS) is characterized by an irresistible need to move the legs while sitting or lying at night with insomnia as a frequent consequence. Human RLS has been associated with abnormalities in the endogenous opioid system, the dopaminergic system, the iron regulatory system, anemia, and inflammatory and auto-immune disorders. Our previous work indicates that mice lacking all three subtypes of opioid receptors have a phenotype similar to that of human RLS. To study the roles of each opioid receptor subtype in RLS, we first used mu opioid receptor knockout (MOR KO) mice based on our earlier studies using postmortem brain and cell culture. The KO mice showed decreased hemoglobin, hematocrit, and red blood cells (RBCs), with an appearance of microcytic RBCs indicating anemia. Together with decreased serum iron and transferrin, but increased ferritin levels, the anemia is similar to that seen with chronic inflammation in humans. A decreased serum iron level was also observed in the wildtype mice treated with an MOR antagonist. Iron was increased in the liver and spleen of the KO mice. Normal circadian variations in the dopaminergic and serotoninergic systems were absent in the KO mice. The KO mice showed hyperactivity and increased thermal sensitivity in wakefulness primarily during what would normally be the sleep phase similar to that seen in human RLS. Deficits in endogenous opioid system transmission could predispose to anemia of inflammation and loss of circadian variations in dopaminergic or serotonergic systems, thereby contributing to an RLS-like phenotype.

Keywords: RRID:AB_10956736; RRID:AB_2261889; RRID:AB_621846; RRID:AB_621847; RRID:AB_631362; RRID:AB_641107; RRID:AB_668816; anemia of inflammation; circadian variations in monoamine systems; hyperactivity; iron deficiency; mu opioid receptor; thermosensory test.

Figure 1.

Concentrations of iron, ferritin and transferrin in the serum. (A) The MOR KO mice (n=6) showed a decreased level of iron in serum compared with the WT (n=4). (B) The MOR KO mice (n=6) had an increased level of ferritin in serum compared with the WT (n=4). (C) The MOR KO (n=9) had a decreased level of transferrin in their serum compared with WT littermates (n=4). Data were analyzed by the unpaired Student’s t-test and are presented as the means plus the standard deviations (SDs). * p≤0.05; ** p<0.01. Only males were used in the experiment.

Figure 2.

Iron concentrations in tissues. (A) No change in the striatal iron level in the MOR KO mice (n=11) compared with the WTs (n=10). (B) The MOR KO mice (n=11) showed an increased iron level in the spleen compared with the WTs (n=10). (C) Male MOR KO mice (n=6), had an increased level of iron in their livers compared with the WT littermates (n= 5). Data were analyzed by the mixed model ANOVA and are presented as the means plus the SDs. * p≤0.05. Both male and female were used in the experiment. Detailed analyses are in Table 2.

Figure 3.

Iron concentrations in serum of mice injected with naloxonazine (n=12) or saline (n=9). (A) The iron level decreased with naloxonazine injection. (B, C) Ferritin and transferrin levels did not have significant changes after the drug injection. Data in Figure 3A were analyzed by the GENMOD with a gamma distribution. Data in B and C were analyzed by the mixed model ANOVA. Scatter plots represent the means plus the SDs. * p≤0.05. Only males were used in the experiment.

Figure 4.

Circadian variations in the striatal dopaminergic and serotonergic systems. (A, B, C) DA, DOPAC, and HVA levels increased in the WT but did not change in the MOR KO mice, during the night (WT, n=10; KO, n=12) compared with the day (WT, n=8; KO, n=9). (D, E) The ratio of DOPAC to DA or 3-MT to DA decreased in the WT during the night but did not change in the MOR KO mice compared with the ratios of the day. (F) The ratio of 5-HIAA to 5- HT increased in the WT mice during the night compared with the day but did not change in the MOR KO mice. Data were analyzed by the mixed model ANOVA and are presented as the means plus the SDs. DOPAC, 3,4-Dihydroxyphenylacetic acid; DA, Dopamine; HVA, Homovanillic acid; 3-MT, 3-Methoxytyramine; 5-HIAA, 5-Hydroxyindoleacetic acid; 5-HT, Serotonin. Bars represent the means plus the SDs. * p≤0.05; ** p<0.01. p values have been adjusted for multiple comparisons using the Benjamini-Hochberg-Yekutieli false discovery rate [FDR (p < 0.05)]. Only males were used in the experiment. Detailed analyses are in Table 3.

Figure 5.

Wheel running test. (A) The MOR KO mice (n=5) had a significant increase in voluntary activity during the light phase (2 days) compared with the WT littermates (n=5). The hourly activity is presented next to the scatter plot. (B) MOR KO mice (n=5) did not have significant changes in the voluntary activity during the dark phase (2 nights) compared with the WT littermates (n=5). However, MOR KO mice had significantly lower activity levels at 6:00 PM 3:00 AM and 4:00 AM. Data were analyzed by the GENMOD with a negative binominal distribution and are presented as medians with 95% confidence intervals (CIs). The hourly activity levels are presented as means plus the SDs. Significant p values are marked above the individual time point. * p≤0.05; ** p<0.01. Only males were used in the experiment. Detailed analyses are in Table 4.

Figure 6.

Tail-flick test. (A) The MOR KO mice (n=14, 3 repeats) had decreased latency in response to the heat stimuli compared with the WT mice (n=14, 3 repeats). (B) The increased pain sensitivity of the MOR KO mice (n=6, 3 repeats) compared to the WTs (n=5, 3 repeats) only appeared at midday, but not at midnight. After the drug injection, there was no difference between the two groups. Data were analyzed by the GENMOD with a gamma distribution and are presented as medians with 95% CIs. * p≤0.05; ** p<0.01. Only males were used in the experiment. Each mouse was tested three times. Each time was coded as trial1, trial2, and trial3, respectively. When data was processed, the “trial” was treated as a repeated measurement and nested under the Animal_ID. Detailed analyses are in Table 5. Age and weight were considered as continuous variables, and their relationships were further analyzed in Supplementary Figure 3.