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Meeting Abstracts  |   February 1998
Identification of a Genetic Region in Mice that Specifies Sensitivity to Propofol 
Author Notes
  • (Simpson) Assistant Professor, Departments of Anesthesiology and Pharmacology, University of Colorado Health Sciences Center.
  • (Rikke, Corley) Research Associate, Institute for Behavioral Genetics, University of Colorado.
  • (Costello) Technician, Institute for Behavioral Genetics, University of Colorado.
  • (Johnson) Professor, Institute for Behavioral Genetics, University of Colorado, and Veterans Administration Medical Center, Denver.
Article Information
Meeting Abstracts   |   February 1998
Identification of a Genetic Region in Mice that Specifies Sensitivity to Propofol 
Anesthesiology 2 1998, Vol.88, 379-389. doi:
Anesthesiology 2 1998, Vol.88, 379-389. doi:
ELUCIDATING the actions of general anesthetic agents at the molecular level is difficult because of the seemingly endless number of targets with which these agents are associated. [1] One promising approach for identifying molecular pathways directly relevant to anesthetic action is to identify and isolate genes that can modulate anesthetic sensitivity. Because anesthetic sensitivity is measured quantitatively and is likely to be influenced by multiple genes, these genes are also called quantitative trait loci or QTLs; presumably, each QTL corresponds to a single gene. [2] Here we characterize a QTL that modulates central nervous system (CNS) sensitivity to the commonly used anesthetic agent propofol (2,6-diisopropylphenol).
The long-sleep (LS) and short-sleep (SS) mice used in this study were selectively bred by McClearn and Kakihana [3] for high or low (respectively) sensitivity to ethanol. Ethanol sensitivity was measured as the duration of the loss of righting reflex (i.e., the inability of a mouse to turn over after being placed on its back, abbreviated LORR) and differs markedly between LS and SS. Given a dose of 4.1 g/kg ethanol, the LORR for LS mice is about 2 h, whereas the LORR for SS mice is only 15 min. The blood ethanol concentration at the time of regaining righting response also differs markedly, [4,5] but ethanol metabolism does not, [6] thus indicating that LS and SS differ in their neurosensitivity. This differential neurosensitivity is also reflected by differences between LS and SS in their response to ethanol at the level of Purkinje cells, [7,8] intraocular transplants, [9] and gamma-aminobutyric acidAreceptors. [10] The LS and SS lines, therefore, are well suited for studying CNS sensitivity, and no model of ethanol-induced hypnosis has been more intensively analyzed. [11,12] 
In addition to ethanol, the LS and SS mice have been found to be differentially sensitive to various CNS depressants. These agents include chloral hydrate, halogenated ethanols, paraldehyde, urethane, flurazepam, enflurane, etomidate, isoflurane, ketamine, [13–16] and midazolam (V. J. Simpson, unpublished data). The diverse chemical structures represented by these agents suggest that LS and SS have been selected, at least in part, for genes that can modulate sensitivity at a point in a molecular pathway where the actions of multiple classes of agents converge. However, LS and SS do not differ in sensitivity to ether or halothane, [17] indicating that there has not been indiscriminate selection for a developmental difference such as differential motor skills. Whether those agents that cause differential sensitivity between LS and SS also have differential effects similar to ethanol on the Purkinje cells has not been determined.
More recently, LS and SS were shown to be differentially sensitive to propofol-induced LORR. [18] When given the same 20 mg/kg dose retroorbitally, SS mice had an LORR of about 3.5 min (SD = 1.01, SEM = 0.4), and the LORR for LS mice was about 2.2 min longer, with no difference between males and females (Table 1, section 1). The SS mice also regained the righting response at a significantly higher propofol brain level, indicating that resistance is neurologic rather than pharmacokinetic. [18] However, LS and SS did not exhibit differences in gamma-aminobutyric acidA-activated chloride channels, assayed in brain microsac preparations, even though propofol has a potent effect on gamma-aminobutyric acidAreceptors. [19] To further elucidate the difference in propofol sensitivity between LS and SS, we are pursuing a positional cloning strategy.
Table 1. Comparison of Propofol-induced Loss of Righting Reflex for LS, SS, and Mice Derived from ILS and ISS 
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Table 1. Comparison of Propofol-induced Loss of Righting Reflex for LS, SS, and Mice Derived from ILS and ISS 
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Positional cloning involves first positioning or “mapping” the gene(s) to be cloned to a small chromosomal region. Mapping of propofol sensitivity is greatly facilitated [11,12,16] by the availability of LSXSS recombinant-inbred (RI) strains of mice. [20] These mice were derived by crossing LS and SS and then intercrossing their progeny (F1s). Chromosomal recombination in the F1gametes resulted in the next generation (F2) having chromosomes that were a random patchwork of the LS and SS chromosomes. F2progeny were then inbred by brother-sister matings for more than 20 generations, resulting in chromosome pairs that are identical (homozygous) at virtually all loci. [21] Strain distribution patterns of genetic markers throughout the genome indicate which chromosomal regions of each RI strain are derived from LS or SS. The strain distribution patterns for 133 marker loci are published [15,22] and available online (at ). Because RI strains are genetically stable, any newly discovered phenotype difference between LS and SS (such as sensitivity to anesthetics) can be mapped without further genotyping by using these markers to look for allelic associations greater than expected by chance (i.e., genetic linkage). [23] 
A disadvantage of the RIs is that there are so few strains available that often QTLs cannot be mapped with high statistical certainty. [24] This is especially true of traits specified by multiple genes or that are only weakly heritable; therefore, most QTLs discovered using RIs are only provisional, meaning that they must be confirmed by testing additional mice. Such provisional QTLs have been identified for differential LORR sensitivity to sedative hypnotics [25] and ethanol [26] using the LSXSS RIs. Several, but not all, of the provisional QTLs for ethanol-induced LORR have been confirmed. [27,28] 
Confirmation of a QTL typically involves testing a large population of F2mice. Like the RIs, F2mice represent a chromosomal patchwork of the parental strains; however, there is no subsequent inbreeding. In this study, an F2population was constructed using ILS and ISS strains, which are inbred strains derived from LS and SS. [15,20] The ILS and ISS strains still carry all of the differential sensitivity to ethanol of their respective parental lines [29,30] and also appear to carry all of the differential sensitivity to propofol as shown in this study (Table 1, section 1). However, because ILS and ISS are inbred, they are genetically stable and greatly simplify the search for genetic linkage.
Materials and Methods
Origin of Mice
ILS, ISS, and LSXSS RI mice were produced in the Specific Pathogen Free facility at the Institute for Behavioral Genetics, University of Colorado at Boulder. ILS and ISS were derived from LS and SS by 20 rounds of brother-sister mating in the absence of any behavioral selection. [15] The LSXSS RIs were similarly derived without selection by reciprocal intercrosses between LS and SS followed by > 20 generations of brother-sister mating. [15,20] Although the RI inbreeding began with 40 sibling pairs, only 25 strains were extant at the time of this study. Animals were weaned at about 25 days of age and housed one to four per cage with like-sex littermates on a 12-h light/dark cycle. Experimental protocols were approved by animal care and use committees at the University of Colorado at Boulder and University of Colorado Health Sciences Center at Denver. For the LSXSS RIs, 5 - 15 mice of each sex were tested per strain. Because mice of the same RI strain are genetically identical, the difference between testing 5 or 15 mice was minor and was accounted for by differences in the SEM. For the ILS by ISS F2s, 194 mice were phenotyped, but 30 mice were inadvertently discarded without being genotyped; therefore the F2variance of Table 1is based on 194 mice, but only 164 mice were analyzed for genetic mapping. C57BL/6J coisogenic mice having the spontaneous albino mutation c2jwere obtained from Jackson Laboratory, Bar Harbor, Maine.
Phenotypic Assessments
LS and SS have been shown to be differentially sensitive to propofol at all doses tested (ranging from 15–35 mg/kg), and all doses had about equal efficacy in bringing about differential sensitivity. [18] In this study, a dose of 20 mg/kg was selected for its reliability in producing LORR without causing death. Propofol (10 mg/ml in Intralipid, Stuart Chemicals, Wilmington, DE) injections were performed when mice were 60 to 94 days old. F2s had a mean age of 73.9 days (SD, 5) and a mean weight of 22.4 g (SD, 3). Dosages of 20 mg/kg (volumes ranging from 30–60 micro liter) were administered by injection into the retroorbital venous sinus using a 26-gauge, 3/8-inch needle and Hamilton syringe. Room temperature was 23–26 [degree sign] Celsius. Propofol sensitivity was measured as LORR. [18] At injection, mice immediately lost consciousness and were placed on their backs in V-shaped plexiglass troughs until recovery of the righting reflex. Animals were judged to have regained righting reflex when they turned over three times in less than 1 min. Full recovery after regaining consciousness was usually immediate. The RI and F2mice were naive to any previous injections and were injected only once with propofol. The LORRs of the (ISS c/c x C57BL/6 c2j/C) F1mice represent the mean of two injections spaced 7 days apart. The mean of the second injection was not significantly different from the mean of the first injection (P = 0.36; analysis of variance, two-tailed significance). The influence of tyrosine brain levels on LORR was tested by intraperitoneal injection of tyrosine (200 mg/kg) or saline (control) immediately before or 30 min before the propofol injections. Brain levels of propofol at awakening (BLA) for the RIs were determined by gas chromatography of pooled samples of brain cortex, as previously described by Simpson and Blednov. [18] 
Marker Assessments
DNA was extracted and purified from spleen, kidney, or liver using a Super-Quik Gene DNA Isolation Kit (AGTC Research, Denver, CO). Mit simple-sequence length polymorphism (SSLP) primers were obtained from Research Genetics (Huntsville, AL) and the polymerase chain reaction was carried out as previously described. [22] These primers are genetic markers that detect alleles at specific chromosomal locations differing in their polymerase chain reaction product length due to different numbers of cytosine and adenine (CA) dinucleotide repeats. [31–33] Coat color was scored by eye on living mice; pigmented RI mice were homozygous C/C and albino RI mice were homozygous c/c [34] (the c locus was renamed Tyr because it encodes for tyrosinase [35,36]).
Statistical Analyses
Broad-sense heritability measures dominant, additive, and between-gene interactions, whereas narrow-sense heritability measures only additive interactions. [21] Broad-sense heritability was estimated as 100% x (VF2- VF1)/VF2, where VF2is the variance of the F2 population and VF1is the weighted average of the variances of ILS, ISS, and ILS by ISS F1s. [21] Narrow-sense heritability was estimated as 100% x 0.5 sigma2B/(sigma2w+ 0.5 sigma2B), in which sigma2Bis the between-strain variance (the variance of the strain means minus the weighted average of the within-strain variances, which is then divided by the number of strains) and sigma2wis the weighted average of the within-strain variances. [37] This formula assumes that interactions between genes are negligible. Within-strain variance (sigma2w) is equivalent to environmental variance because the RI strains had undergone > 20 generations of inbreeding and thus genetic variability within strains would be expected to average < 1.4%. [21] In contrast to F2s, in which one half the mice are homozygous and the other half are heterozygous at any given locus, all of the RIs are homozygous at all loci; therefore, the between-strain variance (sigma sup 2B) of the RIs is multiplied by one half to obtain a standardized genetic variance.
Although the heritability estimates were made from RI and F sub 2 mice derived from different parental strains (noninbred LS and SS vs. inbred ILS and ISS, respectively), the parental strains are closely related. This inference is based on the amount of inbreeding (> 60%) at the time of RI derivation. [20] This inbreeding is confirmed by the predominance of ISS and ILS alleles in the RI strain distribution pattern of SSLP markers throughout the genome. [15] It also can be seen from Table 1(section 1) that LS and ILS, SS and ISS have very similar LORRs in response to propofol.
The QTL maps were anchored using a map position for c and D7Mit31 of 44 centimorgans (cM), which is based on the Mouse Genome Database (MGD, World Wide Web URL:). The positions of other SSLP markers were placed relative to c and D7Mit31 based on the Whitehead Institute/MIT Center for Genome Research map (WI/MIT/CGR, World Wide Web URL:), which is the more recently updated and more accurate map regarding the relative position of the SSLP markers. [38] 
The RI map was constructed using an interval-mapping procedure previously described by Markel et al. [26] The F2linkage map was constructed using Mapmaker 3.0 and QTL mapping was done using Mapmaker/QTL Version 1.1. For the F2s, two mice were excluded because LORR was not induced, and four mice were excluded because of LORRs greater than 2 SDs from the mean (15.9, 16.8, 17, and 44.7 min). Including these mice had no effect on the LOD (logarithm of the odds of linkage) profile, but the peaks were reduced by about 0.5 LOD.
Results
Heritability of Propofol Sensitivity
Heritability is an estimate of the relative importance of heredity versus the environment. [21] We estimated heritability for the propofol sensitivity difference between LS and SS using two approaches. The first estimate was based on the variance of LORR for the ILS by ISS F2mice (Table 1, section 3). The phenotypic variance of F2mice is due to heredity and environment. However, the variance of ILS, ISS, or ILS by ISS F1mice (Table 1, sections 1 and 2) is due only to environment, because the mice within each group are genetically identical. Therefore, the F2variance (heredity and environment) minus the average of the variance of the ILS, ISS, and F sub 1 mice (environment) provides an estimate of genetic variance. The ratio of the genetic variance to the total variance (F2variance) is heritability. In this manner, the propofol sensitivity difference between LS and SS was found to be about 55% heritable. In a similar manner, the LSXSS RI strains yielded a heritability estimate of 32%(see Materials and Methods).
We also examined the nonheritable portion of the difference between LS and SS to determine if there might be a maternal effect. Table 1(sections 2 and 4) shows that the F1 and N2(F1s backcrossed with ILS or ISS) offspring of ILS mothers had a mean LORR about 0.7 min longer than the mean LORR of the offspring of ISS mothers. The probability that this difference was due to chance alone was less than 0.1 (P < 0.1, one-tailed test), thereby suggesting that a difference between ILS and ISS mothers could be having a small effect on LORR. The LORR for RIs and F2s did not covary with age, weight, sex, or time of day of injection.
Location of a Quantitative Trait Locus for Propofol Sensitivity
The LS and SS selected lines are completely differentiated at the albino locus (Tyr), with all SS mice being albino and all LS mice being pigmented. When we examined the LSXSS RIs we found that the 11 albino RI strains had a mean LORR of 4.8 min (SEM, 0.3) in response to propofol, whereas the 13 pigmented strains had a significantly (P <0.0001, two-tailed test) longer mean LORR of 9.3 min (SEM, 0.4), with no difference between males and females (Figure 1(A)). (RI 32 was excluded from this analysis because it was still segregating for the albino mutation at that time.) Furthermore, the LORRs of the albino strains did not overlap with the LORRs of the pigmented strains (Figure 1(B)), suggesting a major QTL. The albino strains also had a significantly higher BLA (P < 0.0001;Figure 1(A)), suggesting the the difference in LORR was due to differential CNS sensitivity and not pharmacokinetic differences. Plotting BLA versus LORR for the RI strains (not shown) confirmed a strong negative correlation (Pearson r correlation coefficient, -0.85) that was highly significant (P <0.001).
Figure 1. Duration of loss of righting reflex (LORR) and brain levels of propofol at awakening (BLA) in pigmented and albino LSXSS RI strains. (A) The difference in LORR between males and females was not significant, whereas the difference between pigmented (black bars, 4.8 +/- SEM) and albino RI strains (white bars, 9.3 +/- SEM) was highly significant for males and females (P < 0.0001, two-tailed test). n refers to the number of RI strains. The LORR for each strain was determined from the mean of 5–15 animals of each sex, and the BLA for each strain was determined from the mean of at least three samples. The difference in propofol BLA between pigmented (8.4 +/- 1.2 micro gram/gm) and albino (17.2 +/- 1.9 micro gram/gm) strains is highly significant (P < 0.0001, two-tailed test). The pigmented RI strains (black bars) used for propofol BLA determination were 2, 3, 6, 7, 8, 9, 17, 30 and 36; and the albino RI strains (white bars) used were 4, 10, 18, 20, 23, 25, and 33. Male and female data are combined for propofol BLA. The correlation coefficient r between propofol BLA and LORR for the 16 RI strains for which both measures could be obtained was -0.85 (P < 0.001). Error bars indicate standard error of the mean. (B) The LORR means for the pigmented RI strains are indicated by the filled ovals, and the means for the albino RI strains are indicated by the open ovals. The numbers next to the ovals indicate the RI strain(s) having that mean. RI strain 32 had a mean LORR of 4.4 min (SEM, 0.7) for females and 6.6 min (SEM, 1.1) for males, but it was not included in this analysis because at that time it was still segregating for the albino mutation.
Figure 1. Duration of loss of righting reflex (LORR) and brain levels of propofol at awakening (BLA) in pigmented and albino LSXSS RI strains. (A) The difference in LORR between males and females was not significant, whereas the difference between pigmented (black bars, 4.8 +/- SEM) and albino RI strains (white bars, 9.3 +/- SEM) was highly significant for males and females (P < 0.0001, two-tailed test). n refers to the number of RI strains. The LORR for each strain was determined from the mean of 5–15 animals of each sex, and the BLA for each strain was determined from the mean of at least three samples. The difference in propofol BLA between pigmented (8.4 +/- 1.2 micro gram/gm) and albino (17.2 +/- 1.9 micro gram/gm) strains is highly significant (P < 0.0001, two-tailed test). The pigmented RI strains (black bars) used for propofol BLA determination were 2, 3, 6, 7, 8, 9, 17, 30 and 36; and the albino RI strains (white bars) used were 4, 10, 18, 20, 23, 25, and 33. Male and female data are combined for propofol BLA. The correlation coefficient r between propofol BLA and LORR for the 16 RI strains for which both measures could be obtained was -0.85 (P < 0.001). Error bars indicate standard error of the mean. (B) The LORR means for the pigmented RI strains are indicated by the filled ovals, and the means for the albino RI strains are indicated by the open ovals. The numbers next to the ovals indicate the RI strain(s) having that mean. RI strain 32 had a mean LORR of 4.4 min (SEM, 0.7) for females and 6.6 min (SEM, 1.1) for males, but it was not included in this analysis because at that time it was still segregating for the albino mutation.
Figure 1. Duration of loss of righting reflex (LORR) and brain levels of propofol at awakening (BLA) in pigmented and albino LSXSS RI strains. (A) The difference in LORR between males and females was not significant, whereas the difference between pigmented (black bars, 4.8 +/- SEM) and albino RI strains (white bars, 9.3 +/- SEM) was highly significant for males and females (P < 0.0001, two-tailed test). n refers to the number of RI strains. The LORR for each strain was determined from the mean of 5–15 animals of each sex, and the BLA for each strain was determined from the mean of at least three samples. The difference in propofol BLA between pigmented (8.4 +/- 1.2 micro gram/gm) and albino (17.2 +/- 1.9 micro gram/gm) strains is highly significant (P < 0.0001, two-tailed test). The pigmented RI strains (black bars) used for propofol BLA determination were 2, 3, 6, 7, 8, 9, 17, 30 and 36; and the albino RI strains (white bars) used were 4, 10, 18, 20, 23, 25, and 33. Male and female data are combined for propofol BLA. The correlation coefficient r between propofol BLA and LORR for the 16 RI strains for which both measures could be obtained was -0.85 (P < 0.001). Error bars indicate standard error of the mean. (B) The LORR means for the pigmented RI strains are indicated by the filled ovals, and the means for the albino RI strains are indicated by the open ovals. The numbers next to the ovals indicate the RI strain(s) having that mean. RI strain 32 had a mean LORR of 4.4 min (SEM, 0.7) for females and 6.6 min (SEM, 1.1) for males, but it was not included in this analysis because at that time it was still segregating for the albino mutation.
×
The region around Tyr was genetically mapped using the regression approach described by Haley and Knott, [39] which we previously used in mapping QTLs for alcohol sensitivity. [26] We assessed the genotypes of seven SSLP markers spanning 50 cM of chromosome 7, obtaining peak LOD scores of 19 and 21 for males and females, respectively. The combined LOD score for males and females was about 28 (Figure 2), far exceeding the value of 3.3 recommended for significant linkage in a whole-genome scan. [40] This assignment was highly specific and positioned the QTL to a 2.5-cM region encompassing Tyr with 2-LOD support, indicating that the probability that the QTL being within this region is greater than 0.99. The LOD peak turned out to be at Tyr itself. This QTL, which we have called Lorp1 (Loss of righting due to propofol), explained about 80% of the genetic variance between LS-like and SS-like RI strains.
Figure 2. Genetic map of murine chromosome 7 showing LOD scores for positioning Lorp1 using the LSXSS RIs. Markers include the albino mutation (c), the pink-eyed dilution mutation (p), and six D7Mit simple-sequence length polymorphisms. The dashed line indicates the recommended threshold for statistical significance. Data were combined for males and females.
Figure 2. Genetic map of murine chromosome 7 showing LOD scores for positioning Lorp1 using the LSXSS RIs. Markers include the albino mutation (c), the pink-eyed dilution mutation (p), and six D7Mit simple-sequence length polymorphisms. The dashed line indicates the recommended threshold for statistical significance. Data were combined for males and females.
Figure 2. Genetic map of murine chromosome 7 showing LOD scores for positioning Lorp1 using the LSXSS RIs. Markers include the albino mutation (c), the pink-eyed dilution mutation (p), and six D7Mit simple-sequence length polymorphisms. The dashed line indicates the recommended threshold for statistical significance. Data were combined for males and females.
×
Confirmation of Linkage
We also were interested to see if F2mice derived by intercrossing the ILS and ISS strains would have the same QTL indicated by the LSXSS RIs. A panel of 164 F2s was genotyped with 16 SSLP markers from murine chromosome 7 that included all of the informative markers within 10 cM of Tyr. [38] A peak LOD score of 4.4 was obtained between marker D7Mit31 within Tyr and D7Mit123 less than 2 cM distal to Tyr (Figure 3), thus confirming Lorp1. F2mice homozygous for the SS alleles of D7Mit123 had an LORR of 5.7 min (SEM, = 0.3), heterozygotes had an LORR of 6.5 min (SEM, 0.3), and LS homozygotes an LORR of 8 min (SEM, 0.4). The 2.3-min difference in LORR between mice homozygous for the S allele and those homozygous for the L allele matches the 2.2-min difference previously observed between LS and SS. [18] This difference is consistent with all of the genetic difference between LS and SS for propofol sensitivity having been captured in the ILS and ISS strains. This difference also suggests that most or all of the genetic difference between ILS and ISS is due to Lorp1. Lorp1 explained about 23% of the F2genetic variance.
Figure 3. Confirmation of Lorp1 from F2intercrosses between inbred long-sleep (ILS) and inbred short-sleep (ISS) mice. Markers are D7Mit simple-sequence length polymorphisms. Marker D7Mit31 is within the Tyr gene near the site of the albino mutation, c. The dashed line indicates the recommended threshold for statistical significance. Data were combined for males and females (n = 158).
Figure 3. Confirmation of Lorp1 from F2intercrosses between inbred long-sleep (ILS) and inbred short-sleep (ISS) mice. Markers are D7Mit simple-sequence length polymorphisms. Marker D7Mit31 is within the Tyr gene near the site of the albino mutation, c. The dashed line indicates the recommended threshold for statistical significance. Data were combined for males and females (n = 158).
Figure 3. Confirmation of Lorp1 from F2intercrosses between inbred long-sleep (ILS) and inbred short-sleep (ISS) mice. Markers are D7Mit simple-sequence length polymorphisms. Marker D7Mit31 is within the Tyr gene near the site of the albino mutation, c. The dashed line indicates the recommended threshold for statistical significance. Data were combined for males and females (n = 158).
×
Mapping of the RI BLA data also supported a QTL linked to Tyr, with a statistically significant peak LOD score of 3.3 occurring at the same location as Lorp1 (not shown). RIs homozygous for the L allele (long LORR) of Lorp1 had a propofol BLA of 8.4 (g/g brain), whereas those homozygous for the S allele (short LORR) had a BLA of 17.2 (g/g brain), a highly significant difference (P = 0.001, one-tailed test assuming equal variances) previously seen in the LS and SS lines. [18] That mice with the S allele had a significantly higher BLA indicated that their CNSs were less sensitive to propofol and not that they were better able to metabolize propofol or sequester propofol outside the CNS (as in fat tissue). F2s could not be used to confirm BLA due to the need to pool samples from several mice to meet the minimum sensitivity requirements of the propofol assay (another advantage of RIs).
Test of Involvement of the Albino Phenotype in Determining Propofol Sensitivity
Considering the tight linkage between Lorp1 and Tyr, we asked whether the resistance of SS mice to propofol might result from the albino phenotype. This was tested by crossing ISS (c/c) with C57BL/6 mice from the Jackson Laboratory that were completely inbred but heterozygous for a spontaneous albino mutation (c2j/C). The F1progeny were thus coisogenic, identical at all loci except Tyr; all F sub 1 progeny derive a c allele from the ISS parent and either a c2jor C allele from the other parent. Thus one half of the F1mice are albino (c/c2j) and the half are normally pigmented (c/C). If propofol resistance were due to albinism, then albinos would be expected to have a mean LORR about 0.8 min less than their pigmented littermates based on the ILS by ISS F2results. However, the albino LORR was not significantly less, and a 0.8-min difference was ruled out at the 95% confidence level after accounting for sex (Figure 4(A)).
Figure 4. Propofol loss of righting reflex for (inbred short-sleep [ISS] c/c x C57BL/6 c2j/C) F1s and for inbred long-sleep (ILS) and ISS mice coinjected with tyrosine. (A) ISS mice have the classical albino mutation, whereas the c2jalbino mutation arose spontaneously in C57BL/6 at Jackson Laboratory. Therefore, F1mice are identical at all loci except the albino locus, pigmented mice (black bars) are c/C, and albino mice (white bars) are c/c2j. LORRs of females are significantly shorter than those of males by 1.3 min (P = 0.002, analysis of variance, two-tailed test). However, LORRs of albinos are not significantly shorter than those of pigmented mice (P = 0.35, analysis of variance, one-tailed test). (B) ILS (black bars) and ISS (white bars) mice coinjected with tyrosine (indicated as pr + tyr) also did not exhibit significant differences in LORR (P = 0.78, analysis of variance, two-tailed test) from controls at a concentration of tyrosine (200 mg/kg) previously shown to differentially alter ethanol-induced LORRs in LS and SS. [41] n indicates the number of mice (males and females) tested. Error bars indicate the SEM.
Figure 4. Propofol loss of righting reflex for (inbred short-sleep [ISS] c/c x C57BL/6 c2j/C) F1s and for inbred long-sleep (ILS) and ISS mice coinjected with tyrosine. (A) ISS mice have the classical albino mutation, whereas the c2jalbino mutation arose spontaneously in C57BL/6 at Jackson Laboratory. Therefore, F1mice are identical at all loci except the albino locus, pigmented mice (black bars) are c/C, and albino mice (white bars) are c/c2j. LORRs of females are significantly shorter than those of males by 1.3 min (P = 0.002, analysis of variance, two-tailed test). However, LORRs of albinos are not significantly shorter than those of pigmented mice (P = 0.35, analysis of variance, one-tailed test). (B) ILS (black bars) and ISS (white bars) mice coinjected with tyrosine (indicated as pr + tyr) also did not exhibit significant differences in LORR (P = 0.78, analysis of variance, two-tailed test) from controls at a concentration of tyrosine (200 mg/kg) previously shown to differentially alter ethanol-induced LORRs in LS and SS. [41]n indicates the number of mice (males and females) tested. Error bars indicate the SEM.
Figure 4. Propofol loss of righting reflex for (inbred short-sleep [ISS] c/c x C57BL/6 c2j/C) F1s and for inbred long-sleep (ILS) and ISS mice coinjected with tyrosine. (A) ISS mice have the classical albino mutation, whereas the c2jalbino mutation arose spontaneously in C57BL/6 at Jackson Laboratory. Therefore, F1mice are identical at all loci except the albino locus, pigmented mice (black bars) are c/C, and albino mice (white bars) are c/c2j. LORRs of females are significantly shorter than those of males by 1.3 min (P = 0.002, analysis of variance, two-tailed test). However, LORRs of albinos are not significantly shorter than those of pigmented mice (P = 0.35, analysis of variance, one-tailed test). (B) ILS (black bars) and ISS (white bars) mice coinjected with tyrosine (indicated as pr + tyr) also did not exhibit significant differences in LORR (P = 0.78, analysis of variance, two-tailed test) from controls at a concentration of tyrosine (200 mg/kg) previously shown to differentially alter ethanol-induced LORRs in LS and SS. [41] n indicates the number of mice (males and females) tested. Error bars indicate the SEM.
×
Because Tyr encodes for the enzyme tyrosinase, we also tried to determine whether tyrosine injections might alter LORR. The connection between Tyr and tyrosine levels was appealing because tyrosine is converted to L-dopa in the rate-limiting step of catecholamine synthesis, and tyrosine shares structural similarities with propofol. It also was intriguing that tyrosine availability was previously shown to alter the ethanol-induced LORRs of LS and SS, [41] and a QTL (Lore7) for ethanol-induced LORR has been found to be linked to Tyr. [28] It was found, however, that coinjecting the same dose of tyrosine that influenced ethanol-induced LORR had no influence on propofol-induced LORR for either ILS or ISS mice (Figure 4(B)).
Discussion
Differential sensitivity to propofol between LS and SS has a substantial genetic component, as indicated by testing both LSXSS RIs and ILS by ISS F2s. The F2estimate of heritability was 55%, which is considerably greater than the more limited estimate of 32% from the RIs, which detects only one type of genetic effect (additive). The difference between the two estimates suggests that in the F2s there are additional dominance or between-gene interactions, or both. Part of the difference could be due to the RIs having been derived from noninbred LS and SS mice, whereas the F2s were derived from inbred ILS and ISS mice. However, the difference between LS and ILS and between SS and ISS is small (see Materials and Methods) and thus unlikely to be a major factor.
A QTL, Lorp1, is tightly linked to Tyr in the middle of murine chromosome 7. This QTL accounts for about 80% of the genetic variance in the RIs, indicating that this is the major locus specifying differential propofol sensitivity between LS and SS. The LORR difference of 2.3 min observed between ILS by ISS F2mice homozygous for the S allele of Lorp1 compared with those homozygous for the L allele also is sufficient to account for all of the LORR difference observed between LS and SS. Although the involvement of other genes is suggested by the heritability estimates, their effects were not detected in the LSXSS RIs, and no whole-genome map of F2s has yet been generated.
The LORR of F2s heterozygous at Lorp1 (6.5 min) was more similar to that of S allele homozygotes (5.7 min) than L homozygotes (8 min). Therefore the S allele appears to be partially dominant over the L allele, although a strictly additive model cannot be ruled out. It is curious, however, that heterozygotes and S allele homozygotes were more similar in LORR to their ILS progenitor (7.4 min) rather than ISS (3.8 min). This could be due to a difference in gene interactions, but it also could be due to environmental differences (e.g., room temperature, noise level, injection proficiency) because the F2s were not tested at the same time as ISS and ILS. In this regard, we have observed that the mean LORR of ILS or ISS can vary from day to day by 1–2 min (in our experiments, however, ILS and ISS were almost always tested together on the same day).
Two methods of mapping were used to position Lorp1: RI interval mapping and F2mapping. Unlike the usual situation, the RI mapping proved to be extremely powerful, resulting in an LOD score of 28. Therefore, the identification of Lorp1 was not merely provisional, but rather highly significant. Further, the RI mapping localized Lorp1 to a 99% confidence interval of just 2.5 cM region. By comparison, large-scale F2mapping of QTLs for ethanol-induced LORR resulted in 99% confidence intervals that were an order of magnitude larger. [28] 
Given the RI mapping result, it was surprising that the F2result did not approach the same significance or sharpness of LOD peak. This probably occurred for several reasons. First, the number of F sub 2 s used (164) is relatively small, much smaller than the number used (approximately 1,000) to confirm ethanol sensitivity QTLs. [28] Second, Lorp1 is a major QTL nearly equivalent to a single-gene effect. Therefore, the ability to test an RI strain repeatedly to reduce environmental variance can drive even a single recombination event between the QTL and a tightly linked marker to very high statistical significance, thus excluding the region beyond that marker. Such independent, repeated testing cannot be done with an F2population. Third, the QTL effect size (the amount of the genetic effect resulting from variation at a particular locus) between SS-like and LS-like RIs was 4.5 min compared with only 2.3 min between F2homozygotes. This suggests additional gene interactions in the F2s that attenuated the influence of Lorp1; however, it also could reflect environmental differences given that RIs and F2s were not tested at the same time.
The LOD score for BLA peaked at the same location as Lorp1, suggesting that Lorp1 affects neurosensitivity. However, the LOD for BLA was much lower at 3.3 than the LOD for Lorp1 at 28. This is most likely due to the smaller number of BLA measurements, a higher coefficient of variation (SD/mean) for BLA, and not including RI strain 1 having a recombination close to Tyr. The difference also could suggest that the LORR difference is not entirely determined by BLA, although variation in BLA did explain about 72% of the variation in LORR.
Provisional QTLs for LORR induced by ethanol, propanol, and chloral hydrate also have been linked to Tyr in LS and SS mice. [25,26] The ethanol QTL, confirmed by Markel and Corley, [27] is called Lore7. [28] It is possible that Lore7 and Lorp1 are the same; however, the map position of Lore7 has a 90% confidence interval of 35 cM and thus is not nearly as sharply defined as the position of Lorp1.
Given the very tight linkage between Lorp1 and Tyr, we considered whether albinism could be responsible for the decreased sensitivity of the SS and ISS strains. However, comparison of coisogenic albino and nonalbino F1mice from the ISS x C57BL/6 c2j/C cross argues that albinism per se is not a causal factor. Albinism also seems to be an unlikely factor given that the c allele is completely recessive with respect to albinism, whereas the S allele of Lorp1 is partially dominant or additive. Although the coisogenic result does not rule out an effect unrelated to albinism and unique to the c allele, such an effect seems unlikely given that the c and c2jalleles abolish tyrosinase activity [42–45] and thus would appear to be functionally equivalent in all aspects.
The coisogenic experiments also do not exclude the possibility that Tyr affects LORR due to the C allele of LS. If the C allele is responsible, a simple hypothesis is that this allele leads to an increase in tyrosinase activity that reduces the availability of tyrosine in the brain for other purposes such as synthesizing catecholamines. If it does, then increasing tyrosine levels might alleviate this competition and cause LS mice to have a shorter LORR similar to SS mice. This is exactly the result obtained when LS mice were coinjected with tyrosine and measured for ethanol-induced LORR (SS mice were unaffected). [41] In our study, however, coinjecting the same dose of tyrosine as that used in the ethanol study had no effect on propofol-induced LORR in the LS or SS mice. Although these results still do not rule out unusual effects of Tyr, they argue against Tyr's involvement due to modulating tyrosine levels. These results also imply that, even though propofol and tyrosine are structurally similar, propofol does not act through competing with tyrosine.
Many other genes besides Tyr fall within the 99% confidence interval of 2.5 cM for Lorp1. Assuming an average gene density, 2.5 cM would be expected to harbor about 150 genes. So far, however, only about ten genes have been either physically or genetically mapped to this region with better than 1-cM resolution (MGD map). These genes are Cckbr, encoding the cholecystokinin B receptor; Cht, a coat color gene; eed, essential for embryonic ectoderm development; fit1, essential for normal growth and fertility; jdf, essential for normal juvenile development and fertility; mod2, encoding mitochondrial malic enzyme; mod2r, encoding a regulator of mod2, and Rps17-rs6 and Rps4-rs2, encoding ribosomal-related proteins. Of these, the cholecystokinin B receptor gene is interesting because it encodes a neurotransmitter receptor expressed throughout the CNS that is known to influence arousal and excitability. [46] However, the position of the human CCKBR gene [47] seems to suggest this gene is closer to Omp and Hbb than it is to Tyr and thus just outside the 99% confidence interval. The TYR locus of humans is on chromosome 11, within cytogenetic band q21 [48]; therefore, other genes being identified in this region as part of the Human Genome Project will suggest additional candidates for Lorp1.
Although studies describe lines of mice differentially sensitive to nitrous oxide, [49] diazepam, [50] and opiates, [51] no loci specifying these differences have been mapped. Therefore, Lorp1 is the first locus specifying differential sensitivity to a clinically relevant general anesthetic agent to have been mapped in a mammalian species. In nonmammalian species, single-gene mutants in the nematode Caenorhabditis elegans and in the fruit fly Drosophila melanogaster have been found to be differentially sensitive to the anesthetic effects of halothane, [52–56] and QTL studies in C. elegans [57] have revealed additional loci underlying halothane sensitivity. There is optimism, therefore, that identifying these gene products will pinpoint molecular pathways directly relevant to the action of general anesthetics.
The authors thank M. Beeson, L. Gordon, and A. Keller for technical support; and J. DeFries and B. Bennett for advice; and Drs. C. P. Gibbs and R. A. Deitrich for their support.
REFERENCES
Franks NP, Lieb WR: Molecular and cellular mechanisms of general anesthesia. Nature 1994; 367:607-14.
Tanksley SA: Mapping polygenes. Annu Rev Genet 1993; 27:205-33.
McClearn GE, Kakihana R: Selective breeding for ethanol sensitivity in SS and LS mice, Development of Animal Models as Pharmacogenetic Tools. Edited by GE McClearn, RA Deitrich, VG Erwin. Washington, DC, US Government Printing Office, 1981, pp 147-59.
Heston WDW, Erwin VG, Anderson SM, Robbins H: A comparison of the effects of alcohol on mice selectively bred for differences in ethanol sleep time. Life Sci 1974; 14:365-70.
Erwin VG, Heston WDW, McClearn GE, Deitrich RA: Effects of hypnotics on mice genetically selected for sensitivity to ethanol. Pharmacol Biochem Behav 1976; 4:679-83.
Smolen A, Marks MJ, Smolen TN, Collins AC: Dose and route of administration alter the relative elimination of ethanol by long-sleep and short-sleep mice. Alcohol Clin Exp Res 1986; 10:198-203.
Spuhler K, Hoffer B, Weiner N, Palmer M: Evidence for genetic correlation of hypnotic effects and cerebellar Purkinje neuron depression in response to ethanol in mice. Pharmacol Biochem Behav 1982; 17:569-78.
Basile A, Hoffer B, Dunwiddie T: Differential sensitivity of cerebellar Purkinje neurons to ethanol in selectively outbred lines of mice: Maintenance in vitro independent of synaptic transmission. Brain Res 1983; 264:69-78.
Palmer MR. Sorenson, SM, Freedman R, Olson L, Hoffer B, Seiger A: Differential ethanol sensitivity of intraocular cerebellar grafts in long-sleep and short-sleep mice. J Pharmacol Exp Ther 1982; 222:480-7.
Wafford KA, Burnett DM, Dunwiddie TV, Harris RA: Genetic differences in the ethanol sensitivity of GABA sub A receptors expressed in Xenopus oocytes. Science 1990; 49:291-3.
Crabbe JC: Genetic animal models in the study of alcoholism. Alcohol Clin Exp Res 1989; 13:120-7.
Crabbe JC, Belknap JK, Buck KJ: Genetic animal models of alcohol and drug abuse. Science 1994; 264:1715-23.
DeFiebre NC, Marley RJ, Wehner JM, Collins AC: Lipid solubility of sedative-hypnotic drugs influences hypothermic and hypnotic responses of long-sleep and short-sleep mice. J Pharm Exp Ther 1992; 263:232-40.
Marley RJ, Miner LL, Wehner JM, Collins AC: Differential effects of central nervous system depressants in long-sleep and short-sleep mice. J Pharmacol Exp Ther 1986; 238:1028-33.
Markel PD, Bennett B, Beeson MA, Gordon L, Simpson VJ, Johnson TE: Strain distribution patterns for genetic markers in the LSXSS recombinant-inbred series. Mam Genome 1996; 7:408-12.
Simpson VJ, Johnson TE: Genetic models in the study of anesthetic drug action. Internat Rev Neurobiol 1996; 39:223-41.
Baker R, Melchoir C, Deitrich: The effect of halothane on mice selectively bred for differential sensitivity to alcohol. Pharmacol Biochem Beh 1980; 12:691-5.
Simpson VJ, Blednov Y: Propofol produces differences in behavior but not chloride channel function between selected lines of mice. Anesth Analg 1996; 82:1-5.
Tanelian DL, Kosek P, Mody I, MacIver MB: The role of the GABA sub A receptor/chloride channel complex in anesthesia. Anesthesiology 1993; 78:757-76.
DeFries JC, Wilson JR, Erwin VG, Petersen DR: LS X SS recombinant inbred strains of mice: Initial characterization. Alcohol Clin Exp Res 1989; 13:196-200.
Falconer DS: Introduction to quantitative genetics. New York, Longman, 1981, pp 83-8, 112-5.
Markel PD, Johnson TE: Initial characterization of STS markers in the LSXSS series of recombinant inbred strains. Mam Genome 1994; 5:199-202.
McClearn GE, Plomin R, Gora-Maslak G, Crabbe JC: The gene chase in behavioral science. Psychol Sci 1991; 2:222-9.
Belknap JK: Mitchell, SR, O'Toole LA, Helms ML, Crabbe JC: Type I and Type II error rates for quantitative trait loci (QTL) mapping studies using recombinant inbred strains. Behav Genet 1996; 26:149-60.
Christensen SC, Johnson TE, Markel PD, Clark VJ, Fulker DW, Corley RP, Collins AC, Wehner JM: Quantitative trait locus analyses of sleep-times induced by sedative-hypnotics in LSXSS recombinant inbred strains of mice. Alcohol Clin Exp Res 1996; 20:543-50.
Markel PD, Fulker DW, Bennett B, Corley RP, DeFries JC, Erwin VG, Johnson TE: Quantitative trait loci for ethanol sensitivity in the LSXSS recombinant inbred strains: Interval-mapping. Behav Genet 1996; 26:447-58.
Markel PD, Corley RP: A multivariate analysis of repeated measures: Linkage of the albinism gene (Tyr) to a QTL influencing ethanol-induced anesthesia in laboratory mice. Psychiatr Genet 1994; 4:205-10.
Markel PD, Bennett B, Beeson M, Gordon L, Johnson TE: Confirmation of quantitative trait loci for ethanol sensitivity in long-sleep and short-sleep mice. Genome Res 1997; 7:92-9.
Markel PD, DeFries JC, Johnson TE: Ethanol-induced anesthesia in inbred strains of long-sleep and short-sleep mice: A genetic analysis of repeated measures using censored data. Behav Genet 1995; 25:67-73.
Markel PD, DeFries JC, Johnson TE: Use of repeated-measures in an analysis of ethanol-induced loss of righting reflex in inbred long-sleep and short-sleep mice. Alcohol Clin Exp Res 1995; 19:299-304.
Copeland NG, Gilbert DJ, Jenkins NA, Nadeau JH, Eppig JT, Maltais LS, Miller JC, Dietrich WF, Steen RG, Lincoln SE, Weaver A, Joyce DC, Merchant M, Wessel M, Katz H, Stein LD, Reeve MP, Daly MJ, Dredge RD, Marquis A, Goodman N, Lander ES: Genome maps IV. Science 1993; 262:67-82.
Dietrich WF, Miller JC, Steen RG, Merchant M, Damron D, Nahf R, Gross A, Joyce DC, Wessel M, Dredge RD, Marquis A, Stein LD, Goodman N, Page DC, Lander ES: A genetic map of the mouse with 4,006 simple sequence length polymorphisms. Nature Genet 1994; 7:220-5.
Dietrich WF, Miller J, Steen R, Merchant MA, Damron-Boles D, Husain Z, Dredge R, Daly MJ, Ingalls KA, O'Connor TJ, Evans CA, DeAngelis MM, Levinson DM, Kruglyak L, Goodman N, Copeland NG, Jenkins NA, Hawkins TL, Stein L, Page DC, Lander ES: A comprehensive genetic map of the mouse genome. Nature 1996; 380:149-52.
Jackson IJ, Bennett DC: Identification of the albino mutation of mouse tyrosinase by analysis of an in vitro revertant. Proc Natl Acad Sci U S A 1984; 87:7010-14.
Beerman F, Ruppert S, Hummler E, Bosch FX, Muller G, Ruther U, Schutz G: Rescue of the albino phenotype by introduction of a functional tyrosinase gene into mice. Embo J 1990; 9:2819-26.
Yokoyama T, Silversides DW, Waymire KG, Kwon BS, Takeuchi T, Overbeek PA: Conserved cysteine to serine mutation in tyrosinase is responsible for the classical albino mutation in laboratory mice. Nucleic Acids Res 1990; 18:7293-8.
Hays WL: Statistics for Psychologists. New York, Holt, Rinehart and Winston, 1963, pp 422-3.
Rikke BA, Johnson DK, Johnson TE: Murine albino-deletion complex: High-resolution microsatellite map and genetically anchored YAC framework map. Genetics 1997; 147:787-99.
Haley CS, Knott SA: A simple regression method for mapping quantitative trait loci in line crosses using flanking markers. Heredity 1992; 69:315-24.
Lander E, Kruglyak L: Genetic dissection of complex traits: Guidelines for interpreting and reporting linkage results. Nature Genet 1995; 11:241-7.
French TA, Clay KL, Weiner N: Role of brain tyrosine availability in mediating differences in ethanol sensitivity in long-sleep and short-sleep mice. J Pharm Exp Ther 1989; 250:556-64.
Townsend DW, Witkop Jr CJ, Mattson J: Tyrosinase subcellular distribution and kinetic parameters in wild type and c-locus mutant C57BL/6J mice. J Exp Zool 1981; 216:113-19.
Barber JL, Townsend D, Olds DP, King RA: Dopachrome oxido-reductase; A new enzyme in the pigment pathway. J Invest Dermatol 1985; 83:145-9.
Halaban R, Moellmann G, Tamura A, Kwon BS, Kuklinska E, Pomerantz SH, Lerner AB: Tyrosinases of murine melanocytes with mutations at the albino locus. Proc Natl Acad Sci U S A 1988; 85:7241-5.
Granholm NH, Opbroek AJ, Harvison GA, Kappenman KE: Tyrosinase activity (TH, DO, PAGE-defined isozymes) and melanin production in melanocytes of 3, 6, 9, and 12-day regenerating hair-bulbs of lethal yellow (A sup y /a), black (a/a) and albino (a/a; c sup 2j /c sup 2j) mice (C57BL/6J). Pigment Cell Res 1990; 3:233-42.
Boden P, Hall MD, Hughes J: Cholecystokinin receptors. Cell Mol Neurobiol 1995; 15:545-59.
Huppi K, Siwarski D, Pisegna JR, Wank S: Chromosomal localization of the gastric and brain receptors for cholecystokinin (CCKAR and CCKBR) in human and mouse. Genomics 1995; 25:727-9.
Brilliant MH, Williams RW, Conti CJ, Angel RJ, Oakey RJ, Holdener BC: Mouse chromosome 7. Mam Genome 1994; 5:S104-23.
Koblin DD, Dong DE, Deady JE, Eger EI II: Selective breeding alters murine resistance to nitrous oxide without alteration in synaptic membrane lipid composition. Anesthesiology 1980; 52:4101-7.
Gallaher EJ, Hollister LE, Gionet SE, Crabbe JC: Mouse lines selected for genetic differences in diazepam sensitivity. Psychopharmacology 1987; 93:25-30.
Katz RJ: The albino locus produces abnormal responses to opiates in the mouse. Eur J Pharmacol 1980; 68:229-32.
Sedensky MM, Meneely PM: Genetic analysis of halothane sensitivity in Caenorhabditis elegans. Science 1987; 236:952-4.
Morgan PG, Sedensky M, Meneely PM: Multiple sites of action of volatile anesthetics in Caenorhabditis elegans. Proc Natl Acad Sci U S A 1990; 87:2965-9.
Krishnan KS, Nash HA: A genetic study of the anesthetic response: mutants of Drosophila melanogaster altered in sensitivity to halothane. Proc Natl Acad Sci U S A 1990; 87:8632-6.
Campbell DB, Nash HA: Use of Drosophila mutants to distinguish among volatile general anesthetics. Proc Natl Acad Sci U S A 1994; 91:2135-9.
Lin M, Nash HA: Influence of general anesthetics on a specific neural pathway in Drosophila melanogaster. Proc Natl Acad Sci U S A 1996; 93:10446-51.
van Swinderen B, Shook DR, Ebert RH, Cherkasova VA, Johnson TE, Reis RJS, Crowder CM: Quantitative trait loci controlling halothane sensitivity in Caenorhabditis elegans. Proc Natl Acad Sci U S A 1997; 94:8232-7.
Figure 1. Duration of loss of righting reflex (LORR) and brain levels of propofol at awakening (BLA) in pigmented and albino LSXSS RI strains. (A) The difference in LORR between males and females was not significant, whereas the difference between pigmented (black bars, 4.8 +/- SEM) and albino RI strains (white bars, 9.3 +/- SEM) was highly significant for males and females (P < 0.0001, two-tailed test). n refers to the number of RI strains. The LORR for each strain was determined from the mean of 5–15 animals of each sex, and the BLA for each strain was determined from the mean of at least three samples. The difference in propofol BLA between pigmented (8.4 +/- 1.2 micro gram/gm) and albino (17.2 +/- 1.9 micro gram/gm) strains is highly significant (P < 0.0001, two-tailed test). The pigmented RI strains (black bars) used for propofol BLA determination were 2, 3, 6, 7, 8, 9, 17, 30 and 36; and the albino RI strains (white bars) used were 4, 10, 18, 20, 23, 25, and 33. Male and female data are combined for propofol BLA. The correlation coefficient r between propofol BLA and LORR for the 16 RI strains for which both measures could be obtained was -0.85 (P < 0.001). Error bars indicate standard error of the mean. (B) The LORR means for the pigmented RI strains are indicated by the filled ovals, and the means for the albino RI strains are indicated by the open ovals. The numbers next to the ovals indicate the RI strain(s) having that mean. RI strain 32 had a mean LORR of 4.4 min (SEM, 0.7) for females and 6.6 min (SEM, 1.1) for males, but it was not included in this analysis because at that time it was still segregating for the albino mutation.
Figure 1. Duration of loss of righting reflex (LORR) and brain levels of propofol at awakening (BLA) in pigmented and albino LSXSS RI strains. (A) The difference in LORR between males and females was not significant, whereas the difference between pigmented (black bars, 4.8 +/- SEM) and albino RI strains (white bars, 9.3 +/- SEM) was highly significant for males and females (P < 0.0001, two-tailed test). n refers to the number of RI strains. The LORR for each strain was determined from the mean of 5–15 animals of each sex, and the BLA for each strain was determined from the mean of at least three samples. The difference in propofol BLA between pigmented (8.4 +/- 1.2 micro gram/gm) and albino (17.2 +/- 1.9 micro gram/gm) strains is highly significant (P < 0.0001, two-tailed test). The pigmented RI strains (black bars) used for propofol BLA determination were 2, 3, 6, 7, 8, 9, 17, 30 and 36; and the albino RI strains (white bars) used were 4, 10, 18, 20, 23, 25, and 33. Male and female data are combined for propofol BLA. The correlation coefficient r between propofol BLA and LORR for the 16 RI strains for which both measures could be obtained was -0.85 (P < 0.001). Error bars indicate standard error of the mean. (B) The LORR means for the pigmented RI strains are indicated by the filled ovals, and the means for the albino RI strains are indicated by the open ovals. The numbers next to the ovals indicate the RI strain(s) having that mean. RI strain 32 had a mean LORR of 4.4 min (SEM, 0.7) for females and 6.6 min (SEM, 1.1) for males, but it was not included in this analysis because at that time it was still segregating for the albino mutation.
Figure 1. Duration of loss of righting reflex (LORR) and brain levels of propofol at awakening (BLA) in pigmented and albino LSXSS RI strains. (A) The difference in LORR between males and females was not significant, whereas the difference between pigmented (black bars, 4.8 +/- SEM) and albino RI strains (white bars, 9.3 +/- SEM) was highly significant for males and females (P < 0.0001, two-tailed test). n refers to the number of RI strains. The LORR for each strain was determined from the mean of 5–15 animals of each sex, and the BLA for each strain was determined from the mean of at least three samples. The difference in propofol BLA between pigmented (8.4 +/- 1.2 micro gram/gm) and albino (17.2 +/- 1.9 micro gram/gm) strains is highly significant (P < 0.0001, two-tailed test). The pigmented RI strains (black bars) used for propofol BLA determination were 2, 3, 6, 7, 8, 9, 17, 30 and 36; and the albino RI strains (white bars) used were 4, 10, 18, 20, 23, 25, and 33. Male and female data are combined for propofol BLA. The correlation coefficient r between propofol BLA and LORR for the 16 RI strains for which both measures could be obtained was -0.85 (P < 0.001). Error bars indicate standard error of the mean. (B) The LORR means for the pigmented RI strains are indicated by the filled ovals, and the means for the albino RI strains are indicated by the open ovals. The numbers next to the ovals indicate the RI strain(s) having that mean. RI strain 32 had a mean LORR of 4.4 min (SEM, 0.7) for females and 6.6 min (SEM, 1.1) for males, but it was not included in this analysis because at that time it was still segregating for the albino mutation.
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Figure 2. Genetic map of murine chromosome 7 showing LOD scores for positioning Lorp1 using the LSXSS RIs. Markers include the albino mutation (c), the pink-eyed dilution mutation (p), and six D7Mit simple-sequence length polymorphisms. The dashed line indicates the recommended threshold for statistical significance. Data were combined for males and females.
Figure 2. Genetic map of murine chromosome 7 showing LOD scores for positioning Lorp1 using the LSXSS RIs. Markers include the albino mutation (c), the pink-eyed dilution mutation (p), and six D7Mit simple-sequence length polymorphisms. The dashed line indicates the recommended threshold for statistical significance. Data were combined for males and females.
Figure 2. Genetic map of murine chromosome 7 showing LOD scores for positioning Lorp1 using the LSXSS RIs. Markers include the albino mutation (c), the pink-eyed dilution mutation (p), and six D7Mit simple-sequence length polymorphisms. The dashed line indicates the recommended threshold for statistical significance. Data were combined for males and females.
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Figure 3. Confirmation of Lorp1 from F2intercrosses between inbred long-sleep (ILS) and inbred short-sleep (ISS) mice. Markers are D7Mit simple-sequence length polymorphisms. Marker D7Mit31 is within the Tyr gene near the site of the albino mutation, c. The dashed line indicates the recommended threshold for statistical significance. Data were combined for males and females (n = 158).
Figure 3. Confirmation of Lorp1 from F2intercrosses between inbred long-sleep (ILS) and inbred short-sleep (ISS) mice. Markers are D7Mit simple-sequence length polymorphisms. Marker D7Mit31 is within the Tyr gene near the site of the albino mutation, c. The dashed line indicates the recommended threshold for statistical significance. Data were combined for males and females (n = 158).
Figure 3. Confirmation of Lorp1 from F2intercrosses between inbred long-sleep (ILS) and inbred short-sleep (ISS) mice. Markers are D7Mit simple-sequence length polymorphisms. Marker D7Mit31 is within the Tyr gene near the site of the albino mutation, c. The dashed line indicates the recommended threshold for statistical significance. Data were combined for males and females (n = 158).
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Figure 4. Propofol loss of righting reflex for (inbred short-sleep [ISS] c/c x C57BL/6 c2j/C) F1s and for inbred long-sleep (ILS) and ISS mice coinjected with tyrosine. (A) ISS mice have the classical albino mutation, whereas the c2jalbino mutation arose spontaneously in C57BL/6 at Jackson Laboratory. Therefore, F1mice are identical at all loci except the albino locus, pigmented mice (black bars) are c/C, and albino mice (white bars) are c/c2j. LORRs of females are significantly shorter than those of males by 1.3 min (P = 0.002, analysis of variance, two-tailed test). However, LORRs of albinos are not significantly shorter than those of pigmented mice (P = 0.35, analysis of variance, one-tailed test). (B) ILS (black bars) and ISS (white bars) mice coinjected with tyrosine (indicated as pr + tyr) also did not exhibit significant differences in LORR (P = 0.78, analysis of variance, two-tailed test) from controls at a concentration of tyrosine (200 mg/kg) previously shown to differentially alter ethanol-induced LORRs in LS and SS. [41] n indicates the number of mice (males and females) tested. Error bars indicate the SEM.
Figure 4. Propofol loss of righting reflex for (inbred short-sleep [ISS] c/c x C57BL/6 c2j/C) F1s and for inbred long-sleep (ILS) and ISS mice coinjected with tyrosine. (A) ISS mice have the classical albino mutation, whereas the c2jalbino mutation arose spontaneously in C57BL/6 at Jackson Laboratory. Therefore, F1mice are identical at all loci except the albino locus, pigmented mice (black bars) are c/C, and albino mice (white bars) are c/c2j. LORRs of females are significantly shorter than those of males by 1.3 min (P = 0.002, analysis of variance, two-tailed test). However, LORRs of albinos are not significantly shorter than those of pigmented mice (P = 0.35, analysis of variance, one-tailed test). (B) ILS (black bars) and ISS (white bars) mice coinjected with tyrosine (indicated as pr + tyr) also did not exhibit significant differences in LORR (P = 0.78, analysis of variance, two-tailed test) from controls at a concentration of tyrosine (200 mg/kg) previously shown to differentially alter ethanol-induced LORRs in LS and SS. [41]n indicates the number of mice (males and females) tested. Error bars indicate the SEM.
Figure 4. Propofol loss of righting reflex for (inbred short-sleep [ISS] c/c x C57BL/6 c2j/C) F1s and for inbred long-sleep (ILS) and ISS mice coinjected with tyrosine. (A) ISS mice have the classical albino mutation, whereas the c2jalbino mutation arose spontaneously in C57BL/6 at Jackson Laboratory. Therefore, F1mice are identical at all loci except the albino locus, pigmented mice (black bars) are c/C, and albino mice (white bars) are c/c2j. LORRs of females are significantly shorter than those of males by 1.3 min (P = 0.002, analysis of variance, two-tailed test). However, LORRs of albinos are not significantly shorter than those of pigmented mice (P = 0.35, analysis of variance, one-tailed test). (B) ILS (black bars) and ISS (white bars) mice coinjected with tyrosine (indicated as pr + tyr) also did not exhibit significant differences in LORR (P = 0.78, analysis of variance, two-tailed test) from controls at a concentration of tyrosine (200 mg/kg) previously shown to differentially alter ethanol-induced LORRs in LS and SS. [41] n indicates the number of mice (males and females) tested. Error bars indicate the SEM.
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Table 1. Comparison of Propofol-induced Loss of Righting Reflex for LS, SS, and Mice Derived from ILS and ISS 
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Table 1. Comparison of Propofol-induced Loss of Righting Reflex for LS, SS, and Mice Derived from ILS and ISS 
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