Stereotypic behaviors in mice selectively bred for high and low methamphetamine-induced stereotypic chewing.

RATIONALE: At high doses, methamphetamine produces repetitive stereotypic behaviors, and the degree to which this occurs is heritable. OBJECTIVES: Mice of a B6D2F2 genetic background were selectively bred for four generations for high (HMA) and low (LMA) numbers of stereotyped chewing episodes measured for 1 min at 33 min post-injection following 10 mg/kg methamphetamine (changed to 7 mg/kg for the high line and 15 mg/kg for the low line in the third selected generation to avoid ceiling and floor effects, respectively). We sought to determine whether stereotypic behaviors other than number of repetitive chewing episodes were altered by the selective breeding process. METHODS: HMA and LMA mice of the third and fourth selected generations were tested for chewing stereotypy, for a number of other stereotypic behaviors previously observed in rodents, and for several other non-stereotypic responses to methamphetamine. Testing in the third selected generation was conducted by observing behaviors on videotape following 7 mg/kg methamphetamine. In the fourth selected generation, mice were also tested in automated activity monitors following 10 mg/kg methamphetamine and in climbing chimneys following 16 mg/kg methamphetamine. Dose-response curves with doses of 1, 2, 3.5, 7, 10, and 15 mg/kg methamphetamine were constructed for the most commonly observed behaviors. RESULTS: LMA mice, which exhibited low stereotyped chewing, exhibited high stereotyped circling and climbing, and the reverse was true for these behaviors for HMA mice. For most of the other behaviors measured, there were drug effects but no differences between selected lines. CONCLUSIONS: These results suggest that these three stereotyped behaviors, chewing, circling, and climbing, at least partly share the same mechanisms, and therefore are influenced by at least some of the same genes, since animals selectively bred for low methamphetamine-induced stereotyped chewing exhibited high amounts of circling and climbing when given methamphetamine. This also suggests that the other stereotypic behaviors that we measured do not occur by the same genetically determined mechanisms as stereotypic chewing.

Quantitative trait loci influencing morphine antinociception in four mapping populations.

Analgesia (pain reduction, or antinociception) is a classical and clinically important effect of morphine administration, and in rodent models sensitivity to morphine has been shown to be strongly influenced by genotype. For example, several studies have reported marked differences in morphine antinociception between the insensitive C57BL/6 (B6) and sensitive DBA/2 (D2) inbred mouse strains on the hot-plate assay. This prompted the present genome-wide search for quantitative trait loci (QTLs) that are chromosomal sites influencing the magnitude of antinociception, by using four mapping populations derived from the B6 and D2 progenitor inbred strains. These four were the BXD recombinant inbred (RI) strain set, an F2 (B6D2F2) population, short-term selective breeding for antinociception from a B6D2F2 founding population, and incipient or completed congenic strains. In the BXD RI set and in the B6D2F2, a genome-wide search identified 10-12 provisional QTLs at a nominal p <.05. The other populations were subsequently used as confirmation steps to test each of the provisional QTL regions. Based on all available mapping populations, four QTLs emerged as significant (p <.00005) on proximal Chromosome (Chr) 1 (females only), proximal Chr 9 (females only), mid Chr 9, and proximal Chr 10. The Chr 10 QTL comaps to the same region as the micro-opioid receptor gene (Oprm); this receptor is a known mediator of morphine's antinociceptive effects. The Chr 1 QTL was evident only in females and comapped with the kappa-opioid receptor gene, Oprk.

Identification of a sex-specific quantitative trait locus mediating nonopioid stress-induced analgesia in female mice.

It is increasingly appreciated that the sexes differ in their perception of noxious stimuli and in their responsivity to exogenous and endogenous analgesic manipulations. We previously reported the existence of qualitative sex differences in the neurochemical mediation of nonopioid (i.e., naloxone-insensitive) stress-induced analgesia (SIA) produced by forced swims and suggested that female mice possess a sex-specific SIA mechanism. This female-specific system is now known to be estrogen-dependent, to be ontogenetically organized, and to vary with reproductive status; however, its neurochemical identity remains obscure. In an attempt to identify candidate genes underlying SIA in both sexes, we performed a two-phase quantitative trait locus (QTL) mapping experiment using the BXD/Ty recombinant inbred (RI) set derived from DBA/2J (D2) and C57BL/6J (B6) inbred mouse strains and (B6xD2)F2 hybrid mice derived from these same progenitors. All mice were subjected to 3 min forced swims in 15 degrees C water; nociceptive sensitivity on the 54 degrees C hot-plate assay was assessed immediately before and 2 min after cessation of the swim. We report the localization of a QTL statistically associated with SIA magnitude [p = 0.00000012; logarithm of the odds (LOD) = 6.1] in female mice only. This female-specific QTL, which we name Fsia1, is located on chromosome 8 at 52-84 cM from the centromere and accounts for 17-26% of the overall trait variance in this sex. The present data provide further evidence of the existence of a female-specific SIA mechanism and highlight the important role of both genetic background and gender in the inhibition of pain.

Genetic sensitivity to hot-plate nociception in DBA/2J and C57BL/6J inbred mouse strains: possible sex-specific mediation by delta2-opioid receptors.

The inbred mouse strains, DBA/2J (D2) and C57BL/6J (B6), display differential sensitivity to acute, thermal nociception as measured on the hot-plate (HP) assay. In an ongoing quantitative trait locus (QTL) mapping study designed to reveal genomic loci showing genetic linkage to HP sensitivity, a putative QTL on chromosome 4 (50-80 cM from the centromere) has been identified that appears to account for variability in this trait in male, but not female mice. An obvious candidate gene located in this same chromosomal region is Oprd1, which encodes the murine delta-opioid receptor. In an attempt to evaluate whether Oprd1 represents this sex-specific QTL for HP sensitivity, we tested D2 and B6 mice of both sexes for HP latencies (hindpaw-lift, -lick or -flutter) following systemic injections of saline, or the opioid receptor antagonists naloxone (NAL; 0.1 and 10 mg/kg), nor-binaltorphimine (nor-BNI; 5 mg/kg), naltrindole (NTI; 5 mg/kg), 7-benzylidenenaltrexone (BNTX; 0.7 mg/kg), or naltriben (NTB; 1 mg/kg). High-dose (10 mg/kg) NAL lowered HP latencies in D2, but not B6 mice, suggesting that the higher HP latencies exhibited by D2 mice reflect opioid mechanisms. HP latencies in both strains and both sexes were unaffected by pretreatment with low-dose (0.1 mg/kg) NAL or nor-BNI, suggesting that neither mu nor kappa receptors affect basal nociceptive sensitivity. The delta-receptor antagonist, NTI, and the delta2-specific antagonist, NTB, (but not the delta1-specific antagonist, BNTX) effectively lowered HP latencies in a strain- and sex-dependent manner: D2 male > B6 male > D2 female > B6 female. These data support the possibility that Oprd1 is a QTL mediating HP sensitivity in mice, and more generally illustrate the important roles of genetic background and gender in the perception of pain.

Quantitative trait loci affecting methamphetamine responses in BXD recombinant inbred mouse strains.

Individual differences in most behavioral and pharmacological responses to abused drugs are dependent on both genetic and environmental factors. The genetic influences on the complex phenotypes related to drug abuse have been difficult to study using classical genetic analyses. Quantitative trait locus (QTL) mapping is a method that has been used successfully to examine genetic contributions to some of these traits by correlating allelic variation in polymorphic genetic markers of known chromosomal location with variation in drug-response phenotypes. We evaluated several behavioral responses to multiple doses of methamphetamine (METH) in C57BL/6J (B6), DBA/2J (D2), and 25 of their recombinant inbred (BXD RI) strains. Stereotyped chewing, horizontal home cage activity, and changes in body temperature after 0, 4, 8, or 16 mg/kg METH, as well as stereotyped climbing behavior after 16 mg/kg METH, were examined. Associations (p < 0.01) between METH sensitivity and allelic status at multiple microsatellite genetic markers were subsequently determined for each response. QTLs were provisionally identified for each phenotype, some unique to a particular behavior and others that appeared to influence multiple phenotypes. Candidate genes suggested by these analyses included several that mapped near genes relevant for the neurotransmitters acetylcholine and glutamate. The locations of QTLs provisionally identified by this analysis were compared with QTLs hypothesized in other studies to influence methamphetamine- and cocaine-related phenotypes. In several instances, QTLs appeared to overlap, which is consistent with idea that common neural substrates underlie some responses to psychostimulants.

Short-term selective breeding as a tool for QTL mapping: ethanol preference drinking in mice.

Short-term selective breeding starting from an F2 intercross of two inbred strains is a largely unexploited but potentially useful tool for quantitative trait locus (QTL) mapping. The selection lines can also serve as a valuable confirmation test of recombinant inbred (RI) QTL results when the same two progenitor strains are used. Starting from an F2 from a C57BL/6J (B6) X DBA/2J (D2) cross (B6D2F2), this approach was used in a population of approximately 72 mice per generation bidirectionally selected for two-bottle choice 10% ethanol (alcohol) preference for four generations. The high-preference line diverged significantly from the low line in the first generation with a realized heritability of .32. By generation 4, the preference ratios in the high line were double those seen in the low line. Regions of the genome previously implicated by BXD RI QTL analysis as containing QTLs were searched using microsatellite markers. The test for the presence of QTLs was based on the divergence of marker allele frequencies in the two oppositely selected lines significantly exceeding that expected from random (genetic) drift and allele frequency estimation error. Combining the BXD and two-way selection line results, the most probable QTL was found on chromosome 3 (near the adhl locus; LOD approximately 2.9), other probable QTLs were found with LOD 2.4-2.6.

Type I and type II error rates for quantitative trait loci (QTL) mapping studies using recombinant inbred mouse strains.

Effective mapping strategies for quantitative trains must allow for the detection of the more important quantitative trait loci (QTLs) while minimizing false positives. Type I (false-positive) and Type II (false-negative) error rates were estimated from a computer simulation of QTL mapping in the BXD recombinant inbred (RI) set compromising 26 strains of mice, and comparisons made with theoretical predictions. The results are generally applicable to other RI sets when corrections are made for differing strain numbers and marker densities. Regardless of the number or magnitude of simulated QTLs contributing to the trait variance, the p value necessary to provide adequate protection against both Type I (alpha=.0001) and Type II (beta=.2) errors, a QTL would have to account for more than half of the between-strain (genetic) variance if the BXD or similar set was used alone. In contrast, a two-step mapping strategy was also considered, where RI strains are used as a preliminary screen for QTLs to be specifically tested (confirmed) in an F2 (or other) population. In this case, QTLs accounting for approximately 16% of the between-strain variance could be detected with an 80% probability in the BXD set when alpha = 0.2. To balance the competing goals of minimizing Type I and II errors, an economical strategy is to adopt a more stringent alpha initially for the RI screen, since this requires only a limited genome search in the F2 of the RI-implicated regions (approximately 10% of the F2 genome when p < .01 in the RIs). If confirmed QTLs do not account in the aggregate for a sufficient proportion of the genetic variance, then a more relaxed alpha value can be used in the RI screen to increase the statistical power. This flexibility in setting RI alpha values is appropriate only when adequate protection against Type I errors comes from the F2 (or other) confirmation test(s).

Localization to chromosome 10 of a locus influencing morphine analgesia in crosses derived from C57BL/6 and DBA/2 strains.

A quantitative trait locus (QTL) was detected and mapped to proximal chromosome 10 near the markers Mpmv5 and D10Mit51 with a strong influence on morphine-induced analgesia in the BXD recombinant inbred (RI) strains and in an F2 cross (B6D2F2) between the BXD progenitor strains, C57BL/6 and DBA/2. A LOD score of 3.9 (p < .00002) was seen for analgesia using the hot plate assay. Naloxone Bmax was also associated with this chromosome region in BXD RI mice. The mu opioid receptor gene (Oprm) has recently been mapped to this same chromosome region. The observation that several morphine-related traits and naloxone Bmax appear to be partly determined by this presumed single locus is consistent with the hypothesis that the mu opioid receptor gene, or one of its modulators, is the basis for the QTL.

Mu-opiate receptor binding is up-regulated in mice selectively bred for high stress-induced analgesia.

Pain perception and sensitivity to opiate analgesics strongly depend on genotype. Mice selectively bred for high (HA) and low (LA) swim stress-induced analgesia display markedly divergent morphine analgesia, a difference that appears to be determined by one or at the most two major genes. In an attempt to provide candidate genes mediating the supranormal analgesia displayed by HA mice, we performed mu-opiate receptor binding on 27th generation HA, LA, and control (C) mice using [3H]naloxone. HA mice were found to have significantly higher whole-brain receptor density (Bmax) than LA mice in whole brain homogenates; no significant difference in affinity (Kd) was observed. Quantitative autoradiography confirmed the line difference in whole-brain receptor binding. In the medial thalamus, a brain area implicated in ascending pathways of pain inhibition, HA mice were found to display significantly higher [3H]naloxone binding than C mice (a 64% increase) and LA mice (a 128% increase). No significant line differences were observed in any other brain locus. Thalamic mu receptors may therefore play an important role in a central 'volume control' mechanism of pain inhibition, and underlie individual differences in the responses of mice to opiate analgesic drugs.

Quantitative trait loci (QTL) applications to substances of abuse: physical dependence studies with nitrous oxide and ethanol in BXD mice.

Recombinant inbred (RI) mouse strains were developed primarily as a tool to detect and provisionally map major gene loci--those with effects large enough to cause a bimodal distribution in the trait of interest. This implied that progress toward gene mapping was possible only for gene loci accounting for at least half of the genetic variance. More recently, QTL (quantitative trait loci) approaches have been advanced that do not require bimodal distributions and are thus applicable to a much wider range of phenotypes. They offer the prospect of meaningful progress toward detecting and mapping minor as well as major gene loci affecting any trait of interest, provided there is a significant degree of genetic determination among the RI strains. This paper presents a review of RI gene mapping efforts concerning phenotypes related to drug abuse and presents new data for studies now in progress for nitrous oxide and acute ethanol withdrawal intensity. These two studies exemplify several strengths and limitations of the RI QTL approach.

Voluntary consumption of morphine in 15 inbred mouse strains.

To determine genetic differences in voluntary morphine consumption, 15 commonly used inbred strains of mice were given ad libitum two-bottle choice between saccharin alone or saccharin/morphine in one bottle and water in the other bottle. Subsequently, the saccharin was gradually reduced to zero, leaving only morphine. Independent groups of mice of the same strains were exposed to quinine in a parallel manner to control for the bitter alkaloid taste of morphine. Of the 15 strains, the C57BL/6J strain showed the highest consumption of morphine, both with or without saccharin and greatest consumption of morphine relative to quinine; it also showed only a slight decline in fluid consumption when morphine was added to the saccharin bottle. In marked contrast, the SWR/J strain showed the least consumption of morphine by the same criteria, followed closely by the AKR/J, CE/J, DBA/2J and SJL/J strains. The strain differences for all the morphine drinking measures exceeded an order of magnitude. Strain-specific voluntary morphine/saccharin consumption was not significantly correlated with saccharin consumption alone, but was highly correlated with morphine consumption alone. The results show that these behaviors are under an unusually large degree of genetic determination, and some of the largest strain differences remained essentially the same regardless of whether saccharin was present, or whether quinine was used as a control tastant.

Quantitative trait loci associated with brain weight in the BXD/Ty recombinant inbred mouse strains.

Adult C57BL/6J (B6) male mice had 37% heavier brains than did DBA/2J (D2) mice, while their body weights did not differ. The BXD recombinant inbred (RI) series of 20 strains, derived from a cross between B6 and D2 inbred strains, was used as the initial screen to determine significant associations between male brain weight and brain:body weight ratio, with allelic variation at 360 known marker gene loci. For brain weight, this yielded five candidate chromosome regions, each reflecting a possible quantitative trait locus (QTL) site affecting brain weight. The second step was to test as many of these five as possible using standard (non-RI) inbred strain data for brain weight previously reported in the literature. For this purpose, only strains possessing the same alleles as the B6 or D2 strains were used. Sufficient data to test two of the five candidate QTL were available. Of these, one was strongly supported as a site affecting brain weight--the D7rp2 region of chromosome 7. For the brain to body weight ratio, four chromosome regions emerged as significantly associated in the BXD series, but none were amenable to testing due to a lack of allelic information for the standard inbred strains. However, two of these regions showed highly significant associations (p less than 0.001, single test) that merit consideration as QTL sites for future testing. These two are the Hba region on chromosome 11 and the D17Tu7 region on chromosome 17. The genetic correlation between brain and body weight was low (r = 0.28), indicating that these two traits are largely genetically independent in the BXD RI series.

Single-locus control of saccharin intake in BXD/Ty recombinant inbred (RI) mice: some methodological implications for RI strain analysis.

The sac locus, with a major effect on saccharin preference, was discovered by Fuller (1974) in C57BL/6J (B6), DBA/2J (D2), and derived crosses, and is now supported in the BXD/Ty recombinant inbred (RI) series by a marked bimodal distribution in saccharin preference among 20 strains. The B6 allele led to increased saccharin preference compared to the D2 allele. Since the search for bimodal distributions reflecting major gene loci is an essential part of RI strain analysis, a new statistical method is proposed to test for bimodality, and comparisons are made to previously proposed methods. Another new RI method, quantitative trait loci (QTL) analysis, allows provisional detection and mapping of minor as well as major gene loci. Using this method as a screen, significant associations with saccharin preference were suggested with marker loci on portions of six chromosomes. One of these, the D12nyu1 locus on chromosome 12, was independently supported in a panel of standard (non-RI) inbred strains also tested for saccharin preference. It is unclear whether this reflects the sac locus.