Progesterone's effects to reduce anxiety behavior of aged mice do not require actions via intracellular progestin receptors.

RATIONALE: Aging is associated with reduced secretion of, and down-regulation of receptors for, progesterone (P); yet, P's effects when administered to younger and older animals have not been systematically investigated. Some of P's antianxiety effects may be due to its conversion to 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) and its subsequent actions as a positive modulator at GABAA receptor complexes (GBRs). OBJECTIVES: We investigated whether P administration can decrease anxiety behavior of progestin receptor (PR) knockout (PRKO) or wild-type control mice. METHODS: P (10 mg/kg) or vehicle (propylene glycol) were administered subcutaneously to intact, female or male wild-type or PRKO mice that were either 9-12 or 18-24 months of age. Behavior in tasks that assess spontaneous activity (activity monitor and roto-rod), free exploration of a novel environment (open field, elevated plus maze, and elevated zero maze), and conflict behavior (mirror chamber, dark-light transition, and punished drinking) were examined 1 h after injection. RESULTS: P significantly decreased anxiety behavior of both PRKO and wild-type mice. P did not alter motor behavior but increased central entries in the open field, time in the open quadrants of the elevated zero maze, time in the mirrored chamber, time in the light compartment of the dark-light transition, and punished drinking in young and old mice. P-administered mice had higher levels of hippocampal 3alpha,5alpha-THP and GABA-stimulated chloride flux than did vehicle-administered PRKO or wild-type mice. CONCLUSIONS: The effects of P to decrease anxiety behavior of younger and older mice do not require classic PRs and may involve actions of 3alpha,5alpha-THP at GBRs.

Genetic analysis of the psychomotor stimulant effect of ethanol.

Genetic influences on the psychomotor stimulant effect of ethanol may be a key feature of abuse liability. While earlier work has shown the activational effects of ethanol to be under the influence of a relatively uncomplicated additive genetic system, preliminary data from our laboratory suggested the possibility of nonadditive genetic variance. In the present study, a full Mendelian cross was conducted to further characterize gene action and search for quantitative trait loci (QTL) influencing the psychomotor stimulant properties of ethanol. We tested 3062 mice of the six Mendelian cross genotypes (P1, P2, F1, F2, BC1 and BC2) derived from a cross between the C57BL/6J (B6) and C3H/HeJ (C3H) inbred strains of mice. On day 1, mice were injected with saline, put in a holding cage for 5 min, then placed in an activity monitor for 5 min. On day 2, mice were injected with 1.5 g/kg ethanol, and activity again monitored for 5 min. Analysis showed the expected activation in the C3H strain and little activation in the B6 strain, with no effect of sex. Biometrical genetic analysis showed a best-fit model that included the mean (m), additive effect (a), and an epistatic parameter (i = homozygote by homozygote interaction). Analysis showed good evidence for QTL on chromosomes 1 (logarithm of odds (LOD) 3.4-7.5, 88-100 cM), 6 (LOD 9.1-10.4, 46-50 cM) and 15 (LOD 7.3-8.8, 28-32 cM). While the regions on chromosomes 1 and 6 have previously been implicated in several different ethanol-related phenotypes, this is the first report of a QTL influencing the psychomotor stimulant properties of ethanol on chromosome 15. Other studies have identified QTL in this region of chromosome 15 mediating locomotor activation caused by other psychostimulants, including cocaine, amphetamine and phencyclidine.

Behavioural testing of standard inbred and 5HT(1B) knockout mice: implications of absent corpus callosum.

Rapid advances in biotechnology have created new demands for tests of mouse behaviour having both high reliability and high throughput for mass screening. This paper discusses several statistical and psychological factors pertinent to replication of results in different laboratories, and it considers the question of which inbred strains are best for test standardization. In this context, the problem of absent corpus callosum in the 129 strains is addressed with data from a recent study of six diverse tests of behaviour, and it is shown that effects of absent corpus callosum are usually nonsignificant and/or very small. Whether any 129 substrain is to be included in the list of standard strains depends on the goal of the standardization--collecting diverse phenotypic data on most available strains by a few expert investigators (the gold standard) or refining behavioural tests in order to establish a normal range of behaviour that can be used to judge a wider range of strains or even an individual mouse.

Genetics of mouse behavior: interactions with laboratory environment.

Strains of mice that show characteristic patterns of behavior are critical for research in neurobehavioral genetics. Possible confounding influences of the laboratory environment were studied in several inbred strains and one null mutant by simultaneous testing in three laboratories on a battery of six behaviors. Apparatus, test protocols, and many environmental variables were rigorously equated. Strains differed markedly in all behaviors, and despite standardization, there were systematic differences in behavior across labs. For some tests, the magnitude of genetic differences depended upon the specific testing lab. Thus, experiments characterizing mutants may yield results that are idiosyncratic to a particular laboratory.

Quantitative trait loci analysis affecting contextual conditioning in mice.

In an extensive backcross of mice between C3H/HeJ (C3H) and C57BL/6J (B6), we sought to map genes that influence learning and memory as measured by performance in a contextual fear-conditioning paradigm. Our results indicate that there are several genetic regions that have a strong influence on performance in this paradigm. The strongest influences map to the proximal and distal ends of chromosome 1 (lod scores of 5.14 and 4.76, respectively). Other chromosomal regions (chromosomes 3, 7, 8, 9 and 18) were also identified as candidates for regions containing genes influencing contextual fear conditioning, with lod scores ranging from 1.8 to 2.7.

Classical and neoclassical approaches to the genetic analysis of alcohol-related phenotypes.

Theoretical descriptions are given for two breeding methods in animal genetics that might be of use in alcohol research. These methods are marker-based selection and marker-based development of congenic strains, both using DNA markers such as polymerase chain reaction-detectable polymorphisms as the criteria for breeding. Such designs would utilize these markers as indicators of adjacent Quantitative Trait Loci (QTL) that are influential on alcohol-related phenotypes. Issues in the logic and implementation of these methods, such as proximity of the markers and the QTL allele, are explored. A third method, development of congenic strains with phenotypic screening, is also described. This method is currently being used to create two sets of congenic lines on a C57BL/6 inbred mouse background. The criterion phenotype is locomotor activation to 1.5 g/kg (i.p.) ethanol. Data are reported on the success of transferring the activation phenotype from two strains, DBA/2Abg and MOLD/Rk-Abg, onto the nonactivated C57BL/6Abg background. The value of these methods in alcohol research is outlined with regard to both identification of relevant genes and for their use as tools in basic research on mechanism of alcohol action.

Biometrical genetic analysis of ethanol's psychomotor stimulant effect.

Hereditary influences on psychomotor stimulant effects of ethanol (ETOH) were studied in a classical Mendelian cross of DBA/2Abg (D2) and C57BL/6Abg (B6) inbred mouse strains. A dose-response study with nine doses (0-3.5 g/kg ip) indicated that B6 mice lack the activational limb of the biphasic curve (< or = 1.5 g/kg), as assessed in a 15-min test. D2 mice ran greater distances and ran faster, at doses up to 2.5 g/kg. B6 mice showed no increments in speed or distance at these doses that activated D2 mice. Several other indices reinforced the conclusion of ETOH-induced behavioral activation in D2 mice and lack there of in B6 mice (traditional photobeam interruptions--horizontal counts; center distance; vertical movements; inactive time; as well as derived indices of running speed and average length of each movement). The F1, F2, and backcross generations produced dose-response curves that showed additive inheritance of the activational response to doses below 1.5 g/kg. A second study (n = 1446) examined response to 1.5 g/kg with a within-subjects design in the full Mendelian cross. This study verified the completely additive mode of inheritance for the total distance measure suggested in the dose-response study, and showed that sex linkage and sex differences were not present. Narrow sense heritability of the ETOH activation response (indexed by total distance) was calculated at 0.35 and approximately 3 loci were estimated to be responsible for the B6/D2 difference. The other phenotypes (described above) also showed strongly additive genetic control.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic influences on locomotor activating effects of ethanol and sodium pentobarbital.

The paradoxical capability of sedative-hypnotics to produce behavioral disinhibition varies among genotypes. In DBA/2 mice ethanol (ETOH) produced strong locomotor stimulation with the peak of the biphasic curve at 1.5 g/kg IP. C57BL/6 mice showed no activation, and F1S were intermediate. These characterizations held for a variety of behavioral indices derived from 15 min tests, such as distance, speed, and rest time, at doses in the 0-2.0 g/kg range. Analogous studies with sodium pentobarbital (0-40 mg/kg) yielded a similar pattern of strain differences in locomotor stimulation. In contrast, loss of righting reflex durations (60 mg/kg PENTO, IP) were similar in the two strains, indicating dissociation of activating and sedative effects. In complementary studies, long- and short-sleep mice, which were bred for differences in soporific effects of ETOH, showed similar activation profiles at ETOH doses up to 1.5 g/kg and PENTO doses up to 30 mg/kg. These studies provide support for an hypothesis of common genetic control of the activation effect for ETOH and PENTO.

Differential activating effects of ethanol in C57BL/6Abg and DBA/2Abg mice.

A characteristic pattern of ETOH-induced locomotor activation in the DBA/2Abg strain (D2), small activation or sedation in the C57BL/6Abg (B6), and an intermediate position of the F1s was found using a between-group design and 1.5 g/kg ETOH. This pattern was consistent for a variety of behavioral indices not previously examined, including distance, rest time, movement speed and length, as well as the traditional horizontal counts. Using a within-subject, multiple day, repeated-testing procedure, the same three genotypes were also assessed after manipulating drug administration order, where ETOH exposure (1.5 g/kg) was on either the first or second test day. Another experiment examined the effect of lighting level on the response to 1.5 g/kg ETOH using a within-subjects approach. Neither the testing order nor lighting condition had any major influence on the magnitude of activation as measured by locomotor activity (distance). The pattern of additive genotypic influences exhibited by the B6, D2, and F1 mice is remarkably resistant to these contextual and procedural manipulations.

The effects of fludrocortisone acetate and deoxycorticosterone acetate on salt appetite in mice.

Three genotypes of mice were shown to increase their intake of 3% sodium chloride (NaCl) following administration of fludrocortisone acetate (25 mg/kg), a synthetic steroid having strong mineralocorticoid/glucocorticoid activity. However, treatment with deoxycorticosterone acetate (DOCA) (10 mg/kg), a corticosteroid with predominantly mineralocorticoid activity, did not increase 3% NaCl consumption by the stock tested. In contrast, both fludrocortisone and DOCA have been shown to be effective activators of sodium appetite in rats. The essential importance of glucocorticoids in potentiating mineralocorticoid activation of sodium hunger in mice is suggested.

Comparisons of subcolonies of selectively bred long-sleep and short-sleep mice.

The Albany subcolony of the selectively bred Long- and Short-Sleep mice was directly compared to the original Colorado colony. As expected from the additional selection applied to the Colorado colony, small differences in the selection phenotype, loss of the righting reflex duration following ethanol treatment, were observed in the Short-Sleep line. However, no colony differences existed in three other indices of ethanol effects. Clear line differences in the shape of the locomotor activity dose-response curve, thermoregulatory effects of ethanol, and ethanol elimination rate replicated earlier findings, but these differences were similar in the two colonies. These data argue for stability of the polygenic control system and provide a picture of remarkable similarity of the two sublines, which were separated by more than 30 generations.

Selective breeding, congenic strains, and other classical genetic approaches to the analysis of alcohol-related polygenic pleiotropisms.

Dimensions of behavioral sensitivities to alcohol in mice are under control of polygenic systems of relatively small size. The mode of inheritance of these phenotypes is frequently additive, with no evidence of dominance, epistasis, or sex linkage. The utility of classical breeding methodologies, such as selection, for assessment of genetic correlations is reviewed. A distinction is drawn between pleiotropisms in these polygenic systems, and the statistical concept of a genetic correlation. Development of congenic strains is argued to be a powerful alternative methodology, heretofore unused in alcohol pharmacogenetics. Using the phenotype of behavioral activation produced by a low dose of ethanol, we describe the production of an activated congenic strain on the non-activated background of the C57BL/6 mouse strain. Through five generations of repeated backcrossing, from a genetically heterogenous stock, "activational" alleles are being successfully transferred to the C57BL/6 background. Theoretical issues in the creation of congenic strains in potentially polygenic systems are covered, including number of effective loci and heritability.

Locomotor activity responses to ethanol in selectively bred long- and short-sleep mice, two inbred mouse strains, and their F1 hybrids.

Locomotor activity responses to sub-hypnotic doses of ethanol (ETOH) were assessed in selected lines of mice (LS and SS), inbred strains, and their F1 hybrids. Data were obtained as photocell beam interruptions in a 15-min test for a dose range of 0 to 2.75 or 3.5 g/kg for LS and SS mice, respectively. Biphasic dose-response curves were obtained for LS and SS mice with the SS mice showing a sedative limb of the dose-response curve shifted to the right. The effects of 2.0 g/kg ETOH were also assessed in a diallel cross of the selected LS/SS lines and C57BL/6Abg and MOLD/RkAbg inbred strains. The 2.0 g/kg dose produced a wide range of responses, from sedation in C57BL/6Abg mice to extreme activation in SS and MOLD/RkAbg mice, and no effect in LS mice. The responses of F1 hybrids reflected a typical pattern of partial dominance, with heterosis in some crosses. When present, dominance was in the direction of greater locomotor activation. These patterns were confirmed by biometrical genetic analysis of the 4 x 4 diallel cross of the two lines and the two inbred strains. The data indicate that loci in addition to those responsible for selection for sedative sensitivity in LS and SS mice can influence locomotor activation produced by sub-hypnotic ETOH doses, and that SS and MOLD mice show locomotor activation for different genetic reasons.

Genetic analyses of the biphasic nature of the alcohol dose-response curve.

Ethanol (ETOH)-induced locomotor activation and depression were studied in 23 genotypes of mice. This included a diallel cross of four inbred strains tested with a range of ETOH doses from 0 to 2.75 g/kg. The diversity in shapes of the biphasic ETOH dose-response curves was both qualitative and quantitative, and additive gene action characterized the genetic control of the dose-response curve. Small dominance effects were typically directional in the direction of more activation, or resistance to sedation. No evidence was found for maternal effects, sex linkage, or epistasis. Sex differences were seen in the increased susceptibility of male mice to locomotor sedation at higher ETOH doses. In the diallel cross, there was no correlation between the degree of activation produced by low ETOH doses and sedation produced by higher doses. This indicates that while considerable genetic influences exist for both activational and sedative domains of ETOH effects, these genetic influences are relatively independent.

Responses to ethanol challenge in long- and short-sleep mice prenatally exposed to alcohol.

Individual sensitivity to alcohol may influence the severity of functional deficits due to prenatal alcohol exposure. To examine this hypothesis, Long-Sleep (LS) and Short-Sleep (SS) mice, selectively bred for differences in ethanol-induced narcosis, were intubated with either 2.9 g/kg ethanol (E) or an isocaloric amount of sucrose (S) twice per day on days 7 through 15 of pregnancy. An untreated control group (C) was maintained for each line. Offspring were fostered to lactating Rockland-Swiss mice at birth. Males and females from each litter were challenged with an acute dose of ethanol (3.8 g/kg) at 30 days of age. Measures of sleep time duration, waking blood ethanol concentrations (BEC), rectal temperatures, heart rate, and ethanol clearance were obtained to examine whether the acute effects of ethanol are altered by prenatal alcohol exposure. Prenatal alcohol exposure did not differentially affect responses to ethanol challenge within either genotype. Ethanol-induced hypothermia, heart-rate depression, and sleep time did differ between genotypes, with LS more affected than SS mice. Ethanol clearance rates were faster for SS than LS mice. These results suggest postnatal pharmacological responses to acute ethanol challenge are not altered by prenatal alcohol exposure in LS and SS mice. Prenatal alcohol-exposed offspring of both mouse genotypes showed lower average heart rate responses than controls, suggesting this measure may be a sensitive indicator of prenatal alcohol effects in mice.

Distinctions among sedative, disinhibitory, and ataxic properties of ethanol in inbred and selectively bred mice.

Three different domains of behavioral action of ethanol (ETOH) were examined in a battery of seven inbred strains and in the selectively bred Long-Sleep (LS) and Short-Sleep (SS) mice. Sedative effects were examined with the loss of the righting reflex test at 3.8 g/kg. The variation among inbred strains was only half the size of the difference between LS and SS mice which were selectively bred for extremes in this phenotype; such a result is expected for phenotypes controlled polygenically. Blood ETOH levels at waking from the narcosis also showed a range of differences among the inbred strains that was less than the LS/SS difference. Ataxia was measured with the grid test, and the inbred strains fell into two groups, resembling the highly ataxic LS line, and the less ataxic SS line. Biphasic effects of ETOH on locomotor activity were strongly genotype dependent. Variation in degree of activation/disinhibition produced by doses up to 1.5 g/kg (IP) ranged from no activation, in the C57BL/6Abg strain which was larger than that seen for SS mice. The patterns of strain differences for both ataxia and activation were highly different from the duration of loss of righting reflex measure, suggesting multiple independent genetically based "sensitivities" to ETOH.

Ethanol teratogenesis in selectivity bred long-sleep and short-sleep mice: a comparison to inbred C57BL/6J mice.

Sensitivity to alcohol may influence the severity of ethanol teratogenesis. To examine this hypothesis, the teratogenic effects of ethanol were compared in Long-Sleep (LS) and Short-Sleep (SS) mice, selectively bred for differences in ethanol-induced narcosis. Inbred C57BL/6J (B6) mice were included to confirm previously reported teratogenic effects using our own treatment regimen and standard assessment technique. Intragastric administration of ethanol (5.8 g/kg) on Days 9 and 10 of pregnancy resulted in growth retardation and an increase in prenatal mortality in LS litters but not in SS litters. Therefore, alcohol sensitivity plays a role in the severity of prenatal alcohol effects. B6 mice showed more ethanol teratogenicity than either LS or SS mice, even though maternal blood ethanol levels were similar across genotypes. This result suggests genetic variations other than alcohol sensitivity also influence ethanol teratogenesis.

Genotype-dependent effects of GABAergic agents on sedative properties of ethanol.

Two lines of mice, selectively bred for differential sensitivity to the soporific effects of ethanol (ETOH), were administered GABAergic drugs in an effort to evaluate a role for GABA in ETOH sensitivity. ETOH sensitive Long-Sleep mice (LS) showed potentiated ETOH sedation when administered bicuculline, muscimol and aminooxyacetic acid (AOAA). ETOH-insensitive SS mice exhibited reduced ETOH sedation in the presence of the antagonists, bicuculline and picrotoxin, and potentiated sedation in the presence of muscimol and AOAA. These changes in narcosis duration were interpreted as central effects, since blood ethanol levels at waking from ETOH sedation varied with GABAergic drug treatment. Picrotoxin antagonized pentobarbital-induced narcosis in both lines, but to a greater extent in SS mice. These and other experiments with a genetically heterogeneous stock suggest GABA involvement in genotype-dependent ETOH sensitivity, but do not support a simple role of GABA receptor involvement.

Convulsant properties of GABA antagonists and anticonvulsant properties of ethanol in selectively bred long- and short-sleep mice.

The convulsant potency of bicuculline, a GABA antagonist, was shown to be greater in Short-Sleep (SS) mice than in Long-Sleep (LS) mice. LS mice, selectively bred for lengthy ethanol-induced narcosis, had longer latencies to myoclonus and clonus following administration of bicuculline and picrotoxin than did ethanol-resistant SS mice. SS mice were also more susceptible to pentylenetetrazol-induced myoclonus, but not clonus. F1 hybrids showed bicuculline seizure sensitivity intermediate to the two parent lines. Ethanol weakly inhibited bicuculline-induced myoclonus in both LS and SS mice. Clonus was clearly antagonized by ethanol in both lines, but to a similar degree. These data provide evidence for a GABAergic role in genotype-dependent sensitivity to ethanol.