Tamara J. Phillips

Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2

The actions of corticotropin-releasing hormone (Crh), a mediator of endocrine and behavioural responses to stress, and the related hormone urocortin (Ucn) are coordinated by two receptors, Crhr1 (encoded by Crhr) and Crhr2 (refs 4,5). These receptors may exhibit distinct functions due to unique tissue distribution and pharmacology. Crhr-null mice have defined central functions for Crhr1 in anxiety and neuroendocrine stress responses. Here we generate Crhr2−/− mice and show that Crhr2 supplies regulatory features to the hypothalamic-pituitary-adrenal axis (HPA) stress response. Although initiation of the stress response appears to be normal, Crhr2−/− mice show early termination of adrenocorticotropic hormone (Acth) release, suggesting that Crhr2 is involved in maintaining HPA drive. Crhr2 also appears to modify the recovery phase of the HPA response, as corticosterone levels remain elevated 90 minutes after stress in Crhr2−/− mice. In addition, stress-coping behaviours associated with dearousal are reduced in Crhr2–/– mice. We also demonstrate that Crhr2 is essential for sustained feeding suppression (hypophagia) induced by Ucn. Feeding is initially suppressed in Crhr2−/− mice following Ucn, but Crhr2−/− mice recover more rapidly and completely than do wild-type mice. In addition to central nervous system effects, we found that, in contrast to wild-type mice, Crhr2−/− mice fail to show the enhanced cardiac performance or reduced blood pressure associated with systemic Ucn, suggesting that Crhr2 mediates these peripheral haemodynamic effects. Moreover, Crhr2−/− mice have elevated basal blood pressure, demonstrating that Crhr2 participates in cardiovascular homeostasis. Our results identify specific responses in the brain and periphery that involve Crhr2.

Mice lacking dopamine D4 receptors are supersensitive to ethanol, cocaine, and methamphetamine

The human dopamine D4 receptor (D4R) has received considerable attention because of its high affinity for the atypical antipsychotic clozapine and the unusually polymorphic nature of its gene. To clarify the in vivo role of the D4R, we produced and analyzed mutant mice (D4R-/-) lacking this protein. Although less active in open field tests, D4R-/- mice outperformed wild-type mice on the rotarod and displayed locomotor supersensitivity to ethanol, cocaine, and methamphetamine. Biochemical analyses revealed that dopamine synthesis and its conversion to DOPAC were elevated in the dorsal striatum from D4R-/- mice. Based on these findings, we propose that the D4R modulates normal, coordinated and drug-stimulated motor behaviors as well as the activity of nigrostriatal dopamine neurons.

Alcohol preference and sensitivity are markedly reduced in mice lacking dopamine D2 receptors.

Although dopaminergic transmission has been strongly implicated in alcohol self-administration, the involvement of specific dopamine receptor subtypes has not been well established. We studied the ethanol preference and sensitivity of D2-receptor-deficient mice to directly evaluate whether dopamine D2 receptors contribute to alcohol (ethanol) consumption. We report a marked aversion to ethanol in these mice, relative to the high preference and consumption exhibited by wild-type littermates. Sensitivity to ethanol-induced locomotor impairment was also reduced in these mutant mice, although they showed a normal locomotor depressant response to the dopamine D1 antagonist SCH-23390. These data demonstrate that dopamine signaling via D 2 receptors is an essential component of the molecular pathway determining ethanol self-administration and sensitivity.

Identifying genes for alcohol and drug sensitivity: recent progress and future directions

New methods for identifying chromosomal regions containing genes that affect murine responses to alcohol and drugs have been used to identify many provisional quantitative trait loci (QTLs) since 1991. By 1998, 24 QTLs had been definitively mapped (P<5x10(-5)) to specific murine chromosomes, which indicates the presence of a relevant gene or genes at each location. The syntenic (homologous) region of the human genome for these genes is often known. For many mapped QTLs, candidate genes with relevant neurobiological function lie within the mapped region. Data that implicate candidate genes for specific responses include studies of knockout animals. Current strategies for gene identification include the use of congenic strains containing QTL regions introduced from another strain. There is increasing emphasis on gene-gene and gene-environment interactions in such studies.

Quantitative Trait Loci Affecting Peak Bone Mineral Density in Mice

Peak bone mass is a major determinant of risk of osteoporotic fracture. Family and twin studies have found a strong genetic component to the determination of bone mineral density (BMD). However, BMD is a complex trait whose expression is confounded by environmental influences and polygenic inheritance. The number, locations, and effects of the individual genes contributing to natural variation in this trait are all unknown. Experimental animal models provide a means to circumvent complicating environmental factors, and the development of dense genetic maps based on molecular markers now provides opportunities to resolve quantitative genetic variation into individual regions of the genome influencing a given trait (quantitative trait loci, QTL). To begin to identify the heritable determinants of BMD, we have examined genetically distinct laboratory mouse strains raised under strict environmental control. Mouse whole‐body bone mineral content by dual‐energy X‐ray absorptiometry (DXA) correlated strongly with skeletal calcium content by ashing, and peak whole‐body BMD by DXA in female mice occurred at ∼80–90 days of age. We therefore determined mean body weight and peak whole body BMD values in 12‐week‐old female mice from a panel of 24 recombinant inbred (RI) BXD strains, derived from a cross between C57BL/6 and DBA/2 progenitors. The distribution of body weight and BMD values among the strains clearly indicated the presence of strong genetic influences on both of these traits, with an estimated narrow sense heritability of 60% and 35%, respectively. The patterns of differences in body weight and peak whole body BMD in the BXD strains were then integrated with a large database of genetic markers previously defined in the RI BXD strains to generate chromosome map sites for QTL. After correction for redundancy among the significant correlations, QTL analysis of the BXD RI strain series provisionally identified 10 chromosomal sites linked to peak bone mass development in the female. Several of the identified sites map near genes encoding hormones, structural proteins, and cell surface receptors that are intricately involved in skeletal homeostasis. Four QTL for body weight were also identified. One of these loci was also strongly linked to inherited variation in BMD. This finding suggests that body weight and peak BMD may be influenced by linked genes or perhaps by common genes with pleiotropic effects. Our phenotyping in the RI BXD strains has allowed us to map a number of specific genetic loci strongly related to the acquisition of peak BMD. Confirmation of these findings will likely result in the understanding of which genes control skeletal health.

Behavioral sensitization to drug stimulant effects in C57BL/6J and DBA/2J inbred mice.

Common features shared by addictive drugs have been difficult to identify. One ubiquitous effect of these drugs is psychomotor stimulation. Further, repeated exposure commonly results in sensitization to drug stimulant effects. This study evaluates sensitization to drugs from several drug classes in C57BL/6J and DBA/2J inbred strain mice. DBA/2J mice showed sensitized responses to ethanol and methamphetamine, whereas C57BL/6J mice developed sensitization to morphine and methamphetamine. Strain susceptibilities to ethanol- and morphine-induced sensitization closely paralleled their sensitivities to the acute stimulant effects of these drugs; this was not the case for methamphetamine. The relative sensitivities of DBA/2J and C57BL/6J mice were not consistent across drugs, suggesting that the stimulant and sensitized responses to these drugs may be mediated by at least partially divergent neural mechanisms.

Behavioral sensitization to ethanol: genetics and the effects of stress.

Some aspects of drug abuse syndromes may be influenced by sensitization to some drug effects. This enhancement of drug effect has been associated with prior drug exposure and with exposure to stressful stimuli. It has been postulated that sensitization to psychomotor stimulant drug effects influences sensitivity to drug reward. The drugs of abuse best characterized for sensitization phenomena include cocaine, amphetamine, and morphine. In general, ethanols molecular mechanisms of action have been difficult to define relative to drugs with known receptor or transporter binding sites and, likewise, ethanol sensitization has been less thoroughly examined. Evidence supporting the existence of behavioral sensitization to ethanol, for genetic differences in the occurrence of ethanol sensitization, and for the influence of corticosterone on the development of ethanol sensitization is reviewed herein. There appear to be different genetic determinants of acute drug sensitivity and sensitization. Cross-sensitization between stress and ethanol suggest a potential role for hypothalamic-pituitary-adrenal (HPA) axis associated changes in ethanol sensitization, consistent with mechanisms likely contributing to sensitization to other abused drugs. Furthermore, glucocorticoid receptors appear to mediate both ethanol- and stress-induced sensitization to ethanol. A biological link between drug reward and drug sensitization involving HPA axis hormones may exist and, thus, study of the sensitization process may elucidate mechanisms relevant to drug abuse.

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 recornbinant inbred (RI) QTL results when the same two progenitor strains are used. Starting from an F2 from a C57BL/6J (B6) × DBA/2J (D2) cross (B6D2F2), this approach was used in a population of ~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 heritabittty 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 AdhI locus; LOD ~2.9), other probable QTLs were found with LOD 2.4–2.6.

Behavioral Studies of Genetic Differences in Alcohol Action

Many investigations of alcohol (ethanol) sensitivity have addressed the question of genetic determination using genetically defined animal populations, particularly mouse and rat stocks. Several reviews have appeared within the last decade that summarized much of the genetic rodent research on alcohol-related behavior (Belknap, 1980; Crabbe and Belknap, 1980; Horowitz and Dudek, 1983; Deitrich and Spuhler, 1984; Crabbe et al., 1985a; Phillips et al., 1989a). Our goal in this chapter is to review, summarize, and critique new information in the field, indicate how and why conclusions drawn from earlier reviews may have changed, and attempt to derive a more comprehensive picture of the contribution of genetic animal studies to research on alcohol’s behavioral effects.

Genetic determinants of sensitivity to ethanol in inbred mice

Mice from 15 inbred strains differed in sensitivity to ethanol-induced effects on open-field activity, hypothermia, rotarod ataxia, and anesthesia. Sensitivities to the different behavioral responses were generally uncorrelated. This suggests that the genetic determinants of behavioral sensitivity to one domain of ethanol effects are unrelated to those determining other responses. On the other hand, some variables were genetically related. For example, those strains sensitive to the loss of righting reflex induced by higher doses of ethanol showed reduced activity in the open field at lower doses and were more sensitive to ethanol-induced decreases in rearing. More generally, the pattern of results suggests that genetically influenced sensitivity to ethanol is not a monolithic phenomenon. Rather, it is specific to the particular response variable studied.