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The Alcohol Tolerant and Alcohol Non-Tolerant rats (AT, ANT) were selectively bred for ethanol-induced ataxia as measured on the inclined plane. Here we report on a quantitative trait locus (QTL) study in an F(2) intercross population derived from inbred AT and ANT (IAT, IANT) and a follow-up study of congenics that were bred to examine one of the mapped QTLs. Over 1200 F(2) offspring were tested for inclined plane sensitivity, acute tolerance on the inclined plane, duration of the loss of righting reflex (LORR) and blood ethanol at regain of the righting reflex (BECRR). F(2) rats that were in the upper and lower 20% for inclined plane sensitivity were genotyped with 78 SSLP markers. Significant QTLs for inclined plane sensitivity were mapped on chromosomes 8 and 20; suggestive QTLs were mapped on chromosomes 1, 2 and 3. Highly significant QTLs for LORR duration (LOD = 12.4) and BECRR (LOD = 5.7) were mapped to the same locus on chromosome 1. Breeding and testing of reciprocal congenic lines confirmed the chromosome 1 LORR/BECRR QTL. A series of recombinant congenic sub-lines were bred to fine-map this QTL. Current results have narrowed the QTL to an interval of between 5 and 20 Mb. We expect to be able to narrow the interval to less than 5 Mb with additional genotyping and continued breeding of recombinant sub-congenic lines.

Previous studies have identified quantitative trait loci (QTL) in the inbred high and low alcohol-sensitive rat (IHAS1 and ILAS1) strains. The original development of the strains involved selection for ethanol sensitivity based on duration of the loss of the righting reflex (LORR) after a standard dose of ethanol. This paper confirms some of these QTL using a short-term selection procedure based on the difference between the blood ethanol level at LORR and regain of the righting response. An F(2) population of rats was developed by a reciprocal cross of IHAS1 and ILAS1 rats. Selection for five generations was carried out using delta-blood ethanol concentration (dBEC) as the selection trait, where dBEC=BECLR (BEC at loss of righting reflex)-BECRR (BEC at regain of righting reflex). The lines were labeled tolerant (TOL) or sensitive (SENS). Approximately one-third of the offspring for each generation in each line were genotyped using DNA markers that had been previously found to be linked to QTL on chromosomes 1, 2, 5, 12, and 13. By the fifth generation of selection, the lines showed a very large difference in dBEC, BECRR, and duration of LORR; BECLR showed little segregation during the selection, and latency to lose the righting reflex showed none. IHAS allele frequency increased in the SENS line for markers on chromosomes 1, 5, 12, and 13 while ILAS allele frequency increased in the TOL line. These results were in good agreement with the two previous QTL studies. On chromosome 2, the selection resulted in an accumulation of ILAS alleles in both lines. This study provides independent confirmation of the location of QTL on chromosomes 1, 5, 12, and 13 for ethanol sensitivity. It also suggests that genetic differences in duration of LORR are mediated primarily by the dBEC phenotype.

Rationale: Genetically influenced alcohol sensitivity is thought to be an important risk factor for the development of alcoholism. An effective first step for identifying genes that mediate variation in alcohol sensitivity is through quantitative trait loci (QTL) mapping in model organisms., Objective: Fourteen provisional QTLs related to alcohol sensitivity were previously mapped in an F2 derived from the IHAS1 and ILAS1 rat lines. The objective of the current study was to confirm those QTLs in an independently derived F2 and in congenics that were bred for two of the loci., Materials and Methods: IHAS1 X ILAS1 F2 (n=450) were tested for alcohol-induced loss of righting reflex (LORR), blood ethanol concentration at regain of righting reflex (BECRR), sensitivity and acute tolerance on the Rotarod, and neurotensin receptor density (NTR1). Rats were genotyped at the 14 candidate loci and QTL mapping was conducted. Reciprocal congenic strains were bred for loci on chromosomes 2 and 5 and tested for LORR and BECRR., Results: Four LORR QTLs were mapped at the suggestive or significant level (chromosomes 2, 5, 12, and 13). BECRR was mapped to chromosomes 5, 12, and 13 either in the original or current experiment. Results of the congenic experiment also support QTLs for LORR and BECRR on chromosomes 2 and 5. QTLs for NTR1 density and behavior on the Rotarod were not confirmed., Conclusions: QTL mapping in crosses derived from the IHAS1 and ILAS1 has successfully identified loci related to alcohol sensitivity. Recombinant congenics are now being bred to more finely map the confirmed QTLs.

Background: Genetic variance in initial sensitivity to ethanol has been implicated as a risk factor for the development of alcoholism. Identification of the genes that confer differential initial sensitivity is an important goal for the development of new treatment strategies and for a comprehensive understanding of the mechanism of ethanol's action. Quantitative trait loci (QTL) mapping for initial sensitivity and other ethanol-related behavioral traits in model organisms has become an important first step for the ultimate identification of genes that contribute to variation in ethanol responses., Methods: An F(2) intercross was made from the Inbred High and Low Alcohol Sensitivity rat lines (IHAS and ILAS). The F(2) rats were tested for duration of the loss of righting reflex test (LORR); blood ethanol concentration at regain of righting reflex (BECrrr); BEC at the first time to reach criterion on the rotarod after 1.6 g/kg of ethanol (BEC1); acute functional tolerance on the rotarod (AFT); and high-affinity neurotensin receptor (NTR1) density in the nucleus accumbens (NAc), caudate putamen (CP), and ventral midbrain (VMB). A full genome scan with an average marker spacing of 16.8 cM for interval QTL mapping was conducted on the F(2) rats (N = 363)., Results: Seven significant or suggestive QTL were detected for LORR, one for BECrrr, three for BEC1, two for NTR1 binding in the CP, and one for binding in the NAc, but none were mapped for AFT or NTR1 binding density in the VMB. Effect size of the seven LORR QTL, the trait for which the parental strains were selected, ranged from 3 to 4%, with all accounting for approximately 22% of the total phenotypic variation. One of the LORR QTL on chromosome 2 (approximately 87 cM) was significant, and a second QTL on chromosome 5 (approximately 37 cM) was suggestive for both LORR and BECrrr., Conclusions: The results indicate that segregating populations derived from the IHAS and ILAS strains can be used for mapping ethanol sensitivity QTL. The chromosome 2 LORR QTL may confer variation in ethanol metabolism, whereas the chromosome 5 LORR/BECrrr QTL likely mediates central nervous system ethanol sensitivity. The small number or absence of QTL for BEC1, AFT, and NTR1 receptor density suggests that genetic variation for these traits is minimal in the IHAS/ILAS strains and/or the effect size of QTL for these traits is too small to be mapped efficiently in this sample of F(2) rats. The ultimate identification of genes underlying these alcohol sensitivity QTL will contribute to our understanding of the actions of alcohol in the central nervous system if not to a deeper understanding of the genetic risk factors for alcoholism.

The Alcohol Tolerant (AT) and Alcohol Nontolerant (ANT) rats, selectively bred for ethanol-induced ataxia on the inclined plane at ALKO in Finland, were moved to the University of Colorado in 1998. The selection phenotype was tested on generation 60 animals in Colorado. In week one, ataxia was measured on the inclined plane 30 minutes after an intraperitoneal dose of 2 g/kg 15% w/v ethanol. Differences in ethanol-induced ataxia between the AT and ANT lines at the University of Colorado were similar to those in the original lines in Finland. In week two, ataxia was measured on the inclined plane at 5 and 30 minutes, and tolerance was measured as the time to regain the original angle of sliding. The AT rats rapidly developed tolerance to 2 g/kg ethanol on the inclined plane; tolerance development was significantly slower in the ANT rats. In week three, the animals were tested for the duration of loss of righting reflex (LORR) and blood ethanol concentration at regain of the righting reflex (BECRRR) following a dose of 3.5 g/kg. The AT rats had a significantly higher BECRRR than did the ANT rats, but did not differ in LORR. A separate experiment with previously untreated rats demonstrated that naïve animals of the two lines did not differ in BECRRR or LORR. AT and ANT rats were genotyped for the mutation that occurs in the gene for the alpha6 subunit of the GABAA receptor, a natural mutation that is known to affect benzodiazepine responses. All ANT animals tested carried the mutant allele, whereas some AT families carried the mutation and others were wild type. There was no effect of the mutation in AT rats for any of the phenotypes that were tested. After several generations of brother-sister mating, the AT and ANT lines were more than 90% inbred as determined by genotyping. One AT (wild-type) line and one ANT (mutant) line were selected for breeding an F2 intercross generation of 1200 animals. They were phenotyped for sensitivity and tolerance to ethanol on each of three consecutive weeks. Order of testing had a modest effect on some of the phenotypes: when tested during the third week as compared to weeks one or two, BECRRR was increased, 30-minute sensitivity was increased, and development of acute tolerance was increased. Statistically significant correlations were found between tolerance and sensitivity at both 5 and 30 minutes, and between LORR and BECRRR. The smaller (or absence of) significant correlations between others of the phenotypes indicate(s) that they are most likely controlled by different sets of genes., (Copyright 2004 Plenum Publishing Corporation)

In previous work, we identified genetic correlations between cAMP accumulation in the cerebellum and sensitivity to the incoordinating effects of ethanol. A genetic correlation suggests that common genes underlie the phenotypes investigated. One method for provisionally identifying genes involved in a given phenotypic measure is quantitative trait locus (QTL) analysis. Using a panel of 30 BXD recombinant inbred strains of mice and the progenitors (DBA/2J and C57BL/6J), and the dowel test for ataxia, we measured the blood ethanol concentrations at the time an animal first fell from the dowel and acute functional tolerance (AFT), and investigated cAMP signaling in the cerebellum. Cyclic AMP accumulation was measured in whole-cell preparations of cerebellar minces from individual mice under basal or stimulated conditions. We conducted a genome-wide QTL analysis of the behavioral and biochemical measures with >2000 genetic markers to identify significant associations. Western blot and comparative sequencing analysis were used to compare cAMP response element binding protein (CREB) levels and protein-coding sequence, respectively. QTL analyses correlating strain means with allelic status at genetic markers identified several significant associations (p < 0.01). Analysis of variance revealed an effect of strain on behavioral and biochemical measures. There was a significant genetic correlation between initial sensitivity and basal cAMP accumulation in the cerebellum. We identified 6 provisional QTLs for initial sensitivity on four chromosomes, 6 provisional QTLs for AFT on four chromosomes, and 11 provisional QTLs for cAMP signaling on nine chromosomes. Two loci were found to overlap for measures of initial sensitivity and for cAMP signaling. Given the genetic correlation between initial sensitivity and basal cAMP accumulation, we investigated candidate genes in a QTL on chromosome 1. Comparative sequence analysis was performed, and protein levels were compared between C57 and DBA mice for Creb1. No significant differences were detected in coding sequence or protein levels for CREB. These results suggest that although ethanol sensitivity and cAMP signaling are determined by multiple genes, they may share certain genetic codetermination.

Background: In previous studies, genetic correlations were observed between hypnotic sensitivity to ethanol and high-affinity neurotensin receptor (NTS1) binding. Provisional quantitative trait loci (QTLs) were identified for these traits, and some of these QTLs were found on common chromosomal regions. In continued efforts to examine the relationship between NTS1 binding capacity and hypnotic sensitivity to ethanol, studies were designed to confirm correlations between NTS1 densities in the brain, duration of ethanol-induced loss of righting reflex (LORR), and blood ethanol concentrations at regain of righting reflex (BECRR). Another purpose of the study was to confirm QTLs for these traits., Methods: ILS X ISS F2 mice and HAS X LAS F2 rats as well as the progenitors were tested for LORR, BECRR, and NTS1 densities. Phenotypic correlations were calculated between LORR and BECRR and between these measures and NTS1 densities in striatum from both mice and rats. The F2 mice were genotyped by using polymorphic markers for five previously reported QTLs for LORR to confirm QTLs for BECRR and NTS1 densities in striatum, ventral midbrain, and frontal cortex., Results: Phenotypic correlations were found between LORR and BECRR (r = -0.66 to -0.74, p < 10(-9)) and between these measures and NTS1 densities in striatum (r = 0.28-0.38, p < 10(-2)) from both mice and rats. QTLs for LORR and BECRR (lod score = 2-6) were found in common regions of chromosomes 1, 2, and 15. By using the combined results from a previous LSXSS RI study and the current results, a suggestive QTL (lod score = 3.1) for striatal NTS1 receptor densities was found on chromosome 15 at approximately 60 cM, in the same region as the chromosome 15 LORR/BECRR QTL., Conclusions: The results are in agreement with previously reported correlations and QTLs for NTS1 receptor densities and measures of hypnotic sensitivity to ethanol in mice and extend those correlations to another species, the rat. These findings support a role for NTS1 in genetically mediated differences in hypnotic sensitivity to ethanol.

Evidence indicates that sensitivity to ethanol is a good predictor of the development of alcoholism. Thus, identification of neuronal processes that regulate ethanol sensitivity has been the subject of much recent research. The present studies were designed to further test the hypothesis that neurotensinergic processes mediate, in part, hypnotic sensitivity to ethanol. Single doses of haloperidol were administered to lines of rats [selectively bred for high and low sensitivity (HAS and LAS, respectively) to hypnotic effects of ethanol] to produce increases in neurotensin (NT) levels in brain regions. At 20 h after administration, haloperidol produced dose-dependent increases in NT immunoreactivity levels in nucleus accumbens (NA) and caudate putamen (CP) in both HAS and LAS lines. Levels of NT in NA and CP returned to control values at 48 h after 4 mg/kg haloperidol. These studies used two measures of hypnotic sensitivity to ethanol: duration of loss of righting reflex (sleep time) and blood ethanol concentration at regain of righting reflex (BECRR). At 20 h, but not 48 h, after haloperidol treatment, both HAS and LAS rats displayed increases in ethanol-induced sleep time with concomitant decreases in BECRR. Pentobarbital-induced sleep time was not increased 20 h after administration of 4 mg/kg haloperidol; however, hypnotic sensitivity to both pentobarbital and ethanol was increased by acute (30-min) pretreatment with 1 mg/kg. These results suggest that NT levels in NA, acting via NT receptors, enhance hypnotic sensitivity to ethanol, but not pentobarbital.

Acute functional tolerance (AFT) manifests as rapid adaptation during a single ethanol exposure, leading to a decrease in the behavioral response to ethanol. In order to investigate the genetic and environmental components of the development of AFT, mice were selectively bred in replicate from HS/Ibg mice. High (HAFT) and low (LAFT) acute functional tolerance selected lines were bred to differ in the rate of development and magnitude of AFT to ethanol's intoxicating effects using a static dowel-balancing task. In the present set of experiments, HAFT and LAFT mice were tested for development of AFT on a fixed-speed rotarod using a protocol similar to that for which they were selected. HAFT mice developed greater AFT to ethanol than did LAFT mice. In a separate experiment, other mice from these lines were tested for initial sensitivity and the development of chronic tolerance to ethanol-induced hypothermia, and ethanol-induced incoordination in the grid test. Previous research has detected possible common genetic control of these phenotypes. No differences between lines were found in initial sensitivity to ethanol or in the development or magnitude of chronic tolerance in either test. These experiments show that genetic factors influencing the development of acute tolerance to ethanol-induced intoxication are at least partially distinct from those influencing initial sensitivity and the development of chronic tolerance to ethanol-induced hypothermia and incoordination. Furthermore, these experiments show that AFT measured by the stationary dowel generalizes to AFT measured by the fixed-speed rotarod.

Background: Genetically based risk for development of alcoholism in humans seems to be related to initial sensitivity and/or acute tolerance to ethanol. The genetic basis for the development of tolerance has received less attention than other ethanol-related behaviors. We have selected lines of mice, according to genetics, which are differentially sensitive to the initial hypnotic effect of ethanol (Short Sleep and Long Sleep, SS and LS) and other lines that differentially develop acute functional tolerance to ethanol (High and Low Acute Functional Tolerance, HAFT and LAFT). We review reports of the relationship between initial sensitivity and two forms of tolerance as measured using different behavioral measures and different time scales. The goal of the study was to investigate alcohol tolerance as measured by different behavioral tests conducted over different time periods and relate these variables to hypnotic sensitivity., Methods: We investigated the phenotypic and genotypic relationships between different measures of tolerance to ethanol in the SS and LS mice. We used two measures of tolerance: (a) The time an animal can remain on a stationary dowel or roto-rod at 5-min intervals up to 30 minutes after a single low dose of ethanol (Acute Single Dose Tolerance, ASDT-dowel or ASDT-roto-rod); and (b) The difference in blood ethanol levels taken when a mouse could repeatedly regain balance on a stationary dowel or roto-rod after successive doses of ethanol (Acute Functional tolerance, AFT-dowel or AFT-roto-rod). The time course in AFT was much longer, up to 2 hours. We carried out the same studies on the High and Low Acute Functional Tolerance (HAFT and LAFT) mice., Results: SS and LS mice differ in hypnotic sensitivity as measured by sleep time, and they differ in all forms of acute tolerance that were measured except in AFT-dowel. Although there were phenotypic correlations between AFT-dowel and ASDT-roto-rod in the Heterogeneous Stock (HS) of mice, provisional Quantitative Trait Loci (determined with Recombinant Inbred mice from a SS X LS cross) for the two phenotypes did not overlap, which indicated that there was little or no genetic correlation between the measures. HAFT and LAFT mice do not differ in hypnotic sensitivity as measured by sleep time measurements nor in ataxic sensitivity as measured on the dowel. The HAFT and LAFT mice both developed tolerance when tested in the 30-minute time frame, but the differences between the lines was largely in the rate of development of tolerance and not the amount developed. On the other hand, when tolerance was measured over 2 hr on the dowel or roto-rod, the HAFT and LAFT animals developed different levels of tolerance., Conclusions: We concluded that measures of tolerance depended on both the time of ethanol's action and the behavioral task used. It seemed that the measures of tolerance used in this study had different genetic bases in mice. Presumably, tolerance will also vary in humans depending on the behavioral measure, and tolerance will also have different genetic bases for the different behavioral measures in humans.

Background: It has been proposed that development of tolerance to the behavioral effects of ethanol depends on the degree of impairment produced by the drug; that is, more sensitive individuals should develop greater tolerance. Tests of this hypothesis with respect to acute functional tolerance have produced contradictory results. We tested the hypothesis by examining the genetic relationship between initial sensitivity and acute functional tolerance in the LSXSS recombinant inbred mice., Methods: We tested mice for initial sensitivity to the ataxic effects of 1.75 g/kg of ethanol in a stationary dowel balance test by determining blood and brain ethanol concentrations at fall. Acute tolerance to the ataxic effects of ethanol was determined by measuring blood ethanol concentration (BEC) at regain of dowel balance ability after the first injection (BEC1RB) and after a second ethanol injection of 2.0 g/kg (BEC2RB). Acute tolerance was quantified by the difference in ethanol concentration at the two regains of balance (BEC2RB - BEC1RB) or by the difference between the second regain and one of the initial sensitivity measures (BEC2RB - initial sensitivity)., Results: Four different measures of initial sensitivity were taken: two that used BEC values and two that used forebrain or hindbrain ethanol concentrations. We calculated acute tolerance values by using each of these initial sensitivity measures plus BEC2RB. No evidence of a genetic relationship between initial sensitivity and acute tolerance was found, which suggests that these are two independent phenomena with respect to stationary dowel balance., Conclusions: Three conclusions can be drawn from this work: (1) Orbital sinus BEC at early time points (<5 min postinjection) may or may not accurately reflect brain EC in mice, dependent on genotype; (2) there is no genetic relationship between initial sensitivity and acute tolerance to stationary dowel ataxia in the LSXSS RIs; and (3) sex-specific factors affect low-dose ethanol responses on the stationary dowel.

Genetic regulation of acute tolerance to ethanol may be associated with ethanol consumption and other ethanol-related behaviors in rodents. We have used lines of mice, selectively bred for high and low acute functional tolerance (HAFT and LAFT, respectively) to ethanol-induced loss of balance to test this hypothesis. Replicate HAFT and LAFT lines differ in AFT to ethanol-induced loss of balance by 4.4- and 5-fold, respectively. Frequency distributions and mean AFT scores for those lines, F(1), and backcrosses show a dominance for the HAFT phenotype. Time courses for acquisition and decay showed that AFT to ethanol-induced loss of balance developed rapidly, could be maintained up to 6 h with repeated doses, and decayed 6 h after peak tolerance and discontinuance of ethanol administration. The lines did not differ in initial sensitivity as measured by brain ethanol concentration at loss of balance, indicating that initial sensitivity and AFT to loss of balance were not coselected traits. Surprisingly, HAFT versus LAFT lines did not differ in development of AFT to loss of righting response, or hypothermia, indicating different mechanisms or neuronal systems mediate genetic influences on these measures. Voluntary ethanol consumption was low in both of the replicate lines, but HAFT lines consumed greater amounts of ethanol than LAFT lines. The HAFT and LAFT lines developed AFT to pentobarbital-induced loss of balance, however, there were no line differences in rates or extent of the AFT development. These results show that genetic regulation of AFT development is drug- as well as response-specific.

We recently conducted a dose-response study of the effects of cocaine on several activity measures in the panel of BxD/Ty recombinant inbred mice. Animals were tested in an automated activity chamber over 2 days with i.p. saline on day 1 and i.p. cocaine on day 2, at one of four doses, 5, 15, 30 or 45 mg kg(-1). The monitor recorded total distance traveled, nosepokes in a holeboard, repeated movements and time spent by an individual in proximity to the centre of the apparatus. Dose-response curves for locomotor activation, i.e. the difference between cocaine and saline scores, showed that for all strains tested, scores increased 5-30 mg kg(-1). With few exceptions, locomotor activity at 45 mg kg(-1) was not significantly higher than that at 30 mg kg(-1). Repeated movement scores showed patterns similar to locomotor activity and nosepokes tended to be progressively inhibited by increasing doses of cocaine. Recombinant inbred strain mean distributions for all behaviours and at all doses exhibited continuous, rather than discrete variation, thus providing evidence of multiple-gene effects on cocaine-related behaviours. Quantitative trait loci (QTL) analysis pointed to several chromosomal locations associated with variations in cocaine-related behaviours and some are either identical or close to QTL reported by others. In separate groups of animals, densities of dopamine D1, and D2 receptors and dopamine uptake transporters were measured in the medial prefrontal cortex, caudate-putamen, nucleus accumbens and ventral midbrain. In all areas, all measures showed distributions consistent with polygenic influence and were associated with QTL. Of particular interest was our finding of a large segment on chromosome 15, which is related to dopamine receptor densities and cocaine-related behaviours.

Background: C57BL/6 inbred mice prefer alcohol whereas DBA/2 mice avoid it. We describe the construction of congenic strains of mice in which DBA/2 alleles for alcohol avoidance were placed on a C57BL/6 background using phenotypic selection., Methods: Mice were primed to drink 10% v/v ethanol in water for 2 days before a two-bottle choice paradigm. N2 males who demonstrated an alcohol-avoidance phenotype were backcrossed to B6 females to construct 15 independent lines., Results: Eight of these lines were lost due to failure to breed or absence of males with an alcohol-avoidance phenotype. The remaining sublines were split to form a total of 21 sublines. In the N7 and N9 generations, a genome scan located provisional quantitative trait loci (QTLs) on chromosomes 1, 2, 3, 6, and 9. Progeny testing confirmed QTLs on chromosomes 1 and 2., Conclusions: The QTL on chromosome 2 overlaps the 95% confidence interval of Alcp1 whereas that on chromosome 1 is new and has been called Alcp5. Marker-assisted selection was used in the N9 and subsequent generations to maintain the congenic lines and produce congenic strains.

A considerable investment in genetic mapping is necessary to confirm linkages of quantitative trait loci for complex traits such as ethanol sensitivity, a significant predictor of alcoholism. Before embarking on such intensive mapping efforts in large intercrosses, we suggest an approach based on genetic marker data in recombinant strains that yield a rationale for selecting a battery of related phenotypes for confirmation studies of quantitative trait loci action. Using this approach with selected strains of mice, we retrospectively consider the relationship between ethanol sensitivity and neurotensin levels in several mammalian brain regions.

C57BL/6J (B6) inbred mice are well known to drink large amounts of alcohol (ethanol) voluntarily and to have only modest ethanol-induced withdrawal under fixed dose conditions. In contrast, DBA/2J (D2) mice are "teetotallers" and exhibit severe ethanol withdrawal. Speculation that an inverse genetic relationship existed between these two traits was substantiated by meta-analysis of existing data collected in multiple genetic models, including large panels of standard and recombinant inbred strains, their crosses, and selectively bred mouse lines. Despite methodological differences among laboratories in measurement of both preference drinking and withdrawal, a nearly universal finding was that genotypes consuming large amounts of 10% ethanol (calculated as g/kg/day) during two-bottle choice preference drinking were genetically predisposed to low withdrawal scores in independent studies after either acute or chronic ethanol treatment. Conversely, low-drinking genotypes had higher withdrawal severity scores. The genetic relationship appears to be strongest in populations derived from B6 and D2, where data from more genotypes (BXD RIs, B6D2F2s, BXD RI F1s, and B6D2F2-derived selectively bred lines) were available for analysis. Gene mapping studies in these populations identified four chromosome regions [on Chromosomes (Chrs) 1, 2, 4, and 15] where genes might potentially influence both traits. Among genotypes with greater genetic diversity (for example, a panel of standard inbred strains or selectively bred lines), the relationship was less pronounced. Thus, reduced susceptibility to the development of high alcohol use may be supported by increased genetic susceptibility to ethanol withdrawal symptoms.

It has been speculated that tolerance to alcohol involves some form of neuronal plasticity that is similar to or the same as that mediating learning and memory. To investigate this possibility further, we tested the hypothesis that acute functional tolerance (AFT) to alcohol is genetically correlated to a Pavlovian learning task: fear conditioning. Mice selectively bred for differences in ability to acquire AFT were tested for fear conditioning. Subjects received a mild footshock paired to a broadband clicker and were tested 24 hr later for their freezing response to the conditioning chamber (context), to an altered chamber, and to the clicker. Both the original and replicate lines selected for high AFT (HAFT) were found to freeze significantly more than those selected for low AFT (LAFT) in response to the context and to the clicker. In a second experiment, an F2 population derived from the C57BL/6 (B6) and DBA/2 (D2) mouse strains were tested first for fear conditioning, followed 3 weeks later by AFT testing. AFT was defined as the difference between blood alcohol levels determined at the time of regain balance on a dowel rod first after 1.75 g/kg of ethanol and again after a subsequent dose of 2.0 g/kg. Consistent with results from HAFT and LAFT, freezing to context was found to be significantly positively correlated to AFT (r = 0.38, p = 0.04) in the F2 mice. The results suggest that co-variation in fear conditioning and AFT may be mediated by one or more of the same or at least tightly linked genes. Further dissection of this correlation may reveal neuronal mechanisms common to both AFT and fear conditioning.

The goal of research presented in this report was to investigate the possibility that differences in beta-ED levels could account for some portion of genetically mediated variation in locomotor activity. Subjects representing six of the LSXSS RI mouse strains were activity tested in a novel environment. Significant effects of strain and time as well as a significant strain by time interaction were detected. Beta-ED levels were estimated from several brain regions and the pituitary of naive and activity-tested mice. Significant strain effects were detected in all brain regions but not in the pituitary. There was no overall effect of exposure to novelty, but some strains showed either a decrease or an increase in beta-ED levels in the septum, amygdala, and midbrain. A significant genetic correlation between adaptation to the novel environment (locomotor activity at 30 min subtracted from locomotor activity at 5 min) and beta-ED levels in the septum was observed. Estimates of hypothalamic mRNA for the beta-ED precursor POMC revealed no effect of strain. Finally, locomotor activity was tested following doses of the mu-opioid antagonist naltrexone. Out of six strains tested (naive to the apparatus), naltrexone dose dependently attenuated locomotor activity in only the strain that showed the highest control level of activity. The results suggest that beta-ED levels influence novelty-induced locomotor activity, but that other important genotype-dependent factors are involved.

The C57BL/6, DBA/2, and recombinant inbred (RI) strains derived from them (BxD RIs) are the most frequently studied mouse strains with regard to genetic regulation of voluntary ethanol consumption (YEC). We have studied VEC in an alternate genetic model provided by the LSxSS RIs. These RI strains exhibit phenotypic extremes in VEC comparable to the C57BL/6 and DBA/2 mice and genotype-dependent sex differences in drinking behavior. A correlational analysis between various ethanol-related behaviors suggests genetic independence of VEC from high-dose neurosensitivity (sleep time), acute ethanol tolerance, hypothermia, and low-dose locomotor activity. A search for quantitative trait loci identified a number of putative quantitative trait loci (QTL), three of which are identical to those previously reported for 10% ethanol drinking in the BxD RIs. We also find a significant correlation between low-affinity neurotensin receptor densities (NTRL) in the frontal cortex and VEC, and more common QTL between these two phenotypes than expected by chance. This suggests a role for frontal cortex NTRL in regulating voluntary ethanol intake.

The purpose of this study was to examine the pharmacokinetics of cocaine in two inbred mouse strains, C57BL/6 (B6) and DBA/2 (D2). Male and female mice were administered 30 mg kg(-1) cocaine IP and killed after 5, 15, 30, or 60 minutes postinjection. Brains were removed quickly and assayed for total brain cocaine concentration. Quantification of cocaine was conducted using gas chromatography and mass spectrometry. The results of this study revealed a strain difference in total brain cocaine kinetics. Specifically, we observed that at 5 min onward, B6 mice cleared cocaine from the brain with a t1/2 estimated at 22.3 min, while distribution in D2 mice appeared to be incomplete until 15 min with a subsequent t1/2 estimated at 11.2 min. These results show that despite faster clearance by D2 mice, the prolonged time to distribution in this strain may help explain why D2 mice show initial greater locomotor activation by cocaine, compared to B6s.

We recently conducted a study of the behavioral effects of combined cocaine and ethanol in genetically defined mice. Male and female C57BL/6 (B6) and DBA/2 (D2) were tested in an automated activity monitor on 2 consecutive days. On day 1, all animals received an IP injection of sterile saline and were placed into the activity monitor for 30 min. Behaviors measured were total distance traveled, stereotypy, nosepokes, and wall-seeking. On day 2, all animals were tested again for 15 min following injection of one of the following: saline, 10% v/v ethanol at 2.0 g kg(-1) or 2.0 g kg(-1) ethanol plus 5, 15, or 30 mg kg(-1) cocaine. Cocaine alone at the same doses was injected into separate groups of animals. For the B6 strain, the overall effect of ethanol was to reduce cocaine-induced locomotor stimulation; no consistent effect of ethanol on cocaine-induced locomotion was observed in D2 mice. Cocaine-induced inhibition of nosepokes in both strains and sexes was partially reversed by ethanol. Ethanol also partially reversed cocaine-elevated stereotypy in both strains and both sexes. In B6 mice, cocaine-increased wall seeking tended to be reversed by coadministration of ethanol, whereas no consistent pattern was observed in the D2s. Results from this study suggest that the several measures affected by cocaine (locomotor activity, stereotypy, exploration, thigmotaxis) were, in turn, differentially affected by concurrent treatment with ethanol. Furthermore, our results point to genetic-based differences in ethanol's effects on cocaine-related behaviors. We address the implications for combined ethanol and cocaine use in humans.

The finding that stress-induced locomotor activity exhibited a significant strain x time interaction in the LS x SS RI strains prompted examination of QTL influencing this behavior as a function of time. The degree of genetic determination for locomotor activity was 0.26 for the first 5 min and decreased to 0.16 for the last 5 min of a 30-min test but the number of genetic factors stayed relatively constant (three or four) across time. A QTL point analysis revealed a total of 15 QTL, 5-8 per 5-min time block. Few of the QTL were detected across all time points and different combinations of QTL were evident for each time period. Five of the QTL were in common with those reported by other investigators for similar behaviors. The results suggest that locomotor behavior is under a greater degree of genetic control during the initial part of the test but environmental factors become increasingly important as the test progresses. Furthermore, different genetic factors appear to be mediating genetic variation in locomotor behavior at any given time point.

Various lines of evidence suggest that a polymorphism in the gene for proopiomelanocortin, Pomc1, might account for some portion of the genetic variance for open-field activity in the LS x SS RI strains. To test this hypothesis, approximately 1600 bp of Pomc1 was sequenced from genomic DNA of seven of the LS x SS strains. Two distinct alleles containing a total of five single-base pair differences were detected. A substitution was found in the coding region causing a Pro-to-Ser conversion, two substitutions occurred in the 3' untranslated region of the mRNA, and a substitution and a deletion were detected in the 3' untranscribed flanking region. The fragment containing the coding region substitution was sequenced in an additional 15 of the LS x SS strains. A total of 12 strains contained one form of the allele, while 10 had the other. The strain distribution pattern of open-field activity scores between the two allels suggests that these alleles do not contribute to the genetic variation of open-field activity in the LS x SS RI strains.

We have analyzed LSXSS recumbinant inbred for ethanol-induced activity using 2.0 g/kg ethanol and a new method we call ethanol activation slope. The ethanol activation slope provides a robust dose-response measure of ethanol activation, independent of both activity after saline and the inhibitory effects of ethanol on locomotor activity. These behavioral data were used in a quantitative trait locus analysis to map chromosomal loci involved in ethanol-induced locomotor activity. We tentatively identified seven loci that mediate the low-dose stimulatory effect of ethanol and six loci involved in locomotion after 2.0 g/kg ethanol. Only one of the loci are in common between the two behaviors. We also compared the behavioral quantitative trait locus to those previously identified that are involved in regulating central nervous system neurotensin levels and neurotensin receptor densities. Six chromosomal regions were identified that regulate at least one central nervous system neurotensin measure and an ethanol-induced locomotor behavior. The identification of loci controlling both central nervous system neurotensin levels or neurotensin receptor densities and ethanol-induced locomotor activity strengthens the proposal that neurotensin regulates, in part, ethanol-induced behaviors and central nervous system sensitivity to ethanol.

Genetic correlations were found between high-affinity neurotensin receptor (NTR(H)) densities and NT-immunoreactivity (NT-ir) levels in specific brain regions and sensitivity to hypnotic and hypothermic effects of ethanol in LSXSS recombinant inbred strains of mice. Simple sequence length polymorphisms were used to identify quantitative trait loci (QTL) influencing hypnotic and hypothermic sensitivity to ethanol, NTR(H) and low-affinity neurotensin receptor densities and NT-ir levels in LSXSS recombinant inbred strains. Common QTL for NTR(H) receptor densities, NT-ir levels and these ethanol actions were identified. One of the QTL (chromosome 2, 80 cM) for NTR(H) density and hypnotic sensitivity is linked to the NTR(H) gene, Ntsr. Also, QTL for NTR(H) density were found in common with confirmed QTL for hypnotic sensitivity on chromosomes 1 (43 cM), 11 (57 cM) and 15 (56 cM) and with an unconfirmed QTL on chromosome 3 (19 cM). Two common QTL for NT-ir levels, but not NTR(H) or low-affinity neurotensin receptor receptors, and ethanol-induced hypothermia were observed on chromosomes 4 (43 cM) and 6 (41 cM). Two common QTL for NT-ir levels and sleep time were identified on chromosomes 3 (19 cM) and 9 (55 cM). Common QTL indicate that genes regulating NT receptor and/or NT-ir expression may be the same as those regulating sensitivity to ethanol.

Rapid adaptation to central nervous system inhibitory effects of ethanol is observed in animals and humans and this acute functional tolerance (AFT) is influenced by genotype in rodents. Studies have been conducted to identify neurochemical processes influencing AFT to ethanol, but little is known regarding genetic regulation of AFT or genetic influences on processes that mediate acquisition of AFT. Our study was designed to develop, by selective breeding, lines of mice that differ in acquisition of AFT to ethanol; such mouse lines will be valuable in identifying the neuroadaptive processes mediating AFT. AFT is defined as the difference in blood ethanol concentration (BEC) at regaining balance on a stationary dowel rod after two consecutive doses of ethanol, 1.75 followed by 2.0 g/kg. Starting with a genetically heterogeneous foundation stock (HS/lbg), seven generations of selective breeding has been completed for high (HAFT1), low (LAFT1), and control lines and four generations have been completed for the replicate HAFT2 and LAFT2 lines. The lines do not differ in initial sensitivity to ethanol; however, the means for AFT scores differ by 2.3- and 4.3-fold for females and males, respectively (106.5 vs. 46.5 mg ethanol/dl blood) for females and 106.2 vs. 24.8 mg/dl for males). Frequency distributions for HAFT1 and LAFT1 show only modest overlap in AFT scores. The lines differ in rates of acquisition of AFT, but not in rates of ethanol clearance. Heritabilities were 0.04 and 0.26 for HAFT1 and LAFT1 lines, respectively, indicating that the selection was asymmetrical. Evidence is provided indicating that practice during intoxication has little effect on acquisition of AFT in HAFT1 and LAFT1 lines.

We are mapping the genes (quantitative trait loci or QTLs) that are responsible for individual differences in ethanol sensitivity, measured as the duration of loss of righting, reflex (LORR) and blood ethanol concentrations upon recovery of the righting reflex (BEC). The Long-Sleep (LS) and Short-Sleep (SS) selected lines of mice manifest an 18-fold difference in LORR and serve as a rodent model for ethanol sensitivity. The LS x SS recombinant inbred (RI) series, developed from LS and SS lines, are an important resource for QTL mapping of ethanol-related responses. The current report summarizes the initial QTL analysis of LORR and BEC in the LS x SS strains and compares the results of correlational analysis with an interval-mapping approach. The data provide strong evidence for QTLs that influence ethanol sensitivity on mouse chromosomes 1 and 2 and possible QTLs on chromosomes 1, 3, 4, 5, 6, 7, 12, 13, 16, and 18. These results are compared to those from an F2 cross which confirms QTLs on chromosomes 1, 2, 4, and 18.

Previous studies of neurotensin (NT) levels and NT receptor densities in specific brain regions of mice selectively bred for differences in sensitivity to ethanol have shown that NTergic processes may mediate some actions of ethanol. In the present study, we have determined the levels of NT and NT receptor densities in specific brain regions of HAS and LAS rats that have been selectively bred for differences in sensitivity to ethanol-induced loss of righting response. Regional differences in NT levels were observed in brains from both HAS and LAS rats and values in hypothalamus, ventral midbrain, and nucleus accumbens from female rats were 25 to 75% higher than levels in corresponding regions from male rats. However, there were no significant line differences in NT-ir levels in corresponding regions from HAS and LAS animals. High-affinity binding (NTH Bmax values), measured by Scatchard analyses, were higher in ventral midbrain from HAS males than from LAS males. NTH receptor densities were higher in HAS males than in HAS females; sex differences were not observed in the LAS line. There were no significant line or sex differences between HAS and LAS in low-affinity (NTL) Bmax values in any brain region. In HAS females, subhypnotic doses of ethanol produced a decrease in NT levels in nucleus accumbens, whereas, hypnotic doses caused an increase in NT levels. Likewise, hypnotic doses elicited increases in NT levels in hypothalamus of female HAS and LAS, but not in ventral midbrain or caudate putamen. These results are consistent with low dose activation of mesolimbic and nigrostriatal dopaminergic neurons in which NT is colocalized with dopamine and with high dose inhibition of these pathways.

It has been postulated that increased dopamine (DA) activity in the medial prefrontal cortex (mPFC) exerts an inhibitory influence over DA release in the nucleus accumbens and, thus, also over locomotor activity. Experiments were designed to examine the role of mPFC DA and neurotensin (NT), a neuropeptide which interacts with DA, in spontaneous locomotor activity. LS/IBG mice were injected bilaterally with either GBR-12909, a selective DA uptake blocker, the DA D1 receptor antagonist R-(+)-SCH-23390, the DA D2 receptor antagonist epidepride, NT or a combination of drugs. GBR-12909 produced a U-shaped dose-response curve with a maximum inhibition of 47% of control. Postmortem tissue levels of DA, 5-hydroxytryptamine, norepinephrine and their major metabolites were determined after microinjections of GBR-12909. Tissue levels of these compounds were not significantly affected by GBR-12909. However, the ratios of homovanilic acid/DA and homovanilic acid + 3,4-dihyroxyphenylacetic acid/DA were significantly decreased, whereas the 5-hydroxyindoleacetic acid/5-hydroxytryptamine ratio was not affected by GBR-12909, suggesting a selective effect on DAergic processes. By itself, R-(+)-SCH-23390 had no effect on locomotor activity except at a very high dose which caused locomotor inhibition (49% of control). Epidepride caused a dose-dependent inhibition of locomotor activity with a maximum inhibition of 49% of control. When coinjected with an inhibitory dose of GBR-12909, both epidepride and R-(+)-SCH-23390 attenuated the GBR-12909 effect in a dose-dependent manner. A broad range of doses of NT was found to have no consistent effect on locomotor activity. However, when coinjected with an inhibitory dose of GBR-12909, NT attenuated the GBR-12909-induced inhibition in a dose-dependent manner. The results suggest that stimulation of DA receptors in the mPFC, both DA D1 and DA D2 receptors mediates locomotor inhibition. Furthermore, stimulation of NT receptors appears to antagonize the effects of DA.

We recently conducted a set of two experiments to investigate the possible co-operation between genetics and exposure to novelty on the putative locomotor inhibiting effects of low doses of cocaine in male and female C57BL/6 and DBA/2 mice. Experiment one examined the effects of three low doses of cocaine (0.1, 0.5, and 1.0 mg/kg) on locomotion, exploration, stereotypy and wall-seeking in an automated activity monitor. Testing occurred on two consecutive days, with subjects receiving an IP injection of saline on day one, and one dose of cocaine on day 2 (S-C). Immediately following injection, subjects were placed into automated activity monitors, where four behaviors were recorded; total distance, nosepokes, stereotypy and margin time. Using this S-C injection regimen, we found significant decreases in measures of total distance and stereotypy when compared to saline in both male and female C57 mice. Experiment two was designed to determine if the observed decrease in locomotor activity was the result of low-dose cocaine or pre-exposure to the test procedure and apparatus. All conditions and procedures were identical to those in experiment one, with the exception of the injection regimen. In this experiment, we injected all subjects IP with 0.1 mg/kg cocaine on day one, followed by saline on day two (C-S). Additionally, a group of subjects receiving saline on both days (S-S) served as the control. In contrast to experiment one results, cocaine produced locomotor activation. Furthermore, significant sex and strain differences were found in both experiments. The results of our experiments suggest that the behavioral effects of low doses of cocaine are markedly influenced by both the genetic constitution of the experimental animal and by familiarity with the test apparatus.

The development of genetic models to help explain individual differences in sensitivity to and susceptibility to misuse certain CNS active substances, like ethanol and psychostimulants, spans a brief, thirty-plus years. The first animal models involved inbred strains and selected lines of mice and rats and predicted genetic-based differential sensitivity to ethanol and its misuse in humans found a few years later. With drugs like cocaine, tracking genetic differences in sensitivity and misuse liability in humans is difficult because of legal problems. Genetically-defined animals, however, have shown most if not all of cocaine-related behavioural, neurophysiological and toxicological effects to evince wide variation with most effects being influenced by several genes. Thus, we argue that animal and human studies of individual differences in drug sensitivity be studied from both quantitative and molecular genetic approaches. For the former, new techniques involving recombinant inbred strains of rodents, genetic correlational analysis and quantitative trait loci analysis are particularly useful, especially as genetic synteny between rodents and humans becomes better described. Also, because drug effects are highly labile to environmental conditions as well as genetic-based individual differences, multivariate, systems level studies should be developed to provide more complete descriptive and mechanistic views of a multifaceted problem.

Neurotensin (NT), a tridecapeptide that satisfies criteria as a neurotransmitter, mimics many actions of ethanol, and evidence indicates that some of the acute effects of ethanol are mediated in part by NT. Recent studies have shown that chronic ethanol treatment produced a downregulation of NT receptors in mesolimbic brain regions of long sleep (LS) mice and that reduced NT binding capacity was associated with acquisition and decay of tolerance to ethanol-induced locomotor inhibition and hypothermia in these mice. The present study was undertaken to determine whether cross-tolerance develops between NT and ethanol and whether chronic NT infusion produces NT receptor downregulation. Animals chronically treated with ethanol were tolerant to NT-mediated locomotor inhibition at a dose of 1.8 pmol NT, ICV, and were tolerant to NT-induced hypothermia at 1.8 and 6.0 pmol NT. Following repeated injections or continuous infusion of NT ICV, LS mice showed tolerance to both NT- and ethanol-induced hypothermia and locomotor inhibition. Indeed, ethanol doses that are hypnotic in control mice (2.8 g/kg) were not effective in abolishing locomotor activity following chronic NT administration. Results with chronic saline infusion ICV indicate that stress alters sensitivity to ethanol-induced hypothermia. Chronic infusion of NT ICV produced a region-specific downregulation of high-affinity NT receptors in the striatum. The results demonstrate that cross-tolerance develops between NT and ethanol, and further support a role for neurotensinergic systems in the actions of ethanol.

Many centrally acting drugs affect neurotensin (NT) systems by increasing levels of the peptide in specific brain regions. If these changes represent increases in extracellular NT levels, then changes in NT receptors would be expected. The focus of this study was to examine the effects of continuous exposure of NT receptors to agonist. Continuous infusion of NT (0.6 or 6 nmol/h) into the lateral ventricle via an osmotic minipump for 3 days caused a significant increase (over saline infusion) in total and low-affinity NT receptor density in the cerebellum of LS mice. High-affinity NT receptor density was increased in the frontal cortex. Seven days of NT infusion (6 nmol/h) caused no changes in NT receptor density.

A summary of pharmacogenetic studies designed to test the hypothesis that NT receptors might mediate or regulate some of the actions of ethanol is presented. Indeed, there are significant genetic correlations between ethanol-induced locomotor activation and high-affinity NT receptor densities in the FC. The results suggest that further studies are needed to determine the role of NT receptors in the FC and MPFC in regulating locomotor activity. In other studies, chronic ethanol treatment, under conditions that produced tolerance to ethanol and caused NT receptor downregulation in the NA and VMB, caused tolerance to locomotor inhibitory effects of centrally administered NT and blunted the effects of intra-VTA NT on dopamine metabolism in the NA and CP. The results show a relationship between NT receptor densities and pharmacological effects of ethanol.

C57BL/6 and DBA/2 mice were used to examine genetic differences in locomotor activating effects of acute cocaine administration and to determine whether differences were mediated by dopaminergic systems. C57BL/6 mice were less activated than DBA/2 mice at 5 and 10 min after 10 and 15 mg/kg cocaine. HPLC analysis showed equivalent brain cocaine concentrations in the two strains at 5 and 10 min after 10, 15, or 20 mg/kg doses. The selective dopamine uptake inhibitor, GBR 12909, at 5 and 7.5 mg/kg, produced greater locomotor activation in DBA/2 mice than in C57BL/6 mice. However, binding studies with the selective dopamine uptake ligand [3H]GBR 12935, revealed no between-strain difference in Kd or Bmax in caudate putamen (CP) or nucleus accumbens (NA) membranes. Competition assays using unlabeled dopamine to compete for [3H]GBR 12935 binding in CP or NA membranes showed no between-strain difference by brain region. The specific D1 or D2 antagonists, SCH 23390 or epidepride, respectively, produced dose-dependent decreases in locomotor activity but there were no between-strain differences. However, epidepride, at a dose of 0.003 mg/kg, completely reversed cocaine-induced (15 mg/kg) activation in both strains. These findings show that C57BL/6 and DBA/2 mice differ in dopamine-related behaviors and suggest that dopaminergic processes may mediate genetic differences in cocaine sensitivity.

Previous studies have shown that long sleep (LS) and short sleep (SS) mice, which were selectively bred for differences in brain sensitivity to ethanol, differ in neurotensin (NT) receptor densities in specific brain regions. The present study was designed to determine whether these receptor differences mediate differences in the effects of NT on the phosphoinositide (PI) second messenger system in four brain regions from LS and SS mice. Baseline and NT- or carbachol-stimulated PI hydrolysis were Ca(2+)-dependent. Stimulation of PI hydrolysis by NT or carbachol was region specific; NT effect was approximately equal in ventral midbrain (VMB) and entorhinal cortex (EC) with slightly less stimulation in nucleus accumbens (NA) and no effect in the cerebellum (CE). Carbachol-enhanced PI hydrolysis was greatest in the VMB followed by EC and NA with no stimulation in the CE. There were no between line (LS vs. SS) differences in carbachol effects, but stimulation by NT was greater in EC and NA from LS than from SS mice. Ethanol enhanced NT-stimulated, but not carbachol-stimulated, PI metabolism in SS and LS NA brain slices. Results with levocabastine, an inhibitor of low-affinity NT receptor (NTL) binding, suggest that NT may stimulate PI hydrolysis via NTL, as well as high-affinity receptors.

Studies were conducted to determine whether genetic differences in behavioural effects of cocaine in C57BL/6 and DBA/2 mice might be mediated by strain differences in dopamine and serotonin transporters and dopamine D1 and D2 receptors in specific brain regions. Binding characteristics of [3H]CFT, a cocaine analogue, in the presence of either GBR12909, a dopamine uptake blocker or fluoxetine, a serotonin uptake blocker and binding of [3H]-paroxetine, a specific serotonin uptake receptor antagonist, were evaluated. We observed regional differences in [3H]CFT binding parameters in the presence of GBR12909 or fluoxetine, but no strain differences by brain region were observed. There were no differences in [3H]paroxetine binding characteristics between corresponding brain regions from C57BL and DBA/2 mice. The D1 antagonist, [3H]SCH23390 and the D2 ligands [3H]sulpiride or [125I]epidepride were used to determine dopamine receptor characteristics. Regional differences were found in [3H]SCH23390 and [3H]sulpiride, with higher affinities and lower densities in frontal cortex compared to striatum; with no differences in [3H]SCH23390 binding in corresponding tissues from C57BL and DBA/2 brains. There were strain-related differences in [3H]sulpiride and in [3H]epidepride binding in striatal membranes with higher densities in C57BL than in DBA/2. Our findings suggest striatal D2 receptor differences are possibly involved in genetic differences in cocaine-related behaviours.

We recently conducted an experiment to investigate the possible cooperation between genetic makeup and differential housing on cocaine self-administration in male and female C57BL/6J and DBA/2J mice. Cocaine self-selection was measured in a two-choice test with one choice being cocaine-HCl solution of 40 mg% in tap water and the other choice being plain tap water. Housing conditions began at weaning (21-23 days of age) and consisted of group housed (GH) with 2-3 mice per cage, and isolated housed (IH) with 1 mouse per cage. The results of this study revealed overall strain, sex and housing differences, with C57BL/6Js consuming more cocaine solution than DBA/2J subjects, females consuming more cocaine solution than males, and group housed consuming more than isolate housed subjects. In a second study, the effect of differential housing on open-field locomotor activity was investigated. Testing was conducted on two consecutive days, with subjects receiving an IP injection of saline on day 1, and 15 mg/kg cocaine HCl on day 2. Four behaviors were recorded, including: total distance, nosepokes, stereotypy, and margin time. Overall, the results revealed significant strain differences for stereotypy and nosepokes, and males were found to be more activated by cocaine than females. Additionally, DBA males tended to be differentially affected by housing condition, with IH showing suppressed locomotor activity as compared to GH subjects. Last, significant strain by housing interactions occurred in nosepokes and stereotypy time.

We investigated the effects of cocaine on multiple activity measures and cocaine self-selection in C57BL/6Ibg and DBA/2Ibg mice. Male mice were tested in an automated activity monitor at three doses of cocaine, 5, 15 and 30 mg kg-1. Activity measures included locomotion, rearings, stereotyped movements and wall-seeking. Testing was conducted on 2 days with saline injection, i.p. on day one and cocaine i.p. injected on day two. We also tested other mice of both strains for cocaine ingestion in a two-choice test, pairing tap water with 40 mg% cocaine HCl in tap water. Two separate groups of mice received 15 or 30 mg kg-1 of cocaine i.p., killed at 5 min and brain cocaine levels were determined by HPLC. Cocaine produced dose-related increases in locomotion in both strains, with a delay in initial activation noticed at 30 mg kg-1 in C57s but not in DBAs. In DBAs, cocaine suppressed rearings and increased stereotyped movements while having no consistent effect on either behaviour in C57s. At all doses, cocaine produced moderate increases in proximity to the wall in DBAs and 30 mg kg-1 produced pronounced wall-seeking in C57s. At 15 and 30 mg kg-1 DBAs tended to have higher levels of cocaine in whole brain than did C57s. Finally, C57s consumed significantly more cocaine than did the DBAs.

Levels of endogenous neurotensin (NT-IR) in the LS x SS RI strains differed by 3.0-, 4.7-, 5.4-, and 6.9-fold in the ventral midbrain (VMB), hypothalamus (HY), nucleus accumbens (NA), and caudate putamen (CP), respectively. Frequency distributions and estimates of the number of genes indicate that differences in NT-IR are polygenically influenced. The NT-IR levels in NA and CP were significantly correlated, but levels in the VMB did not correlate with those in the NA or CP. Specific binding to either low (NTL)- or high (NTH)-affinity receptors as measured in the absence or presence of levocabastine differed significantly in brain regions from among LS X SS mouse strains. Results indicate a polygenic influence mediating the differences in receptor densities and suggest differences in genetic regulation of NTL and NTH receptors.