N-methyl-D-aspartate receptor responses are differentially modulated by noncompetitive receptor antagonists and ethanol in inbred long-sleep and short-sleep mice: behavior and electrophysiology

BACKGROUND: Short-sleep (SS) mice exhibit higher locomotor activity than do long-sleep (LS) mice when injected with low doses of ethanol or the noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist MK-801 (dizocilpine). SS mice also have higher densities of brain NMDARs. However, two strains of LS X SS recombinant inbred (RI) mice also show differential activation to ethanol and MK-801, but have similar numbers of NMDARs. Here we used inbred LS (ILS) and SS (ISS) mice to investigate further the relationship between NMDARs and sensitivity to the stimulant effects of low doses of ethanol. METHODS: Open field activity and spontaneous alternations were measured after saline or drug injection. [3H]MK-801 binding parameters were determined in hippocampus, cortex, striatum, and nucleus accumbens. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region of hippocampal slices. RESULTS: Systemic injection of either ethanol or MK-801 increased locomotor activity to a greater extent in ISS mice than in ILS mice. The competitive NMDAR antagonist 2-carboxypiperazin-4-yl-propyl-1-lphosphonic acid (+/- CPP) depressed activity of ILS, but not ISS, mice. No strain differences were observed in spontaneous alternations or in the number or affinity of NMDARs in the brain regions examined. Likewise, the magnitudes of hippocampal NMDAR-mediated fEPSPs were similar in ILS and ISS mice and were inhibited to the same extent by a competitive NMDAR antagonist. However, both ethanol and the NMDAR NR2B receptor antagonist ifenprodil inhibited the late component of hippocampal NMDAR fEPSPs to a greater extent in ISS, than in ILS, mice. CONCLUSIONS: Differential ethanol- and MK-801-induced behavioral activation in ILS and ISS mice was not associated with differences in NMDAR number. Nonetheless, pharmacological differences in hippocampal NMDAR responsiveness suggest that ISS mice express NMDARs that have a greater sensitivity to noncompetitive, but not competitive, NMDAR antagonists. These differences, which may reflect differences in NMDAR subunit composition, could underlie the differential responsiveness to low doses of ethanol in ILS and ISS mice.     

Differences in NMDA Receptor Antagonist-Induced Locomotor Activity and [3H]MK-801 Binding Sites in Short-Sleep and Long-Sleep Mice

Short-Sleep (SS) and Long-Sleep (LS) mice differ in initial sensitivity to ethanol. Ethanol acts as an antagonist at N-methyl d -aspartate receptors (NMDARs). Therefore, we tested whether SS and LS mice also differ in initial sensitivity to NMDAR antagonists. Systemic injection (intraperitoneal) of either the noncompetitive NMDAR antagonist MK-801 (dizocilpine) or the competitive NMDAR antagonist 2-carboxypiperazin-4-yl-propyl-1-phosphonic acid (CPP) produced similar results. At lower drug doses, SS mice showed greater loco-motor activation than LS mice; and at higher doses, SS mice continued to be activated whereas LS mice became sedated. Brain levels of [³H]MK-801 were 40% higher in SS, compared with LS, mice. However, blood levels of [³H]MK-801 and [³H]CPP and brain levels of [³H]CPP were similar in the two lines. NMDARs were measured using quantitative autoradiographic analysis of in vitro [³H]MK-801 binding to SS and LS mouse brains. Significantly higher (20 to 30%) receptor densities were observed in the hippocampus and cerebral cortex of SS mice. Our results support the hypothesis that SS and LS mice differ in initial sensitivity to NMDAR antagonists and suggest that the line differences in the dose-response relationships for MK-801- and CPP-induced locomotor activity are qualitatively similar to those reported for ethanol. Differences in pharmacokinetics and number of NMDARs may contribute to, but are unlikely to entirely account for, the differential behavioral responsiveness of SS and LS mice to MK-801 and CPP.     

MK-801 blocks the development of behavioral sensitization to the ethanol

BACKGROUND: Studies have indicated that MK-801 (a noncompetitive N-methyl-D-aspartate receptor antagonist) participates in the long-term neural changes responsible for sensitization to stimulant drugs. It is known that repeated administration of low doses of ethanol sensitizes animals to its stimulant effect. In this work we investigated whether MK-801 alters the development of behavioral sensitization to ethanol. METHODS: Groups of male Swiss mice were treated with saline or ethanol (2.0 g/kg) plus saline or MK-801 (0.25 mg/kg) for 21 days. On day 25, all animals received an ethanol challenge injection (2.0 g/kg). We measured locomotor activity on days 1, 7, 14, 21) and 25. In addition, we assessed the effects of different doses of MK-801 on the response to a low dose of ethanol (2.0 g/kg). RESULTS: Ethanol-treated mice developed sensitization to the locomotor-stimulating effect of the drug, whereas those concomitantly receiving ethanol and MK-801 did not. All doses of MK-801 that were used stimulated the locomotor activity of both ethanol and saline-treated animals. CONCLUSIONS: The findings support the hypothesis that N-methyl-D-aspartate receptors have an important role in the development of sensitization to drugs of abuse.     

Role of major NMDA or AMPA receptor subunits in MK-801 potentiation of ethanol intoxication

Background: The glutamate system plays a major role in mediating EtOH’s effects on brain and behavior, and is implicated in the pathophysiology of alcohol‐related disorders. N‐methyl‐D‐aspartate receptor (NMDAR) antagonists such as MK‐801 (dizocilpine) interact with EtOH at the behavioral level, but the molecular basis of this interaction is unclear. Methods: We first characterized the effects of MK‐801 treatment on responses to the ataxic (accelerating rotarod), hypothermic and sedative/hypnotic effects of acute EtOH administration in C57BL/6J and 129/SvImJ inbred mice. Effects of another NMDAR antagonist, phencyclidine, on EtOH‐induced sedation/hypnosis were also assessed. Gene knockout of the NMDAR subunit NR2A or l ‐alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate GluR1 or pharmacological antagonism of the NMDAR subunit NR2B (via Ro 25‐6981) was employed to examine whether inactivating any one of these glutamate signaling molecules modified MK‐801’s effect on EtOH‐related behaviors. Results: MK‐801 markedly potentiated the ataxic effects of 1.75 g/kg EtOH and the sedative/hypnotic effects of 3.0 g/kg EtOH, but not the hypothermic effects of 3.0 g/kg EtOH, in C57BL/6J and 129/SvImJ mice. Phencyclidine potentiated EtOH‐induced sedation/hypnosis in both inbred strains. Neither NR2A nor GluR1 KO significantly altered basal EtOH‐induced ataxia, hypothermia, or sedation/hypnosis. Ro 25‐6981 modestly increased EtOH‐induced sedation/hypnosis. The ability of MK‐801 to potentiate EtOH‐induced ataxia and sedation/hypnosis was unaffected by GluR1 KO or NR2B antagonism. NR2A KO partially reduced MK‐801 + EtOH‐induced sedation/hypnosis, but not ataxia or hypothermia. Conclusions: Data confirm a robust and response‐specific potentiating effect of MK‐801 on sensitivity to EtOH’s intoxicating effects. Inactivation of three major components of glutamate signaling had no or only partial impact on the ability of MK‐801 to potentiate behavioral sensitivity to EtOH. Further work to elucidate the mechanisms underlying NMDAR × EtOH interactions could ultimately provide novel insight into the role of NMDARs in alcoholism and its treatment.     

Ethanol Attenuates the HFS-Induced, ERK-Mediated LTP in a Dose-Dependent Manner in Rat Striatum

Background: The striatum has been implicated to play a role in the control of voluntary behavior, and striatal synaptic plasticity is involved in instrumental learning. Ethanol is known to alter synaptic plasticity, in turn altering the behavior of human and animals. However, it remains unclear whether the striatum plays a role in the effects of ethanol on the central nervous system. The objective of this investigation was to study the effects of acute perfusion of ethanol on long‐term potentiation (LTP) to elucidate the mechanisms of addictive drugs in the striatum. In addition, we investigated the contribution of intracellular extracellular signal regulated protein kinase (ERK) signaling pathway to corticostriatal LTP induction. Methods: The stimulation evoked population spikes (PS) were recorded from the dorsomedial striatum (DMS) slices of rat using the extracellular recording technique. The LTP in DMS slices was induced by high‐frequency stimulation (HFS). The ERK level of the DMS was assessed with the Western blot technique. Results: U0126, the inhibitor of ERK, eliminated or significantly attenuated the LTP induced by HFS of the PS in the DMS. MK801 and APV, N‐methyl‐d ‐aspartic acid receptor (NMDAR) antagonists, inhibited the induction of striatal LTP, and HFS‐induced ERK activation decreased in the slices treated with MK801 in the DMS. Clinically relevant concentrations of ethanol (22 to 88 mM) dose‐dependently attenuated the HFS‐induced striatal LTP and ERK activation in this brain region. Conclusions: The LTP of the PS in the DMS is, at least partly, mediated by the ERK pathway coupling to NMDARs. Ethanol attenuated the HFS‐induced, ERK‐mediated LTP in a dose‐dependent manner in this brain region. These results indicate that ethanol may change the synaptic plasticity of corticostriatal circuits underlying the learning of goal‐directed instrumental actions, which is mediated by an intracellular ERK signaling pathway associated with NMDARs.     

MK-801-induced locomotor activity in long-sleep x short-sleep recombinant inbred mouse strains: correlational analysis with low-dose ethanol and provisional quantitative trait loci

BACKGROUND: Low doses of the N-methyl-D-aspartate receptor (NMDAR) antagonist MK-801 (dizocilpine) or ethanol increase locomotor activity to a lesser extent in long-sleep (LS), than in short-sleep (SS), mice. LS mice also have fewer brain [3H]MK-801 binding sites than SS mice. In this study, LSXSS recombinant inbred (RI) mice were used to investigate whether different NMDAR densities contribute to differential MK-801 activation and whether common genes are involved in initial sensitivity to MK-801-and ethanol-induced activation. METHODS: Locomotor activity was measured for 90 min after saline or MK-801 injection. Quantitative autoradiographic analysis of [3H]MK-801 binding was used to measure densities of NMDARs in seven brain regions. The ethanol (1-2 g/kg) activation scores from Erwin and colleagues (1997) were used for correlational analysis, as was their method for quantitative trait loci (QTL) analysis. RESULTS: Both saline and MK-801 (0.3 mg/kg, given intraperitoneally) induced a continuum of locomotor responses across the LSXSS RI strains. There was a 4-fold range of MK-801 difference scores (MK-801 score-saline baseline), with the RI 9 and RI 4 strains representing low and high responders, respectively. Dose-response experiments with these two strains confirmed that 0.3 mg/kg MK-801 produced significant activation, similar to previous results with LS and SS mice. However, unlike previous LS/SS results, lower densities of NMDARs were not observed in the RI 9 than in the RI 4 mouse brains. No significant genetic correlations were observed between MK-801-induced and ethanol-induced responses in the LSXSS RI mice. Two provisional MK-801 activation QTLs were identified (p < 0.01) on chromosomes 11 and 19, neither in common with those mapped for ethanol activation. CONCLUSIONS: Different densities of brain NMDARs are unlikely to account for the differential activation of LSXSS RI mice by MK-801. Additionally, in the RI mice either separate sets of genes regulate low dose MK-801- and ethanol-induced locomotor responses or the overlapping subset of genes controlling these two behaviors is small (< or =10%).     

Norepinephrine transporter: a candidate gene for initial ethanol sensitivity in inbred long-sleep and short-sleep mice

BACKGROUND: Altered noradrenergic neurotransmission is associated with depression and may contribute to drug abuse and alcoholism. Differential initial sensitivity to ethanol is an important predictor of risk for future alcoholism, making the inbred long-sleep (ILS) and inbred short-sleep (ISS) mice a useful model for identifying genes that may contribute to alcoholism. METHODS: In this study, molecular biological, neurochemical, and behavioral approaches were used to test the hypothesis that the norepinephrine transporter (NET) contributes to the differences in ethanol-induced loss of righting reflex (LORR) in ILS and ISS mice. RESULTS: We used these mice to investigate the NET as a candidate gene contributing to this phenotype. The ILS and ISS mice carry different DNA haplotypes for NET, showing eight silent differences between allelic coding regions. Only the ILS haplotype is found in other mouse strains thus far sequenced. Brain regional analyses revealed that ILS mice have 30 to 50% lower [3H]NE uptake, NET binding, and NET mRNA levels than ISS mice. Maximal [3H]NE uptake and NET number were reduced, with no change in affinity, in the ILS mice. These neurobiological changes were associated with significant influences on the behavioral phenotype of these mice, as demonstrated by (1) a differential response in the duration of ethanol-induced LORR in ILS and ISS mice pretreated with a NET inhibitor and (2) increased ethanol-induced LORR in LXS recombinant inbred (RI) strains, homozygous for ILS in the NET chromosomal region (44-47 cM), compared with ISS homozygous strains. CONCLUSIONS: This is the first report to suggest that the NET gene is one of many possible genetic factors influencing ethanol sensitivity in ILS, ISS, and LXS RI mouse strains.     

Differential effects of ethanol on gamma-aminobutyric acid-A receptor-mediated synaptic currents in congenic strains of inbred long and short-sleep mice

BACKGROUND: Ethanol enhances gamma-aminobutyric acid (GABA)A receptor-mediated responses in the brain, and this enhancement is greater in a mouse line behaviorally more sensitive to ethanol (long sleep) than in a line (short sleep) behaviorally less ethanol sensitive (assayed by loss of righting; sleep time). Quantitative trait locus (QTL) analysis of inbred long sleep (ILS) and inbred short sleep (ISS) phenotypes revealed four chromosomal regions (Lore1, Lore2, Lore4, and Lore5) that together account for approximately 50% of ethanol-induced sleep-time variance. Congenic strains were generated, each of which is homozygous for one of four ISS Lore QTLs on the ILS background. These congenic mouse strains are ideally suited for asking which QTL regions might correlate with other phenotypes that differ between ILS and ISS mice. Here we used the congenics to investigate altered GABAA responses to ethanol. METHODS: Evoked GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) were measured by whole-cell voltage-clamp recording procedures in CA1 pyramidal neurons in hippocampal brain slices. RESULTS: GABAA IPSC responses in hippocampal brain slices from ILS mice were significantly enhanced by 80 mM ethanol, whereas those from ISS mice were not affected. ILS.Lore2 and ILS.Lore5 congenic strains were significantly enhanced by 80 mM ethanol, similar to the background (control) ILS mice. However, ethanol had no significant effect on GABAA responses in ILS.Lore1 and ILS.Lore4 congenic mice, similar to the ISS mice, thus reflecting the influence of ISS alleles on the ILS phenotype. CONCLUSIONS: Our results suggest that alleles located in the Lore1 and Lore4 QTL regions confer ethanol sensitivity of GABAA receptor-mediated IPSCs. Thus, for these QTLs, GABAA IPSCs may represent an endophenotype of sedative/hypnotic sensitivity to ethanol. Although the Lore2 and Lore5 QTL regions have a significant effect on sleep time, they do not play a significant role in the differential ethanol enhancement of GABAA IPSCs between ILS and ISS mice.     

Delay discounting predicts behavioral sensitization to ethanol in outbred WSC mice

Background: Alcoholic individuals discount the value of future rewards more steeply than social drinkers, which is viewed as symptomatic of higher levels of impulsivity. However, the mechanisms underlying this difference are unknown. This study examined 2 hypotheses about the relationship between discounting and ethanol's effects in mice: (1) steep discounters are less sensitive to the initial stimulant‐like effects of ethanol and (2) steep discounters exhibit greater behavioral adaptation to stimulant effects with repeated ethanol exposure. Methods: An adjusting amount procedure was used to assess discounting as a function of delay in ethanol‐naïve genetically heterogeneous WSC mice. Mice chose between a small amount of sucrose solution delivered immediately and 19.5 μL delivered following a delay (0, 2, 4, 8, or 12 seconds, varied between sessions). Within sessions, the amount (μL) of immediate sucrose was adjusted until animals became indifferent between the immediate and specific delayed reward. Hyperbolic discount functions were fitted to quantify the degree of delay discounting. Then, in a within‐subjects design over 13 days, mice received a pattern of daily injections of saline or ethanol, and after certain treatments their locomotor activity was assessed for 15 minutes. Results: Animals with steeper discount functions (greater impulsivity) tended to exhibit less locomotor stimulation on their initial exposure to ethanol. However, steeper discounting was positively associated with increases in locomotor activity after repeated exposure (sensitization), indicating that steep discounters showed higher levels of sensitization to the stimulating effects of ethanol. Conclusions: These results suggest 2 behavioral effects of ethanol, associated with an increased risk for alcohol abuse, that are associated with variations in delay discounting.     

Interaction between S-propranolol and ethanol in mice selectively bred for ethanol sensitivity: the inbred short- and long-sleep mice

BACKGROUND: We have studied the effect of a beta-adrenergic blocking agent, S-propranolol, on the response of mice to anesthetic doses of ethanol. We used the selectively bred short and long sleep (ISS and ILS) mice. These mice were selected for their differential sensitivity to anesthetic doses of ethanol and then inbred. The study was prompted by the finding that the effect of ethanol on the firing rate of cerebellar Purkinje cells is modulated by beta-adrenergic input. In addition, this firing rate depression by ethanol is highly correlated with the anesthetic potency of ethanol. We were attempting to find a behavioral correlate of this effect of beta-adrenergic agents in the ISS and ILS mice. METHODS: We studied the effect of S-propranolol plus ethanol on the sleep time and blood ethanol at awakening in the inbred ILS and ISS mice. We administered anesthetic doses of ethanol with and without S-propranolol. We conducted studies of the rate of disappearance of ethanol in the presence of S-propranolol and carried out sleep time and metabolic studies with mice in an incubator held at 32 to 33 degrees C. RESULTS: We found that S-propranolol caused a prolonged anesthetic time brought about by ethanol but only in ISS mice. There was no significant difference in the blood ethanol levels at awakening with or without S-propranolol, indicating that S-propranolol had no effect on the brain sensitivity. Subsequently, we showed that this was due to a profound hypothermia caused by a combination of S-propranolol and ethanol. This was greater in the ISS mice because a larger dose of ethanol was required for the anesthetic effect of ethanol. The effect on ethanol disappearance rate, temperature drop, and anesthesia time all were largely reversed by placing the animals in an incubator at 32 to 33 degrees C. CONCLUSIONS: Profound hypothermia lowers the ethanol disappearance rate when both S-propranolol and ethanol are given. The effect of S-propranolol is likely due to the blockade of beta-adrenergic receptors that prevents thermogenic responses to the hypothermia brought about by ethanol. The results indicated that there might be a genetic effect controlling the hypothermic response to the combination of S-propranolol and ethanol. Further experiments to investigate this are reported in a subsequent article. We could find no evidence of a central nervous system effect of S-propranolol on the hypnotic actions of ethanol in these strains of mice.     

Proopiomelanocortin Peptides Are Not Essential for Development of Ethanol-Induced Behavioral Sensitization

Background: Behavioral sensitization is a result of neuroadaptation to repeated drug administration and is hypothesized to reflect an increased susceptibility to drug abuse. Proopiomelanocortin (POMC) derived peptides including β‐endorphin and α‐melanocyte stimulating hormone have been implicated in development of behavioral sensitization and the reinforcing effects of alcohol and other drugs of abuse. This study used a genetically engineered mouse strain that is deficient for neural POMC to directly determine if any POMC peptides are necessary for the development of ethanol‐induced locomotor sensitization. Methods: Adult female mice deficient for POMC in neurons only (Pomc^?/?Tg/Tg, KO) and wildtype (Pomc^+/+Tg/Tg, WT) littermates were injected once daily with either saline or ethanol (i.p.) for 12 to 13 days. On ethanol test day (day 13 or 14) all mice from both treatment groups received an i.p. injection of ethanol immediately before a 15‐minute analysis of locomotor activity. Blood ethanol concentration (BEC) was measured on ethanol test day immediately following the test session. Baseline locomotor activity was measured for 15 minutes after a saline injection 2 days later in both groups. Results: There was no significant difference in BEC between genotypes (WT = 2.11 ± 0.06; KO = 2.03 ± 0.08 mg/ml). Both WT and nPOMC‐deficient mice treated repeatedly with ethanol demonstrated a significant increase in locomotor activity on test day when compared to repeated saline‐treated counterparts. In addition, mice of both genotypes in the repeated saline groups showed a significant locomotor stimulant response to acute ethanol injection. Conclusions: Central POMC peptides are not required for either the acute locomotor stimulatory effect of ethanol or the development of ethanol‐induced locomotor sensitization. While these peptides may modulate other ethanol‐associated behaviors, they are not essential for development of behavioral sensitization.     

Evidence That the Lore-1 Region Specifies Ethanol-Induced Activation in Addition to Sedative/Hypnotic Sensitivity to Ethanol

BACKGROUND: Low-dose ethanol-induced activation (LDA) and initial sensitivity to alcohol are both predictors of alcohol abuse in human populations. Our hypothesis is that one or more genes specifying hypnotic sensitivity also specify LDA. We tested this hypothesis by using congenic mice derived from the inbred long-sleep (ILS) and inbred short-sleep (ISS) strains, which carry an ILS region introgressed onto an ISS background. METHODS: LDA was assessed by assigning mice randomly to receive one of five doses of ethanol ranging from 1.2 to 2.4 g/kg. On day 1, animals were injected with saline and placed in a brightly lit activity monitor for 30 min, after which they were returned to their home cages. On day 2, mice were injected with ethanol (20% w/v), their activity was monitored for a 30-min period, and LDA was determined by subtracting day 1 activity. The blood ethanol concentration of each animal was then assessed at 30 min by retro-orbital collection of 25 microl of blood. RESULTS: Ethanol had a significant effect on the activity of ISS mice, but ILS mice showed no activation at any dose, similar to the activities of the outbred lines. All three congenic strains were activated at several doses. Lore-2 and Lore-5 were not ILS-like (less active than ISS) at any dose. In contrast, ISS.ILS-Lore-1 congenics (carrying an ILS-derived Lore-1 allele on the ISS background) were significantly less activated than the ISS controls at 1.8 and 2.4 g/kg of ethanol. CONCLUSIONS: The Lore-2 and Lore-5 congenic regions do not affect LDA. In contrast, the Lore-1 congenic region carries one or more genes specifying both initial hypnotic sensitivity to ethanol and LDA.     

Alcohol withdrawal-induced hippocampal neurotoxicity in vitro and seizures in vivo are both reduced by memantine

Background: The ethanol withdrawal (EWD) syndrome is typically treated using benzodiazepines such as diazepam. However there is concern that benzodiazepines may not prevent neurotoxicity associated with EWD. Antagonists of glutamate/N‐Methyl‐D‐Aspartate receptors (NMDARs) such as MK801 have been shown to be effective against both EWD‐induced neurotoxicity in vitro and seizures in vivo. However, most of these agents have adverse side effects. An exception is the moderate affinity NMDAR channel blocker memantine, used in Alzheimer’s dementia. The present studies examined the ability of memantine to protect against EWD‐related toxicity in vitro and seizures in vivo. Methods: Organotypic hippocampal slice cultures from neonatal rat pups were treated starting at 15 days in vitro with 100 mM ethanol for 10 days followed by a 24‐hour EWD period. During the 24‐hour EWD period cultures were treated with memantine (15 or 30 μM). MK801 (10 μM) was utilized as a positive control. For the in vivo studies, the ability of memantine (2, 5, 10, and 15 mg/kg) to reduce convulsions was analyzed in Swiss‐Webster mice using the handling induced convulsion test paradigm. Results: In vitro studies demonstrated that memantine is effective at blocking EWD‐induced neurotoxicity. In vivo experiments showed that memantine also significantly reduced convulsions induced by EWD in mice. Conclusions: Memantine may be of therapeutic value during alcohol detoxification by virtue of its having neuroprotective effects in addition to anti‐seizure activity. The potential role of memantine in treatment of alcoholism is deserving of further study.     

Forward,relaxed, and reverse selection for reduced and enhanced sensitivity to ethanol's locomotor stimulant effects in mice

BACKGROUND: Rarely have trait markers for alcoholism risk been identified. However, relative sensitivity to the arousing effects of ethanol and sensitivity to ethanol's sedative effects have been distinguished as potentially valuable behavioral risk factors. Both traits are genetically influenced and have been modeled in mice by measuring sensitivity to ethanol-induced locomotor stimulation and hypnosis. Reverse selection was performed to examine the hypothesis that forward selection for differential sensitivity to ethanol's locomotor stimulant effects resulted in homozygous fixation of selection trait-relevant alleles and to test the hypothesis that common genes influence ethanol's stimulant and sedative effects. METHODS: Bidirectional selective breeding was completed for enhanced (FAST mice) and reduced (SLOW mice) sensitivity to ethanol's locomotor stimulant effects. Selection was terminated (relaxed), and the lines were tested to detect genetic drift. Reverse selection for enhanced sensitivity to ethanol-induced stimulation in SLOW mice and reduced sensitivity in FAST mice was performed for 16 generations. Forward and reverse selected lines were tested for sensitivity to ethanol's sedative effects by measuring duration of ethanol-induced loss of righting reflex. RESULTS: Differential sensitivity to the sedative effects of ethanol emerged with selection for differential ethanol stimulation, indicating a common genetic influence on these traits. SLOW mice developed greater sensitivity to ethanol's sedative effects relative to FAST mice. Reverse selection, never before reported for a pharmacogenetic trait, was effective in eliminating most of the difference in stimulant sensitivity between the FAST and SLOW lines and also eliminated the difference in loss of righting reflex duration. CONCLUSIONS: Residual heterozygosity persisted at trait-relevant loci even at the selection plateau, possibly due to heterosis, natural selection favoring heterozygosity, or epistatic phenomena involving differences in the sets of genes regulating the high- versus low-sensitivity traits. They also suggest that some common genes influence sensitivity to ethanol's locomotor stimulant and sedative effects.     

GPR37 associates with the dopamine transporter to modulate dopamine uptake and behavioral responses to dopaminergic drugs

The orphan G protein-coupled receptor 37 (GPR37) is a substrate of parkin; its insoluble aggregates accumulate in brain samples of Parkinson's disease patients. We report here that GPR37 interacts with the dopamine transporter (DAT) and modulates DAT activity. GPR37 and DAT were found colocalized in mouse striatal presynaptic membranes and in transfected cells and their interaction was confirmed by coimmunoprecipitation assays. Gpr37-null mutant mice showed enhanced DAT-mediated dopamine uptake in striatal membrane samples, with a significant increase in the number of plasma membrane DAT molecules. The null mutant mice also exhibited a decrease in cocaine-induced locomotor activity and in catalepsy induced by dopamine receptor antagonists. These results reveal the specific role of GPR37, a putative peptidergic G protein-coupled receptor, in modulating the functional expression of DAT and the behavioral responses to dopaminergic drugs.     

Use of Repeated Measures in an Analysis of Ethanol-Induced Loss of Righting Reflex in Inbred Long-Sleep and Short-Sleep Mice

We present a repeated-measures analysis of ethanol-induced loss of righting reflex (LRR) in Inbred Long-Sleep (ILS) and Inbred Short-Sleep (ISS) strains of mice and their F₁ and F₂ cross progeny. Mice were administered a 4.1 g/kg intraperitoneal dose of ethanol at two times, 7–10 days apart. Repeatability is nonsignificant in ILS, ISS, and F, mice, but is highly significant (0.47, p < 0.01) in the F₂ mice. Mean LRR does not differ between trials 1 and 2, with the exception of the ISS strain in which the interaction of sex with LRR sensitization is significant. This two-trial method leads to increased accuracy of genotype assessment for pharmacological or behavioral traits where trial 1 does not influence the outcome of trial 2. The repeated-measures design facilitates novel analyses of the duration of LRR, and results suggest that most environmental variance for LRR is due to nonreplicable influences.     

Possible pleiotropic effects of genes specifying sedative/hypnotic sensitivity to ethanol on other alcohol-related traits

BACKGROUND: Initial sensitivity to ethanol is a predictor of alcohol abuse that has been studied extensively in both human and animal populations. Selection for initial sensitivity to the sedative/hypnotic effects of ethanol resulted in the long-sleep and short-sleep lines of mice. Some of the genes selected in these lines could also specify differential responses in other ethanol-related phenotypes and, perhaps, for other drugs of abuse. We assessed congenic mice carrying a single quantitative trait locus (QTL) from the inbred long-sleep (ILS) or inbred short-sleep (ISS) strain on the reciprocal background for a number of ethanol- and pentobarbital-related phenotypes. METHODS: Each congenic strain was tested for ethanol elimination rates at 4.1 g/kg, ethanol-induced ataxia at 2.0 g/kg, ethanol-induced hypothermia at 4.1 g/kg, and pentobarbital-induced loss of righting reflex (LORR) at 60 mg/kg. Additionally, the ILS.ISS congenics were tested for low-dose ethanol-induced activation (LDA) at five doses ranging from 0.6 to 1.2 g/kg ethanol, and the ISS.ILS congenics were tested for LDA at 1.8 g/kg of ethanol. RESULTS: There was little difference in the ethanol elimination rate between congenics and background strains, although a modest sex effect was found, with the females eliminating ethanol more rapidly than the males. We were unable to replicate previous differences found in LDA for the congenic on the ISS background, because none of the congenics differed from controls for LDA. congenics showed a differential effect of pentobarbital-induced LORR in the expected directions. The congenics on the ISS background showed more ethanol-induced ataxia than the ISS controls. Additionally, the hypothermic response seems affected by and and maybe others. CONCLUSIONS: At least two regions carrying a QTL specifying sensitivity to high doses of ethanol cospecify altered sensitivity in other measures of alcohol action. Specifically, these QTLs clearly affect ethanol-induced hypothermia and pentobarbital-induced LORR and possibly ethanol-induced ataxia.     

Reciprocal congenics defining individual quantitative trait Loci for sedative/hypnotic sensitivity to ethanol

BACKGROUND: We have identified four major genes or quantitative trait loci (QTLs) that determine duration of loss of righting reflex (LORR), induced by sedative doses of ethanol: Lore1, Lore2, Lore4, and Lore5. Together these genes explain more than 50% of the phenotypic variance for sensitivity to the sedative/hypnotic effects of ethanol between the Inbred Long Sleep (ILS) and Inbred Short Sleep (ISS) strains of mice. The derivation of these strains is reviewed here. METHODS: Each QTL has been bred onto the opposite background (ILS or ISS) through 10 rounds of backcrossing by using QTL-marker-assisted counter selection to produce reciprocal congenic strains. Mice were genotyped for markers that flanked each of the QTLs. Selection for the donor at the desired QTL, and against donor markers at the other four QTLs, allowed rapid fixation of the genetic background. Phenotypic assessment in the ISS-recipient congenic strains was conducted throughout the backcross. RESULTS: By the N5 generation, phenotypic assessments failed to detect significant effects in some sublines; these sublines were discarded and positive lines split to create new replicate sublines. In the N10, all sublines retained the phenotypic difference between heterozygotes and ISS homozygotes; however, the expected additive effect was not found in the Lore1 congenics. On the ILS background, each Lore was captured, as shown by the expected differential LORR. Two strains on the ILS background, and one on the ISS, exhibited the differential effect on blood ethanol concentration associated with the donor strain. CONCLUSIONS: Congenic strains represent an important resource for confirmation of previously identified QTLs, for identification and mapping of additional phenotypes, and for exclusion of candidate genes. QTL-marker-assisted selection rapidly stabilized the genetic background within four generations (based on phenotypic assessments); however, phenotypic selection during the backcrossing to generate congenic strains did not contribute to the successful capture of the ISS QTLs.