Locomotor activity induced by noncompetitive NMDA receptor antagonists versus dopamine transporter inhibitors: opposite strain differences in inbred long-sleep and short-sleep mice

Background: The actions of ethanol in the brain involve multiple neuroreceptor systems, including glutamatergic N‐methyl‐D‐aspartate receptor (NMDAR) channels. In a novel environment, both ethanol and the noncompetitive NMDAR antagonist MK‐801 stimulate locomotor activity to a lesser extent in inbred long‐sleep (ILS) mice compared with inbred short‐sleep (ISS) mice. The behaviorally activating effects of noncompetitive NMDAR antagonists are thought to involve increased monoamine neurotransmission. Thus, in this study, we sought to determine whether: (1) habituation to the behavioral environment alters the differential locomotor‐stimulant effects of noncompetitive NMDAR antagonists in ILS and ISS mice and (2) the differential behavioral sensitivity of ILS and ISS mice to noncompetitive NMDAR antagonists is mediated through direct inhibition of the dopamine transporter (DAT). Methods: Open field locomotor activity was measured following acute systemic injection of saline or drug. [³H]DA uptake parameters were determined in striatal synaptosomes prepared from drug‐naïve mice. Results: Habituation to the testing environment abolished the strain differences in saline‐induced locomotor activity. However, ethanol‐ as well as MK‐801‐treated ILS mice still exhibited reduced locomotor activity compared with ISS mice, suggesting that a drug‐environment interaction is not the primary explanation for the strain differences. The noncompetitive NMDAR antagonists phencyclidine and ketamine also induced significantly lower locomotor activity in ILS than in ISS mice. In contrast, the DAT inhibitors cocaine and GBR 12909 and the DA releaser amphetamine induced greater locomotor activity in ILS than in ISS mice, a strain difference opposite that of the noncompetitive NMDAR antagonists. Furthermore, the differential behavioral effect found with DAT inhibitors was not mediated by differences in the affinity nor number of striatal DATs between ILS and ISS mice. Conclusions: Our results support the conclusion that the differential locomotor‐stimulant effects of ethanol and noncompetitive NMDAR antagonists in ILS and ISS mice are not mediated through direct inhibition of DAT.     

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%).     

Acamprosate,MK-801, and ifenprodil inhibit neurotoxicity and calcium entry induced by ethanol withdrawal in organotypic slice cultures from neonatal rat hippocampus

BACKGROUND: The antirelapse drug acamprosate has previously been reported to inhibit activating effects of polyamines on -methyl-D-aspartic acid receptor (NMDAR) function. Because increased synthesis of polyamines has been suggested as a mechanism for potentiation of NMDAR function during ethanol withdrawal, we evaluated the effects of acamprosate, MK-801, and ifenprodil in a cell culture model of ethanol withdrawal-induced neurotoxicity. METHODS: Organotypic hippocampal cultures from 8-day-old neonatal rats were maintained in vitro for 23 days before experimental use. The ethanol withdrawal model consisted of exposing cultures to ethanol (70-100 mM) for 4 days before being "withdrawn" into Calcium-Locke's buffer for 1 hr and then into minimal medium for 23 hr. Uptake of (45)CaCl(2) and propidium iodide by damaged cells was assessed 1 hr and 24 hr after the start of ethanol withdrawal, respectively. Additional studies examined effects of exposure to NMDA (50 microM) or spermidine (100 microM) on withdrawal-induced hippocampal damage. Last, these studies examined the ability of the sodium salt of acamprosate (Na-acamprosate, 200 microM), ifenprodil (50 microM), or MK-801 (30 microM) to inhibit neurotoxicity and (45)Ca(2+) entry produced by these insults. RESULTS: Ethanol withdrawal was associated with significantly greater toxicity and (45)Ca(2+) entry, relative to controls. Exposure to spermidine and NMDA during ethanol withdrawal further increased neurotoxicity and (45)Ca(2+) entry. Acamprosate, ifenprodil, and MK-801 almost completely prevented ethanol withdrawal-induced toxicity and (45)Ca(2+) entry. Acamprosate also reduced spermidine-induced neurotoxicity during ethanol withdrawal but was ineffective against NMDA-induced toxicity or (45)Ca(2+) entry at this time. CONCLUSIONS: The results support the contention that acamprosate, like ifenprodil, interacts with polyamines and that these compounds may be effective in reducing consequences of ethanol withdrawal. NMDAR activation is also strongly implicated in ethanol withdrawal neurotoxicity, but whether acamprosate causes any of these effects in this preparation directly via the NMDAR remains uncertain.     

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.     

Prenatal ethanol exposure modifies [3H]MK-801 binding to NMDA receptors: spermidine and ifenprodil

BACKGROUND: It has been suggested that abnormalities seen in fetal alcohol syndrome are linked with NMDA receptor malfunction. Our laboratory has previously shown that prenatal ethanol treatment decreases [3H]MK-801 binding density at postnatal day 21, when NMDA receptor subunit protein levels were unaltered. Thus, the focus of the present study was to examine whether prenatal ethanol modifies native NMDA receptor levels. METHODS: Cerebral cortices were taken from offspring born to three treatment groups of pregnant Sprague Dawley(R) rats: an ethanol group given an ethanol liquid diet during the gestational period, a pair-fed control group that received a liquid diet without ethanol, and an ad libitum group fed rat chow and tap water. Western blot studies were carried out at postnatal days 1, 7, 14, and 21 to examine total protein expression of NR1 and NR1b splice variants. NR2 subunit levels were examined by [3H]MK-801 binding studies using spermidine, an endogenous polyamine, and ifenprodil, a selective NR2B antagonist. RESULTS: [3H]MK-801 binding density was significantly reduced in prenatal ethanol-treated groups compared with ad libitum and pair-fed control groups. Spermidine increased [3H]MK-801 binding, although potentiation by spermidine was not significantly different among all three experimental groups. Furthermore, no significant differences in total protein expression of NR1 or NR1b splice variants were observed in cortical membrane homogenates at postnatal days 1 through 21. [3H]MK-801 binding in the presence of ifenprodil showed that prenatal ethanol treatment significantly decreased low-affinity ifenprodil binding. High-affinity ifenprodil binding was reduced in both pair-fed and ethanol-treated groups. CONCLUSIONS: These results suggest that prenatal ethanol treatment reduces [3H]MK-801 binding and that this reduction may be due to a decrease in NR2A subunits.     

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.     

Alteration of [3H]MK-801 Binding Associated with the JV-Methyl-d-Aspartate Receptor Complex by Acute Ethanol in Rat Cortex and Hippocampus In Vitro

We investigated the effect of ethanol on specific binding of [³H]MK-801 to the intrachannel phencyclidine (PCP) receptor site, as an index of change in the functional response of the N-methyl-d -Aspartate (NMDA)-associated ion channel. Saturation binding experiments were performed on synaptic membrane homogenates from adult rat cortex and hippocampus. [³H]MK-801 binding assays were conducted under conditions of basal, 10 μm glutamate, or 10 μm glutamate + 30 μm d -serine, with and without 50 or 100 mm ethanol. Association experiments of [³H]MK-801 binding (5 nm) were conducted under conditions of 0 or 10 μm glutamate, with varying concentrations of glycine (0.01, 0.10, and 10 μm) with and without 100 mm ethanol. Ethanol (50 and 100 mm ) significantly decreased the percentage of high-affinity (open-channel state) MK-801 receptors with a concomitant increase in percentage of low-affinity receptors, but did not change high- and low-affinity constants of the two binding states. An ethanol-induced increase in the closed-channel receptor density in basal and activated conditions was suggested by the saturation experiments. Association experiments further explained this finding, in that ethanol (100 mm ) significantly decreased fast component (open-channel) [³H]MK-801 binding in conditions of glycine (0.01–10 μm ) only and activated conditions of glutamate + glycine (0.01–0.10 μm ). However, the observed fast and slow kinetic rate constants of [³H]MK-801 binding, as well as total specific binding (fast + slow components), were not altered. Thus, ethanol seems to act as a noncompetitive antagonist upon the gating mechanism of, and ligand access to, the NMDA-coupled ion channel. These findings support previous observations of ethanol selectively reducing NMDA-activated calcium influx, and reducing the frequency and duration of ion channel opening in electrophysiological studies. Similar to previous reports on NMDA-stimulated calcium influx and [³H]MK-801 binding, glycine (at the maximal concentration of 10 μm ), in the presence of 10 μm glutamate, was found to reverse ethanol inhibition of fast component binding.