Manipulations of extracellular Loop 2 in α1 GlyR ultra-sensitive ethanol receptors (USERs) enhance receptor sensitivity to isoflurane, ethanol, and lidocaine, but not propofol.

We recently developed ultra-sensitive ethanol receptors (USERs) as a novel tool for investigation of single receptor subunit populations sensitized to extremely low ethanol concentrations that do not affect other receptors in the nervous system. To this end, we found that mutations within the extracellular Loop 2 region of glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs) can significantly increase receptor sensitivity to micro-molar concentrations of ethanol resulting in up to a 100-fold increase in ethanol sensitivity relative to wild-type (WT) receptors. The current study investigated: (1) Whether structural manipulations of Loop 2 in α1 GlyRs could similarly increase receptor sensitivity to other anesthetics; and (2) If mutations exclusive to the C-terminal end of Loop 2 are sufficient to impart these changes. We expressed α1 GlyR USERs in Xenopus oocytes and tested the effects of three classes of anesthetics, isoflurane (volatile), propofol (intravenous), and lidocaine (local), known to enhance glycine-induced chloride currents using two-electrode voltage clamp electrophysiology. Loop 2 mutations produced a significant 10-fold increase in isoflurane and lidocaine sensitivity, but no increase in propofol sensitivity compared to WT α1 GlyRs. Interestingly, we also found that structural manipulations in the C-terminal end of Loop 2 were sufficient and selective for α1 GlyR modulation by ethanol, isoflurane, and lidocaine. These studies are the first to report the extracellular region of α1 GlyRs as a site of lidocaine action. Overall, the findings suggest that Loop 2 of α1 GlyRs is a key region that mediates isoflurane and lidocaine modulation. Moreover, the results identify important amino acids in Loop 2 that regulate isoflurane, lidocaine, and ethanol action. Collectively, these data indicate the commonality of the sites for isoflurane, lidocaine, and ethanol action, and the structural requirements for allosteric modulation on α1 GlyRs within the extracellular Loop 2 region.

Differential effects of propofol and ethanol on P2X4 receptors expressed in Xenopus oocytes.

The current study investigated the effects of propofol on P2X4 receptors expressed in Xenopus oocytes using two-electrode voltage clamp. We also tested the effects of 100 mM ethanol on the same oocytes used to test propofol. Propofol potentiated ATP-gated currents in a concentration dependent manner in P2X4 receptors. In agreement with our previous findings, ethanol inhibited P2X4 receptors. The opposite effects of propofol and ethanol on P2X4 receptor function suggest that these anesthetics act via different sites/mechanisms in P2X receptors as has been suggested for GABA(A) and glycine receptors.

Pressure-sensitive and -insensitive coupling in gamma-aminobutyric acid(A) receptors.

RATIONALE: Previous behavioral and biochemical studies suggest that allosteric coupling processes initiated by benzodiazepines, barbiturates and neuroactive steroids can be sub-categorized on the basis of their sensitivities to antagonism by increased atmospheric pressure. However, biochemical evidence supporting this hypothesis was limited to single concentration studies in long sleep (LS) mice. OBJECTIVE: The present paper addresses these issues by extending biochemical investigation of pressure effects on allosteric modulators across a range of concentrations that allosterically enhance gamma-aminobutyric acid (GABA)A receptor function and alter behavior using two mouse genotypes. In addition, the effects of pressure on ligand binding were explored to further investigate the mechanism of pressure antagonism of allosteric modulation. METHODS: The effects of 12 times normal atmospheric pressure (ATA) of helium-oxygen gas (heliox) on allosteric modulation of GABA(A) receptor function and [3H]flunitrazepam binding was tested in LS and C57BL mouse brain membranes (microsacs) using chloride flux and high-affinity binding assays. RESULTS: In both genotypes, exposure to 12 ATA heliox antagonized the allosteric enhancement of GABA(A) receptor function by flunitrazepam (0.1-10 microM) and pentobarbital (0.1-50 microM) but did not affect allosteric modulation by 3alpha-hydroxy-5beta-pregnan-20-one (0.1-1 microM). Pressure did not affect benzodiazepine receptor affinity (Kd) or the number of benzodiazepine receptors (Bmax). THE RESULTS: (1) confirm that there are differences in sensitivity to pressure antagonism of allosteric coupling among GABA(A) allosteric modulators; (2) demonstrate that these differences are not concentration or genotype dependent; (3) add evidence that pressure antagonizes allosteric modulation by uncoupling the receptor and (4) support the hypothesis that allosteric modulation of receptor function can be sub-categorized on the basis of sensitivity to pressure antagonism.

Direct evidence for a cause-effect link between ethanol potentiation of GABA(A) receptor function and intoxication from hyperbaric studies in C57, LS, and SS mice.

BACKGROUND: This article uses a direct ethanol antagonist, increased atmospheric pressure, to further test the causative link between ethanol potentiation of gamma-aminobutyric acid (GABA) type A receptor function and ethanol's behavioral effects. This was done by determining whether initial biochemical findings in long-sleep (LS) mice extended to other genotypes and whether the previously reported insensitivity of short-sleep (SS) mice to pressure antagonism of ethanol-induced loss of righting reflex extended to a nonselected ethanol-induced behavior. METHODS: The effects of 12 times normal atmospheric pressure of helium-oxygen gas (heliox) versus ethanol (25-200 mM) potentiation of GABA-activated Cl- uptake in brain membranes (microsacs) from C57, LS, and SS mice were tested by using a 36Cl- flux assay. The effects of pressure versus ethanol's (2 g/kg) anticonvulsant effect in SS mice were tested by using time to onset of isoniazid-induced myoclonic seizures. RESULTS: Exposure to 12 times normal atmospheric pressure heliox antagonized ethanol potentiation of GABA-activated Cl- uptake in all three genotypes across a range of ethanol concentrations that cause ethanol's behavioral and anesthetic effects. Pressure did not affect baseline receptor function. The threshold for initiating ethanol potentiation differed between genotypes in accordance with their behavioral sensitivities to ethanol (C57 and LS, < or =25 mM; SS, >50 mM). Pressure antagonized ethanol's anticonvulsant effect in SS mice. CONCLUSIONS: The results add important direct evidence supporting the hypothesis that ethanol potentiation of GABA(A) receptor function is an initial action of ethanol causing its behavioral effects. These findings also provide insight into possible effects of selective breeding on GABA(A) receptor function.

Ethanol enhances GABAA receptor function in short sleep and long sleep mouse brain membranes.

BACKGROUND: SS and LS mice have been used to explore the genetic and neurochemical bases for differences in sensitivity to ethanol. The present study investigated the effects of ethanol on GABAA receptor function in microsacs from these genotypes. The purpose was to test a key element of the hypothesis that differences between these lines in sensitivity to ethanol-induced enhancement of GABAA receptor function underlie their selected differences in sensitivity to ethanol-induced loss of righting reflex (LORR). METHODS: The effects of ethanol on GABA-activated 36Cl- uptake in brain membranes (microsacs) isolated from male SS and LS mice were tested using a chloride flux filtration assay. RESULTS: Ethanol significantly enhanced GABA-activated 36Cl- uptake in SS microsacs at concentrations of 100-300 mM. Ethanol did not significantly affect GABA-activated chloride uptake in this preparation at concentrations of 25 and 50 mM. Ethanol significantly enhanced GABA-activated 36Cl- uptake in LS microsacs at concentrations of 25-100 mM, but not at 200 mM. CONCLUSION: The present studies are the first to show a statistically significant effect of ethanol on GABA-activated chloride uptake in both SS and LS mice with a clear difference between the genotypes in threshold. The relative threshold differences between SS and LS microsacs in sensitivity to ethanol indicate that selection for resistance to ethanol-induced LORR in SS mice has shifted the ethanol-GABAA receptor concentration-response curve to the right. The findings add key evidence that supports a cause-effect relationship between sensitivity to ethanol-induced potentiation of GABAA receptor function and genetically determined sensitivity to ethanol's behavioral effects.

Effects of posttraining ethanol on an appetitive task.

The present study examined the effects of posttraining ethanol administration upon retention of an appetitive task using a variety of retention behaviors associated with the task. Male C57BL/6J mice were individually trained to find a cheese pellet placed in the corner of an open field. Five behavioral measures were used including locomotor activity counts, rearings, grooming episodes, approaches to the cheese pellet, and latency to consume the cheese pellet. Immediately after training, mice were injected intraperitoneally with saline or 2.0 g/kg of ethanol and then returned to their home cage in which four "intruder" mice were added for 2 h after training. On subsequent testing days (1, 6, 14, and 51 days posttraining), mice were returned to the original training environment and the five behaviors were measured. Both saline- and ethanol-treated mice habituated to the initially novel test environment at similar rates as indicated by decreased exploratory behavior (locomotor activity and rearings). In contrast, a divergence in the latency to consume the cheese pellet was observed: Saline-treated mice behaved as though the cheese was rewarding (decreased latency to eat the pellet), while the ethanol group behaved as though the cheese was aversive (increased latency to eat the pellet). Taken with previous studies, these results demonstrate that posttraining ethanol can have strikingly different effects on retention depending on the task, the measure of retention used, and the underlying neural structures involved.

In vivo and in vitro hyperbaric studies in mice suggest novel sites of action for ethanol.

The present study uses increased atmospheric pressure as an ethanol antagonist to test the hypothesis that allosteric coupling pathways in the GABA(A) receptor complex represent initial sites of action for ethanol. This was accomplished using behavioral and in vitro measures to determine the effects of pressure on ethanol and other GABAergic drugs in C57BL/6 and LS mice. Behaviorally, exposure to 12 times normal atmospheric pressure (ATA) of a helium-oxygen gas mixture (heliox) antagonized loss of righting reflex (LORR) induced by the allosteric modulators ethanol and pentobarbital, but did not antagonize LORR induced by the direct GABA agonist 4,5,6,7-tetrahydroisoxazolo-pyridin-3-ol (THIP). Similarly, exposure to 12 ATA heliox antagonized the anticonvulsant effects verses isoniazid of ethanol, diazepam and pentobarbital. Biochemically, exposure to 12 ATA heliox antagonized potentiation of GABA-activated 36Cl-uptake by ethanol, flunitrazepam and pentobarbital in LS mouse brain preparations, but did not alter GABA-activated 36Cl- uptake per se. In contrast to its antagonist effect versus other allosteric modulators, pressure did not antagonize these behavioral or in vitro effects induced by the neuroactive steroid, 3alpha-hydroxy-5beta-pregnan-20-one (3alpha,5beta-P). These findings add to evidence that pressure directly and selectively antagonizes drug effects mediated through allosteric coupling pathways. The results fit predictions, and thus support the hypothesis that allosteric coupling pathways in GABA(A) receptors represent initial sites of action for ethanol. Collectively, the results suggest that there may be common physicochemical and underlying structural characteristics that define ethanol sensitive regions of receptor proteins and/or their associated membranes that can be identified by pressure within (e.g., GABA(A)) and possibly across (e.g., GABA(A), NMDA, 5HT3) receptors.

Direct antagonism of ethanol's effects on GABA(A) receptors by increased atmospheric pressure.

Previous studies have shown that exposure to 12 times normal atmospheric pressure of helium-oxygen gas (heliox) directly antagonizes a range of ethanol's acute and chronic behavioral effects. The present study extends the investigation to the biochemical level by investigating the effects of pressure on ethanol-induced potentiation of GABA(A) receptor function in mouse membrane vesicles (microsacs). Exposure to 12 atmospheric pressure heliox significantly antagonized ethanol (25 to 100 mM) potentiation of GABA-activated 36Cl- uptake, but did not significantly alter baseline GABA(A) receptor function measured by the response of the system to GABA (10 to 100 microM), bicuculline (3 and 100 microM), or picrotoxin (100 microM). These findings add essential support for the hypothesis that hyperbaric exposure is a direct ethanol antagonist that can be used as a tool to help identify ethanol's initial cellular and molecular sites of action that cause its behavioral effects. Taken in context with previous behavioral studies, the present results also provide important new evidence for a cause-effect relationship between ethanol potentiation of GABA(A) receptor function and ethanol's anesthetic and behavioral effects.

Morphine-6-glucuronide-induced locomotor stimulation in mice: role of opioid receptors.

Morphine-6beta-glucuronide is a major metabolite of morphine with potent analgesic actions. To explore the importance of this opiate when administered as a drug by its own or in morphine action, we studied the locomotor activity response to morphine and morphine-6-glucuronide in drug-naive C57 BL/6JBom mice. The effects of administration of the two opiates on a battery of 7 different locomotor activities were studied and compared to saline controls. A dose of 20 micromol/kg morphine-6-glucuronide resulted in more locomotion than the same dose of morphine, while at higher doses (up to 120 micromol/kg), similar increases for most locomotor behaviours were recorded for both drugs. Pretreatment with naltrindole indicated that the delta-receptors play an equivalent but minor role in mediating both morphine-6-glucuronide and morphine hyperlocomotion. Administration of high naltrexone doses (10 mg/kg) completely abolished the locomotor stimulation induced by both opiates. However, at intermediate naltrexone doses of 0.25 and 0.5 mg/kg, morphine-induced behaviours was completely inhibited while morphine-6-glucuronide induced behaviours demonstrated partial resistance to naltrexone inhibition. The mu1-specific receptor antagonist naloxonazine caused 75% reduction of morphine induced behaviours and only 50% inhibition of morphine-6-glucuronide induced behaviors. Taken together our observations indicated general similarity but also marked differences between morphine and morphine-6-glucuronide with respect to opiate receptors mediating the locomotor stimulatory effect.

Use of inhalation to study the effect of ethanol and ethanol dependence on neonatal mouse development without maternal separation: a preliminary study.

This study explored the use of ethanol inhalation as a model to study the effects of ethanol and ethanol dependence on neonatal brain development in mice without maternal separation. In these experiments two day old Swiss Webster mice with their mothers were put in an inhalation chamber and continuously exposed to ethanol vapors for 12 days. The results indicate that: (a) the neonates developed substantial blood ethanol levels (160 to 290 mg/dl); (b) the mothers had minimal blood ethanol concentrations (BECs < 10mg/dl); (c) no mortality was observed during ethanol exposure; (d) physical dependence to ethanol was produced in the neonates, as evidenced by typical withdrawal symptoms.; (e) exposure to ethanol vapors did not affect the weight gain of the neonates indicating that nutrition and suckling ability was not significantly altered; the body weight of the mothers were also not affected; (f) 12 days of neonatal ethanol exposure significantly reduced whole brain and cerebellar weights on postnatal day 45 as compared to the controls; (g) neonatal ethanol exposure resulted in behavioral changes on postnatal day 40 to 41. Twelve days of ethanol exposure significantly impaired habituation, but did not alter spontaneous locomotion and (h) ethanol sensitivity on postnatal day 45 measured by Loss of Righting Reflex (LORR) was not affected. Although further studies are necessary, the results demonstrate that exposure to ethanol vapors can cause high BECs in the neonates without causing meaningful BECs in the mothers. Collectively, the results indicate that the ethanol inhalation technique can be used to investigate the effects of ethanol and ethanol dependence on neonatal development in mice during the rodent equivalent of the human third trimester.

Effects of ethanol and temperature on NMDA receptor function in different mouse genotypes.

The present study investigated whether temperature-related changes in NMDA receptor sensitivity to ethanol might play a role in mediating the effects of body temperature on behavioral sensitivity to ethanol or in determining genotypic differences in sensitivity to ethanol. We accomplished this by determining the effects of ethanol on three different mouse genotypes (C57, LS, and SS) on two types of NMDA receptor-mediated responses at 30 degrees and 35 degrees C: (i) extracellularly recorded synaptic potentials elicited in the CA1 region of the in vitro hippocampal slice preparation by stimulation of the Schaffer-commisural pathway in the presence of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blocker, 6,7-dinitroquinoxaline-2,3-dione, and low magnesium concentration; and (ii) increase in [3H]MK-801 binding elicited by glutamate in telencephalic membrane preparations. Ethanol significantly decreased NMDA receptor-mediated excitatory postsynaptic potential (EPSP) amplitude and area in the three genotypes. In C57, the effect of ethanol on NMDA receptor-mediated EPSP amplitude and area was more pronounced at 30 degrees C, compared with that at 35 degrees C. In most cases, there was a good correlation between the effects of ethanol on EPSP amplitude and area. The order of sensitivity between the three genotypes was C57 = LS > SS at 35 degrees C and C57 > LS = SS at 30 degrees C. Similarly, ethanol significantly decreased glutamate-stimulated [3H]MK-801 binding in membrane fractions. The effect of ethanol was temperature-dependent, because ethanol produced more inhibition at 30 degrees C than at 35 degrees C in all genotypes. The effect of ethanol on MK-801 binding was concentration-dependent, and the sensitivity to 100 mM ethanol of the genotypes at 35 degrees C was LS > SS = C57, whereas it was SS > LS = C57 at 30 degrees C. Collectively, the results demonstrate that temperature is an important variable that can influence NMDA receptor sensitivity to ethanol measured via electrophysiological and binding techniques, and that temperature can influence relative sensitivity of NMDA receptors to ethanol between mouse genotypes. Furthermore, the findings indicate that temperature-induced changes in sensitivity of NMDA receptors to ethanol may play a role in mediating the effects of body temperature on behavioral sensitivity to ethanol in LS, but not C57 and SS mice.

Effect of 12 atmospheres helium-oxygen on the response of mice to convulsant drugs.

The mechanism by which 12 atm abs of a helium-oxygen gas mixture (heliox) antagonizes behavioral effects of ethanol is unknown. Although the threshold for pressure-reversal of general anesthesia and expression of the high pressure neurologic syndrome (HPNS) is well above 12 atm abs in mice, the ethanol antagonism by 12 atm abs heliox could result from similar underlying excitatory effects. To investigate this possibility, the behavior of water-injected control mice and the latency to convulsions in drug-injected mice were determined in 1 atm abs air and 12 atm abs heliox. Four convulsant drugs were tested: picrotoxin (2 mg/kg), dl-allylglycine (300 mg/kg), isoniazid (300 mg/kg), and l-methionine-dl-sulfoximine (170 mg/kg). Responses were videotaped to observe behavior and to measure latency to the onset of myoclonus and clonus. Results indicated no observable excitatory effects of 12 atm abs in control mice. The latency to myoclonus was significantly reduced by pressure in allylglycine-treated mice but not in mice treated with the other convulsants. Latency to clonus was not significantly altered by pressure, relative to latency at 1 atm abs heliox, for any drug tested. In conclusion, the present findings indicate that exposure to 12 atm abs heliox is not proconvulsant and, thus, the findings do not support the hypothesis that 12 atm abs heliox antagonizes ethanol indirectly via an increase in central nervous system excitability.

Low level hyperbaric antagonism of diazepam's locomotor depressant and anticonvulsant properties in mice.

Exposure to 12 atmospheres absolute (12 ATA) helium oxygen gas (heliox) (low level hyperbaric exposure) antagonizes the behavioral effects of ethanol and n-propanol, but not morphine. These and other results indicate that the mechanism of the antagonism is direct (pharmacodynamic) and selective. Our study further investigates the selectivity of low level hyperbaric antagonism by testing its effectiveness against diazepam, a high affinity binding drug that acts via allosteric modulation of GABAA receptors. C57BL/6J mice received injections i.p. of vehicle or diazepam, and were then exposed to 1 ATA air, 1 ATA heliox or 12 ATA heliox. Exposure to 12 ATA heliox antagonized the locomotor depressant effect of 4 and 6 mg/kg, but not 8 mg/kg diazepam. Hyperbaric exposure also antagonized the anticonvulsant effect of 8 and 24 mg/kg, but not 4 mg/kg, diazepam vs. 300 mg/kg isoniazid. Exposure to 12 ATA heliox did not significantly affect blood concentrations of diazepam or its metabolite n-desmethyl diazepam. The pharmacological characteristics of the antagonism (direct, surmountable, rightward shift in diazepam's dose-response curve) closely matched those seen in previous studies for hyperbaric antagonism of ethanol. The results add to the evidence that low level hyperbaric exposure is a direct, mechanistic antagonist that selectively antagonizes drugs that act via perturbation or allosteric modulation of receptor function. Moreover, the results suggest that allosteric coupling pathways, which transduce binding events on ligand-gated ion channels, may represent initial sites of action for ethanol.

Low-level hyperbaric exposure antagonizes locomotor effects of ethanol and n-propanol but not morphine in C57BL mice.

Low-level hyperbaric exposure antagonizes a broad range of behavioral effects of ethanol in a direct, reversible, and competitive manner. This study investigates the selectivity of the antagonism across other drugs. C57BL/6 mice were injected with saline, ethanol, n-propanol, or morphine sulfate, and then were exposed to 1 atmosphere absolute (ATA) air, 1 ATA helium-oxygen gas mixture (heliox), or 12 ATA heliox. Locomotor activity was measured from 10 to 40 min following injection. N-propanol produced a dose-dependent depression of locomotor activity from 1.0 g/kg. Morphine produced a dose-dependent stimulation of locomotor activity at doses of 3.75-12.0 mg/kg. Exposure to 12 ATA heliox significantly antagonized the locomotor depressant effects of 1.0 g/kg n-propanol and 2.5 g/kg ethanol, without significantly affecting blood concentrations of these drugs measured at 40 min postinjection. Exposure to 12 ATA heliox did not significantly antagonize the locomotor-stimulating effects of the two morphine doses tested (3.75 and 7.5 mg/kg). These findings suggest that exposure to 12 ATA heliox antagonizes the behavioral effects of intoxicant-anesthetic drugs like ethanol and n-propanol, which are believed to act via perturbation or allosteric modulation of functional proteins, but does not antagonize the effects of drugs like morphine, which act via more direct mechanisms. This demonstration of selective antagonism adds important support for the hypothesis that low-level hyperbaric exposure is a direct mechanistic ethanol antagonist, with characteristics similar to a competitive pharmacological antagonist.

Low-level hyperbaric antagonism of ethanol's anticonvulsant property in C57BL/6J mice.

This study investigated the ability of hyperbaric exposure to antagonize ethanol's anticonvulsant effect on isoniazid (INH)-induced seizures. Drug-naive, male C57BL/6 mice were injected intraperitoneally with saline, 1.5, 2.0, or 2.5 g/kg ethanol followed immediately by an intramuscular injection of 300 mg/kg of INH. The mice were then exposed to either 1 atmosphere absolute (1 ATA) air, 1 ATA helium-oxygen gas mixture (heliox), or 12 ATA heliox at temperatures that offset the hypothermic effects of helium. Ethanol increased the latency to onset of myoclonus in a dose-dependent manner. Exposure to 12 ATA heliox antagonized ethanol's anticonvulsant effect at 2.0 and 2.5 g/kg, but not at 1.5 g/kg. Ethanol also increased the latency to onset of clonus in a dose-dependent manner beginning at 2.0 g/kg. Exposure to 12 ATA heliox antagonized this anticonvulsant effect. When exposed to 12 ATA heliox, the blood ethanol concentrations at time to onset of myoclonus were significantly higher in mice treated with 2.5 g/kg of ethanol as compared with blood ethanol concentrations of mice exposed to 1 ATA air. These findings extend the acute behavioral effects of ethanol known to be antagonized by hyperbaric exposure and support the hypothesis that low-level hyperbaric exposure blocks or reverses the initial action(s) of ethanol leading to its acute behavioral effects.

Temperature dependence of ethanol depression in mice: dose response.

Manipulation of body temperature during intoxication significantly alters brain sensitivity to ethanol. The current study tested the generality of this effect within the hypnotic dose range. Drug naive, male C57BL/6J mice were injected with 3.2, 3.6, or 4.0 g/kg ethanol (20% w/v) and were exposed to 1 of 7 designated temperatures from 13 degrees to 34 degrees C to manipulate body temperature during intoxication. Rectal temperature at return of righting reflex (RORR) was significantly, positively correlated with loss of righting reflex (LORR) duration and significantly, negatively correlated with blood ethanol concentration (BEC) at RORR at all three doses. These results indicate that increasing body temperature during intoxication increased ethanol sensitivity in C57 mice at all three doses tested and demonstrate the generality of temperature dependence across hypnotic doses in these animals. Interestingly, the LORR duration was dose-dependent at each ambient temperature, but the degree of body temperature change and the BEC at RORR were not dose-dependent. Overall, these results emphasize the importance of body temperature as a variable in ethanol research.

Morphine-6-glucuronide: a potent stimulator of locomotor activity in mice.

The present study tested the hypothesis that morphine glucuronides have stimulant properties by studying their effects on locomotor activity in mice. Drug-naive C57BL/6J male mice were injected with saline, morphine, morphine-6-glucuronide (M6G) or morphine-3-glucuronide (M3G). In some experiments, mice were injected with saline or naloxone 5 min prior to drug treatment. Injection of 40 mg/kg morphine or M6G, but not M3G, significantly increased activity versus saline. The extent of activation induced by M6G was markedly higher than for morphine. Subsequent dose-response studies across a somewhat lower dose range using equimolar doses of morphine and M6G (3-80 mumoles/kg) found that both drugs significantly increased locomotor activity beginning at 20 mumoles/kg. M6G increased locomotor activity from 1.3 to 2.1 times more than for equimolar doses of morphine. Pretreatment with naloxone (10 mg/kg) completely abolished the locomotor stimulation induced by 32 mumoles/kg morphine and M6G. These findings present evidence that M6G is an active metabolite of morphine which has behaviorally stimulating effects and may play an important role in mediating the reinforcing properties of morphine in humans.

Low-level hyperbaric antagonism of ethanol-induced locomotor depression in C57BL/6J mice: dose response.

This study characterized the antagonistic effects of hyperbaric exposure on the dose-response curve for ethanol-induced depression of locomotor activity. Drug-naive, male C57BL/6 mice were injected intraperitoneally with saline, 1.5, 2.0, 2.5, or 3.0 g/kg ethanol, and were exposed to 1 atmosphere absolute (ATA) air or 12 ATA helium-oxygen gas mixtures (heliox) at temperatures that offset the hypothermic effects of ethanol and helium. Locomotor activity was measured 10-30 min after injection. In addition, the effects of exposure to 12 ATA heliox on blood ethanol concentrations were tested in a separate group of mice injected with 2.5 g/kg ethanol. Ethanol produced a dose-dependent depression of locomotor activity beginning at 2.0 g/kg. Exposure to 12 ATA heliox completely antagonized the locomotor depressant effects of 2.0 and 2.5 g/kg ethanol and partially blocked the effects of 3.0 g/kg. Activity in mice given 1.5 g/kg ethanol was not significantly affected at 1 ATA air, but was significantly increased at 12 ATA heliox. Low-level hyperbaric exposure shifted the ethanol dose-response curve to the right with a resultant increase in the ED50 of ethanol for locomotor depression from 2.6 to 3.3 g/kg. Exposure to 12 ATA heliox did not alter blood ethanol concentrations in mice injected with 2.5 g/kg ethanol. These findings with 12 ATA heliox present key new evidence for the hypothesis that low-level hyperbaric exposure acts directly, with a pattern analogous to a competitive, mechanistic antagonist of ethanol.

Genetically determined differences in the antagonistic effect of pressure on ethanol-induced loss of righting reflex in mice.

Hyperbaric exposure antagonizes ethanol's behavioral effects in a wide variety of species. Recent studies indicating that there are genetically determined differences in the effects of body temperature manipulation on ethanol sensitivity suggested that genotype might also influence the effects of hyperbaric exposure on ethanol intoxication. To investigate this possibility, ethanol injected long sleep (LS)/Ibg (2.7 g/kg), short sleep (SS)/Ibg (4.8 g/kg), 129/J (2.9 g/kg), and C57BL/6J (3.6 g/kg) mice were exposed to one atmosphere absolute (ATA) air or to one or 12 ATA helium-oxygen (heliox) at ambient temperatures selected to offset ethanol and helium-induced hypothermia. Hyperbaric exposure significantly reduced loss of righting reflex (LORR) duration in LS, 129, and C57 mice, but not in SS mice. A second experiment found that hyperbaric exposure significantly reduced LORR duration and increased the blood ethanol concentration (BEC) at return of righting reflex (RORR) in LS mice, but did not significantly affect either measure in SS mice. These results indicate that exposure to 12 ATA heliox antagonizes ethanol-induced LORR in LS, 129 and C57 mice, but not in SS mice. Taken with previous results, the present findings suggest that the antagonism in LS, 129, and C57 mice reflects a pressure-induced decrease in brain sensitivity to ethanol and that the lack of antagonism in SS mice cannot be explained by pressure-induced or genotypic differences in ethanol pharmacokinetics.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between brain temperature during intoxication and ethanol sensitivity in LS and SS mice.

The present study characterized the relationship between brain temperature, rectal temperature, and ethanol sensitivity in the selectivity bred long-sleep (LS) and short-sleep (SS) mice. Radiotelemetric brain probe implanted and nonimplanted LS/lbg and SS/lbg male mice were injected with 2.5 and 4.9 g/kg ethanol, respectively, before exposure to ambient temperatures of 15 degrees C, 22 degrees C, or 34 degrees C. Ambient temperature significantly affected rectal temperature, brain temperature, and ethanol sensitivity, measured by impairment of righting reflex. Brain and rectal temperatures at return of righting reflex (RORR) were highly correlated. In SS mice brain and rectal temperatures at RORR were significantly positively correlated with loss of righting reflex (LORR) duration and significantly negatively correlated with blood ethanol concentration (BEC) at RORR. In LS mice rectal temperature at RORR was significantly negatively correlated with LORR duration, while both brain and rectal temperature at RORR were significantly positively correlated with BEC at RORR. The strength of the correlations and r2 values generated from linear regression analysis indicates that body temperature during intoxication can explain up to 52% of the variability in ethanol sensitivity in SS mice, but only 19% of the variability in ethanol sensitivity in LS mice. The correlational analyses are consistent with previous results based on comparisons between rectal temperature and ethanol sensitivity and extend to direct brain temperature measurement the evidence that decreasing temperature during intoxication decreases ethanol sensitivity in SS mice and increases ethanol sensitivity in LS mice.(ABSTRACT TRUNCATED AT 250 WORDS)