Repeated Ethanol Injections Research Articles

Effects of Repeated Ethanol Exposures on NMDA Receptor Expression and Locomotor Sensitization in Mice Expressing Ethanol Resistant NMDA Receptors.

Evidence from a large number of preclinical studies suggests that chronic exposure to drugs of abuse, such as psychostimulants or ethanol induces changes in glutamatergic transmission in key brain areas associated with reward and control of behavior. These changes include alterations in the expression of ionotropic glutamate receptors including N-methyl-D-aspartate receptors (NMDAR) that are important for regulating neuronal activity and synaptic plasticity. NMDA receptors are inhibited by ethanol and reductions in NMDA-mediated signaling are thought to trigger homestatic responses that limit ethanol's effects on glutamatergic transmission. Following repeated exposures to ethanol, these homeostatic responses may become unstable leading to an altered glutamatergic state that contributes to the escalations in drinking and cognitive deficits observed in alcohol-dependent subjects. An important unanswered question is whether ethanol-induced changes in NMDAR expression are modulated by the intrinsic sensitivity of the receptor to ethanol. In this study, we examined the effects of ethanol on NMDAR subunit expression in cortical (orbitofrontal, medial prefrontal), striatal (dorsal and ventral striatum) and limbic (dorsal hippocampus, basolateral amygdala) areas in mice genetically modified to express ethanol-resistant receptors (F639A mice). These mice have been previously shown to drink more ethanol than their wild-type counterparts and have altered behavioral responses to certain actions of ethanol. Following long-term voluntary drinking, F639A mice showed elevations in GluN2A but not GluN1 or GluN2B expression as compared to wild-type mice. Mice treated with repeated injections with ethanol (2–3.5 g/kg; i.p.) showed changes in NMDAR expression that varied in a complex manner with genotype, brain region, subunit type and exposure protocol all contributing to the observed response. F639A mice, but not wild-type mice, showed enhanced motor activity following repeated ethanol injections and this was associated with differences in NMDAR subunit expression across brain regions thought to be involved in drug sensitization. Overall, while the results of the study suggest that NMDARs with reduced sensitivity to ethanol favor the development of locomotor sensitization, they also show that intrinsic ethanol sensitivity is not the sole determinant underlying changes in NMDAR expression following repeated exposures to ethanol.

The effect of repeated exposure to ethanol on pre-existing fear memories in rats.

RationaleThere is a high degree of comorbidity between alcohol use disorder and post-traumatic stress disorder (PTSD), but little is known about the interactions of ethanol with traumatic memories.ObjectivesUsing auditory fear conditioning in rats, we asked if repeated exposure to ethanol could modify the retrieval of fear memories acquired prior to ethanol exposure.MethodsFollowing auditory fear conditioning, Sprague-Dawley rats were given daily injections of ethanol (1.5 g/kg) or saline over 5 days. Two days later, they were given 20 trials of extinction training and then tested for extinction memory the following day. In a separate experiment, conditioned rats were given repeated ethanol injections and processed for c-Fos immunohistochemistry following a fear retrieval session.ResultsTwo days following the cessation of ethanol, the magnitude of conditioned fear responses (freezing and suppression of bar pressing) was significantly increased. This increase persisted the following day. Waiting 10 days following cessation of ethanol eliminated the effect on fear retrieval. In rats conditioned with low shock levels, repeated exposure to ethanol converted a sub-threshold fear memory into a supra-threshold fear memory. It also increased c-Fos expression in the prelimbic prefrontal cortex, paraventricular thalamus, and the central and basolateral nuclei of the amygdala, areas implicated in the retrieval of fear memories.ConclusionsThese results suggest that repeated exposure to ethanol may exacerbate pre-existing traumatic memories.

Correlation between ethanol behavioral sensitization and midbrain dopamine neuron reactivity to ethanol.

Repeated ethanol injections lead to a sensitization of its stimulant effects in mice. Some recent results argue against a role for ventral tegmental area (VTA) dopamine neurons in ethanol behavioral sensitization. The aim of the present study was to test whether in vivo ethanol locomotor sensitization correlates with changes in either basal- or ethanol-evoked firing rates of dopamine neurons in vitro. Female Swiss mice were daily injected with 2.5 g/kg ethanol (or saline in the control group) for 7 days and their locomotor activity was recorded. At the end of the sensitization procedure, extracellular recordings were made from dopaminergic neurons in midbrain slices from these mice. Significantly higher spontaneous basal firing rates of dopamine neurons were recorded in ethanol-sensitized mice relative to control mice, but without correlations with the behavioral effects. The superfusion of sulpiride, a dopamine D2 antagonist, induced a stronger increase of dopamine neuron firing rates in ethanol-sensitized mice. This shows that the D2 feedback in dopamine neurons is preserved after chronic ethanol administration and argues against a reduced D2 feedback as an explanation for the increased dopamine neuron basal firing rates in ethanol-sensitized mice. Finally, ethanol superfusion (10-100 mM) significantly increased the firing rates of dopamine neurons and this effect was of higher magnitude in ethanol-sensitized mice. Furthermore, there were significant correlations between such a sensitization of dopamine neuron activity and ethanol behavioral sensitization. These results support the hypothesis that changes in brain dopamine neuron activity contribute to the behavioral sensitization of the stimulant effects of ethanol.

Higher long-lasting ethanol sensitization after adolescent ethanol exposure in mice

Due to their maturing brain, adolescents are suggested to be more vulnerable to the long-term consequences of chronic alcohol use. Increased sensitization to the stimulant effects of ethanol is a possible consequence of ethanol exposure during adolescence. The aim of this study was to characterize the long-term alterations in the stimulant effects of ethanol and in the rate of ethanol sensitization in mice pre-exposed to ethanol during adolescence in comparison to mice pre-exposed to ethanol in adulthood. Adolescent and adult female Swiss mice were injected with saline or ethanol (2.5 or 4 g/kg) during 14 consecutive days. After a 3-week period of ethanol abstinence, mice were tested as adults before and after a second exposure to daily repeated ethanol injections. All mice pre-exposed to ethanol as adults or adolescents showed higher stimulant effects when re-exposed to ethanol 3 weeks later. However, this enhanced sensitivity to the stimulant effects of ethanol was of significantly higher magnitude in mice repeatedly injected with high ethanol doses (4 g/kg) during adolescence. Furthermore, the increased expression of ethanol stimulant effects in these mice was maintained even after a second procedure of ethanol sensitization. Adolescence is a critical period for the development of a sensitization to ethanol stimulant properties providing that high intermittent ethanol doses are administered. These results might contribute to explain the relationship between age at first alcohol use and risks of later alcohol problems and highlight the dangers of repeated consumption of high alcohol amounts in young adolescents.

Changes in extracellular levels of glutamate in the nucleus accumbens after ethanol-induced behavioral sensitization in adolescent and adult mice

Repeated administration of low doses of ethanol gradually increases locomotor responses to ethanol in adult Swiss mice. This phenomenon is known as behavioral sensitization. However, we have shown that adolescent Swiss mice show either behavioral tolerance or no sensitization after repeated ethanol injections. Although the mesolimbic dopamine system has been extensively implicated in behavioral sensitization, several studies have demonstrated an important role of glutamatergic transmission in this phenomenon. In addition, relatively few studies have examined the role of developmental factors in behavioral sensitization to ethanol. To examine the relationship between age differences in behavioral sensitization to ethanol and the neurochemical adaptations related to glutamate within nucleus accumbens (NAc), in vivo microdialysis was conducted in adolescent and adult Swiss mice treated with ethanol (1.8 g/kg) or saline for 15 days and subsequently challenged with an acute dose (1.8 g/kg) of ethanol 6 days later. Consistent with previous findings, only adult mice demonstrated evidence of behavioral sensitization. However, ethanol-treated adolescent mice demonstrated a 196.1 ± 40.0% peak increase in extracellular levels of glutamate in the NAc after ethanol challenge in comparison with the basal values, whereas ethanol-treated adult mice demonstrated a 52.2 ± 6.2% reduction in extracellular levels of glutamate in the NAc after ethanol challenge. These observations suggest an age-dependent inverse relationship between behavioral and glutamatergic responses to repeated ethanol exposure.

Selective mu- and kappa-opioid receptor antagonists administered into the nucleus accumbens interfere with rapid tolerance to ethanol in rats

Previous findings have shown that intra-accumbens injection of naltrexone, a non-selective opioid antagonist, blocks the acquisition of rapid tolerance to ethanol in rats. This study investigates the effects of intra-accumbens injection of the selective mu-, delta-, and kappa-opioid antagonists, respectively, naloxonazine, naltrindole, and nor-binaltorphimine, on rapid tolerance to ethanol. Male Wistar rats with guide cannulae directed to the shell or the core portions of the nucleus accumbens received a microinjection of naloxonazine (2-4 microg), naltrindole (2-4 microg), nor-binaltorphimine (2.5-5 microg), or vehicle. After 5 min, each group was divided in two groups that received ethanol (2.7 g/kg i.p.) or saline. Rats were then tested for motor coordination on the tilting plane apparatus. Twenty four hours later, all rats received a challenge dose of ethanol (2.7 g/kg i.p.) and were tested on the tilt plane again. Repeated injections of ethanol caused a reduction in motor impairment suggesting the development of tolerance. However, rats injected with 4 microg naloxonazine into either core or shell portions of the nucleus accumbens did not exhibit tolerance when challenged with ethanol on day 2. Rats treated with 5 microg nor-binaltorphimine into accumbens core plus intraperitoneal saline on day 1 showed reduced motor impairment when challenged with ethanol on day 2, suggesting cross-tolerance to ethanol. Taken together, our results suggests that mu-opioid receptors in both shell and core portions of the nucleus accumbens, and possibly kappa-opioid in the core, participate in the modulation of rapid tolerance to ethanol.

Percutaneous ethanol injection therapy: a surgery‐sparing treatment for primary hyperparathyroidism

To describe our 3-year experience in the long-term efficacy and safety of percutaneous ethanol injection therapy (PEIT), as an alternative to surgery for the management of patients with primary hyperparathyroidism (p-HPT). Prospective study with a mean follow-up of 19.6 +/- 10.6 months. Our study population included 19 consecutive high risk patients with p-HPT, who met the criteria for surgery. Under ultrasonic guidance, ethanol (95%) was injected into parathyroid glands with a volume of >or= 0.15 cm(3). With the aim of normalizing intact parathormone (iPTH) values, repeated ethanol injections were carried out, in an interval of 2 weeks, until normalization of iPTH was reached or until no residual blood supply was detected by ultrasound in the gland. Biochemical parameters were monitored throughout the study. At 6-month follow-up, normalization of iPTH levels (10-65 ng/l) was achieved in 11 (58%) patients (responders). Of the eight remaining patients (nonresponders), six patients had reduced (but not normalized) iPTH levels and two patients required parathyroid surgery. Seventeen (11 responders and 6 nonresponders) of the 19 patients (89.5%) became normocalcaemic (serum Ca <or= 2.57 mmol/l) and remained so for a mean follow-up of 21 months (8-39 months). The odds ratio (P < 0.05) of response vs. no response to PEIT was 16.7-fold for pretreatment iPTH < 200 vs. > 200 ng/l. The only complication was a transient dysphonia noticed in three patients. PEIT is a safe and effective nonsurgical treatment for patients with p-HPT, who are unsuitable for surgical intervention.

Parametric analysis of the development and expression of ethanol-induced behavioral sensitization in female Swiss mice: effects of dose, injection schedule, and test context

Repeated administrations of ethanol induce a progressive and enduring increase in its locomotor stimulant effects, a phenomenon termed behavioral sensitization that has not been systematically characterized. The aim of the present studies was to characterize the development and expression of ethanol sensitization in female Swiss mice by examining (1) the doses of ethanol that induce behavioral sensitization, (2) the doses of acute ethanol challenges that are necessary to express behavioral sensitization, (3) the effects of the intervals between administrations, and (4) the context dependency of ethanol sensitization. Mice were i.p. injected for 8 days with various ethanol doses, and locomotion was recorded for 5 min. Two days after the last sensitization session, ethanol sensitization was tested in 30-min test sessions. Mice repeatedly injected with 2.5 g/kg ethanol showed a progressive (200-300%) increase in locomotor activity. In response to a 2.5 g/kg ethanol challenge, the mice repeatedly treated with doses above 1.5 g/kg showed a significant sensitization. Following the induction of sensitization with the maximally effective sensitizing dose (2.5 g/kg), mice showed greater activation after challenges with 1.5, 2.0, 2.5, and 3.0 g/kg ethanol. The intervals (24, 48, or 96 h) between ethanol injections did not affect the induction or expression of sensitization. Finally, sensitization to 2.5 g/kg ethanol was expressed regardless of the context in which it was induced. Female Swiss mice develop a robust context-independent sensitization after repeated ethanol injections at all doses above 1.5 g/kg, including highly sedative doses such as 4 g/kg.

Involvement of protein kinase A in ethanol-induced locomotor activity and sensitization

Rationale Mutant mice lacking the RIIβ subunit of protein kinase A (regulatory subunit II beta −/−) show increased ethanol preference. Recent evidence suggests a relationship between heightened ethanol preference and susceptibility to ethanol-induced locomotor sensitization. It is currently unknown if protein kinase A signaling modulates the stimulant effects and/or behavioral sensitization caused by ethanol administration. To address this question, we examined the effects of repeated ethanol administration on locomotor activity RIIβ −/− and littermate wild-type (RIIβ +/+) mice on multiple genetic backgrounds. Methods Over three consecutive days, mice were given single i.p. saline injections and immediately placed in a locomotor activity apparatus to establish a composite baseline for locomotor activity. Next, mice maintained on a hybrid 129/SvEv×C57BL/6J or pure C57BL/6J genetic background were given 10 i.p. ethanol injections before being placed in the activity apparatus. Each ethanol injection was separated by 3–4 days. To determine if changes in behavior were specific to ethanol injection, naïve mice were tested following repeated daily saline injections. The effects of ethanol injection on locomotor behavior were also assessed using an alternate paradigm in which mice were given repeated ethanol injections in their home cage environment. Results Relative to RIIβ +/+ mice, RIIβ −/− mice, regardless of genetic background, consistently showed significantly greater ethanol-induced locomotor activation. RIIβ −/− mice also showed increased sensitivity to ethanol-induced locomotor sensitization resulting from repeated administration, an effect that was dependent on genetic background and testing paradigm. Increased locomotor activity by RIIβ −/− mice was specific to ethanol injections, and was not related to altered blood ethanol levels. Conclusions These data provide novel evidence implicating an influence of protein kinase A signaling on ethanol-induced locomotor activity and behavioral sensitization. The observation that RIIβ −/− mice are more sensitive to the effects of repeated ethanol administration suggests that normal protein kinase A signaling limits, or is protective against, the stimulant effects of ethanol and the plastic alterations that underlie behavioral sensitization.

Imbalance between neuroexcitatory and neuroinhibitory amino acids causes craving for ethanol

Long-term exposure to ethanol leads to an imbalance in different excitatory and inhibitory amino acids. When ethanol consumption is reduced or completely stopped, these imbalances in different amino acids and neurotransmitters are behaviorally expressed in the form of ethanol withdrawal. Glutamate, a major excitatory amino acid, and GABA, a major inhibitory amino acid, are responsible, at least partly, for ethanol withdrawal symptoms. The hypofunction of GABA A receptors and enhanced function of NMDA receptors are suggested to be responsible for the increase in the behavioral susceptibility during ethanol withdrawal. This imbalance between receptors may be exacerbated by repeated withdrawal. Because multiple and repeated periods of chronic ethanol consumption and withdrawal often occur in alcohol abusers, animal studies on the neurochemical changes in different amino acids following chronic ethanol treatment (CET) that is interrupted by repeated ethanol withdrawal episodes may be of clinical relevance for the development of treatment strategies. Brain glutamate increases during the first cycle of ethanol withdrawal, and this increase is much higher during the third cycle of ethanol withdrawal. The elevated glutamate released in the hippocampus during the first cycle of ethanol withdrawal episode was exacerbated in subsequent withdrawal episodes. Acamprosate, a drug used during human alcohol detoxification, is able to completely block the glutamate increase observed during the first as well as the third withdrawal of ethanol. In ethanol-naïve rats, there was no change in the glutamate microdialysate content after an acute ethanol injection. However, when repeated ethanol injections were cued with a vinegar stimulus that had previously been associated with the same ethanol injection, a significant increase in glutamate microdialysate content was assayed. Furthermore, when the cue was omitted, the ethanol injection induced no changes in glutamate microdialysate content in rats that had been previously ethanol conditioned. By comparison, a saline injection had no effect on extracellular glutamate concentration in rats naïve for ethanol as well as in rats daily administered with repeated ethanol injections that were not paired with the cue. It appears probable that these conditioned responses by extracellular glutamate concentrations may participate in the environmental cue-induced conditioned cravings for ethanol that are thought to be related to the high frequency of relapse in detoxified alcoholics.

Importance of magnesium ions in development of tolerance to ethanol: studies on cultured cerebral vascular smooth muscle cells, type-2 astrocytes and intact rat brain

This study was designed to examine the roles of intracellular free magnesium ion concentration ([Mg2+]i) in ethanol-induced intoxication and development of tolerance in cultured canine cerebral vascular smooth muscle cells and astrocytes as well as intact rat brain. The basal, resting level of [Mg2+]i in cerebrovascular cells was 732.5 ± 82.4 μM. Exposure of cultured canine cerebral vascular smooth muscle cells to ethanol (10 and 25 mM) for 24 h reduced the concentrations of [Mg2+]i to 521.1 ± 59.6 μM, and 308.2 ± 37.8μM, respectively. However, exposure of these cultured vascular cells to the same concentrations of ethanol, after initial pretreatment with ethanol for 24 h, failed to interfere with the levels of [Mg2+]i. Measurement of [Mg2+]i at 48 h and 72 h indicated that the decreased levels of [Mg2+]i induced by ethanol at 24 h treatment returned toward baseline. Similar experiments were performed in cultured type-2 astrocytes isolated from neonatal rat brain. The basal level of [Mg2+]i in type-2 astrocytes was about 125 μM. Incubation of these cells with 10 mM ethanol for 10 min resulted in a 27% reduction in the level of [Mg2+]i, whereas incubation with 25 mM ethanol resulted in almost a 50% reduction in [Mg2+]i. The decreased levels of [Mg2+]i lasted around 30 min, until the measurement finished. Continuous incubation of these cultured astrocytes, with ethanol (either 10 mM or 25 mM), for more than 24 h, indicated that the concentrations of [Mg2+]i in type-2 astrocytes were equivalent to those at basal, resting levels. In vivo31P-NMR spectroscopy, performed on intact rat brains, indicated that an initial administration of 4 mg/kg ethanol (∼20–25 mM blood alcohol level) resulted (after 20–40 min of exposure) in severe deficits in whole brain [Mg2+]i (550 ± 33 μM to 358 ± 24 μM). Repeated injections of ethanol (4 mg/kg) over the next 24–72 h resulted in progressively diminishing effects on brain [Mg2+]i. These experimental data indicate that chronic ethanol treatment can induce a tolerance to depletion of [Mg2+]i in cerebrovascular smooth muscle cells, type-2 astrocytes as well as intact rat brain. The results suggest that [Mg2+]i might play a major role in alcohol-induced tolerance in the brain.

Long-term results after percutaneous minimally invasive procedure treatment of symptomatic simple renal cysts.

To assess the effectiveness and long-term results of two repeated ethanol injections in the treatment of symptomatic renal cysts. Sixty-eight patients 47 to 75 years old with 77 large (6.3-14.8 cm; mean 8.62 cm) symptomatic cysts were included in this study. Of the 68 patients, in 4 cases there were renal cysts associated with renal calculi. An 8FR Pigtail catheter was inserted into the cyst under ultrasound guidance followed by aspiration of cystic fluid. Two repeated ethanol 95% injections were performed every 24 hours. Patients were followed-up by ultrasonography for a period of 12-48 months (mean 30). In two patients the cyst puncture was not feasible because of poor visualization of puncture site while in one patient there was communication of cystic cavity with the pelvocaliceal system. In these patients surgical resection of cysts was performed. Complete and partial regression rates were 57/68 (83.82%) and 8/68 (11.76%) respectively. In four patients with renal stones and renal cysts, extracorporeal shock wave lithotripsy (ESWL) was successfully executed I month after sclerotherapy. Our results suggest that percutaneous aspiration followed by two repeated ethanol injections is highly effective on reducing recurrence of simple renal cysts.

ACUTE ETHANOL EXPOSURE DECREASES THE ANALGESIC POTENCY OF MORPHINE IN MICE

Chronic (7 days), forced ethanol drinking can decrease the analgesic potency of opioid agonists in mice. In the present study, the effect of short-term ethanol treatment was examined using forced ethanol access and ethanol injection protocols. Mice were given forced access to 1, 3 or 7% (v/v) ethanol for 24 hr and then tested for s.c. morphine analgesia using the tailflick assay. Controls had access to water. Another group of mice was injected i.p. with 2.5 g/kg ethanol or water 4 times over a 21 hr period and tested 3 hr after the final injection for morphine analgesia. Other mice were injected once i.p. with 1, 2 or 3 g/kg ethanol or water and tested 24 hr later using the tailflick. In the forced access study, ethanol dose-dependently decreased morphine's analgesic potency with the highest dose (7%) producing a 1.6-fold shift in the ED 50. This decrease in morphine potency was similar to that found in a related study using 7% ethanol for 7 days (1.8-fold shift). Repeated ethanol injections significantly reduced the analgesic potency of morphine (1.9-fold shift), whereas, a single injection of 1, 2 or 3 g/kg ethanol did not alter the potency of morphine. Control studies indicated that neither 24 hr water nor food deprivation affected morphine potency. Overall, these data show that sustained exposure to ethanol over a 24 hr period will dose-dependently decrease morphine's analgesic potency. © 1998 Elsevier Science Inc.

Neurotransmitter interactions in the striatum and hypothalamus of mice after single and repeated ethanol treatment.

In single treatment study, ethanol was administered intraperitoneally to ICR mice (about 34 g) in the amounts of 1.0, 2.0, 3.0 or 4.0 g/kg body weight. The 3,4-dihydroxyphenylacetic acid (DOPAC) + homovanillic acid (HVA) concentration in the striatum was elevated with 3.0 and 4.0 g/kg of ethanol. In the hypothalamus, the DOPAC, HVA and 5-hydroxyindoleacetic acid concentrations were increased after injection of 3.0 and 4.0 g/kg of ethanol. Furthermore, the acetylcholine (ACh) and gamma-aminobutyric acid (GABA) concentrations were also increased following the injection of 1.0, 2.0, 3.0 and 4.0 g/kg. To study the effects of repeated administration, mice were injected intraperitoneally with 1.0 or 2.0 g/kg of ethanol once daily for 7 days. The DOPAC + HVA level in the striatum was elevated after injection of 1.0 and 2.0 g/kg of ethanol. The GABA and ACh concentrations in the hypothalamus were decreased after repeated injections of ethanol. These results suggest that ethanol significantly alters the utilization of dopamine, ACh and GABA in the hypothalamus. This may partially explain why ethanol has such profound effects on emotional behavior and mood.

Intratumor Ethanol Injection Therapy for Solitary Minute Hepatocellular Carcinoma

To evaluate the effect of percutaneous ethanol injection given alone, we studied disease recurrence and prognosis in 37 patients with solitary hepatocellular carcinoma up to 2 cm. The patients were classified as Child's class A, 22; B, 11; and C, 4. During follow-up for periods ranging from 10 to 44 (mean, 26) months, 15 of the 37 patients (40.5%) had disease recurrence. The 1-, 2-, and 3-year recurrence rates were 37, 49, and 53%, respectively (Kaplan-Meier method). Significant risk factors for recurrence included the hypervascularity and the histological differentiation of the tumor (p < 0.01). Of those 15 patients, two had local recurrence, eight had nodular recurrence (fewer than two nodules), and five had widespread multinodular recurrence. The patients with local or nodular recurrence then received repeated injections of ethanol, while those with multinodular recurrence received transarterial embolization or chemolipiodolization therapy as possible. Seven of the 37 patients died during follow-up. The 1-, 2-, and 3-year survival rates were 95, 81, and 70%, respectively (Kaplan-Meier method). No factor was significant in prognosis. We observed a significant relationship between recurrence pattern and prognosis. Patients with a widespread multinodular recurrence had a significantly shorter survival (p < 0.01). In summary, ethanol injections given alone appeared to be effective in treating small and solitary hepatocellular carcinomas. Repeated injection of ethanol of the site of recurrence was useful in prolonging survival.

The NMDA receptor antagonist dizocilpine differentially affects environment-dependent and environment-independent ethanol tolerance.

Antagonists of the N-methyl-D-aspartate subtype of glutamate receptor have been reported to block the development of tolerance to various effects of ethanol and opiates, using paradigms in which tolerance is believed to be governed by learning. There is considerable evidence to implicate the N-methyl-D-aspartate receptor in learning processes, and therefore the ability of the antagonists to block tolerance has been attributed to their effects on learning. To evaluate this hypothesis, we compared, in C57BL/6 mice, the effect of the uncompetitive N-methyl-D-aspartate receptor antagonist, dizocilpine, on environment-dependent (associative) tolerance to ethanol, which is governed by learning, and on environment-independent (nonassociative) ethanol tolerance, in which learning plays a minimal role. Environment-dependent tolerance was induced by repeated ethanol injections, and dizocilpine blocked the development of this type of tolerance to the hypothermic and incoordinating effects of ethanol. In contrast, when environment-independent ethanol tolerance was induced by feeding the mice an ethanol-containing liquid diet, dizocilpine treatment had no effect on the development of tolerance to the hypothermic, incoordinating or hypnotic effects of ethanol. The results support the hypothesis that the effect of N-methyl-D-aspartate receptor antagonists on ethanol tolerance reflects the more general role of this receptor in processes involving learning and memory.

Brain cholinergic involvement in the rapid development of tolerance to the hypothermic action of ethanol

1. 1. The enzymes of the cholinergic system have been investigated in discrete brain regions in mice treated with repeated injection of ethanol. Male mice kept under controlled environmental conditions were treated with ethanol (3 g/kg/day) for 3 days. Animals were sacrificed 1 hr after ethanol injections. Brain regions studied were cerebral cortex, cerebellum, midbrain, hypothalamus, medulla oblongata, amygdala, and hippocampus. 2. 2. The administration of a single dose of ethanol resulted in significant increase ( P < 0.05) in choline acetyltransferase (ChAT) activity in all different brain regions. Repeated injections of ethanol at the 2nd and 3rd day did not result in any further rise in ChAT activity of the brain regions studied except for the midbrain. 3. 3. The results also show that acetylcholinesterase (AChE) activities increased significantly ( P < 0.05) in the pons and hippocampus in acutely ethanol intoxicated animals. The repeated injection of ethanol resulted in significant increase in AChE activities of the cortex and the amygdala. Meanwhile, animals developed tolerance to the hypothermic action of ethanol after ethanol third injection. 4. 4. The results of the present investigation indicate that the rapid development of the hypothermic tolerance to ethanol might be mediated by the brain cholinergic system.

Adrenal glucocorticoids as a required factor in the development of ethanol tolerance in mice.

Male C57BL/6J mice were given chronic ethanol treatment by two procedures. One was by a liquid diet containing 6.4% (v/v) ethanol; the other was by repeated injections of ethanol (3.5 g/kg, i.p. twice daily). After 5 days of the liquid diet treatment, sleep time following a challenge dose of ethanol (3.0 g/kg i.p.) was reduced to 10 min as compared with 30 min in the controls not previously exposed to ethanol. After 10 days, none of the ethanol-treated animals slept. Bilateral adrenalectomy (Adx) had no effect on sleep time (34 min). However, the reduction of sleep time in ethanol-treated Adx animals was much less: 19 min after 5 days, and 20 min after 10 days. Replacement with corticosterone in Adx animals restored the reduction of sleep time to the same levels as intact animals, indicating that glucocorticoids are the hormonal factor involved in the Adx effect. By the injection procedure for short-term administration of moderate doses of ethanol, sleep time following the injection of 3.5 g/kg ethanol was reduced from 95 min on day 1 to 57 min on day 3. There was no difference between intact and Adx animals. The results from both treatment procedures suggest that Adx abolished the later, severe stage of tolerance development but not the initial stage. Functional tolerance to ethanol occurring in this advanced stage may be a glucocorticoid-dependent process.

Ethanol-induced hypothermia in mice: Influence of genotype on development of tolerance

Male mice of BALB/c, C57BL, DBA/2 strains and two lines of mice selectively bred for sensitivity to ethanol, Long-Sleep (LS) and Short-Sleep (SS), were tested for ethanol-induced hypothermia following varied doses of ethanol. The results show that the genotype as well as the dose of the drug determines the intensity and the duration of the effect. Repeated injection of ethanol results in the decrease of hypothermia in BALB/c mice and in C57BL, but not in DBA/2 mice, indicating that tolerance as measured in this study may not develop in certain genotypes of mice. The blood ethanol elimination data after repeated injections of ethanol indicate that the metabolic factors do not explain the changes observed in the hypothermic effects of repeated injections of ethanol.

Effect of Ascorbic Acid and Zinc Sulfate on Ethanol Toxicity and Metabolism

SummaryAcute (single dose) and chronic (multiple dose) ethanol intoxication has been produced in rats and mice so that the effects on survival and on ethanol metabolism could be determined in animals pretreated with ascrobic acid and/or zinc sulfate. Only 13 of 40 mice (32%) injected ip with a fixed amount of ethanol on 2 consecutive days survived. In contrast, the survival rates in matched animals pretreated with ascrobic acid (25 mg) or zinc (1 μM) were 100 and 90%, respectively (20 animals per group). Smaller amounts of zinc (0.2 and 0.4 μM) improved the survival after the first injection of ethanol (24 hr), but not after the second (48 hr). Similar observations were made in rats given repeated injections of ethanol over a 4-week period. Serial blood ethanol concentrations were determined in rats pretreated with ascrobic acid, zinc, a combination of ascorbic acid and zinc, pyrazole (an alcohol dehydrogen-ase inhibitor), or saline (control). Blood ethanol concentrations were significantly lower (P < 0.0...