Motor-impairing Effects Of Ethanol Research Articles

Knockin Mice with Ethanol-Insensitive α1-Containing γ-Aminobutyric Acid Type A Receptors Display Selective Alterations in Behavioral Responses to Ethanol

Despite the pervasiveness of alcohol (ethanol) use, it is unclear how the multiple molecular targets for ethanol contribute to its many behavioral effects. The function of GABA type A receptors (GABA(A)-Rs) is altered by ethanol, but there are multiple subtypes of these receptors, and thus far, individual subunits have not been definitively linked with specific behavioral actions. The alpha1 subunit of the GABA(A)-R is the most abundant alpha subunit in the brain, and the goal of this study was to determine the role of receptors containing this subunit in alcohol action. We designed an alpha1 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity and constructed knockin mice containing this mutant subunit. Hippocampal slice recordings from these mice indicated that the mutant receptors were less sensitive to ethanol's potentiating effects. Behaviorally, we observed that mutant mice recovered more quickly from the motor-impairing effects of ethanol and etomidate, but not pentobarbital, and showed increased anxiolytic effects of ethanol. No differences were observed in ethanol-induced hypnosis, locomotor stimulation, cognitive impairment, or in ethanol preference and consumption. Overall, these studies demonstrate that the postsynaptic effects of ethanol at GABAergic synapses containing the alpha1 subunit are important for specific ethanol-induced behavioral effects.

Motor Impairing Effects of Ethanol and Diazepam in Rats Selectively Bred for High and Low Ethanol Consumption in a Limited‐Access Paradigm

Some clinical studies suggest that an initial low-level response in ethanol sensitivity is a good predictor of risk for developing subsequent high levels of ethanol consumption in humans; however, there are some inconsistencies in the data. In experimental research, this association between low ethanol sensitivity and high ethanol intake has not been consistently reported in studies that have used rat lines that have been genetically selected for differences in ethanol intake under continuous access conditions (e.g., UChA versus UchB, P versus NP, AA versus ANA). The present study investigated ethanol sensitivity in high (HARF) and low (LARF) ethanol-preferring rats selectively bred under limited-access conditions. For comparative purposes, motor impairment induced by diazepam was also examined. Motor impairment was assessed using the tilt plane. Ethanol (1.25, 2.0, and 2.5 g/kg, intraperitoneally) was administered to ethanol-naive male and female HARF and LARF rats, and their performance was assessed at t = 0, 30, and 60 min. Blood ethanol levels were measured in a separate group of ethanol-naive rats. Finally, in a separate group of male and female HARF and LARF rats, diazepam-induced (1, 3, and 10 mg/kg, intraperitoneally) motor impairments were evaluated in a similar manner. In the ethanol study, HARF rats showed greater dose-dependent impairments than their LARF counterparts. Male rats exhibited greater sensitivity to ethanol-induced impairment than their female counterparts. These observations were unrelated to sex or line differences in the blood ethanol levels achieved. Similar impairments were observed with diazepam, with HARF rats exhibiting greater motor impairment than LARF rats. The results suggest that selective breeding for high and low ethanol drinking in a limited-access paradigm has led to inherent differences in sensitivity to ethanol- and diazepam-induced motor impairments. The pattern of diazepam-induced impairments suggests possible variations in GABA(A) receptor activity, although more research is necessary to determine such involvement.

Characterizing the ontogeny of ethanol-associated increases in corticosterone

The following experiments were conducted as adjuncts to previous work in an effort to characterize the ontogenetic profile of the elevations in corticosterone after ethanol challenge. In Experiment 1, female and male Sprague–Dawley rats were administered intraperitoneally either a 1.5- or a 4.5-g/kg dose of ethanol on postnatal day (PND) 16, 26, 36, or 56. Blood samples were collected at 40, 80, or 160 min after ethanol injection and analyzed by means of radioimmunoassay for corticosterone levels and correlated with brain alcohol levels (BrALs) determined from brain samples collected at the same time intervals. In Experiment 2, the ethanol dose was varied ontogenetically to equate functional impairment across age, with the use of intraperitoneal doses of ethanol of 3.2, 2.6, or 2.2 g/kg, to induce equivalent amounts of ethanol-induced motor impairment in infant (PND 22), adolescent (PND 28), or adult (PND 60) rats, respectively. Animals were tested on a swim task 15 min after injection, with blood and brain samples collected immediately after the swim and analyzed for corticosterone levels and BrALs as in Experiment 1. Reminiscent of previous reports of an age-related increase in sensitivity to the hypnotic and motor-impairing effects of ethanol, the corticosterone response to an ethanol challenge increased at least through adolescence, with sex differences emerging by PND 26 and becoming more pronounced in adulthood. To the extent that corticosterone release is involved in the reinforcing effects of drugs, ontogenetic differences in the response of the hypothalamic–adrenal–pituitary (HPA) axis to ethanol could contribute to the excessive alcohol consumption often observed during adolescence.

Characterizing the ontogeny of ethanol-associated increases in corticosterone

The following experiments were conducted as adjuncts to previous work in an effort to characterize the ontogenetic profile of the elevations in corticosterone after ethanol challenge. In Experiment 1, female and male Sprague–Dawley rats were administered intraperitoneally either a 1.5- or a 4.5-g/kg dose of ethanol on postnatal day (PND) 16, 26, 36, or 56. Blood samples were collected at 40, 80, or 160 min after ethanol injection and analyzed by means of radioimmunoassay for corticosterone levels and correlated with brain alcohol levels (BrALs) determined from brain samples collected at the same time intervals. In Experiment 2, the ethanol dose was varied ontogenetically to equate functional impairment across age, with the use of intraperitoneal doses of ethanol of 3.2, 2.6, or 2.2 g/kg, to induce equivalent amounts of ethanol-induced motor impairment in infant (PND 22), adolescent (PND 28), or adult (PND 60) rats, respectively. Animals were tested on a swim task 15 min after injection, with blood and brain samples collected immediately after the swim and analyzed for corticosterone levels and BrALs as in Experiment 1. Reminiscent of previous reports of an age-related increase in sensitivity to the hypnotic and motor-impairing effects of ethanol, the corticosterone response to an ethanol challenge increased at least through adolescence, with sex differences emerging by PND 26 and becoming more pronounced in adulthood. To the extent that corticosterone release is involved in the reinforcing effects of drugs, ontogenetic differences in the response of the hypothalamic–adrenal–pituitary (HPA) axis to ethanol could contribute to the excessive alcohol consumption often observed during adolescence.

Functional Ethanol Tolerance in Drosophila

In humans, repeated alcohol consumption leads to the development of tolerance, manifested as a reduced physiological and behavioral response to a particular dose of alcohol. Here we show that adult Drosophila develop tolerance to the sedating and motor-impairing effects of ethanol with kinetics of acquisition and dissipation that mimic those seen in mammals. Importantly, this tolerance is not caused by changes in ethanol absorption or metabolism. Rather, the development of tolerance requires the functional and structural integrity of specific central brain regions. Mutants unable to synthesize the catecholamine octopamine are also impaired in their ability to develop tolerance. Taken together, these data show that Drosophila is a suitable model system in which to study the molecular and neuroanatomical bases of ethanol tolerance.

Chronic ethanol treatment and GABA A receptor a6 subunit gene expression: a study using a6 subunit-deficient mice

Chronic alcohol administration increases the expression of cerebellum-specific GABA A receptor α6 subunit mRNA, protein and selective autoradiographical fingerprint on rat and mouse brain sections. We have tested whether the α6 gene is activated by chronic alcohol administration (daily p.o. injection of 2 g/kg during the first 3 days and 2.5 g/kg during the next 17 days) that produced tolerance in the rotarod test to motor impairment by acute challenge of ethanol (2 g/kg, i.p.). We utilized a mouse line engineered to express E. coli β-galactosidase enzyme and an unfunctional truncated α6 subunit under the control of the α6 gene promoter. Chronic ethanol treatment failed to alter the cerebellar β-galactosidase activity when compared with no treatment and isocaloric sucrose treatment in groups of α6 subunit-deficient mice. The results suggest that tolerance to motor-impairing effects of ethanol can be achieved in the absence of α6 subunit-containing GABA(A) receptors, but that the reported upregulation of α6 gene transcription by ethanol treatment requires functional α6 subunits.

Effect of naltrexone on acute tolerance to ethanol in UChB rats

The effect of naltrexone (a non-selective opioid receptor antagonist) on both alcohol consumption in a voluntary selection situation and acute tolerance to the motor impairment effect of ethanol was examined in female high alcohol-drinking (UChB) rats using the tilting plane test. In experiment 1, the effect of naltrexone on alcohol consumption was studied in UChB rats which were given a daily 1-h period access to a 10% (v/v) ethanol solution with food and water always available. Naltrexone in doses of 5 or 10 mg/kg intaperitoneal (i.p.) caused dose-dependent reduction in voluntary alcohol intake by 45% and 66%, respectively, without altering daily water intake. In experiment 2, the effect of naltrexone (5 mg/kg i.p.) on acute tolerance to motor impairment effect of a dose of 2.3 g ethanol/kg i.p. was examined. A comparison of control (C) and naltrexone (Nal) UChB groups indicated that naltrexone slowed the recovery of the motor activity and reduce acute tolerance development at comparable ethanol levels in cerebral blood. These results suggest a contribution of the opioid system to acute tolerance to ethanol.

Lack of Tolerance to Ethanol-Induced Motor Impairment on Accelerod Performance in Rats

The effect of ethanol on rats was investigated at increasing rates of acceleration for bar rotation speed. Ethanol was given to rats by a liquid diet starting with 2.4% ethanol (v/v) for 3 days. Then the ethanol concentration was increased to 4.8% (v/v) for 3 days and finally to 7.2% (v/v) for 15 days. Accelerod performance was recorded before and throughout 20 days of ethanol intake. Mean blood ethanol levels were 266.34 ± 13.11 and 285.20 ± 9.77 mg/dl on the 7th and 15th days of ethanol (7.2% v/v) consumption, respectively, as measured in a parallel group of animals. Ethanol produced significant concentration-dependent impairments in the accelerod performance of rats. The motor impairment effect of ethanol was most prominent in the test using the greatest rate of acceleration (from 0 to 79 rpm within 2 min). The impairment effect of ethanol on accelerod performance occurred throughout the period of ethanol exposure. Our results indicate that motor impairment on the accelerod performance test produced by an ethanol liquid diet depends on the concentration of ethanol and the rate of acceleration. In addition, under free-access conditions accelerod performance may not be a suitable behavioral test for detecting tolerance development to ethanol in rats.

Rapid Tolerance and Crosstolerance to Motor Impairment Effects of Benzodiazepines, Barbiturates, and Ethanol

Motor impairment (tilt-plane test) test was used to assess the phenomenon of rapid tolerance and crosstolerance to benzodiazepines, barbiturates, and ethanol. The motor impairment responses to benzodiazepines (chlordiazepoxide and diazepam) and to various barbiturates (pentobarbital, phenobarbital, and barbital) were significantly reduced on day 2 in rats that had been treated on day 1 with benzodiazepines and barbiturates, respectively, compared to the control group treated with saline on day 1. Benzodiazepine treatment on day 1 resulted in rapid crosstolerance to the motor impairment effects of ethanol on day 2. Benzodiazepine treatment, however, did not result in rapid crosstolerance to the three barbiturates (pentobarbital, barbital, and phenobarbital) tested. In contrast to the lack of rapid crosstolerance to barbiturates after treatment with benzodiazepines, barbiturate treatment clearly conferred rapid crosstolerance to benzodiazepines and to ethanol. This asymmetry of rapid crosstolerance raises the possibility that benzodiazepines and barbiturates invoke tolerance by mechanisms that are not wholly identical. Therefore, tolerance to the broad range of actions of barbiturates would include crosstolerance to the effects of benzodiazepines, whereas tolerance to benzodiazepines would include only a weak or partial crosstolerance to some of the effects of barbiturates.

Differential development of acute tolerance to the motor impairment and anticonvulsant effects of ethanol.

The development of acute tolerance to the motor impairment and anticonvulsant effects of ethanol was examined. Acute tolerance to the motor impairment effect of ethanol was shown by a decrease in the degree of intoxication, as measured on the moving belt task, at higher blood ethanol levels ranging from 206 to 256 mg/dl. There was no evidence of acute tolerance to the anticonvulsant effect of ethanol in rats tested over the same time period. These results indicate that, like chronic tolerance, acute tolerance to ethanol develops at different rates for different effects of the drug. The fact that chronic tolerance to the anticonvulsant effect of ethanol has been well documented raises doubts about the assumption that similar physiological changes underlie acute and chronic tolerance to a drug effect, and support the idea that the relationship between acute and chronic tolerance is more complex than previously thought.

Influence of intoxicated practice on the development of acute tolerance to the motor impairment effect of ethanol.

The influence of practice while under intoxication was tested on the development of acute tolerance to the motor impairment effect of ethanol. In experiment 1, the motor impairment effect induced by an IP injection of 1.8 g/kg ethanol was quantified after various intervals in separate groups of animals. Lower impairment scores were observed in rats tested at 30 and 45 min after ethanol administration than in those tested at 15 min. In group that was tested repeatedly after ethanol administration, intoxication decreased more rapidly and to a greater extent. The same phenomenon was observed in experiment 2 when a higher dose of ethanol (2.2 g/kg) and later testing (60-180 min after ethanol administration) were employed. To maintain constant blood ethanol levels, those tested at later times received a supplementary dose of ethanol. Impairment scores were lower in rats tested at later times than in those tested earlier. Again, the impairment scores for the practice group decreased more rapidly and to a greater extent. Blood ethanol levels among various groups were essentially the same. Acute tolerance to ethanol can develop without opportunity for practice while under intoxication. Intoxicated practice, however, can facilitate acute tolerance development.

Role of initial sensitivity and genetic factors in the development of tolerance to ethanol in AT and ANT rats

The role of initial sensitivity and genetic factors in the development of tolerance to ethanol were examined in rats selected for low (AT) and high (ANT) sensitivity to the motor impairment effect of ethanol. Following chronic ethanol treatment (5 g/kg PO, daily for 20 days), the AT and ANT rats acquired tolerance to the motor impairment effect of ethanol at a similar rate. The AT rats, however, acquired tolerance to the hypothermic effect of ethanol at a higher rate than the ANT rats. Such ethanol treatment did not produce any metabolic tolerance to ethanol in these animals. Since there is no difference in the initial response to the hypothermic effect of ethanol between the AT and ANT rats, the observed differences in the rate of tolerance development might be related to a direct genetic factor. The similar rate of tolerance development to the motor impairment effect of ethanol between the two lines was attributed to an interaction between an indirect (initial sensitivity) and a genetic factor in tolerance development.

GABA turnover in the brain of rat lines developed for differential ethanol-induced motor impairment

The role of GABAergic neurons in the differential sensitivity to ethanol between the AT (Alcohol Tolerant) and ANT (Alcohol Nontolerant) rat lines developed for low and high degree of motor impairment from ethanol, was studied by comparing the effect of ethanol (2 or 4 g/kg, IP) on GABA turnover in different regions of the brain in these rat lines. GABA turnover was estimated from the accumulation of GABA after inhibition of GABA aminotransferase with aminooxyacetic acid (AOAA, 50 mg/kg, IP) given 10 min after administration of ethanol. The rats were killed two hours after the AOAA treatment with focused microwaves. The concentrations of GABA, aspartate, glutamate, glutamine and taurine were analyzed with HPLC. The saline-treated ANT rats were found to have a higher concentration of GABA in the striatum and a higher rate of GABA accumulation in the cerebellum than the AT rats. Ethanol suppressed the accumulation of GABA in both lines, but the suppression was significantly greater in the AT rats than in the ANT rats. In specific regions, this line difference was significant in the cerebral cortex and cerebellum with the higher ethanol dose. No line differences were found in the brain or tail blood ethanol concentration. AOAA increased the concentration of glutamine, decreased that of aspartate and glutamate, and did not modify that of taurine. The AOAA-induced changes in the concentrations of these amino acids were, however, minor relative to those found in the concentrations of GABA. The results that GABAergic mechanisms are involved in the differential sensitivity to the motor-impairing effects of ethanol between the AT and ANT rats.

Roles of intoxicated practice in the development of ethanol tolerance.

The development of tolerance to the motor impairment effect of ethanol was examined in separate groups of rats receiving and not receiving intoxicated practice. Tolerance to the motor impairment effect of ethanol developed whether or not rats received intoxicated practice during chronic ethanol treatment. Depending on the treatment dosage and test dose, intoxicated practice might enhance the level of tolerance attained. Tolerance to other effects of ethanol (hypothermia and narcosis) developed as a function of the treatment dosage. Intoxicated practice on the moving belt did not modify the development of tolerance to these effects of ethanol. Tolerance to the motor impairment effect of ethanol, however, was retained much longer in the intoxicated practice group following the termination of ethanol treatment.

Effect of GABAergic drugs on motor impairment from ethanol, barbital and lorazepam in rat lines selected for differential sensitivity to ethanol

The effect of GABAergic drugs on the motor-impairing effects of ethanol, barbital, and lorazepam were studied in the ethanol-sensitive ANT (Alcohol Nontolerant) and ethanol-insensitive AT (Alcohol Tolerant) rat lines, selected for differential ethanol-induced motor impairment on the tilting plane. The basic population from which these rat lines were derived, the mixed (M) line, was also included in the study. The ANT rats were more sensitive to the intoxicating effects of ethanol, barbital, and lorazepam than the AT and M rats at the dose ranges tested. Picrotoxin antagonized motor impairment from all three drugs. Flumazenil (Ro 15-1788) antagonized only the effects of lorazepam, and isoniazid did not modify motor impairment induced by any of the three drugs. These results confirm that the selection of AT and ANT lines has not been specific to ethanol, and that it has increased sensitivity to ethanol, barbital, and lorazepam in the ANT rats rather than decreasing it in the AT rats relative to the M rats. The finding that picrotoxin counteracted motor impairment from ethanol, barbital, and lorazepam support the view that the GABA A receptor complex is important in mediating the intoxicating effects of these drugs. These results also suggest that the genetically-determined difference in sensitivity to ethanol between the rat lines involves GABAergic mechanisms, but it remains to be determined whether any part of the GABA A receptor itself has been affected by the selection program.

Factors involved in the differential response to ethanol, barbital and pentobarbital in rats selectively bred for ethanol sensitivity

Two lines of rats, 'least affected' (LA) and 'most affected' (MA), had been selectively bred for their differential sensitivity to ethanol. Both males and females of the LA strain were observed to be less sensitive than their MA counterparts to the acute hypnotic and motor-impairing effects of ethanol. However, a lower ethanol metabolic rate of the MA males suggests that both CNS and metabolic factors contribute to their enhanced sensitivity to ethanol. By contrast, no differences were observed between the LA and MA males with respect to the hypnotic and subhypnotic effects of pentobarbital or to the clearance of this drug. MA females were more sensitive only to the hypnotic effects of pentobarbital, probably because of a smaller apparent volume of distribution. No strain difference was observed in the hypnotic effect or clearance of barbital. These observations suggest that, in spite of a differential sensitivity to ethanol, the LA and MA lines do not differ in their response to the barbiturates tested.

Effect of p-chlorophenylalnine on the loss and maintenance of tolerance to ethanol.

Rats were rendered tolerant to the motor-impairing effects of ethanol by daily oral administration. Subsequently, ethanol was withdrawn and the effect of p-chlorophenylalanine (p-CPA) on tolerance loss was examined. In two separate studies it was demonstrated that p-CPA, in a dosage regimen that produces extensive depletion of brain serotonin (5-HT), accelerated tolerance loss. These experiments suggest that at least part of p-CPA's inhibitory effect on net tolerance development to ethanol can be accounted for by its accelerating effect on tolerance loss; however, an inhibitory effect on tolerance acquisition cannot be excluded. On the other hand, once tolerance was established, p-CPA did not affect the maintenance of tolerance to ethanol.

Acute tolerance to the motor impairment effects of ethanol

Rats were trained to avoid electric shock by running treadmill-fashion along a moving belt. When the animals were consistently performing at a 95% avoidance level, the effects of saline and 1.5, 2.0, and 2.5 g/kg ethanol on motor performance were assessed in three 2-min trials 23 min following IP administration. Results indicated a strong relationship between plasma alcohol concentration and the mean time off the belt for the three trials. Further, the technique differentiated a dose-dependent impairment in responding. In addition, the results indicated that the motor impairment effects diminished for all dose levels over the 7-min test period. It is unlikely that the improved performance across the three trials is due to changes in metabolism or increased duration of exposure to the drug. The data suggest that the moving belt test is sensitive to practice effects and that a behavioral augmentation to the motor impairment effects of ethanol can occur within several minutes.