LAS Rats Research Articles

Developmental alterations of ethanol sensitivity in selectively bred high and low alcohol sensitive rats

Initial sensitivity and acute tolerance to ethanol have been implicated as risk factors in the development of alcoholism in humans. These behaviors were investigated in rats selectively bred for differences in hypnotic sensitivity following their first dose of ethanol in two different experiments. In Experiment 1, developmental profiles of the association between initial sensitivity and acute tolerance induced by a single exposure to ethanol were examined using male and female high, low, and control alcohol sensitive (HAS, LAS, and CAS) rats. Dose–response curves were constructed for duration of the loss of the righting reflex and for blood ethanol concentration (BEC) at the regain of the righting reflex. Animals were tested with a single ethanol dose ranging from 1.5 to 5.0 g/kg at either 15, 25, 40, 70, 120, or 180 days of age (DOA). For each group, acute tolerance to ethanol was estimated by the slope of the regression line using dose of ethanol and mean BEC at regain. In general, all rat lines showed an increase in hypnotic sensitivity to ethanol with age. To a large degree, the lower sensitivity observed in 15 and 25 DOA HAS and LAS rats was associated with an increase in the development of acute ethanol tolerance relative to older rats. Divergence of the LAS and CAS lines was evident by 25 DOA and remained stable with advancing age. However, HAS rats did not differ significantly from CAS rats until 40 DOA, after which the magnitude of the difference continued to increase with age. In Experiment 2, rats were treated with alcohol at 25, 70, or 180 DOA. Rats at 70 or 180 DOA required less ethanol to disrupt their motor coordination on a rotating dowel (rotarod). Blood ethanol levels were determined at the loss and subsequent regain of the ability to negotiate the rotarod. Total duration of inability to negotiate the rotarod also was recorded. HAS rats were less able to remain on a rotarod while under the influence of alcohol relative to LAS and CAS rats regardless of age. However, no evidence of acute tolerance was observed in this experiment and, in fact, there was evidence of reverse tolerance in that all animals had lower BEC values at regain of ability than they did at loss.

Relationship of Brain Ethanol Metabolism to the Hypnotic Effect of Ethanol. II: Studies in Selectively Bred Rats and Mice

To clarify the role of brain acetaldehyde in the hypnotic effect of ethanol, we compared the ethanol-oxidizing capacity (rate of acetaldehyde accumulation) and catalase and aldehyde dehydrogenase activity in the brains of animals genetically selected for different sensitivities to the hypnotic effect of ethanol. We used high, low, or control alcohol-sensitive rats (HAS, LAS, and CAS) and short- and long-sleep mice (SS and LS), as well as SS x LS recombinant inbred mice with known strain differences in mean duration of ethanol-induced sleep. We studied the rate of accumulation of acetaldehyde from ethanol in brain homogenates of these animals and correlated those values with their hypnotic sensitivity to ethanol. Acetaldehyde accumulation from ethanol was significantly higher in the brain homogenates from HAS rats and LS mice with high sensitivity to the hypnotic effect of ethanol in vivo, compared with LAS rats and SS mice with low sensitivity to ethanol. A correlation was found between the duration of ethanol-induced sleep and the in vitro rate of accumulation of ethanol-derived acetaldehyde in the brains of recombinant SS x LS mice strains. There was no correlation of sleep time with brain catalase levels. There were no line differences in brain catalase or aldehyde dehydrogenase or in alcohol or aldehyde dehydrogenase activity in livers of LAS, CAS, and HAS rats or in SS and LS mice. A correlation between the brain acetaldehyde accumulation, but not catalase levels, and the central effect of ethanol was demonstrated in animals genetically differing in initial sensitivity to the hypnotic effect of ethanol.

Role of GABA in the Actions of Ethanol in Rats Selectively Bred for Ethanol Sensitivity

Rats from the N/Nih heterogeneous stock have been selectively bred for high (HAS) or low (LAS) initial sensitivity to injected ethanol as measured by duration of the loss of the righting reflex. The selection for ethanol sensitivity in these lines apparently has reached a maximum. These lines are useful to elucidate the central nervous system mechanisms of the genetic differences between the lines and also provide clues to the mechanisms of ethanol’s action. We have found that: 1) ethanol, etomidate, and ketamine but not propofol produce different sleep times and brain levels of the drug on awakening between these two lines; 2) only ethanol, etomidate, and ketamine produced significant differences between the HAS and LAS rats in GABA-mediated stimulation of chloride uptake into brain microsacs; 3) GABA, propofol, and etomidate decreased the K d for flunitrazepam binding to whole-brain membranes but equally in both lines. Neither ethanol nor ketamine had an effect; 4) only GABA, ethanol, and etomidate increased the K d for TBPS binding and only GABA decreased B max of TBPS binding. As with the previous selection for ethanol sensitivity in mice (short and long sleep) these lines of rats have very marked line differences in GABA-mediated events, and these are correlated with the sedative effects of ethanol. From these and previous studies we know that the major differences between selected lines of mice and rats are that the mouse lines are not differentially sensitive to halothane or pentobarbital while the rat lines are. However, the mouse lines are differentially sensitive to propofol and the rat lines are not. These data should be useful in dissecting the actions of ethanol at the GABA A receptor.

Cardiovascular Effects of Ethanol in Rats Selectively Bred for High or Low Sensitivity to the Hypnotic Effects of Ethanol

The cardiovascular effects of acute ethanol administration were studied in rats selectively bred for high (HAS) or low sensitivity (LAS) to the acute hypnotic effect of ethanol. In chronically cannulated, conscious animals, the intravenous administration of 1 g/kg of ethanol caused moderate hypotension and tachycardia in both HAS and LAS rats. The phenylephrine-induced reflex bradycardia and the baroreflex slope were significantly reduced by ethanol only in HAS and not in LAS rats. In urethane-anesthetized HAS rats, the baroreflex inhibitory action of ethanol was present under control conditions, but it was absent after depletion of endogenous GABA by treatment of the rats with 100 mg/kg of 3-mercaptopropionate. These findings confirm the role of endogenous GABA in the baroreflex inhibitory action of ethanol, and are compatible with the documented differential sensitivity of the GABAA, receptor complex to ethanol in HAS and LAS rats.

Neurotensin levels and receptors in HAS and LAS rat brains: Effects of ethanol

Previous studies of neurotensin (NT) levels and NT receptor densities in specific brain regions of mice selectively bred for differences in sensitivity to ethanol have shown that NTergic processes may mediate some actions of ethanol. In the present study, we have determined the levels of NT and NT receptor densities in specific brain regions of HAS and LAS rats that have been selectively bred for differences in sensitivity to ethanol-induced loss of righting response. Regional differences in NT levels were observed in brains from both HAS and LAS rats and values in hypothalamus, ventral midbrain, and nucleus accumbens from female rats were 25 to 75% higher than levels in corresponding regions from male rats. However, there were no significant line differences in NT-ir levels in corresponding regions from HAS and LAS animals. High-affinity binding (NTH B max values), measured by Scatchard analyses, were higher in ventral midbrain from HAS males than from LAS males. NTH receptor densities were higher in HAS males than in HAS females; sex differences were not observed in the LAS line. There were no significant line or sex differences between HAS and LAS in low-affinity (NT L) B max values in any brain region. In HAS females, subhypnotic doses of ethanol produced a decrease in NT levels in nucleus accumbens, whereas, hypnotic doses caused an increase in NT levels. Likewise, hypnotic doses elicited increases in NT levels in hypothalamus of female HAS and LAS, but not in ventral midbrain or caudate putamen. These results are consistent with low dose activation of mesolimbic and nigrostriatal dopaminergic neurons in which NT is colocalized with dopamine and with high dose inhibition of these pathways.

Differential open field reactivity in HAS and LAS rats

To identify potential differences in emotional reactivity in high (HAS) and low (LAS) alcohol-sensitive rats in open field tests, 20 ethanol naive, 19th generation HAS and 20 LAS rats from the University of Colorado's Alcohol Research Center each received three consecutive daily 20-min exposures to a 1-sq m open field. The results of repeated-measures ANOVA and Keuls tests, performed on each of five concurrently recorded behaviors, indicated that, compared to HAS rats, LAS animals exhibited significantly greater latencies to begin ambulation, elevated ambulation, and rearing scores that failed to habituate over 3 days, and an increasing intersession bolus count. In addition, significant positive correlations occurred between days 2 + 3 (but not day 1) ambulation and bolus counts in LAS but not HAS rats, and between day 1 ambulation and (i) rearings and (ii) center square entries in HAS but not LAS rats. Together with other cited data, these results provide support for a hypothesis of relatively greater emotional reactivity in LAS rats and illustrate the need for multiple measures and sessions in evaluating open field behavior.

Selective breeding of rats differing in sensitivity to the effects of acute ethanol administration.

Selective breeding of rats for sensitivity to the anesthetic effects of ethanol is being carried out with rats derived from the genetically heterogeneous N/Nih stock. Thirteen generations of within family selection have been achieved with replicate high (HAS), low (LAS) and control alcohol sensitive (CAS) lines. Significant separation between lines on sleep time and blood ethanol concentration (BEC) at awakening following ethanol administration has been achieved. In general, the results obtained so far replicate the findings with short (SS) and long (LS) sleep mice. One exception is that the high alcohol sensitivity rats (HAS) also appear more sensitive to pentobarbital relative to LAS rats. This finding is opposite to that which occurs with SS and LS mice where the low ethanol sensitive SS mice appear more sensitive to pentobarbital than the LS mice.

Gamma‐Aminobutyric Acid‐Activated Chloride Channels in Rats Selectively Bred for Differential Acute Sensitivity to Alcohol

Effects of various sedative hypnotic agents on GABA-mediated chloride flux were evaluated in whole brain membrane vesicles (microsacs) prepared from rats selectively bred for high (HAS) and low sensitivity (LAS) to an acute hypnotic dose of alcohol. The HAS rats were more sensitive to the effects of pentobarbital, phenobarbital, flunitrazepam, and ethanol on GABA-mediated chloride flux compared with the LAS rats. No differences between the lines in GABA-stimulated chloride flux were observed. Modulation of 1-[3H]-phenyl-4-butyl-2,6,7-trioxabicyclo(2.2.2)octane ([3H]-TBOB) and [3H]-diazepam binding also was measured. The lines did not differ in inhibition of [3H]-TBOB binding by pentobarbital, phenobarbital, muscimol or picrotoxin. Although the lines displayed almost identical KD and Bmax for [3H]-diazepam binding, the GABA agonist, muscimol, was a more potent stimulator of [3H]-diazepam binding in membranes prepared from HAS rats than from LAS rats. These findings are discussed in light of previous work using other selected lines.