Where are the mu receptors that mediate opioid analgesia? An autoradiographic study in the HAR and LAR selection lines.

One line (strain) of mouse has been selectively bred in our laboratory for 15 generations to exhibit a very high sensitivity to levorphanol-induced analgesia on the hot plate assay (HAR or high antinociceptive response line). Concurrently, a second line (LAR or low antinociceptive response line) has been bred in the opposite direction, i.e., to exhibit a very low sensitivity under the same conditions. This has resulted in a 7-fold difference in sensitivity between HAR and LAR mice as a result of changes in gene frequency. Receptor autoradiographic studies with 3H-DAGO were carried out in the central gray to find receptor populations differing greatly in density between HAR and LAR mice to parallel their in vivo sensitivity differences: such receptors would then be implicated in mediating in vivo analgesia. The caudal portions of the dorsal raphe nucleus (DRN) showed 1.5- to 2-fold differences in density of mu sites, while the periaqueductal gray (PAG) showed relatively small differences. These results strongly suggest that mu receptors in a portion of the DRN are involved in mediating analgesia due to systemically administered opioids in this population of mice.

Hexokinase redistribution in vivo.

Heterogenous stock mice in addition to mice selectively bred to maximally differ in their severity of alcohol withdrawal seizures (withdrawal seizure-resistant (WSR) and withdrawal seizure-prone (WSP] were used to provide evidence in favor of the importance of the rapidly changing distribution of brain hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) (HK). An ischemic response at 15, 30, 60 and 120 s after killing showed a decreasing cerebellar cytosolic HK concentration of 31%, 15%, 14% and 10% while the cerebral concentrations were 23%, 13%, 13% and 14%, respectively. WSR and WSP mice given an acute i.p. dose of 4 g/kg of alcohol showed opposite HK responses. Cytosolic HK in WSR mice decreased 18.5%, while WSP mice showed an increase of 20.3% over paired saline-injected controls. When ischemia was allowed to proceed in WSP mice following an in vivo alcohol treatment, cytosolic HK decreased in parallel to mice not given alcohol. These data suggest that alcohol can cause an HK redistribution in vivo which could play a role in the differing sensitivities of WSR and WSP mice to alcohol related seizures.

Ethanol and diazepam withdrawal convulsions are extensively codetermined in WSP and WSR mice.

Selective breeding was used to produce lines of mice which differ markedly in their genetically-mediated vulnerability to handling-induced convulsions (HIC) associated with the ethanol withdrawal syndrome. These are known as the ethanol withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) selection lines. As a result of 5 generations of selective breeding with ethanol, a 3.4-fold difference between WSP and WSR mice was seen in HIC associated with ethanol withdrawal. When diazepam was used as the dependence-producing drug, a 2.4-fold difference emerged. After 6 more generations of selective breeding with ethanol, an approximate 10-fold difference was seen with ethanol, while with diazepam, this difference in HIC scores was also about 10-fold. This close parallel between ethanol and diazepam indicates that physical dependence on both drugs, as indexed by handling-induced convulsions, is extensively codetermined by the same genes, and thus by the same mechanisms, in these selectively-bred mice.

Ethanol and nitrous oxide produce withdrawal-induced convulsions by similar mechanisms in mice.

Twenty generations of selective breeding were used to produce lines (strains) of mice which differ markedly from one another in ethanol physical dependence development as indexed by handling-induced convulsions (HIC) induced by withdrawal from ethanol. These withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) selection lines now differ by over 10-fold in HIC scores after equivalent exposure to intoxicating levels of ethanol via inhalation. Since handling-induced convulsions can be readily elicited following withdrawal from nitrous oxide, we sought to determine if the very large differences in ethanol withdrawal-induced HIC bred into these selection lines would generalize to nitrous oxide. Following a 60 min exposure to 75% nitrous oxide (in O2), a greater than 10-fold difference in HIC scores, and a 2-fold difference in tremor incidence was seen upon withdrawal in WSP vs. WSR mice. These findings closely parallel those seen with ethanol, and demonstrate that a large degree of commonality exists in the genes and the mechanisms determining these withdrawal signs. HIC elicited by nitrous oxide withdrawal were readily suppressed by ethanol, and HIC elicited by ethanol withdrawal were promptly suppressed by 75% nitrous oxide in WSP mice. Nitrous oxide also suppressed HIC and tremor associated with nitrous oxide withdrawal.

DSLET (D-ser2-leu5-enkephalin-Thr6) produces analgesia on the hot plate by mechanisms largely different from DAGO and morphine-like opioids.

Fifteen generations of selective breeding were used to produce lines (strains) of mice which differ markedly from one another in levorphanol-induced antinociception on the hot plate assay. These are the high antinociceptive response (HAR) and low antinociceptive response (LAR) selection lines, which now differ by over 5-fold in the i.p. dose of levorphanol doubling control (no drug or saline) latency scores. We sought to determine if these large genetically-mediated differences in antinociceptive sensitivity bred into these selection lines with i.p. levorphanol would generalize equally to a series of enkephalin analogues known to differ in their selectivity for mu and delta opioid receptors. DAGO (D-ala2, MePhe4, Gly-ol5 enkephalin), a highly mu selective agent, produced a 67-fold difference between HAR and LAR mice in the slopes of the dose-response curves on the hot plate assay, while DSLET (D-ser2, leu enkephalin Thr6), a delta selective agent, only produced a 5.4-fold difference via the i.c.v. route. DADLE (D-ala, D-ser enkephalin) a slightly delta preferring ligand, was found to be intermediate (17.4-fold difference). These findings demonstrate that selective breeding has been quite successful in altering those genes which control analgesia due to mu selective agents, while relatively little change has occurred in those genes which control analgesia due to delta agonists. Thus, analgesia mediated by the former has been genetically dissociated from analgesia mediated by the latter, implying that DAGO has mechanisms of action largely dependent of DSLET on the hot plate assay. These findings are consistent with the contention that the mu receptor mediates analgesia produced by DAGO, while a different receptor (presumably delta) mediates much of the analgesic effects of DSLET.

Selective breeding for levorphanol-induced antinociception on the hot-plate assay: commonalities in mechanism of action with morphine, pentazocine, ethylketocyclazocine, U-50488H and clonidine in mice.

Selective breeding (selection) was used to bidirectionally alter gene frequencies affecting levorphanol antinociception on the hot-plate assay in mice. After 12 generations of selective breeding, the high antinociceptive response line exhibited about 7 times steeper dose-response curve than did the low antinociceptive response line whereas only small differences were seen with saline alone. The authors sought to determine whether these large, genetically mediated differences in sensitivity bred into the high and low antinociceptive response lines (lineages) with levorphanol would also be evident with other analgesics. Should this occur with any particular drug, this would imply common mechanisms of action between that drug and levorphanol mediated by common gene action. This was found to be the case with morphine, but progressively less similarity to levorphanol was seen with other analgesics with the following rank order: morphine greater than pentazocine greater than ethylketocyclazocine greater than U-50488H greater than clonidine. Thus, the mechanisms of action for the latter compounds are different from levorphanol in varying degrees. The role of sedation produced by some of these drugs was also evaluated and was found to be independent of the antinociceptive effects. Thus, the latter was not confounded by the former in these genetic lines of mice.

Genetic differences in anticonvulsant sensitivity in mouse lines selectively bred for ethanol withdrawal severity.

WSP (withdrawal seizure-prone) mice exhibit approximately 10-fold more severe withdrawal convulsions than WSR (withdrawal seizure-resistant) mice after identical chronic ethanol exposure. Although WSP and WSR mice do not differ in threshold for seizures elicited by electroconvulsive shock (ECS), WSR mice are more sensitive to ethanol-induced elevation of ECS seizure thresholds. The current experiments demonstrated that WSR mice showed more ECS-induced seizure threshold elevation than WSP mice when tested after the administration of C1-C5 straight-chain alcohols. Whereas the brain concentrations of the C1 and C2 alcohols did not differ between the lines, WSP mice tended to have higher brain concentrations than WSR mice of the C3-C5 alcohols, even though they exhibited the smaller behavioral response in all cases. Thus, the difference between WSP and WSR mice was one of neurosensitivity and could not be attributed to pharmacokinetic differences. The WSR line was also more sensitive to ethchlorvynol, methyprylon, barbital, phenobarbital, pentobarbital, diazepam, valproic acid and phenytoin in this test. Examining loss of righting reflex (RR), we found that WSP and WSR mice did not differ in ED50, latency to lose RR or duration of loss of RR. Thus, the genetic anticonvulsant sensitivity difference is not simply a genetic difference in sensitivity to central nervous system depression between the lines. In summary, WSR mice were more sensitive to the anticonvulsant effects of a variety of compounds than WSP mice, suggesting that some genes influence both ethanol withdrawal seizures and ethanol's anticonvulsant effects.

Sample preparation for inositol measurement: Sep-Pak C18 use in detergent removal.

The enzymatic-fluorometric method of myo-inositol quantitation by L.C. MacGregor and F.M. Matschinsky (1984, Anal. Biochem. 141, 382-389) has proved to be very useful in accurately measuring small amounts of myo-inositol. Although this procedure is quite satisfactory, relatively high concentrations of detergents, salts, NADH, and malate interfere. To extend the usefulness of the MacGregor and Matschinsky method we report here an extraction procedure which removed the interfering substances, yet allowed the recovery of close to 100% of the inositol. The procedure involved first passing the sample through a Sep-Pak C18 cartridge to remove detergent and then through a mixed-bed resin to remove the ionic constituents. The procedure with the Sep-Pak C18 cartridge is applicable to a wide variety of biological samples requiring detergent removal.