Anticonvulsive effects of kappa-opioid receptor modulation in an animal model of ethanol withdrawal

Although the neurochemical mechanisms contributing to alcohol withdrawal seizures are poorly understood, withdrawal seizures probably reflect neuronal hyperexcitability resulting from adaptation to chronic alcohol. Altered kappa-Opioid receptor (KOP-R) signaling has been observed in multiple seizure types; however, a role for this system in ethanol withdrawal seizures has not been systematically characterized. We hypothesized that pharmacological manipulations of the KOP-R would alter withdrawal in mice selectively bred for differences in ethanol withdrawal severity. Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice were made physically dependent using chronic ethanol vapor inhalation, and the effects of the KOP-R antagonist nor-binaltorphimine or agonist U-50,488H on withdrawal severity were examined. Pretreatment with nor-binaltorphimine significantly increased handling-induced convulsion (HIC) severity in withdrawing WSR mice, with no observable effects in withdrawing WSP mice. In contrast, U-50,488H significantly decreased HIC severity in WSP mice, with no effects in WSR mice. During extended withdrawal (i.e. hours 12+), a rebound hyperexcitability was observed in WSP mice given agonist. Thus, administration of a KOP-R antagonist increased withdrawal severity in mice normally resistant to withdrawal seizures, while a KOP-R agonist reduced convulsion severity in animals susceptible to withdrawal seizures. These observations are consistent with differences in the KOP-R system observed in these lines at the molecular level, and suggest the KOP-R system may be a promising therapeutic target for management of ethanol withdrawal seizures. Finally, these findings underscore the importance of determining the potential for rebound increases in withdrawal severity during later withdrawal episodes.     

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.     

Relationship of membrane physical properties to alcohol dependence in mice selected for genetic differences in alcohol withdrawal

Genetic selection based on severity of withdrawal seizures following inhalation of ethanol vapor has produced two lines of mice, WSR (withdrawal seizure resistant) and WSP (withdrawal seizure prone), that differ markedly in withdrawal signs. In the present study, we report that these mice also differed in the severity of withdrawal seizures following consumption of an ethanol-containing liquid diet but did not differ in ethanol intake. In contrast to ethanol withdrawal seizures, the lines displayed similar sensitivity to electrical- or pentylenetetrazole-induced seizures. These results suggest that the lines differ in the development of physical dependence on ethanol rather than seizure sensitivity per se. Because decreased synaptic membrane fluidity has been associated with ethanol dependence, we used fluorescence polarization of diphenylhexatriene and trimethylammonium-diphenylhexatriene to evaluate membrane fluidity in WSP and WSR mice fed lab chow, an ethanol-containing liquid diet, or an isocaloric sucrose-containing liquid diet. Fluidity of brain synaptic membranes was identical for WSP and WSR mice fed lab chow. The control liquid diet did not alter membrane fluidity, and the ethanol diet decreased fluidity equally for WSP and WSR mice. Thus, the genetic difference in development of ethanol dependence found in these lines was not reflected in the physical properties of brain membranes.     

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.     

Genetic selection for ethanol withdrawal severity: Differences in replicate mouse lines

We report an ongoing within-family selective breeding project for the severity of handling-induced withdrawal seizures in mice made physically dependent on ethanol by inhalation. Two Withdrawal Seizure Prone (WSP) and two Withdrawal Seizure Resistant (WSR) lines have been subjected to five generations of selection, and two control (WSC) lines are maintained. Each WSP line had more severe and each WSR line had less severe withdrawal convulsions than its respective WSC line. Differences relative to control lines were more pronounced in the WSP lines and were not due to differences in effective dose of ethanol. Heritabilities were higher in the WSP lines than in the WSR lines. These lines will be useful for studying physiological determinants of ethanol dependence and withdrawal.     

Selected line difference in sensitivity to a GABAergic neurosteroid during ethanol withdrawal

The neurosteroid allopregnanolone (ALLO) is a potent positive modulator of gamma-aminobutyric acid(A) (GABA(A)) receptors. Earlier work indicates that sensitivity to the anticonvulsant effect of ALLO was enhanced during ethanol (EtOH) withdrawal in rats and in C57BL/6 mice, an inbred strain with mild EtOH withdrawal. In contrast, ALLO sensitivity was reduced during EtOH withdrawal in DBA/2 mice, an inbred strain with severe EtOH withdrawal. Thus, the present studies examined ALLO sensitivity during EtOH withdrawal in another animal model of EtOH withdrawal severity, the Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) selected lines. Male mice were exposed to EtOH vapor or air for 72 h. During peak withdrawal, animals were injected with ALLO [0, 3.2, 5, 10 or 17 mg/kg, intraperitoneally (i.p.)] and tested for their sensitivity to the anticonvulsant effect. In separate studies, potentiation of GABA-stimulated chloride uptake by ALLO (10 nm to 10 microm) was assessed in microsacs prepared from mouse brain mice during peak withdrawal. Notably, WSP mice were cross-tolerant to the anticonvulsant effect of ALLO during EtOH withdrawal (i.e. significant decrease in the efficacy of ALLO) when compared with values in air-exposed mice. In contrast, sensitivity to the anticonvulsant effect of ALLO was unchanged during EtOH withdrawal in the WSR line. Functional sensitivity of GABA(A) receptors to ALLO was significantly decreased during EtOH withdrawal in WSP mice in a manner consistent with the change in behavioral sensitivity to ALLO. These findings suggest that mice selectively bred for differences in EtOH withdrawal severity are differentially sensitive to ALLO during EtOH withdrawal.     

Modulation of γ-Aminobutyric AcidA Receptor-Operated Chloride Channels by Benzodiazepine Inverse Agonists Is Related to Genetic Differences in Ethanol Withdrawal Seizure Severity

To determine whether genetic differences in development of ethanol dependence are related to changes in gamma-aminobutyric acidA (GABAA) receptor function, we measured 36Cl- uptake by brain cortical membrane vesicles from withdrawal seizure prone and withdrawal seizure resistant (WSP/WSR) mice treated chronically with ethanol. Muscimol-stimulated chloride flux was not different between WSP and WSR mice before or after ethanol treatment. Also, augmentation of muscimol action by flunitrazepam or inhibition of muscimol action by the inverse agonists Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]- [1,4]benzodiazepine-3-carboxylate) and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) was not different for ethanol-naive WSP and WSR mice. However, chronic ethanol administration enhanced the inhibitory actions of DMCM and Ro 15-4513 on membranes from WSP but not WSR mice. Conversely, chronic ethanol treatment attenuated the action of flunitrazepam on membranes from WSR but not WSP mice, suggesting that the actions of benzodiazepine agonists and inverse agonists are under separate genetic control. These genetic differences in actions of DMCM and Ro 15-4513 indicate that sensitization to benzodiazepine inverse agonists produced by chronic ethanol treatment may be related to development of withdrawal seizures and suggest that differences in the GABA/benzodiazepine receptor complex represent alleles that have segregated during the selection of the WSP/WSR mice.     

Effects of lorazepam tolerance and withdrawal on GABAA receptor operated chloride channels in mice selected for differences in ethanol withdrawal severity.

Withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) mice were treated with 5 mg/kg lorazepam for 7 days via implanted osmotic mini pumps. Following chronic drug treatment, brains were assayed for GABA-mediated chloride flux (GABA-Cl-). Under control (drug naive) conditions, brain membranes prepared from WSP and WSR lines did not differ in flunitrazepam or ethanol stimulation of GABA-mediated 36Cl- uptake, but the WSP lines were more sensitive to inhibition of 36Cl- flux by the inverse agonist, FG-7142. Membranes from lorazepam tolerant WSP and WSR mice were resistant to flunitrazepam- and ethanol-stimulation of GABA-Cl-. Withdrawal from chronic treatment, by an acute injection with the benzodiazepine antagonist RO15-1788, returned flunitrazepam stimulation of GABA-Cl- to near control levels in WSR membranes but not in WSP membranes and restored ethanol modulation of the channel to control levels in both lines. Inhibition of chloride flux by the benzodiazepine partial inverse agonist, FG-7142 was greater in membranes from WSP mice compared with WSR mice. Tolerance to lorazepam increased sensitivity of the WSR membranes to FG-7142 without altering the response in the WSP line. Again, withdrawal restored the Cl- flux response to FG-7142 back to near control levels. Lorazepam tolerance lowered [3H]-flunitrazepam binding affinity slightly only in the WSR strain with no change in binding number. Withdrawal from chronic lorazepam treatment produced no significant change in binding affinity or number. The initial genotypic differences in benzodiazepine inverse agonist sensitivity, may be related to the selection for withdrawal seizure severity. Chronic administration of lorazepam reduces the coupling between the benzodiazepine agonist site and the chloride channel and concomitantly increases coupling between the channel and the inverse agonist site, while withdrawal resets the receptor coupling back to control response levels. However, for the WSP line, this drug environment dependent shift in channel coupling bias appears to be deficient compared with the WSR line.     

Chronic Ethanol Treatment Alters Brain Levels of γ-Aminobutyric AcidA Receptor Subunit mRNAs: Relationship to Genetic Differences in Ethanol Withdrawal Seizure Severity

Chronic ethanol treatment is known to alter the function of the gamma-aminobutyric acidA (GABAA) benzodiazepine receptor complex. To determine if genetic differences in development of ethanol dependence are related to expression of GABAA receptor subunits, we measured whole brain levels of mRNA for the alpha 1, alpha 3, alpha 6, gamma 2s, gamma 2L, and gamma 3 receptor subunits in withdrawal seizure-prone and -resistant (WSP and WSR, respectively) mice fed an ethanol-containing liquid diet or a control diet. Brain poly(A)+ RNA was converted to cDNA and amplified by the polymerase chain reaction using primers conserved among GABAA receptor subunits. Quantification was carried out by densitometric analysis of Southern blots generated using subunit-specific probes. Chronic ethanol treatment decreased the content of alpha 1 mRNA in WSP but not WSR mice and decreased the content of alpha 6 mRNA in WSR but not WSP mice. The content of gamma 3 mRNA was increased by chronic ethanol in both lines. In untreated mice, the WSP line had lower levels of alpha 3 and alpha 6 mRNA than the WSR line. Thus, a decrease in the content of alpha 1 mRNA is most clearly linked with development of withdrawal signs, although the amounts of alpha 6 and alpha 3 may also be important in the genetic differences between WSP and WSR mice. In contrast, levels of mRNA for gamma 2S and gamma 2L subunits do not appear to be altered in ethanol dependence.     

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.     

Increased CNS sensitivity to flurothyl as a measure of physical dependence in mice following morphine,phenobarbital, and ethanol treatment

Male C57BL/6 mice were administered either morphine, phenobarbital or ethanol in their drinking water in order to make them dependent. During withdrawal onset of myoclonus, clonus and tonus was evaluated with flurothyl, a convulsant inhalant. Morphine, phenobarbital and ethanol treated subjects displayed significantly lower latencies for myoclonic and clonic convulsive behavior when compared with their respective controls. The increased CNS excitability observed is characteristic of physical dependence and the flurothyl technique employed is discussed in light of its broad applicability to a number of agents characterized by their dependence producing properties.     

Allosteric Regulation of the N-Methyl-d-Aspartate Receptor-Linked Ion Channel Complex and Effects of Ethanol in Ethanol-Withdrawal Seizure-Prone and -Resistant Mice

Abstract: The effects of ethanol, glycine, and spermidine on the specific binding of [³H]MK-801 were characterized in Triton-treated membranes prepared from the hippocampus and cortex of ethanol-withdrawal seizure-prone (WSP) and -resistant (WSR) mice. Glycine, an allosteric agonist at the NMDA receptor-linked ion channel complex, caused an increase in specific [³H]MK-801 binding to hippocampal membrane preparations. There were no significant differences in EC₅₀ values between the selected lines for the effect of glycine (WSP, 391.7 ± 48.4 nM; WSR, 313.4 ± 77 nM) in the presence of 10 µM NMDA or in the maximal response to the agonist (WSP, 1.75 ± 0.26 pmol/mg of protein; WSR, 1.67 ± 0.22 pmol/mg of protein). The EC₅₀ values for the spermidine-induced increase in [³H]MK-801 binding in membranes from hippocampus in the absence (WSP, 11.7 ± 0.83 µM; WSR, 9.98 ± 1.29 µM) or in the presence of 10 µM glycine and 10 µM NMDA (WSP, 2.1 ± 0.35 µM; WSR, 2.37 ± 0.42 µM) also did not differ. Similar results were obtained in cortical membranes. Saturation isotherms indicated that there was no difference in the density of [³H]MK-801 binding sites, or in their affinity for the radioligand, between the mouse lines. In addition, administration of ethanol by inhalation (24 h) to WSP and WSR mice did not cause an increase in the density of [³H]MK-801 binding sites, and there was no difference in the density or affinity of binding sites between the mouse lines. Withdrawal from ethanol (6 h), which causes an increase in the severity of handling-induced convulsions in WSP mice, also did not alter the binding site density or affinity for radioligand. The results suggest that the characteristics of the NMDA receptor-linked ion channel complex in the tissue preparations described here do not differ in WSP and WSR mice. Thus, genetic differences in seizure susceptibility during ethanol withdrawal can be dissociated from the total density of hippocampal or cortex NMDA receptors under activating conditions.     

THE ANTICONVULSANT ACTION OF GABA-ELEVATING AGENTS: A RE-EVALUATION

Abstract— The GABA-elevating agents, aminooxyacetic acid, hydrazine, and hydroxylamine, all possessed anticonvulsant properties, although to a widely varying degree. Aminooxyacetic acid was the most efficacious in delaying drug-induced seizures in mice whereas hydroxylamine brought about only a slight delay in the onset of seizures. The anticonvulsant action was observed against various convulsant agents regardless of whether the convulsant mechanism might involve a deranged GABA metabolism (allylglycine, isonicotinic acid hydrazide, hydrazine), an interference with GABA function (picrotoxin) or some other mechanism (pentylenetetrazol). The anticonvulsant action was not related in a simple manner to either GABA levels or glutamic acid decarboxylase (GAD) activities but the anomalous situation whereby seizures occurred when the GABA content of brain was above normal could be resolved on the basis of an expression which included changes in both GABA levels and GAD activity. The possibility was proposed that the anticonvulsant action of aminooxyacetic acid involved two separate mechanisms.     

Chromosomal loci influencing chronic alcohol withdrawal severity

Ethanol (alcohol) withdrawal-induced convulsions are a key index of physical dependence on ethanol and a clinically important consequence of alcohol abuse in humans. In rodent models, severity of withdrawal is strongly influenced by genotype. For example, many studies have reported marked differences in withdrawal severity between the WSR (Withdrawal Seizure Resistant) and WSP (Withdrawal Seizure Prone) mouse strains selectively bred for over 25 generations to differ in chronic withdrawal severity. Therefore, we used an F₂ intercross between the inbred WSP and WSR strains for a genome-wide search for quantitative trait loci (QTLs), which are chromosomal sites containing genes influencing the magnitude of withdrawal. We also used the recently developed HW, RHW (high withdrawal) and LW, RLW (low withdrawal) lines selectively bred for the same trait and in the same manner as the WSP, WSR lines. QTL analysis was then used to dissect the continuous trait distribution of withdrawal severity into component loci, and to map them to broad chromosomal regions by using the Pseudomarker 0.9 and Map Manager QT29b programs. This genome-wide search identified five significant QTLs influencing chronic withdrawal severity on Chromosomes (Chrs) 1 (proximal), 4 (mid), 8 (mid), 11 (proximal), and 14 (mid), plus significant interactions (epistasis) between loci on Chr 11 with 13, 4 with 8, and 8 with 14.     

THE ROLE OF GABA METABOLISM IN THE CONVULSANT AND ANTICONVULSANT ACTIONS OF AMINOOXYACETIC ACID

Abstract— At high dosage levels AOAA acted as a convulsant agent in mice and rats but in lower amounts it was an effective anticonvulsant agent against INH-induced seizures, by tripling the time to the onset of the convulsions. AOAA elevated brain GABA levels as a result of a preferential inhibition of the GABA-T enzyme system but, contrary to previous reports, the activity of the GAD enzyme system was also inhibited, even by relatively low dosage levels of AOAA. The state of excitability of the brain following the administration of AOAA was related, within the limits of the present study, to changes in GAD activity and GABA levels, but additional data are required before the relationship can be properly evaluated.     

Neuropharmacological profile of FrPbAII, purified from the venom of the social spider Parawixia bistriata (Araneae, Araneidae), in Wistar rats

The aims of the present study were to investigate the anticonvulsant activity and behavioral toxicity of FrPbAII using freely moving Wistar rats. Moreover, the effectiveness of this compound against chemical convulsants was compared to that of the inhibitor of the GABAergic uptake, nipecotic acid. Our results show that FrPbAII was effective against seizures induced by the i.c.v. injection of pilocarpine (ED(50) = 0.05 microg/animal), picrotoxin (ED(50) = 0.02 microg/animal), kainic acid (ED(50) = 0.2 microg/animal) and the systemic administration of PTZ (ED(50) = 0.03 microg/animal). The anticonvulsant effect of FrPbAII differed from that of nipecotic acid in potency, as the doses needed to block the seizures were more than 10 folds lower. Toxicity assays revealed that in the rotarod, the toxic dose of the FrPbAII is 1.33 microg/animal, and the therapeutic indexes were calculated for each convulsant. Furthermore, the spontaneous locomotor activity of treated animals was not altered when compared to control animals but differed from the animals treated with nipecotic acid. Still, FrPbAII did not induce changes in any of the behavioral parameters analyzed. Finally, when tested for cognitive impairments in the Morris water maze, the i.c.v. injection of FrPbAII did not alter escape latencies of treated animals. These findings indicate that the novel GABA uptake inhibitor is a potent anticonvulsant with mild side-effects when administered to Wistar rats.     

Effects of iloprost, prostaglandin E1 (PGE1) and prostacyclin (PGI2) on chemically and electrically induced seizures in mice

The effects of iloprost (ZK 36,374), a new chemically stable analogue of prostacyclin (PGI2), on strychnine-, pentylene-tetrazol-, and maximal electroshock-induced seizures were studied in mice. The time from the beginning of the injection of the convulsant or inducing electroshock to the stage of persistent seizures was determined, and lack of tonic hindlimb extension was regarded as inhibition of convulsions. In doses of 8 micrograms--16 micrograms kg-1 iloprost already exhibited an anticonvulsant action by markedly reducing the incidence of seizures and mortality following strychnine, pentylenetetrazol or maximal electroshock. The onset of tonic seizures was also reduced by iloprost. PGE1 and PGI2 were generally effective in 7 to 13 times higher doses than iloprost. It is suggested that the anticonvulsant activity of iloprost, PGE1 and PGI2 might involve a common basic mechanism. Due to its efficacy, iloprost is a useful tool in the investigation of the anticonvulsant action of prostaglandins.     

PD117302, a selective non-peptide opioid kappa agonist, protects against NMDA and maximal electroshock convulsions in rats

The pharmacological profile of PD117302 was studied in three rat models of experimental seizures. It was determined that PD117302 is a potent and efficacious anticonvulsant against NMDA (ED50 = 0.27 mg/kg, i.v.) and MES (ED50 = 16.3 mg/kg, s.c.), but not flurothyl, convulsions. Its anticonvulsant profile was dose- and time-dependent, stereospecific and sensitive to naloxone and the selective kappa opioid antagonist nor-binaltorphimine. Given these findings, we suggest that PD117302 acts via the kappa receptor to modulate seizure protection. Furthermore, in view of its marked ability to block NMDA excitotoxicity (including lethality) it seems possible that this drug, or related compounds, may have potential therapeutic utility as a neuroprotective agent.