Oral administration of glycine and polyamine receptor antagonists blocks ethanol withdrawal seizures

Cessation of chronic administration of orally administered large amounts of ethanol for 7 days resulted in a markedly increased frequency of audiogenic seizures in Sprague-Dawley rats. Oral administration of the novel glycine receptor antagonist, L-701,324, produced a dose-dependent (2.5 and 5.0 mg/kg; –30 min) inhibition of ethanol withdrawal signs when measured about 12 h after withdrawal of the ethanol treatment. Similarly, using the same experimental paradigm, oral administration of the specific polyamine receptor antagonist, eliprodil, caused a dose-related (2.0 and 5.0 mg/kg; –30 min) inhibition of ethanol withdrawal-induced audiogenic seizure activity. The inhibition of ethanol withdrawal seizures produced by L-701,324 and eliprodil, respectively, was obtained at doses which by themselves did not change the locomotor activity in naive Sprague-Dawley rats. The findings that L-701,324 and eliprodil are potent inhibitors of seizure activity induced by cessation of chronic ethanol administration and the fact that they, in contrast to currently available NMDA receptor antagonists, do not produce psychotomimetic and/or sedative effects, suggest that these drugs may represent a new class of therapeutically useful pharmacological agents for the treatment of ethanol withdrawal seizures. Furthermore, since there is evidence that eliprodil produces its pharmacological actions through a specific inhibition of NMDAR1 and/or NMDAR2B subunits, these data may indicate that certain NMDA receptor subunits may be of particular importance for the mediation of seizure activity following the discontinuation of chronic ethanol exposure.     

Electroencephalographic correlates of audiogenic seizures during ethanol withdrawal in mice

C57BL/6Bg mice had silver bead electrodes chronically implanted on the surface of the cortex and had their cortical EEG recorded during audiogenic seizures following ethanol withdrawal. For 7 days, the experimental groups were fed a liquid diet containing 6% v/v ethanol ad lib as the only source of food and water. The control group was fed a similar diet containing an isocaloric amount of sucrose. The cortical EEG's of experimental and control groups before, during, and after treatment were virtually identical. Only the experimental group was susceptible to audiogenic seizures. During audiogenic seizures, the cortical EEG showed no sign of spike waves or paroxysmal activity. This is in contrast to picrotoxin convulsions with these same mice as well as to spontaneous convulsions in animals following ethanol withdrawal. Similar EEG observations have been reported on audiogenic seizures from genetic and acoustically primed susceptibilities. Consequently, we suggest that all audiogenic seizure responses, including those during ethanol withdrawal, are a type of subcortical epilepsy.     

Neurons in the periaqueductal gray are critically involved in the neuronal network for audiogenic seizures during ethanol withdrawal

The periaqueductal gray (PAG) is implicated in the network subserving audiogenic seizures (AGS). AGS are seen during ethanol withdrawal (ETX), and the present study examined effects of focal NMDA receptor blockade in PAG during ETX and PAG neuronal firing changes associated with ETX. Bilateral cannulae or microwire electrodes were chronically implanted into PAG. Ethanol was administered intragastrically at 8-h intervals for 4 days, resulting in AGS susceptibility during ETX. Microinjection of a competitive NMDA receptor antagonist, DL-2-amino-7-phosphonoheptanoic acid (AP7) (2 and 5 but not 1 nmol/side), into the PAG suppressed AGS, in part, reversibly. In microwire experiments spontaneous and acoustically evoked PAG neuronal responses in behaving rats were reduced significantly 1 h after initial administration of ethanol. During ETX, when the animals were susceptible to AGS, significant increases in spontaneous and acoustically evoked PAG neuronal firing occurred. PAG neurons exhibited burst firing 2-4 s prior to the tonic-clonic phase of AGS and tonic repetitive firing during this seizure phase, which ceased during post-ictal depression. Increased NMDA receptor function in PAG may be important to the aberrant PAG neuronal firing in AGS, since previous studies observed upregulation of NMDA receptors during ETX, and the present study observed that focal microinjection of a NMDA antagonist into PAG blocked AGS.     

The effects of chronic ethanol administration on amygdala neuronal firing and ethanol withdrawal seizures

Physical dependence on ethanol results in an ethanol withdrawal (ETX) syndrome including susceptibility to audiogenic seizures (AGS) in rodents after abrupt cessation of ethanol. Chronic ethanol administration and ETX induce functional changes of neurons in several brain regions, including the amygdala. Amygdala neurons are requisite elements of the neuronal network subserving AGS propagation during ETX induced by a subacute "binge" ethanol administration protocol. However, the effects of chronic ethanol administration on amygdala neuronal firing and ETX seizure behaviors are unknown. In the present study ethanol (5g/kg) was administered intragastrically in Sprague-Dawley rats once daily for 28days [chronic intermittent ethanol (CIE) protocol]. One week later the rats began receiving ethanol intragastrically three times daily for 4days (binge protocol). Microwire electrodes were implanted prior to CIE or on the day after CIE ended to record extracellular action potentials in lateral amygdala (LAMG) neurons. The first dose of ethanol administered in the binge protocol following CIE treatment did not alter LAMG neuronal firing, which contrasts with firing suppression seen previously in the binge protocol alone. These data indicate that CIE induces neuroadaptive changes in the ETX network which reduce LAMG response to ethanol. LAMG neuronal responses to acoustic stimuli prior to AGS were significantly decreased during ETX as compared to those before ethanol treatment. LAMG neurons fired tonically throughout the tonic convulsions during AGS. CIE plus binge treatment resulted in a significantly greater mean seizure duration and a significantly elevated incidence of death than was seen previously with the binge protocol alone, indicating an elevated seizure severity following chronic ethanol administration.     

Effect of NMDA- and strychnine-insensitive glycine site antagonists on NMDA-mediated convulsions and learning

Intracerebroventricular (ICV) injection of N-methyl-d-aspartate (NMDA) was shown to induce generalized seizures in mice. The competitive NMDA antagonistsdl-2-amino-5-phosphonovaleroate (dl-AP7) and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP), the NMDA channel blocker antagonist (+)-5-methyl-10,11-dihydro 5H-dibenzo-[a,d] cycloheptan-5,10-imine maleate (MK-801) and the strychnine-insensitive glycine antagonists kynurenic acid (KYNA) and 7-chloro-kynurenic acid (7-Cl-KYNA), when co-administered (ICV) with NMDA, antagonized NMDA-induced generalized seizures. Administration (ICV) ofdl-AP7, CPP and MK-801 resulted in impared learning performance in a passive avoidance task in mice, with ED₅₀ close to the anticonvulsant dose. The glycine antagonists KYNA and 7-Cl-KYNA at high doses significantly failed to affect performance in the same model of learning. The results indicate that compounds acting at the strychnine-insensitive glycine site may have a larger therapeutic window as anticonvulsants than antagonists of the NMDA receptor and channel.     

Benzoazepine derivative as potent antagonists of the glycine binding site associated to the NMDA receptor

A series of benzoazepine derivatives, bearing suitable substituents at the C-3 position, was designed and evaluated by superimposition with the pharmacophore model of the glycine binding site. To fully explore the SAR of this class of compounds and to allow the preparation of new different compounds at the C-3 position, appropriate synthetic routes were set up. The benzoazepines were evaluated in terms of in vitro affinity using [3H]glycine binding assay and in vivo potency by inhibition of convulsions induced by N-methyl-D-aspartate (NMDA) in mice. This further analysis confirmed the preliminary results previously reported and that compound 27 is the most promising compound (Ki=32 nM, ED(50)=0.09 mg/kg, i.v.) in this series. Significant neuroprotective effect was observed after both pre- and post-ischaemia administration in the MCAo model. In particular, after post-ischaemia administration, it was found to be still effective when the administration was delayed up to 6 h after occlusion of the middle cerebral artery.     

Zinc enhances ethanol modulation of the α1 glycine receptor

Glycine receptor function mediates most inhibitory neurotransmission in the brainstem and spinal cord and is enhanced by alcohols, volatile anesthetics, inhaled drugs of abuse, and endogenous compounds including zinc. Because zinc exists ubiquitously throughout the brain, investigations of its effects on the enhancement of GlyR function by alcohols and anesthetics are important to understanding the effects of these agents in vivo. In the present study, the effects of zinc plus ethanol, pentanol, or isoflurane were tested on homomeric α1 glycine receptors to determine if concurrent applications of physiological concentrations of zinc with each of these modulators changed the magnitude of their effects. Homomeric α1 glycine receptors were expressed in Xenopus laevis oocytes, and the two-electrode voltage-clamp technique was used to measure glycine-mediated currents in the presence of combinations of zinc with ethanol, pentanol or isoflurane. The combined effects of zinc plus ethanol were greater than the sum of the effects produced by either compound alone. However, this was not seen when zinc was combined with either pentanol or isoflurane. Chelation of zinc by tricine decreased the effects of sub-maximal, but not maximal, concentrations of glycine, and diminished the magnitude of ethanol enhancement observed. These findings suggest a zinc/ethanol interaction at the α1 GlyR that results in the enhancement of the effects of ethanol action on GlyR function.     

Oral ethanol self-administration in rats is reduced by the administration of dopamine and glutamate receptor antagonists into the nucleus accumbens

The purpose of this study was to assess the role of endogenous dopamine and glutamate systems within the nucleus accumbens in modulating responses for oral ethanol reinforcements (10% w/v) in a free-choice operant task. Pretreatment with both systemic (100 µg/kg) and intra-nucleus accumbens microinjection of fluphenazine (2 and 4 µg), a dopamine receptor antagonist, significantly decreased responding for ethanol, without significantly affecting responses for water. Ethanol self-administration was also attenuated by microinjection into the nucleus accumbens of 2-amino-5-phosphopentanoic acid (AP-5, 3 and 6 µg), a competitive NMDA receptor antagonist. These results suggest that dopamine and glutamate neurotransmission in the nucleus accumbens may regulate ethanol self-administration and its reinforcing effects.     

The effect of phenobarbital and carbamazepine on the ethanol withdrawal reaction in the rat

The effects of phenobarbital (PB) and carbamazepine (CZ) on the ethanol withdrawal reaction in the rat were investigated in a blind study including an untreated control group. Physical ethanol dependence was established by intragastric intubation during a 4-day period. Both the degree of intoxication and the withdrawal reaction were assessed by standardised assessment instruments. Treatment with PB (40–60 mg/kg) and CZ (80–120 mg/kg) was initiated 10 h after the last ethanol dose and continued during the first 24 h of withdrawal. Serum concentrations of the drugs were measured.Both PB and CZ significantly reduced the ethanol withdrawal reaction compared to controls, and PB was significantly more effective than CZ. The degree of drug intoxication signs assessed by the same rating scale as the degree of ethanol intoxication indicated that maximum tolerable drug doses were used.PB probably exerts its treatment effect through the mechanism of cross dependence with ethanol, while CZ may exert a more specific effect on limbic structures responsible for central nervous system excitability.Abbreviations ANOVA analysis of variance - BEC blood ethanol concentration - CIS cumulated intoxication score - CZ carbamazepine - ICS incomplete clonic seizure - PB phenobarbital     

Both agonists and antagonists of the strychnine-insensitive glycine site of N-methyl-D-aspartate receptors modulate polysynaptic excitations in slices of mouse olfactory cortex

Summary In this study, it is reported that bath application of D-serine and, to a lesser extent glycine, potentiated polysynaptic but not monosynaptic excitations evoked in slices of mouse olfactory cortex perfused with solution containing Mg²⁺ (1 mmol/l), picrotoxin and strychnine (both 25 μmol/l). Effects were largely confined to the longer latency components of the field potentials and occurred at amino acid concentrations of between 0.01 and 1 mmol/l. The effects of D-serine and glycine were antagonized by 7-chlorokynurenate and indole-2-carboxylate, antagonists of the glycine regulatory site of the N-methyl-D-aspartate (NMDA) receptor complex. D-Serine (glycine not tested) also potentiated, and 7-chlorokynurenate partially inhibited the longer latency components of the polysynaptic field potentials evoked in slices perfused in the absence of picrotoxin and strychnine. However, neither D-serine nor glycine potentiated responses evoked by the bath application of NMDA. It is concluded that under the present experimental conditions, the glycine regulatory sites of those NMDA receptor involved in the mediation of polysynaptic excitations in the mouse olfactory cortex are not saturated with endogenous glycine.     

4-Substituted-kynurenic acid derivatives: A novel class of NMDA receptor glycine site antagonists

A series of 4-substituted-kynurenic acid derivatives possessing several different substituents at C4-position which are consisted of both a flexible propyloxy chain and an adjunct several type of carbonyl groups has been synthesized and evaluated for theirin vitro antagonist activity at the glycine site on the NMDA receptor. Of them, N-benzoylthiourea15c and N-phenylthiourea15a were found to have the bestin vitro binding affinity with IC₅₀ of 3.95 and 6.04 μM, respectively. On the other hand, in compounds12a∼c and13 the displacement of a thiourea group to an amide or a carbamate caused a significant decrease of thein vitro binding affinity. In the SAR study of the 4-substituted kynurenic acid derivatives, it was realized that the terminal substitution pattern on a flexible C4-propyloxy chain of kynurenic acid nucleus significantly influences on the binding affinity for glycine site; the binding affinity to the NMDA receptor might be increased by the introduction of a suitable electron rich substituent at C4 of kynurenic acid nucleus.     

Positive relationship between the number of prior ethanol withdrawal episodes and the severity of subsequent withdrawal seizures

One factor that has been shown to influence the severity of an ethanol withdrawal syndrome is a history of prior experience with episodes of ethanol withdrawal. It has been hypothesized that the progressive intensification of withdrawal symptoms following repeated bouts of ethanol intoxication and withdrawal may represent the manifestations of a kindling-like process. In mice, repeated episodes of ethanol withdrawal potentiate the severity of subsequent withdrawal seizures, even when the total amount of ethanol intoxication is equated across groups. In the current experiments, mice received 16-h bouts of continuous exposure to ethanol vapor in inhalation chambers separated by 8-h periods of abstinence. The withdrawal response was assessed by scoring handling—induced convulsions. The results demonstrated that a positive relationship exists between the number of prior episodes of ethanol withdrawal and the severity of subsequent withdrawal seizures. This conclusion was supported by both between-subject and within-subject comparisons. The difference in withdrawal severity does not appear to be related to differences in the level of intoxication, since blood ethanol levels immediately preceding withdrawal testing were similar for all groups. Further, the differential withdrawal response exhibited by multiple and single withdrawal groups cannot be explained by a difference in the rate of ethanol elimination. Although the mechanism(s) remain to be determined, taken together, these results provide support for the kindling hypothesis of ethanol withdrawal.     

Potentiation of ethanol withdrawal by prior dependence

Thirty rats were randomly assigned to three groups. Group 1 was given a 21-day exposure to an ethanol (EtOH) liquid diet, while Groups 2 and 3 were given equivalent amounts of an isocaloric non-EtOH liquid diet. Group 1 rats had withdrawal syndromes following EtOH removal. After a two-week recovery period, Groups 1 and 2 were both exposed to an EtOH diet, while Group 3 again received an isocaloric non-EtOH liquid diet. Groups 1 and 2 were withdrawn after 12 days of EtOH exposure and were rated with a behavioral withdrawal rating scale, for which interobserver reliability estimates were determined. Previously dependent (Group 1) rats showed more severe withdrawal syndromes, including a higher incidence of seizures, than rats undergoing their initial withdrawal (Group 2). Studies that do not agree with this finding are discussed. Address for offprint requests: Alcohol Research (3D), Veterans Administration Hospital, Salt Lake City, Utah 84148, U.S.A.     

Dopamine-sensitive adenylate cyclase in homogenates of rat striata during ethanol and barbiturate withdrawal

Summary Repeated administrations of ethanol and of phenobarbital to rats led to characteristic withdrawal symptoms when the drug had been stopped. Since both drugs affect brain dopamine metabolism, the postjunctional sensitivity to dopamine in the corpora striata was tested during ethanol or phenobarbital withdrawal. This was done by studying the dopamine-sensitive adenylate cyclase in homogenates of the corpora striata of ethanol- or phenobarbital-dependent rats. The results demonstrated a slight postjunctional subsensitivity to dopamine in withdrawal from both ethanol and phenobarbital. Both drugs, when added in vitro, did not affect the postjunctional sensitivity to dopamine. The results do not support the hypothesis, at least not in the case of dopamine, that a postjunctional supersensitivity to neurotransmitters is important for withdrawal symptoms after chronic administration of drugs inducing physical dependence.Part of the results were presented at the 17th Spring Meeting of the German Pharmacological Society, Mainz, March 23–26, 1976     

Enhanced ultrasonic vocalization and Fos protein expression following ethanol withdrawal: effects of flumazenil

RATIONALE: The acquisition of a caffeine conditioned flavour preference depends on the caffeine deprivation status of subjects during conditioning. It is not known if the expression of an established flavour preference is also state-dependent. OBJECTIVES: To determine if the expression of a flavour preference conditioned by caffeine is dependent on the level of deprivation at the time of testing. METHODS: In a double-blind placebo controlled study, 44 subjects were given 4 days exposure to a novel flavoured drink following overnight abstinence from caffeine. Half the subjects received caffeine (100 mg) in the drink, while the remainder had placebo (maltodextrin, 100 mg). Subjects rated the pleasantness of the drink each time. On a fifth (test) day, the subjects were given additional caffeine (100 mg) or placebo 2 h before consuming and rating the pleasantness of the drink. RESULTS: Pleasantness ratings for the novel drink increased over the 4 conditioning days in subjects receiving caffeine, but decreased in those given placebo. On day 5, subjects who were trained and tested in the same caffeine deprivation state expressed pleasantness ratings similar to those for the final training day. In contrast, subjects who were trained and tested in different states expressed pleasantness ratings that were significantly different from those of the final training day. CONCLUSIONS: These results suggest that the expression of caffeine conditioned flavour preferences are acutely sensitive to current motivational state, and a number of possible explanations are discussed.     

侧脑室注射八肽胆囊收缩素及其受体拮抗剂对吗啡依赖大鼠戒断症状的影响

目的观察侧脑室给予八肽胆囊收缩素(CCK-8)及其受体拮抗剂慢性干预对吗啡依赖大鼠戒断症状的影响,并在体外观察CCK-8对μ阿片受体结合反应的影响,初步探讨CCK-8对吗啡依赖过程的影响及其相关受体机制。方法建立大鼠吗啡依赖及纳络酮催促戒断模型,侧脑室注射CCK-8及CCK1受体拮抗剂devazepide、CCK2受体拮抗剂LY-288,513慢性干预,观察其对戒断症状的影响;应用放射配基结合实验体外检测CCK-8对μ阿片受体结合特征的影响。结果①吗啡注射前10 min侧脑室注射CCK-8和CCK受体拮抗剂devazepide、LY-288,513慢性干预均能降低吗啡依赖大鼠的戒断症状评分,并可明显改善体重下降、跳跃、齿颤、流涎等戒断症状,与戒断组相比差异均有显著性(P<0.01);②10-8～10-6 mol.L-1 CCK-8可以剂量依赖性地抑制大鼠脑组织中μ阿片受体与其配基的结合(P<0.01),降低μ阿片受体结合反应的Bmax值,而对Kd值无影响;且此抑制作用可被CCK1及CCK2受体拮抗剂翻转(P<0.01)。结论 CCK-8及其受体拮抗剂慢性干预均能减轻吗啡依赖大鼠戒断症状;CCK-8通过抑制μ阿片受体与其配基的结合,降低μ阿片受体的Bmax值,发挥其"抗阿片作用"。     

Effects of acute ethanol intoxication,chronic ethanol intoxication,and ethanol withdrawal on magnesium and calcium metabolism in the rat

Effects of ethanol intoxication and withdrawal on magnesium and calcium metabolism were studied in rats. During acute ethanol intoxication, plasma [Mg²⁺] was increased and plasma [Ca²⁺] decreased. During chronic intoxication, plasma [Mg²⁺] was normalized whereas plasma [Ca²⁺] was persistently subnormal. Ethanol withdrawal was followed by a decrease in plasma [Mg²⁺] and a normalization of plasma [Ca²⁺]. These various changes are probably related to changes in systemic pH and to the biochemical effects of ethanol and ethanol withdrawal on intermediary metabolism. Cerebrospinal fluid [Mg²⁺] was unchanged during intoxication and withdrawal and it was concluded that no etiological role can presently be ascribed to the magnesium ion as far as cerebral signs of ethanol intoxication and withdrawal in the rat are concerned. No consistent changes in erythrocyte [Mg²⁺] were encountered during ethanol intoxication and withdrawal in rats.     

Blockade of the discriminative stimulus effects of ethanol with 5-HT3 receptor antagonists

The ability of selective 5-HT₃ receptor antagonists to block the discriminative stimulus effects of ethanol was investigated in pigeons trained with food reinforcement to discriminate ethanol (1.5 g/kg; IG) from water. The 5-HT₃ receptor antagonists that are substituted tropines, ICS 205-930 (0.1–0.56 mg/kg) and MDL 72222 (3.0–17.0 mg/kg), blocked ethanol-appropriate responding, in a dose-dependent manner, suggesting that some of the discriminative stimulus effects of ethanol are mediated via the 5-HT₃ receptor. The blockade the discriminative stimulus effects of ethanol occurred in the presence of approximately 25–40 mM blood ethanol levels. Furthermore, the ethanol dose-effect function was shifted to the right by increasing doses of MDL 72222, suggesting a surmountable antagonism of the discriminative stimulus effects of ethanol. However, the benzamide zacopride (0.56–1.7 mg/kg), which is also a 5-HT₃ receptor antagonist, did not block the discriminative stimulus effects of ethanol. In addition, the dopaminergic antagonist haloperidol and the 5-HT₂ receptor antagonist ketanserin also failed to block the ethanol discrimination. The results suggest that 5-HT₃ mediated neurotransmission is an important component of ethanol's discriminative stimulus effects, but that the structural characteristics of the selective 5-HT₃ receptor antagonists influence their ability to block this action of ethanol. Furthermore, these findings implicate a significant role of 5-HT₃ activity in the behavioral effects of ethanol that may provide a pharmacological means for therapeutic intervention of alcohol abuse.     

Approaches to the rational design of selective melanocortin receptor antagonists

Introduction: When establishing the physiological roles of specific receptors in normal and disease states, it is critical to have selective antagonist ligands for each receptor in a receptor system with several subtypes. The melanocortin receptors have five subtypes referred to as the melanocortin 1 receptor, melanocortin 2 receptor, melanocortin 3 receptor, melanocortin 4 receptor and melanocortin 5 receptor, and they are of critical importance for many aspects of human health and disease.

Areas covered: This article reviews the current efforts to design selective antagonistic ligands for the five human melanocortin receptors summarizing the currently published orthosteric and allosteric antagonists for each of these receptors.

Expert opinion: Though there has been progress, there are still few drugs available that address the many significant biological activities and diseases that are associated with these receptors, which is possibly due to the lack of receptor selectivity that these designed ligands are currently showing. The authors believe that further studies into the antagonists' 3D conformational and topographical properties in addition to future mutagenesis studies will provide greater insight into these ligands which could play a role in the treatment of various diseases in the future.     

Comparison of the effects of drugs on hyperexcitability induced in hippocampal slices by withdrawal from chronic ethanol consumption

1. The effects of drugs, previously demonstrated to have a range of effects on the behavioural signs of ethanol withdrawal hyperexcitability, were examined in area CA1 in isolated hippocampal slices prepared after withdrawal from chronic ethanol in vivo.
2. The decreases seen after the ethanol treatment in the thresholds for production of single and multiple population spikes were prevented when the dihydropyridine calcium channel antagonist, isradipine, was included in the perfusion medium at 4 μM.
3. Another dihydropyridine, felodipine, which had no activity against withdrawal signs in vivo, did not affect the changes in field potentials, at concentrations up to 10 μM.
4. Diltiazem, which increased withdrawal hyperexcitability in vivo, had no effect on the withdrawal changes in field potentials at 30 μM; higher concentrations affected the control slices.
5. The novel anticonvulsant, gabapentin, at 1 μM but not at 100 nM, significantly decreased the signs of withdrawal hyperexcitability in the hippocampal slices. When the CCK_B antagonist, CI988, was added to the bathing medium, at 1 μM, there were small, but significant decreases in the withdrawal hyperexcitability.
6. The results showed that the actions of these drugs on the changes in the field potentials in isolated hippocampal slices were very similar to their previously demonstrated effects on the convulsive signs of ethanol withdrawal in vivo, but differences were seen in the corresponding comparison with anxiolytic actions in vivo.