Impaired fear conditioning but enhanced seizure sensitivity in rats given repeated experience of withdrawal from alcohol

Repeated experience of withdrawal from chronic alcohol treatment increases sensitivity to seizures. It has been argued by analogy that negative affective consequences of withdrawal also sensitize, but repeated experience of withdrawal from another sedative-hypnotic drug, diazepam, results in amelioration of withdrawal anxiety and aversiveness. We tested whether giving rats repeated experience of withdrawal from alcohol altered their ability to acquire a conditioned emotional response (CER). Male Hooded Lister rats were fed a nutritionally complete liquid diet as their only food source. Different groups received control diet, or diet containing 7% ethanol. Rats receiving ethanol diet were fed for either 24 days (Single withdrawal, SWD), or 30 days, with two periods of 3 days, starting at day 11, and 21, in which they received control diet (Repeated withdrawal, RWD). All rats were fed lab chow at the end of their liquid diet feeding period. Starting 12 days after the final withdrawal, groups of Control, SWD and RWD rats were given pentylenetetrazole (PTZ; 30 mg/kg, i.p.) three times a week, and scored for seizures. The occurrence of two successive Stage 5 seizures was taken as the criterion for full PTZ kindling. Other groups of control, SWD and RWD rats were trained to operate levers to obtain food, and were then exposed, in a fully counterbalanced design, to light and tone stimuli which predicted unavoidable footshock (CS+), or which had no consequences (CS-). Rats consumed approximately 17.5 g/kg/day of ethanol, resulting in blood alcohol levels of approximately 100 mg/dL. Repeated administration of PTZ resulted in increasing seizure scores. RWD rats achieved kindling criterion faster than either Control or SWD rats. No differences were seen in the groups in flinch threshold to footshock (0.3 mA). At a shock intensity of 0.35 mA, Control, but not RWD or SWD rats showed significant suppression to the CS+ CS- presentation did not affect response rates. The three groups differed in their response to pairing the CS+ with increasing shock levels, the Controls remaining more sensitive to the CS+. SWD rats showed significant suppression of lever pressing during CS+ presentations only at 0.45 and 0.5 mA, and RWD rats only at 0.5 mA. Giving rats repeated experience of withdrawal from chronic ethanol results in increased sensitivity to PTZ kindling, but reduces their ability to acquire a CER. Withdrawal kindling of sensitivity to anxiogenic events does not seem to occur under circumstances which give rise to kindling of seizure sensitivity.     

Aversive conditioning following repeated withdrawal from ethanol and epileptic kindling

Repeated withdrawal from ethanol, a procedure which resembles amygdala kindling in increasing seizure sensitivity, impairs the acquisition of fear conditioning (Stephens et al., 2001, Eur. J. Neurosci.,14, 2023-31). In contrast, rats previously kindled by repeated electrical stimulation of basolateral amygdala, or repeated administration of pentylenetetrazol, showed increased suppression of operant responding during the presentation of a stimulus conditioned to footshock when conditioning took place several weeks following the kindling experience. Neither form of kindling nor repeated ethanol withdrawal altered taste aversion conditioning, though rats treated chronically with ethanol and given a single withdrawal experience showed enhanced taste aversion conditioning. These results suggest that, despite evidence suggesting a common neuronal mechanism underlying seizure sensitivity following these types of kindling, they differ in their effects on fear conditioning.     

Repeated withdrawal from ethanol impairs acquisition but not expression of conditioned fear

Repeated withdrawal from ethanol impairs acquisition of conditioned fear [Stephens, D.N., Brown, G., Duka, T. & Ripley, T.L. (2001) Eur. J. Neurosci., 14, 2023-2031]. This study further examined the effect of repeated withdrawal from ethanol on the expression and acquisition of fear conditioning. Following training, presentation of a cue associated with footshock (CS+) resulted in a suppression of operant responding for food reinforcement. In different groups, shock thresholds were manipulated to give weak or severe behavioural suppression. Rats were subsequently chronically treated with ethanol-containing liquid diet either continuously (single withdrawal) or with three withdrawal periods (repeated withdrawal). Ethanol treatment and withdrawal had no effect on conditioned suppression of responding tested 2 weeks after the final withdrawal, at either shock intensity. Nevertheless, extinction of conditioned fear was impaired in the repeated withdrawal group exposed to the higher shock intensity. In the high intensity group, the stimulus-shock association was then reversed, so that the previously neutral conditioned stimulus (CS-) became the CS+. Acquisition of suppression to the new CS+ was significantly less in the animals previously given repeated experience of withdrawal, confirming our previous finding. Thus, repeated withdrawal from ethanol lead to disruption in the acquisition of fear conditioning but had no effect on retrieval of an association formed prior to the ethanol-withdrawal experiences.     

AMPA-receptor involvement in c-fos expression in the medial prefrontal cortex and amygdala dissociates neural substrates of conditioned activity and conditioned reward

Exposure to an environment, previously conditioned to amphetamine (1 mg/kg, i.p.), induced locomotor activity and c-fos expression (a marker for neuronal activation) in the mouse medial prefrontal cortex (mPFC) and amygdala; acute or repeated amphetamine (1 mg/kg, i.p.) administration induced c-fos expression additionally in the nucleus accumbens. An alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-receptor antagonist, 2, 3-dihydroxy-6-nitro-7-sulphamoyl-benzo(f)quinoxaline (NBQX), blocked expression of conditioned activity, and prevented the increase in c-fos expression in mPFC, implicating mPFC AMPAergic transmission in the conditioned component of behavioural sensitization to amphetamine. NBQX failed to block the expression of amphetamine-conditioned place preference, a measure of conditioned reward, or conditioned c-fos expression in the amygdala, an area implicated in the expression of conditioned place preference. These findings indicate that the conditioned components of behavioural sensitization depend on AMPA-receptor-mediated activation in mPFC, but that conditioned reward does not.     

Previous experience of withdrawal from chronic diazepam ameliorates the aversiveness of precipitated withdrawal and reduces withdrawal-induced c-fos expression in nucleus accumbens

Flumazenil (20 mg/kg, i.p.)-precipitated withdrawal from chronic treatment with diazepam (DZP, 15 mg/kg, s.c. in sesame oil for 21 days) resulted in a decreased seizure threshold to the convulsant, pentylenetetrazole (PTZ), infused into the tail vein; withdrawal from 21-day chronic diazepam treatment, interspersed with two periods of drug withdrawal, resulted in a greater decrease in convulsant threshold. A separate experiment showed that consumption of a sucrose solution immediately prior to precipitated withdrawal resulted in a decreased subsequent consumption of the sucrose solution; no such evidence of a conditioned taste aversion (CTA) was seen in mice given prior experience of withdrawal. Thus, prior experience of withdrawal enhanced the effects of a subsequent precipitated withdrawal in increasing seizure sensitivity, but weakened the ability of this withdrawal to serve as an aversive unconditioned stimulus (US). The weakening of the aversive properties of precipitated withdrawal may reflect habituation to the withdrawal stimulus, and was accompanied by a loss of the ability of withdrawal to induce c-fos expression in the shell of the nucleus accumbens, an area sensitive to both novel, and stressful, as well as rewarding stimuli.     

Interactions between chemical and electrical kindling of the rat amygdala

Holtzman rats were implanted with a chemitrode into the left basolateral amygdala, which could then be stimulated electrically (400 μA, 1 s, AC) or chemically by injection of carbachol (1 μl, 2.7 nmoles, sterile, isotonic). Group A received a daily injection of carbachol and developed kindled seizures. Group B received carbachol mixed with equimolar atropine, which blocked seizures and kindling. After 20 injections, both groups were stimulated electrically once a day and kindled at similar rates. Two additional groups received electrical or sham stimulation, followed by carbachol kindlind. No transfer effects were observed. Four additional groups received 27 nmoles of atropine through the chemitrode, followed 15 min later by electrical stimulation, sham stimulation, carbachol injection or saline injection, respectively. Atropine completely blocked carbachol kindling but did not alter the rate of electrical kindling. No difference in the number of QNB binding sites was observed in the amygdala of rats sacrificed two weeks after full electrical kindling. The lack of interaction between electrical and carbachol kindling and the failure of atropine to block electrical kindling of the amygdala suggest that the activation of local muscarinic synapses, while essential for carbachol kindling, is not required for electrical kindling of the rat amygdala.     

Microinjection of a corticotropin-releasing factor antagonist into the central nucleus of the amygdala reverses anxiogenic-like effects of ethanol withdrawal

Previous studies have shown that spontaneous exploration of the Elevated Plus Maze provides a sensitive measure of ‘anxiety’ induced by pharmacological or behavioral stressors. In particular, the percent time spent exploring the open arms of the plus maze is decreased during ethanol withdrawal, and this effect is antagonized by intracerebroventricular administration of 25 μg of alpha-helical CRF, a corticotropin-releasing factor antagonist (H.A. Baldwin et al.,Psychopharmacology, 103 (1991) 227–232). The present study was designed to examine the effect of α-helical CRF infusion within the central nucleus of the amygdala during ethanol withdrawal. Rats were made dependent on ethanol by maintenance on an ethanol-containing liquid diet for 16 days, withdrawn from ethanol and tested on the elevated plus maze at 8 h post-ethanol access. In comparison with pair-fed control rats, ethanol withdrawn subjects spent significantly less percent time exploring the open arms of the plus maze. This decrease in open arm exploration was antagonized by administration of α-helical CRF (250 ng) bilaterally into the central nucleus of the amygdala, but not by intracerebroventricular administration of 250 ng of α-helical CRF. The ability of intra-amygdala α-helical CRF to antagonize decreased open arm exploration is unlikely to be due to changes in motor activity, since general activity on the maze was reduced in all EtOH withdrawal groups. These results suggest that the central nucleus of the amygdala may be an effective site for endogenous CRF systems to mediate anxious behavior associated with ethanol withdrawal.     

GABAA receptors mediate the opposing roles of dopamine and the tegmental pedunculopontine nucleus in the motivational effects of ethanol

Recent work has demonstrated that changes in ventral tegmental area (VTA) GABA_A receptor ion conductance properties are responsible for switching morphine's positive reinforcing properties from a dopamine-independent to a dopamine-dependent pathway when an animal transitions from a non-deprived (minimal drug exposure) to a dependent (chronic drug exposure) and withdrawn state. Here we show that a double dissociation of ethanol's positive reinforcing properties is exactly opposite to that seen with morphine. In C57BL/6 mice, ethanol-conditioned place preferences were blocked in dopamine D2 receptor knockout non-deprived mice, but not by a lesion of the tegmental pedunculopontine nucleus (TPP). On the other hand, TPP lesions, but not a D2 receptor mutation, blocked ethanol-conditioned place preferences in ethanol-dependent and withdrawn mice. The opposite effects of ethanol and opiates can be explained by their proposed actions through a common VTA GABA_A receptor switching mechanism.     

The role of pituitary adenylyl cyclase activating polypeptide in affective signs of nicotine withdrawal

Recent evidence implicates endogenous pituitary adenylyl cyclase activating polypeptide (PACAP) in the aversive effect of nicotine. In the present study, we assessed if nicotine-induced conditioned place preference (CPP) or affective signs of nicotine withdrawal would be altered in the absence of PACAP and if there were any sex-related differences in these responses. Male and female mice lacking PACAP and their wild-type controls were tested for baseline place preference on day 1, received conditioning with saline or nicotine (1 mg/kg) on alternate days for 6 days and were then tested for CPP the next day. Mice were then exposed to four additional conditioning and were tested again for nicotine-induced CPP 24 hr later. Controls were conditioned with saline in both chambers and tested similarly. All mice were then, 96 hr later, challenged with mecamylamine (3 mg/kg), and tested for anxiety-like behaviors 30 min later. Mice were then, 2 hr later, forced to swim for 15 min and then tested for depression-like behaviors 24 hr later. Our results showed that male but not female mice lacking PACAP expressed a significant CPP that was comparable to their wild-type controls. In contrast, male but not female mice lacking PACAP exhibited reduced anxiety- and depression-like behaviors compared to their wild-type controls following the mecamylamine challenge. These results suggest that endogenous PACAP is involved in affective signs of nicotine withdrawal, but there is a sex-related difference in this response.     

Effects of fluoxetine on ethanol withdrawal syndrome in rats

The present study was designed to investigate the effects of fluoxetine, a selective serotonin reuptake inhibitor, on ethanol withdrawal syndrome in rats. Adult male Wistar rats (218-255 g) were subjects. Ethanol (7.2%, v/v) was given to rats by a liquid diet for 21 days. Control rats were pair fed an isocaloric liquid diet containing sucrose as a caloric substitute to ethanol. Fluoxetine (2.5, 5 and 10 mg/kg) and saline were injected to rats intraperitoneally just before ethanol withdrawal. After 2nd, 4th and 6th hour of ethanol withdrawal, rats were observed for 5 min, and withdrawal signs that included locomotor hyperactivity, agitation, stereotyped behavior, wet dog shakes and tremor were recorded or rated. A second series of injections was given at 6 h after the first one, and subjects were then tested for audiogenic seizures. Fluoxetine produced some dose-dependent and significant inhibitory effects on all the signs of ethanol withdrawal during ethanol withdrawal period. Our results suggest that acute fluoxetine treatment has some beneficial effects on ethanol withdrawal in rats. Thus, this drug may be useful for treatment of ethanol withdrawal syndrome.     

Effects of disconnection of amygdala dopamine and nucleus accumbens N‐methyl‐d‐aspartate receptors on ethanol‐seeking behavior in mice

There is a strong interest in harnessing the genetic manipulations that are possible in mice to investigate the functional neural mechanisms modulating the associative processes that control drug‐seeking behavior. However, it is unknown whether intracranial techniques, such as the disconnection procedure commonly used in rats to examine serial connectivity between implicated areas, can be successfully applied to mice. We have previously demonstrated that the expression of ethanol‐seeking behavior in mice is dependent upon amygdala (Amy) dopamine and nucleus accumbens (Acb) N‐methyl‐d ‐aspartate (NMDA) receptor activation ( Gremel & Cunningham, 2009 ). Here, we used a neuropharmacological disconnection procedure to investigate whether dopamine activation of the Amy directly leading to increases in Acb glutamate release and binding of NMDA receptors modulates the expression of ethanol‐seeking behavior. Immediately before testing the expression of an ethanol‐induced conditioned place preference, mice were given an Amy infusion of flupenthixol and either an ipsilateral or contralateral Acb infusion of AP‐5. Although both ipsilateral and contralateral manipulations reduced the expression of ethanol conditioned place preference, in a separate experiment we demonstrated that a unilateral Acb infusion of AP‐5, but not Amy flupenthixol, is sufficient to disrupt preference. The finding of a significant blockade by unilateral AP‐5 into the Acb precludes any conclusions about a unique role for the Amy/Acb neuroanatomical connection in this model of ethanol‐seeking behavior. Further, the current results suggest potential limitations in transferring techniques from rats to mice in order to study serial interactions between neural areas underlying motivated behaviors. Nevertheless, these findings provide evidence showing that Acb NMDA receptors play an important role in the expression of ethanol‐conditioned behavior.     

奎硫平对酒依赖患者稽延性戒断症状及睡眠的影响

目的:评估奎硫平对酒依赖患者稽延性戒断症状的治疗效果及睡眠质量和睡眠结构的影响。方法:选取80例酒依赖患者急性期戒酒治疗后随机分为奎硫平治疗组(研究组)和维生素治疗组(对照组),研究组给予200~400 mg/d奎硫平治疗,对照组给予维生素治疗。采用宾西法尼亚酒精渴求量表(PACS)、视觉模拟渴求量表(VAS)及汉密尔顿抑郁量表(HAMD)、汉密尔顿焦虑量表(HAMA)分别于入组时和治疗4周对两组患者进行评定,同时接受多导睡眠图(PSG)检测。结果:治疗前两组PACS、VAS和HAMD、HAMA评分差异无统计学意义(P0.05),治疗后研究组PACS、VAS和HAMD、HAMA总分较治疗前均显著降低,差异有统计学意义(P0.05)。治疗前两组PSG各指标差异无统计学意义(P0.05),治疗后研究组觉醒次数(AN)减少[(5.93±1.53)vs(6.91±1.60),t=2.559,P=0.000],睡眠效率(SE)升高[(68.3±4.17)vs(60.8±4.20),t=7.316,P=0.000],总睡眠时间(TST)增加[(292.5±25.9)vs(271.7±23.3),t=3.451,P=0.018],差异有统计学意义。睡眠潜伏期(SL)、快眼动睡眠(REM)百分比及REM睡眠潜伏期(RL)等指标两组间差异无统计学意义(P0.05)。对照组治疗前后各指标差异无统计学意义(P0.05)。结论:奎硫平可改善酒依赖患者急性戒断后的心理渴求、焦虑、抑郁等稽延性戒断症状,提高部分睡眠质量。     

Adenosine A2A receptors are involved in physical dependence and place conditioning induced by THC

A2A adenosine and CB1 cannabinoid receptors are highly expressed in the central nervous system, where they modulate numerous physiological processes including adaptive responses to drugs of abuse. Both purinergic and cannabinoid systems interact with dopamine neurotransmission (through A2A and CB1 receptors, respectively). Changes in dopamine neurotransmission play an important role in addictive-related behaviours. In this study, we investigated the contribution of A2A adenosine receptors in several behavioural responses of Delta9-tetrahydrocannabinol (THC) related to its addictive properties, including tolerance, physical dependence and motivational effects. For this purpose, we first investigated acute THC responses in mice lacking A2A adenosine receptors. Antinociception, hypolocomotion and hypothermia induced by acute THC administration remained unaffected in mutant mice. Chronic THC treatment developed similar tolerance to these acute effects in wild-type and A2A-knockout mice. However, differences in the body weight pattern were found between genotypes during such chronic treatment. Interestingly, the somatic manifestations of SR141716A-precipitated THC withdrawal were significantly attenuated in mutant mice. The motivational responses of THC were also evaluated by using the place-conditioning paradigm. A significant reduction of THC-induced rewarding and aversive effects was found in mice lacking A2A adenosine receptors in comparison with wild-type littermates. Binding studies revealed that these behavioural changes were not associated with any modification in the distribution and/or functional activity of CB1 receptors in knockout mice. Therefore, this study shows, for the first time, a specific involvement of A2A receptors in the addictive-related properties of cannabinoids.     

Lack of neuropeptide Y attenuates the somatic signs of opiate withdrawal

Recent evidence suggests that neuropeptide Y (NPY) may be involved in the neurobiological responses to drugs of abuse. This study was designed to assess the possible contribution of NPY to opiate withdrawal behaviors. Here we report that mice lacking the NPY gene show normal conditioned place aversion to opiate withdrawal, but show attenuated opiate withdrawal somatic signs.     

Developmental exposure to an environmental PCB mixture delays the propagation of electrical kindling from the amygdala

Developmental PCB exposure impairs hearing and induces brainstem audiogenic seizures in adult offspring. The degree to which this enhanced susceptibility to seizure is manifest in other brain regions has not been examined. Thus, electrical kindling of the amygdala was used to evaluate the effect of developmental exposure to an environmentally relevant PCB mixture on seizure susceptibility in the rat. Female Long-Evans rats were dosed orally with 0 or 6 mg/kg/day of the PCB mixture dissolved in corn oil vehicle 4 weeks prior to mating and continued through gestation and up until postnatal day (PND) 21. On PND 21, pups were weaned, and two males from each litter were randomly selected for the kindling study. As adults, the male rats were implanted bilaterally with electrodes in the basolateral amygdala. For each animal, afterdischarge (AD) thresholds in the amygdala were determined on the first day of testing followed by once daily stimulation at a standard 200 μA stimulus intensity until three stage 5 generalized seizures (GS) ensued. Developmental PCB exposure did not affect the AD threshold or total cumulative AD duration, but PCB exposure did increase the latency to behavioral manifestations of seizure propagation. PCB exposed animals required significantly more stimulations to reach stage 2 seizures compared to control animals, indicating attenuated focal (amygdala) excitability. A delay in kindling progression in the amygdala stands in contrast to our previous finding of increased susceptibility to brainstem-mediated audiogenic seizures in PCB-exposed animals in response to a an intense auditory stimulus. These seemingly divergent results are not unexpected given the distinct source, type, and mechanistic underpinnings of these different seizure models. A delay in epileptogenesis following focal amygdala stimulation may reflect a decrease in neuroplasticity following developmental PCB exposure consistent with reductions in use-dependent synaptic plasticity that have been reported in the hippocampus of developmentally PCB exposed animals.     

Long-lasting effects of feline amygdala kindling on monoamines, seizures and sleep

This report describes the relationship between monoamines, sleep and seizures before and 1-month after amygdala kindling in young cats (<1 year old; n=8; six female and two male). Concentrations (fmoles of norepinephrine or NE, dopamine or DA and serotonin or 5-HT) were quantified in consecutive, 5-min microdialysis samples (2 microl/min infusion rate) from amygdala and locus ceruleus complex (LC) during four, 6-8-h polygraphic recordings before (n=2) and 1 month post-kindling (n=2); 5-min recording epochs were temporally adjusted to correspond to dialysate samples and differentiated according to dominant sleep or waking state (lasting > or =80% of 5-min epoch) and degree of spontaneous seizure activity (number and duration of focal versus generalized spikes and spike trains and behavioral seizure correlates). Post-kindling records in each cat were divided into two groups (n=1 record each) based on higher or lower spontaneous EEG and behavioral seizure activity and compared to pre-kindling records. We found: (1) before and after kindling, NE and 5-HT but not DA concentrations were significantly lower in sleep than waking at both sites; (2) after kindling, each cat showed cyclic patterns, as follows: (a) higher NE, 5-HT and DA concentrations accompanied increased seizure activity with delayed sleep onset latency and increased sleep fragmentation (reduced sleep state percentages, number of epochs and/or epoch duration) in one recording versus (b) lower monoaminergic concentrations accompanied reduced seizure activity, rapid sleep onset and reduced sleep disruption in the other recording. The alternating, post-kindling pattern suggested "rebound" effects which could explain some controversies in the literature about chronic effects of kindling on monoamines and sleep-waking state patterns.