Previous stress exposure enhances both anxiety-like behaviour and p35 levels in the basolateral amygdala complex: Modulation by midazolam

Stress exposure induces long lasting neurobiological changes in selected brain areas, which could be associated with the emergence of negative emotional responses. In the present study, previously restrained animals exhibited excessive anxiety one day later in the elevated plus maze. We explore whether stress exposure affects the expression levels of cyclin-dependent kinase 5 (Cdk5) and of its activator protein p35, in diverse amygdaloid nuclei. Stress exposure enhanced p35 levels selectively in the basolateral amygdala (BLA). This up-regulation might be functionally associated with the occurrence of exaggerated anxiety since such emotional response was selectively reversed by an intra-BLA infusion of olomoucine, a Cdk5 inhibitor, 15 min prior to the restraint session. Moreover, pre-treatment with midazolam, a benzodiazepine ligand, not only prevented the excessive anxiety but also attenuated the p35 increase in the BLA of stressed rats. In conclusion, we suggest a pivotal role of the Cdk5/p35 complex, specifically in BLA in the excessive anxiety induced by a previous stressful experience.     

Blockade of CRF1 and CCK2 receptors attenuated the elevated anxiety-like behavior induced by immobilization stress

Two highly co-localized neurotransmitters: corticotropin-releasing factor (CRF) and cholecystokinin (CCK), have been implicated in the development of stress-related anxiety disorders. This study was designed to examine the role of CRF1 and CCK2 receptors on the anxiety-like behavior induced by immobilization stress. Our results showed that 30-min immobilization enhanced the anxiety-like behavior in C57BL/6J mice examined in the elevated plus maze (EPM). The combined pretreatment of CR2945 (a CCK2 receptor antagonist) and antalarmin (a CRF1 receptor antagonist) fully blocked this elevated anxiety-like behavior, while the application of CR2945 or antalarmin alone showed only partial effects. The increased expression of CRF1 and CCK2 receptors at protein levels in three anxiety-related brain regions: cortex, hippocampus and hypothalamus, was detected by Western blot. The increased mRNA expression of CCK, CRF, CCK2 and CRF1 receptors was also examined by real-time RT-PCR. Our study demonstrated that the blockade of CRF1 and CCK2 receptors attenuated the elevated anxiety-like behavior induced by immobilization stress, suggestive of the CRF and CCK systems contributing to the development of stress-related anxiety behavior.     

Chronic restraint stress induced neurobehavioral alterations and histological changes in rat

Several lines of research on human and rodent subjects have demonstrated that stress results in multiple negative outcomes, including increased incidence of psychopathologies. Restraint stress in rats is known to adversely affect the physiological, psychological and reproductive axis in rats. Male rats were subjected to restraint stress for 3 hours consecutively for 14 days. The behavioral studies include Elevated Place Maze, Open Field and Morris Water Maze tests. Our results show that chronic restraint stress involved a development of anxiety in EPM, reduced motor activity in OF, impaired memory spatial in MWM tests, and induced change in testicular function, as reflected by significant decrease in plasma level of testosterone, correlate well with the damages in testis. The Results of the present study confirm that chronic restraint stress induced cognitive dysfunction, enhance anxiety like behavior and induced testicular damage in male rats Wistar.     

Anxiolytic effects of ethanol and phenobarbital are abolished in test-experienced rats submitted to the elevated plus maze

Prior test experience compromises the anxiolytic efficacy of benzodiazepines (BZs) either in rats or mice, a phenomenon not exclusive to the elevated plus-maze (EPM) animal model of anxiety, which is referred to as "one-trial tolerance." However, it remains to be determined whether a similar event occurs when testing other drugs that also possess binding-sites on the GABA(A) receptor, such as ethanol and barbiturates. In the present study, we have addressed this issue using maze-naive and maze-experienced (free exploration of the EPM 48 h earlier for 5 min) rats pretreated with ethanol (1.0-1.4 g/kg) or phenobarbital (20-60 mg/kg) and submitted to the EPM. The results confirmed the anxiolytic profile of both drugs, represented by increased open arm exploration and decreased risk assessment behavior, in maze-naive rats. However, in maze-experienced rats, neither ethanol nor phenobarbital anxiolytic effects were observed, suggesting that prior maze experience compromised the drugs' anxiolytic activity. Thus, the "one-trial tolerance" phenomenon might also be extended to other drugs that bind to the GABA(A) receptor complex.     

Antinociceptive effects of elevated plus-maze exposure: influence of opiate receptor manipulations

It has been suggested that anxiety may be a critical factor in certain forms of non-opioid environmental analgesia. In the present study, 5-min exposure to the elevated plus-maze test of anxiety (EPM) induced a mild, though enduring, elevation in tail-flick latencies in male mice. Pretreatment with the opiate antagonist naltrexone (0.1–10.0 mg/kg) failed to block EPM-induced antinociception; indeed, the highest dose actuallyenhanced the response. This effect could not be attributed to intrinsic analgetic activity of naltrexone. Rather, analysis of EPM behaviours suggested that it may have been secondary to an anxiogenic effect of the compound. The involvement of non-opioid substrates in this form of pain inhibition was further supported by the failure of chronic morphine treatment (7 days; 7.5 mg/kg) to alter either the antinociceptive or behavioural response to EPM exposure. Irrespective of treatment history, mice showed a retest EPM profile of enhanced anxiety, with tail-flick data suggesting a major contribution of anticipatory factors. Several important methodological variables are discussed and findings are contrasted with parallel studies on non-opioid defeat analgesia.     

Behavioral profile of mice with impaired cognition in the elevated plus-maze due to a deficiency in neural cell adhesion molecule

The elevated plus-maze (EPM) test is one of the most used tests for screening levels of anxiety in rodents. In the present study, we studied how impaired cognition due to a deficiency in the neural cell adhesion molecule (NCAM) could affect the behavior of mice in the EPM task. NCAM-knockout mice demonstrated impaired learning in both object-recognition and fear-conditioning tasks. Analysis of the behavior of mice in the EPM task using a minute-by-minute method revealed a profound influence of genotype. Wild-type mice demonstrated quick learning of the aversive properties of the open arms during the first few minutes of a single EPM task, whereas NCAM−/− mice were unable to learn the aversive properties of the open arms of EPM. Wild-type mice also demonstrated habituation to the EPM task in a test/retest paradigm whereas NCAM-knockout mice failed to habituate during the second EPM presentation. Our data show that the anxiolytic-like behavior of NCAM-knockout mice is not just related to levels of innate anxiety but also to their inability to recognize potential danger associated with the open arms of the EPM task.     

Effects of interleukin-1beta and lipopolysaccharide on behavior of mice in the elevated plus-maze and open field tests

It has been postulated that infections, inflammatory processes and resulting cytokines may be causative factors in emotional disorders, including depression and anxiety. Support for this possibility has been sought in studies of animal behavior following administration of interleukin-1 (IL-1) and lipopolysaccharide (LPS). However, such treatments induce a variety of behavioral responses, collectively known as sickness behavior, some of which could affect the performance in tests used to assess anxiety and depression. Thus the effects of peripheral administration of IL-1beta and LPS on the behavior of mice were studied in the elevated plus-maze (EPM) and the open field (OF). Mouse IL-1beta (30, 100, 300, and 1000 ng) was injected intraperitoneally 30 or 60 min, and LPS (0.5, 1 and 5 microg) 120 min before the tests. IL-1beta and LPS induced dose-dependent decreases in open arm entries and the time spent on the open arms in the EPM, effects considered to reflect anxiety-like behavior. However, entries to all arms were also reduced in a dose-dependent manner, indicating a decrease in general activity. In the OF, IL-1beta and LPS decreased the number of line crossings in the center of the field, that can also be considered to reflect anxiety-like behavior. However, this effect was accompanied by a similar decrease in line crossings in the periphery, as well as in rears and climbs. Thus the doses of IL-1beta and LPS necessary to induce these effects also decreased locomotor activity in the EPM and OF. Therefore, the behavioral responses induced by IL-1beta and LPS in the EPM and the OF considered to reflect anxiety must be interpreted in the light of this reduction in overall activity. Thus the results do not provide unequivocal support for the suggestion that LPS or IL-1 mediate anxiety. Nevertheless, because infections, endotoxins, and the ensuing cytokines cause alterations in CNS norepinephrine and serotonin, they may contribute to emotionality, and perhaps to anxiety.     

Activation patterns of cells in selected brain stem nuclei of more and less stress responsive rats in two animal models of PTSD - predator exposure and submersion stress

This study had two purposes. First: compare predator and water submersion stress cFos activation patterns in dorsal raphe (DR), locus coeruleus (LC) and periaqueductal gray (PAG). Second: identify markers of vulnerability to stressors within these areas. Rats were either predator or submersion stressed and tested 1.75 h later for anxiety-like behavior. Immediately thereafter, rats were sacrificed and cFos expression examined. In DR, serotonergic cells expressing or not expressing cFos were also counted. Predator and submersion stress increased anxiety-like behavior (in the elevated plus maze- EPM) equally over controls. Moreover, stressed rats spent equally less time in the center of the hole board than handled controls, another indication of increased anxiety-like behavior. To examine vulnerability, rats which were less anxious (LA) and more (highly) anxious (MA) in the EPM were selected from among handled control and stressed animals. LA rats in the stressed groups were considered stress non-responsive and MA stressed rats were considered stress responsive. LA and MA rats did not differ in cFos expression in any brain area, though stressors did increase cFos cell counts in all areas over controls. Intriguingly, the number of serotonergic DR neurons not activated by stress predicted degree of anxiety response to submersion stress only. LA submersion stressed rats had more serotonergic cells than all other groups, and MA submersion stressed rats had fewer serotonergic cells than all other groups, which did not differ. Moreover, these cell counts correlated with EPM anxiety. We conclude that a surplus of such cells protects against anxiogenic effects of submersion, while a paucity of such cells enhances vulnerability to submersion stress. Other data suggest serotonergic cells may exert their effects via inhibition of dorsolateral PAG cells during submersion stress. Findings are discussed with respect to serotonergic transmission in vulnerability to predator stress and relevance of findings for post traumatic stress disorder (PTSD).This article is part of a Special Issue entitled ‘Post-Traumatic Stress Disorder’.     

Elevated anxiety-like behavior following ethanol exposure in mutant mice lacking neuropeptide Y (NPY)

BACKGROUND: Neuropeptide Y (NPY) is a neuromodulator with anxiolytic properties. Recent evidence suggests that NPY modulates neurobiological responses to ethanol. Because withdrawal from ethanol is associated with elevated anxiety-like behavior, and because central NPY modulates anxiety, we assessed anxiety-like behavior in mutant mice lacking normal production of NPY (NPY-/-) and in normal wild-type mice (NPY+/+) 6h after removal of a liquid diet containing 4.5% ethanol. METHODS: NPY-/- and NPY+/+ mice on a pure 129/SvEv genetic background were given 6 days of access to a liquid ethanol diet (ED) or control diet (CD). Six hours before elevated plus maze (EPM) testing, ED was replaced with CD in the ethanol-withdrawn group. RESULTS: Ethanol-withdrawn NPY-/- mice showed significantly less open arm time and total proportion of time spent in the open arm of the EPM relative to ethanol-withdrawn NPY+/+ mice and when compared to NPY-/- and NPY+/+ mice that had access to the CD. On the other hand, ethanol-withdrawn NPY+/+ mice did not show altered EPM behavior relative to controls. CONCLUSIONS: Central NPY is protective against anxiety-like behavior stemming from exposure to and/or withdrawal from ethanol. Targets aimed at NPY receptors may be useful compounds for treating anxiety associated with ethanol dependence.     

Antagonism of AMPA receptors produces anxiolytic-like behavior in rodents: Effects of GYKI 52466 and its novel analogues

Rationale Although emerging number of data supports the role of glutamate receptors and the potential of their antagonists in anxiety disorders, the involvement of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors in anxiety is less well characterized. Objective To evaluate the anxiolytic potential of 2,3-benzodiazepine (2,3BDZ) type AMPA receptor antagonists in various models of anxiety. Materials and methods Whole-cell currents, hippocampal field potentials, elevated plus maze (EPM), meta-chlorophenylpiperazine (mCPP)-induced anxiety model, Vogel test in rats and light–dark test (LD) in mice were used to determine AMPA/kainite receptor properties and anxiolytic-like activity of a series of 2,3BDZ-type compounds. Results The reference compound GYKI 52466 was proved active in two anxiety models in non-sedative doses: minimal effective dose (MED) was especially low in EPM (0.01 mg/kg) GYKI 53405 and GYKI 53655 showed anxiolytic-like activity in two tests (EPM and mCPP). EGIS-8332 was active in EPM and LD while EGIS-9637 showed anxiolytic-like potency in EPM, mCPP and Vogel model. EGIS-10608 was the most effective compound among 2,3BDZs tested in EPM and Vogel models (MEDs are 0.01 and 2.5 mg/kg, respectively). 2,3BDZs were active in anxiety models at doses lower than those produced sedative effects. NBQX showed anxiolytic-like activity in EPM only (3 mg/kg). Conclusions The results show that non-competitive AMPA receptor antagonists can profoundly block anxiety-like behavior in rodents independently from their motor depressant activity. However, the sedative properties at higher doses might limit their therapeutic utility as new anxiolytic drugs.     

Enhanced Cdk5 activity and p35 translocation in the ventral striatum of acute and chronic methamphetamine-treated rats.

The cyclin-dependent kinase Cdk5 and DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa)-dependent signaling have been implicated in the regulation of dopaminergic neurotransmission after chronic cocaine treatment. In this study, we examined if Cdk5 signaling participates in the behavioral and biochemical effect of acute and chronic methamphetamine (METH) treatment. We found that Cdk5 activity and the membrane fraction of p35 protein, a Cdk5 activator, in the ventral striatum increased transiently after an injection of 4 mg/kg METH, while intra-accumbens treatment with a Cdk5 inhibitor, roscovitine, prevented the acute METH-induced locomotor activation. The phosphorylation of DARPP-32 at both Thr75 and Thr34 was differentially regulated after acute METH treatment, but the levels of total Cdk5, p35, and DARPP-32 remained the same. To determine if Cdk5 signaling was associated with behavior sensitization to METH, rats that received repetitive injections of METH (4 mg/kg) for 14 consecutive days were analyzed at withdrawal day 7. The results indicate that Cdk5 activity and p35 translocation in the ventral striatum were upregulated in METH-sensitized rats; treatment with roscovitine in the nucleus accumbens effectively suppressed the 1 mg/kg METH-induced behavioral sensitization. Concomitantly, a decrease in the amount of PP-2A and DARPP-32 phosphorylation at Thr34, but an increase in phosphorylation of DARPP-32/Thr75, was observed in the ventral striatum of sensitized rats. The overall results demonstrate that Cdk5/p35 and downstream signaling in the ventral striatum play a critical role in the effects of acute METH treatment as well as the development of behavioral METH sensitization.     

Cdk5/p35参与了慢性应激诱导的大鼠抑郁样行为

背景:对抑郁症发生机制的研究表明,抑郁症患者表现为海马以及其他脑边缘结构的体积减少和细胞丢失,抗抑郁剂可显著促进抑郁模型动物海马神经元发生,提示神经可塑性机制涉及了抑郁症的发生过程。周期素依赖性蛋白激酶-5(Cdk5)及其活性调节蛋白p35是保持成熟神经系统内边缘系统,尤其是海马中神经元具有潜在可塑性的关键激酶。Cdk5在中枢神经系统发育过程中起着至关重要的作用,主要与神经元迁移、轴突生长和神经递质释放有关,还参与细胞骨架形成、轴突导向、膜转运、突触功能和多巴胺信号转导等多种神经功能调控。然而,在某些病理性因素作用下,Cdk5激酶活性异常升高会引起神经元的过度死亡,导致一些神经退行性疾病的发生。Cdk5在调节神经元的生存方面发挥着重要的作用,那么在抑郁症损伤的海马神经元中,Cdk5是否也参与了神经元的生存过程,从而介导了抑郁症的发生尚未见到报道。目的:本研究将基于抑郁症神经可塑性的研究基础,探讨Cdk5/p35在抑郁症模型大鼠抑郁样行为中的作用,以期进一步阐明抑郁症发生的神经生物学机制,并为临床发现新的治疗抑郁症的有效手段奠定理论基础。方法:采用连续21d的慢性不可预见性的中等强度应激抑郁模型(CMS),以动物的体重变化、蔗糖水偏爱(sucrosep reference)和自发活动能力为指标,考察抑郁模型动物行为学变化;通过测定Cdk5特定底物-组蛋白H1被磷酸化的程度检测大鼠海马部位Cdk5激酶活性,用Westernblot的方法检测p35蛋白的表达;同时在应激过程中,采用微量注射的方法,分别在海马齿状回(DG),CA1及CA3亚区给予Cdk5激酶抑制剂(butyrolactone),观察抑制不同脑区Cdk5激酶对抑郁症模型动物体重,糖水偏爱、自发活动性等抑郁样行为的影响。此外,在慢性应激的同时,连续给予抗抑郁药venlafaxine和mirtazapine,考察抗抑郁剂的治疗作用对海马p35蛋白表达水平的影响。结果:慢性应激大鼠海马部位Cdk5激酶活性显著升高;Western blot结果表明应激大鼠海马DG区胞膜组分p35蛋白的表达水平明显上调,而胞浆p35蛋白表达水平显著降低;相关性分析结果表明海马Cdk5激酶活性与胞膜p35蛋白的表达水平呈明显正相关,而与胞浆p35蛋白的表达水平呈明显负相关;大鼠体重增加值,糖水偏爱与海马胞膜p35蛋白的表达水平呈显著负相关。上述结果表明海马Cdk5激酶活性升高参与了慢性应激诱导的大鼠抑郁样行为。行为学检测结果表明,海马DG区微注射Cdk5激酶抑制剂butyrolactone(50,100ng)可显著逆转慢性应激引起的大鼠抑郁样行为,而在CA1和CA3区微注射butyrolactone(100ng)则对大鼠的抑郁样行为没有明显影响,说明抑制Cdk5激酶活性改善大鼠抑郁样行为具有脑区特异性。在慢性应激实验中发现连续给予抗抑郁药venlafaxine(40mg·kg-1)和mirtazapine(20mg·kg-1)可明显降低DG区胞膜p35蛋白的表达水平,促进p35蛋白由胞膜转运至胞浆,而抗精神病药aripiprazole(5mg.kg-1)对慢性应激引起的DG区胞膜p35蛋白表达增加没有明显影响,说明降低胞膜p35蛋白的表达水平,抑制Cdk5激酶活性是抗抑郁药物特异性的。结论:本研究通过一系列实验证实了Cdk5/p35在慢性应激大鼠抑郁样行为中的作用,抑制Cdk5活性可有效逆转动物的抑郁样行为,抗抑郁剂在发挥治疗作用的同时可抑制Cdk5激酶的异常激活。因此,本研究结果为抑郁症的神经生物学机制研究和临床抗抑郁治疗药物研究开发提供了新的思路。     

Sub-chronic exposure to noise affects locomotor activity and produces anxiogenic and depressive like behavior in rats

Noise is defined as a displeasing and unwanted sound. It is one of the most encountered stressor to which mankind is exposed. Frustration, poor reading, impaired hearing and difficulty in problem solving activities are the common consequences of noise stress. It has been reported to produce atrophy of dendrites and alterations in neurotransmitter levels. Long term exposure to inescapable noise stress induces exhaustion, defeat, annoyance followed by decreased muscle movement, social contacts and mood changes. The present study was aimed to investigate the detrimental effects of noise exposure on behavior of rats and its association with altered neurochemistry. Changes in neurotransmitter levels in different brain regions including hippocampus have been reported following noise exposure and these changes in neurotransmitters levels have also been associated with altered behavior. In the present study, locomotor activity in rats was assessed by open field test (OFT) while anxiety and depressive behavior was monitored by elevated plus maze (EPM) and tail suspension (TST) tests. The results showed that 15 days sub-chronic exposure to noise stress induced anxiety and depression like behavior in male rats. These behavioral deficits observed in the present study suggest that an altered brain serotonergic and dopaminergic activity may be involved in the various psychological disorders following exposure to noise stress.     

Participation of alpha-melanocyte stimulating hormone in ethanol-induced anxiolysis and withdrawal anxiety in rats

Although recent reports underscore a close association between the ethanol consumption and the central melanocortin (MC) system in rats, neurobehavioral component of this association has not been explored. In this study, we investigated the role of alpha-melanocyte stimulating hormone (alpha-MSH) in ethanol (1.5-2 g/kg, i.p.) induced anxiolysis and anxiety-like behavior following withdrawal from prolonged ethanol (9% v/v ethanol, 15 days) consumption, using elevated plus maze (EPM) test in rats. While alpha-MSH (1-5 microg/rat, i.c.v.) showed dose-dependent anxiogenic-like effect, the MC4 receptor antagonist HS014 (1-10 nM/rat, i.c.v.) or antiserum against alpha-MSH (1:500-1:50 dilution, 5 microl/rat, i.c.v.) failed to produce any effect in the EPM test. The anxiolytic-like effect of ethanol was suppressed by central administration of alpha-MSH (0.5 microg/rat, i.c.v.). On the other hand, pretreatment with either HS014 (5 nM/rat, i.c.v.) or antiserum against alpha-MSH (1:100 dilution, 5 microl/rat, i.c.v.) enhanced anxiolytic action of ethanol. Moreover, ethanol withdrawal anxiety was markedly blocked by HS014 (1-10 nM/rat, i.c.v.). These results suggest that alpha-MSH may be implicated in ethanol-induced anxiolysis and withdrawal anxiety. These findings also suggest MC4 receptors as possible therapeutic target for development of drugs to address the ethanol withdrawal-related conditions.     

Behavioral effects of saredutant, a tachykinin NK2 receptor antagonist, in experimental models of mood disorders under basal and stress-related conditions

The present study was made to investigate the role of tachykinin NK2 receptors in the expression of stress-related behaviors in animals. Under basal conditions, intraperitoneal (i.p.) administration of the selective tachykinin NK2 receptor antagonist, saredutant (1 and 3 mg/kg) or diazepam (1 mg/kg) exerted anxiolytic-like effects in rodents, as they reduced grooming score of Wistar male rats tested in the novelty-induced grooming sampling test (NGT) and increased percentage of time and entries in open arms of Swiss male mice tested in the elevated plus maze (EPM) test. After previous exposure to stress-related conditions, as induced by a 2-min forced swim made 5 min prior to the EPM test, saredutant but not diazepam, exhibited anxiolytic-like effects in mice. To study the antidepressant-like activity of tachykinin NK2 receptor antagonist under basal conditions, different groups of rats were injected i.p. with saredutant (2.5, 5 and 10 mg/kg) or the tricyclic antidepressant, clomipramine (50 mg/kg) and tested in the forced swim test (FST), a widely used antidepressant-responsive test. The influence of stress-related conditions was studied in rats subjected to electric foot-shocks (1 mA, 1 s) 24, 5 and 1 h prior to FST, after drugs injection. In the FST, clomipramine decreased the immobility time only under basal conditions, but not after application of acute foot-shocks. To the contrary, saredutant-treated rats also exhibited more active behavior in FST after previous exposure to stressors. These results give further support to the hypothesis that tachykinin NK2 receptors may be a therapeutic target for pharmacological treatment of stress-related diseases, such as anxiety and depression.     

Prior multiple ethanol withdrawals enhance stress-induced anxiety-like behavior: inhibition by CRF1- and benzodiazepine-receptor antagonists and a 5-HT1a-receptor agonist.

Repeated withdrawals from chronic ethanol induce a persistent adaptive change. Further, stress substitutes for the initial two withdrawals of a multiple-withdrawal protocol to sensitize rats to withdrawal-induced anxiety-like behavior ('anxiety'). Therefore, it was tested whether the persistent adaptation induced by multiple-withdrawal exposures allows stress to elicit anxiety after a period of abstinence. Social interaction was used to assess the degree of anxiety induced by 45 min of restraint stress 3, 7, or 14 days after rats were exposed to multiple withdrawals from a chronic 4.5% ethanol diet. Restraint stress reduced social interaction (ie anxiety-like behavior) at 3, but not at 7 or 14 days, after the multiple withdrawals. No anxiety response was observed in animals that received multiple withdrawals without stress or in animals that received stress when exposed only to control liquid diet. Drugs (ie a CRF1-receptor antagonist, a benzodiazepine receptor antagonist, and a 5-HT1A-receptor agonist) previously demonstrated to block the cumulative adaptation, when administered during repeated withdrawals, prevented stress-induced anxiety-like behavior during abstinence. Additionally, these drugs applied prior to stress in the rats previously exposed to the repeated withdrawal protocol, likewise, minimized stress-induced anxiety. The anxiety following stress during abstinence from previous chronic ethanol exposure is indicative of an interaction of stress with the persistent adaptive change caused by repeated withdrawals. Stress eliciting anxiety-like behavior during abstinence from previous ethanol exposures in rats is consistent with stress inducing anxiety during recovery (sobriety) in the alcoholic, a circumstance that can facilitate craving and relapse.     

Repeated lipopolysaccharide (LPS) or cytokine treatments sensitize ethanol withdrawal-induced anxiety-like behavior.

Previous investigations demonstrated that repeated stresses before an ethanol exposure sensitize ethanol withdrawal-induced anxiety-like behavior ('anxiety'). In addition to activating the hypothalamic-pituitary-adrenal axis, acute stress also elevates cytokines in brain. Initially, to test possible cytokine involvement in this stress/withdrawal protocol, cytokines were increased in brain with 2 weekly repeated lipopolysaccharide (LPS) administrations (1000 microg/kg) [corrected] (LPS/withdrawal protocol) or with twice weekly intracerebroventricular (i.c.v.) administrations of the cytokines IL-1 beta, CCL2 (MCP-1) or TNFalpha (cytokine/withdrawal protocol) before exposure and withdrawal from a 5-day cycle of chronic ethanol diet. Both protocols sensitized withdrawal-induced anxiety and confirm cytokine involvement in the sensitized anxiety response. Testing of various doses of LPS (16-1000 microg/kg) and TNFalpha (3-100 ng, i.c.v.) demonstrated the dose-related nature of these protocols to sensitize withdrawal-induced anxiety. The sensitized anxiety was not produced by a single 5-day ethanol diet cycle or by repeated LPS or cytokine treatments alone. Likewise, sensitized anxiety in these protocols could not be attributed to differences in ethanol ingestion. When challenged with a subsequent re-exposure to a 5-day ethanol diet cycle 16 days after completion of the LPS/withdrawal or cytokine/withdrawal protocols, an increase in withdrawal-induced anxiety was observed-an indication of induction of an underlying persistent adaptive change. Finally, just as found previously with the stress/withdrawal protocol, administration of the benzodiazepine receptor antagonist flumazenil before the LPS or TNF treatments prevented anxiety sensitization. Together, these findings indicate that increased cytokine activity induces adaptive change that supports sensitization of ethanol withdrawal-induced anxiety that may be linked to GABA(A)-receptor function.     

Anxiety-induced antinociception in mice: effects of systemic and intra-amygdala administration of 8-OH-DPAT and midazolam

RATIONALE: Mice exhibit antinociception after a single experience in the elevated plus maze (EPM), an animal model of anxiety. OBJECTIVE: This study investigated the mechanisms involved in this form of anxiety-induced antinociception. METHODS: Nociception was evaluated by means of the writhing test in mice confined either to the open or enclosed arms of the EPM. The effects of systemic (naloxone, midazolam and 8-OH-DPAT) or intra-amygdala (8-OH-DPAT, NAN-190 and midazolam) drug infusions were investigated in mice previously treated i.p. with 0.6% acetic acid, an algic stimulus that induces abdominal contortions. The effects of these drugs on conventional measures of anxiety (% entries and % time in open arms) in a standard EPM test were also independently investigated. RESULTS: Open-arm confinement resulted in a high-magnitude antinociception (minimum 85%, maximum 450%) compared with enclosed arm confinement. The opiate antagonist naloxone (1 mg/kg and 10 mg/kg) neither blocked this open arm-induced antinociception (OAIA) nor modified indices of anxiety in EPM. Administration of midazolam (0.5-2 mg/kg, s.c.) increased OAIA and produced antinociception in enclosed confined animals, as well as attenuating anxiety in the EPM. The 5-HT(1A) receptor agonist 8-OH-DPAT (0.05-1 mg/kg, s.c.) had biphasic effects on OAIA, antagonising the response at the lowest dose and intensifying it at the highest dose. In addition, low doses of this agent reduced anxiety in the EPM. Although bilateral injections of 8-OH-DPAT (5.6 nmol/0.4 microl) or NAN-190 (5.6 nmol and 10 nmol/0.4 microl) into the amygdala did not alter OAIA, increased anxiety was observed in the EPM. In contrast, intra-amygdala administration of midazolam (10 nmol and 30 nmol/0.4 microl) blocked both OAIA and anxiety. CONCLUSIONS: These results with systemic and intracerebral drug infusion suggest that 5-HT(1A) receptors localised in the amygdala are not involved in the pain inhibitory processes that are "recruited" during aversive situations. However, activation of these receptors does phasically increase anxiety. Although the intrinsic antinociceptive properties of systemically administered midazolam confounded interpretation of its effects on OAIA, intra-amygdala injections of this compound suggest that benzodiazepine receptors in this brain region modulate both the antinociceptive and behavioural (anxiety) responses to the EPM.