Withdrawal-induced Anxiety-like Behavior Research Articles

Neuronal nicotinic acetylcholine receptor of the central amygdala modulates the ethanol-induced tolerance to anxiolysis and withdrawal-induced anxiety in male rats.

The nicotine acetylcholinergic receptor (nAchR) in the central nucleus of the amygdala (CeA) is known to modulate anxiety traits as well as ethanol-induced behavioral effects. Therefore, the present study investigated the role of CeA nAChR in the tolerance to ethanol anxiolysis and withdrawal-induced anxiety-related effects in rats on elevated plus maze (EPM). To develop ethanol dependence, rats were given free access to an ethanol-containing liquid diet for 10 days. To assess the development of tolerance, separate groups of rats were challenged with ethanol (2 g/kg, i.p.) on days 1, 3, 5, 7 and 10 during the period of ethanol exposure, followed by an EPM assessment. Moreover, expression of ethanol withdrawal was induced after switching ethanol-dependent rats to a liquid diet on day 11, and withdrawal-induced anxiety-like behavior was noted at different post-withdrawal time points using the EPM test. The ethanol-dependent rats were pretreated with intra-CeA (i.CeA) (bilateral) injections of nicotine (0.25 µg/rat) or mecamylamine (MEC) (5 ng/rat) before the challenge dose of ethanol on subthreshold tolerance on the 5th day or on peak tolerance day, that is, 7th or 10th, and before assessment of postwithdrawal anxiety on the 11th day on EPM. Bilateral i.CeA preadministration of nicotine before the challenge dose of ethanol on days 5, 7 and 10 exhibited enhanced tolerance, while injection of MEC, completely mitigated the tolerance to the ethanol-induced antianxiety effect. On the other hand, ethanol-withdrawn rats pretreated i.CeA with nicotine exacerbated while pretreatment with MEC, alleviated the ethanol withdrawal-induced anxiety on all time points. Thus, the present investigation indicates that stimulation of nAChR in CeA negatively modulates the ethanol-induced chronic behavioral effects on anxiety in rats. It is proposed that nAChR antagonists might be useful in the treatment of alcohol use disorder and ethanol withdrawal-related anxiety-like behavior.

The Role of Microglia in Sex- and Region-Specific Blood-Brain Barrier Integrity During Nicotine Withdrawal

BackgroundSmoking is the largest preventable cause of death and disease in the United States, with <5% of quit attempts being successful. Microglia activation and proinflammatory neuroimmune signaling in reward neurocircuitry are implicated in nicotine withdrawal symptomology. Microglia are integral regulators of blood-brain barrier (BBB) functionality as well; however, whether the effects of nicotine withdrawal on microglia function impact BBB integrity is unknown. MethodsMice were treated with chronic nicotine (12 mg/kg/day) and subjected to 48 hours nicotine withdrawal. Regional BBB permeability, together with messenger RNA and protein expression of tight junction proteins, were assessed. PLX5622 chow was used to deplete microglia to evaluate the role of microglia in regulating BBB integrity and nicotine withdrawal symptomology. ResultsFemale mice had higher baseline BBB permeability in the prefrontal cortex and hippocampus than males. Nicotine withdrawal further exacerbated the BBB permeability selectively in the prefrontal cortex of females. These effects were concurrent with prefrontal cortex alterations in a subset of tight junction proteins with increased proinflammatory responses following nicotine withdrawal in females. Depletion of microglia via PLX5622 treatment prevented all these molecular effects and attenuated withdrawal-induced anxiety-like behavior in female mice. ConclusionsThese results are the first to show sex differences in regional BBB permeability during nicotine withdrawal. This represents a possible link to both the reduced smoking cessation success seen in women and women’s increased risk for smoking-related neurovascular disorders. Furthermore, these findings open an avenue for sex-specific therapeutics that target microglia and BBB dysfunction during nicotine withdrawal in women.

Aspirin Effect on Tobacco Smoke Withdrawal-Induced Anxiety In Female Rats

Chronic exposure to cigarette smoke produces neuroinflammation and long-term changes in neurotransmitter systems, especially glutamatergic systems. We examined the effects of cigarette smoke on astroglial glutamate transporters as well as NF-κB expression in mesocorticolimbic brain regions including the prefrontal cortex (PFC) and the nucleus accumbens (NAc). The behavioral consequences of cigarette smoke exposure on withdrawal-induced anxiety-like behavior were assessed using open field (OF) and light/dark (LD) tests. Sprague-Dawley rats were randomly assigned to 5 experimental groups: a control group exposed only to standard room air, a cigarette smoke exposed group treated with saline vehicle, two cigarette smoke exposed groups treated with aspirin (15mg/kg and 30mg/kg, respectively). Lastly, a group treated only with aspirin (30 mg/kg). Cigarette smoke exposure was performed for 2hr/day, 5days/week, for 31days. Behavioral tests were conducted weekly, 24hrs after cigarette smoke exposure, during acute withdrawal. At the end of week 4, rats were given either saline or aspirin 45 min before cigarette exposure for 11 days. Cigarette smoke increased withdrawal-induced anxiety, and 30 mg/kg aspirin attenuated this effect. Cigarette smoke exposure increased the relative mRNA and the protein level for nuclear factor ĸB (NFĸB) in the PFC and the NAc, and aspirin treatment reversed this effect. In addition, cigarette smoke decreased the relative mRNA and the protein levels of glutamate transporter 1 (GLT-1) and the cystine-glutamate transporter (xCT) in the PFC and the NAc, while aspirin treatment normalized their expression. Thus, cigarette smoke caused neuroinflammation, alterations in relative mRNA glutamate transporter expression, and increased anxiety-like behavior, and these effects were attenuated by aspirin treatment.

Nanoassemblies from the aqueous extract of roasted coffee beans modulate the behavioral and molecular effects of smoking withdrawal-induced anxiety in female rats.

Antioxidant-rich plant extracts have demonstrated tremendous value as inflammatory modulators and as nanomaterial precursors. Chronic cigarette smoking alters neurotransmitter systems, particularly the glutamatergic system, and produces neuroinflammation. This study aimed to investigate the behavioral and molecular correlates of cigarette smoking withdrawal-induced anxiety-like behavior in rats, and whether these effects could be mitigated by the administration of antioxidant nanoassemblies prepared by spontaneous oxidation of dark-roasted Arabica coffee bean aqueous extracts. Four experimental groups of female Sprague-Dawley rats were randomly assigned to: (i) a control group that was only exposed to room air, (ii) a COF group that was administered 20mg/kg of the coffee nanoassemblies by oral gavage, (iii) a SMOK group that was exposed to cigarette smoke and was given an oral gavage of distilled water, (iv) and a SMOK + COF group that was exposed to cigarette smoke and administered 20mg/kg of the coffee nanoassemblies. Animals were exposed to cigarette smoke for 2h per day, fivedays per week, with a 2-day withdrawal period each week. At the end of the 4th week, rats began receiving either distilled water or the coffee nanoassemblies before being exposed to cigarette smoke for 21 additional days. Weekly behavioral tests revealed that cigarette smoking withdrawal exacerbated anxiety, while the administration of the coffee nanoassemblies reduced this effect. The effect of cigarette smoking on astroglial glutamate transporters and nuclear factor kappa B (NF-κB) expression in brain subregions was also measured. Smoking reduced the relative mRNA and protein levels of the glutamate transporter 1 (GLT-1) and the cystine/glutamate antiporter (xCT), and increased the levels of NF-κB, but these effects were attenuated by the coffee nanoassemblies. Thus, administration of the antioxidant nanoassemblies decreased the negative effects of cigarette smoke, which included neuroinflammation, changes in glutamate transporters' expression, and a rise in anxiety-like behavior.

Identification of candidate genes for nicotine withdrawal in C57BL/6J×DBA/2J recombinant inbred mice.

Nicotine is the reinforcing ingredient in tobacco. Following chronic exposure, sudden cessation of nicotine use produces negative symptoms of withdrawal that contribute to dependence. The molecular mechanisms underlying nicotine withdrawal behaviors, however, are poorly understood. Using recombinant inbred mice, chronic nicotine was delivered by minipump and withdrawal induced using mecamylamine. Somatic signs of withdrawal, and anxiety-like behavior using elevated plus maze, were then assessed. Interval mapping was used to identify associations between genetic variation and withdrawal behaviors, and with basal gene expression. Differential gene expression following nicotine exposure and withdrawal was also assessed in progenitor mice using microarrays. Quantitative trait loci mapping identified chromosome intervals with significant genetic associations to somatic signs of withdrawal or withdrawal-induced anxiety-like behavior. Using bioinformatics, and association with basal gene expression in nucleus accumbens, we implicated Rb1, Bnip3l, Pnma2, Itm2b, and Kif13b as candidate genes for somatic signs of withdrawal, and Galr1, which showed trans-regulation from a region of chromosome 14 that was associated with somatic signs of withdrawal. Candidate genes within the chromosome 9 region associated with anxiety-like withdrawal behavior included Dixdc1, Ncam1, and Sorl1. Bioinformatics identified six genes that were also significantly associated with nicotine or alcohol traits in recent human genome-wide association studies. Withdrawal-associated somatic signs and anxiety-like behavior had strong non-overlapping genetic associations, respectively, with regions of chromosome 14 and chromosome 9. Genetic, behavioral and gene expression correlations, and bioinformatics analysis identified several candidate genes that may represent novel molecular targets for modulating nicotine withdrawal symptoms.

Effects of waterpipe tobacco smoke and ceftriaxone treatment on the expression of endocannabinoid receptors in mesocorticolimbic brain regions

Chronic tobacco exposure can alter the endocannabinoid (eCB) system, consequently leading to an anxiety state. In this study, we investigated the effects of waterpipe tobacco smoke (WTS) on cannabinoid receptor 1 and 2 (CBR1 and CBR2) gene and protein expression in mesocorticolimbic brain regions. Using elevated plus maze (EPM) and open field (OF) tests, the effects of WTS exposure on withdrawal-induced anxiety-like behavior were examined. The effect of ceftriaxone (CEF), a β-lactam known to upregulate glutamate transporter 1 (GLT-1), on anxiety and the expression of cannabinoid receptors was also determined. Male Sprague-Dawley rats were randomly assigned to four groups: 1) the Control group was exposed only to standard room air; 2) the WTS group was exposed to tobacco smoke and treated with saline vehicle; 3) the WTS-CEF group was exposed to WTS and treated with ceftriaxone; and 4) the CEF group was exposed only to standard room air and treated with ceftriaxone. Rats were exposed to WTS (or room air) for two hours per day, five days per week for a period of four weeks. Behavioral tests (EPM and OF) were conducted weekly during acute withdrawal, 24 h following WTS exposure. Rats were given either saline or ceftriaxone (200 mg/kg i.p.) for five days during Week 4, 30 min prior to WTS exposure. Withdrawal-induced anxiety was induced by WTS exposure but was reduced by ceftriaxone treatment. WTS exposure decreased CBR1 mRNA and protein expression in the NAc and VTA, but not PFC, and ceftriaxone treatment attenuated these effects. WTS exposure did not change CBR2 mRNA expression in the NAc, VTA, or PFC. These findings demonstrate that WTS exposure dysregulated the endocannabinoid system and increased anxiety-like behavior, and these effects were reversed by ceftriaxone treatment, which suggest the involvement of glutamate transporter 1 in these effects.

Prolonged ethanol exposure modulates constitutive internalization and recycling of 5-HT1A receptors.

Alcohol exposure alters the signaling of the serotoninergic system, which is involved in alcohol consumption, reward, and dependence. In particular, dysregulation of serotonin receptor type 1A (5-HT1AR) is associated with alcohol intake and withdrawal-induced anxiety-like behavior in rodents. However, how ethanol regulates 5-HT1AR activity and cell surface availability remains elusive. Using neuroblastoma 2a cells stably expressing human 5-HT1ARs tagged with hemagglutinin at the N-terminus, we found that prolonged ethanol exposure (18h) reduced the basal surface levels of 5-HT1ARs in a concentration-dependent manner. This reduction is attributed to both enhanced receptor internalization and attenuated receptor recycling. Moreover, constitutive 5-HT1AR internalization in ethanol naïve cells was blocked by concanavalin A (ConA) but not nystatin, suggesting clathrin-dependent 5-HT1AR internalization. In contrast, constitutive 5-HT1AR internalization in ethanol-treated cells was blocked by nystatin but not by ConA, indicating that constitutive 5-HT1AR internalization switched from a clathrin- to a caveolin-dependent pathway. Dynasore, an inhibitor of dynamin, blocked 5-HT1AR internalization in both vehicle- and ethanol-treated cells. Furthermore, ethanol exposure enhanced the activity of dynamin I via dephosphorylation and reduced myosin Va levels, which may contribute to increased internalization and reduced recycling of 5-HT1ARs, respectively. Our findings suggest that prolonged ethanol exposure not only alters the endocytic trafficking of 5-HT1ARs but also the mechanism by which constitutive 5-HT1AR internalization occurs.

Ceftriaxone Reduces Waterpipe Tobacco Smoke Withdrawal-induced Anxiety in rats via Modulating the Expression of TNF-α/NFĸB, Nrf2, and GLT-1

Tobacco exposure has been linked to neuroinflammation and adaptive/maladaptive changes in neurotransmitter systems, including in glutamatergic systems. We examined the effects of waterpipe tobacco smoke (WTS) on inflammatory mediators and astroglial glutamate transporters in mesocorticolimbic brain regions including the prefrontal cortex (PFC), nucleus accumbens (NAc) and ventral tegmental area (VTA). The behavioral consequences of WTS exposure on withdrawal-induced anxiety-like behavior were assessed using elevated plus maze (EPM) and open field (OF) tests. Male Sprague-Dawley rats were randomly assigned to 3 experimental groups: a control group exposed only to standard room air, a WTS exposed group treated with saline vehicle, and a WTS exposed group treated with ceftriaxone. WTS exposure was performed for 2 h/day, 5 days/week, for 4 weeks. Behavioral tests (EPM and OF) were conducted weekly 24 h after WTS exposure, during acute withdrawal. During week 4, rats were given either saline or ceftriaxone (200 mg/kg i.p.) 30 min before WTS exposure. WTS increased withdrawal-induced anxiety, and ceftriaxone attenuated this effect. WTS exposure increased the relative mRNA levels for nuclear factor ĸB (NFĸB), tumor necrosis factor-α (TNF-α), and brain-derived neurotrophic factor (BDNF) in the PFC, NAc and VTA, and ceftriaxone treatment reversed these effects. In addition, WTS decreased the relative mRNA of nuclear factor erythroid 2 related factor 2 (Nrf2), glutamate transporter 1 (GLT-1) and cystine-glutamate transporter (xCT) in PFC, NAc and VTA, and ceftriaxone treatment normalized their expression. WTS caused neuroinflammation, alteration in relative mRNA glutamate transport expression, and increased anxiety-like behavior, and these effects were attenuated by ceftriaxone treatment.

Pituitary adenylate cyclase-activating polypeptide (PACAP) modulates dependence-induced alcohol drinking and anxiety-like behavior in male rats

Alcohol use disorder (AUD) is a devastating illness defined by periods of heavy drinking and withdrawal, often leading to a chronic relapsing course. Initially, alcohol is consumed for its positive reinforcing effects, but later stages of AUD are characterized by drinking to alleviate withdrawal-induced negative emotional states. Brain stress response systems in the extended amygdala are recruited by excessive alcohol intake, sensitized by repeated withdrawal, and contribute to the development of addiction. In this study, we investigated one such brain stress response system, pituitary adenylate cyclase-activating polypeptide (PACAP), and its cognate receptor, PAC1R, in alcohol withdrawal-induced behaviors. During acute withdrawal, rats exposed to chronic intermittent ethanol vapor (ethanol-dependent) displayed a significant increase in PACAP levels in the bed nucleus of the stria terminalis (BNST), a brain area within the extended amygdala critically involved in both stress and withdrawal. No changes in PACAP levels were observed in the central nucleus of the amygdala. Site-specific microinfusion of the PAC1R antagonist PACAP(6-38) into the BNST dose-dependently blocked excessive alcohol intake in ethanol-dependent rats without affecting water intake overall or basal ethanol intake in control, nondependent rats. Intra-BNST PACAP(6-38) also reversed ethanol withdrawal-induced anxiety-like behavior in ethanol-dependent rats, but did not affect this measure in control rats. Our findings show that chronic intermittent exposure to ethanol recruits the PACAP/PAC1R system of the BNST and that these neuroadaptations mediate the heightened alcohol drinking and anxiety-like behavior observed during withdrawal, suggesting that this system represents a major brain stress element responsible for the negative reinforcement associated with the "dark side" of alcohol addiction.

Impact of the metabotropic glutamate receptor7 (mGlu7) allosteric agonist, AMN082, on fear learning and memory and anxiety-like behavior

The present study was conducted to evaluate the influence of AMN082, the metabotropic glutamate receptor subtype 7 (mGlu7) allosteric agonist on different stages of memory processes connected with fear conditioning in the passive avoidance (PA) learning task in mice and negative emotional state (anxiety-like) induced by ethanol- and morphine-withdrawal in the elevated plus maze (EPM) test in rats. To perform the PA test, AMN082 (1.25, 2.5 and 5 mg/kg, i. p.) was injected to interfere with (or inhibit) acquisition, consolidation, and retrieval processes. The retention latency in each group was recorded using a step-through passive avoidance task 24 h after training. In turn, in ethanol- and morphine-withdrawal rats, the influence of AMN082 on anxiety-like behavior was estimated in the EPM test 24 h- (ethanol) and 72- h (morphine) after the last dose of repeated drug administrations. In all experimental groups, AMN082 at the dose of 5 mg/kg significantly decreased the step-through latency of long-term memory in the PA task. These AMN082 effects were reversed by MMPIP (10 mg/kg), the antagonist of mGlu7 receptor. AMN082 (2.5 and 5 mg/kg) also decreased ethanol- and morphine withdrawal-induced anxiety-like behavior in the EPM test, and this AMN082 (5 mg/kg) effect was counteracted by MMPIP pretreatment. Taken together, the results show that mGlu7 is involved in fear learning to the context and anxiety-like state connected with unpleasant experiences after ethanol- and morphine withdrawal in rodents. However, it appears that functional dissociation exists between these two AMN082 effects.

Inhibition of the rostromedial tegmental nucleus reverses alcohol withdrawal-induced anxiety-like behavior.

Acute withdrawal from alcohol is associated with a number of unpleasant symptoms that play an important role in preventing recovery and long-term abstinence. Considerable research has focused on the role that neuropeptide systems and the amygdala play in mediating affective symptoms of acute withdrawal, but promising preclinical findings have not translated successfully into the clinic. The rostromedial tegmental nucleus (RMTg) has been implicated in both fear and anxiety. In addition, RMTg neurons exert inhibitory control over midbrain dopamine neurons, the activity of which are suppressed during acute withdrawal. Thus, we hypothesized that the RMTg may play a role in mediating symptoms of acute withdrawal. Using a chronic ethanol vapor exposure paradigm that renders rats physically dependent on ethanol, we observed significant withdrawal-induced enhancement of cFos expression in the RMTg. This was accompanied by a significant increase in somatic symptoms and a decrease in reward sensitivity as measured by intracranial self-stimulation (ICSS). Both measures followed a similar time course to RMTg cFos expression with peak symptom severity occurring 12 h following cessation of ethanol exposure. Heightened anxiety-like behavior was also observed in withdrawn rats at this same time point. RMTg inhibition had no effect on somatic signs of withdrawal or withdrawal-induced changes in reward sensitivity, but significantly attenuated withdrawal-induced anxiety-like behavior. Together, these data demonstrate that the RMTg plays a distinct role in the negative affective state associated with acute withdrawal and may therefore be critically involved in the neurobiological mechanisms that promote relapse during early stages of recovery.

Distinct Roles for Two Chromosome 1 Loci in Ethanol Withdrawal, Consumption, and Conditioned Place Preference.

We previously identified a region on chromosome 1 that harbor quantitative trait loci (QTLs) with large effects on alcohol withdrawal risk using both chronic and acute models in mice. Here, using newly created and existing QTL interval-specific congenic (ISC) models, we report the first evidence that this region harbors two distinct alcohol withdrawal QTLs (Alcw11and Alcw12), which underlie 13% and 3–6%, respectively, of the genetic variance in alcohol withdrawal severity measured using the handling-induced convulsion. Our results also precisely localize Alcw11 and Alcw12 to discreet chromosome regions (syntenic with human 1q23.1–23.3) that encompass a limited number of genes with validated genotype-dependent transcript expression and/or non-synonymous sequence variation that may underlie QTL phenotypic effects. ISC analyses also implicate Alcw11and Alcw12 in withdrawal-induced anxiety-like behavior, representing the first evidence for their broader roles in alcohol withdrawal beyond convulsions; but detect no evidence for Alcw12 involvement in ethanol conditioned place preference (CPP) or consumption. Our data point to high-quality candidates for Alcw12, including genes involved in mitochondrial respiration, spatial buffering, and neural plasticity, and to Kcnj9 as a high-quality candidate for Alcw11. Our studies are the first to show, using two null mutant models on different genetic backgrounds, that Kcnj9 −/− mice demonstrate significantly less severe alcohol withdrawal than wildtype littermates using acute and repeated exposure paradigms. We also demonstrate that Kcnj9 −/− voluntarily consume significantly more alcohol (20%, two-bottle choice) than wildtype littermates. Taken together with evidence implicating Kcnj9 in ethanol CPP, our results support a broad role for this locus in ethanol reward and withdrawal phenotypes. In summary, our results demonstrate two distinct chromosome 1 QTLs that significantly affect risk for ethanol withdrawal, and point to their distinct unique roles in alcohol reward phenotypes.

Preclinical evaluation of the kappa-opioid receptor antagonist CERC-501 as a candidate therapeutic for alcohol use disorders.

Prior work suggests a role of kappa-opioid signaling in the control of alcohol drinking, in particular when drinking is escalated due to alcohol-induced long-term neuroadaptations. Here, we examined the small molecule selective kappa antagonist CERC-501 in rat models of alcohol-related behaviors, with the objective to evaluate its potential as a candidate therapeutic for alcohol use disorders. We first tested the effect of CERC-501 on acute alcohol withdrawal-induced anxiety-like behavior. CERC-501 was then tested on basal as well as escalated alcohol self-administration induced by 20% alcohol intermittent access. Finally, we determined the effects of CERC-501 on relapse to alcohol seeking triggered by both stress and alcohol-associated cues. Control experiments were performed to confirm the specificity of CERC-501 effects on alcohol-related behaviors. CERC-501 reversed anxiety-like behavior induced by alcohol withdrawal. It did not affect basal alcohol self-administration but did dose-dependently suppress self-administration that had escalated following long-term intermittent access to alcohol. CERC-501 blocked relapse to alcohol seeking induced by stress, but not when relapse-like behavior was triggered by alcohol-associated cues. The effects of CERC-501 were observed in the absence of sedative side effects and were not due to effects on alcohol metabolism. Thus, in a broad battery of preclinical alcohol models, CERC-501 has an activity profile characteristic of anti-stress compounds. Combined with its demonstrated preclinical and clinical safety profile, these data support clinical development of CERC-501 for alcohol use disorders, in particular for patients with negatively reinforced, stress-driven alcohol seeking and use.

Distinct Roles of CREB Within the Ventral and Dorsal Hippocampus in Mediating Nicotine Withdrawal Phenotypes.

Addiction to nicotine and the inability to quit smoking are influenced by genetic factors, emphasizing the importance of understanding how genes and drugs of abuse mechanistically impact each other. One well-characterized protein responsible for regulating both response to drugs and gene expression is the transcription factor CREB (cAMP-responsive element binding protein). Previous work indicates that hippocampal-specific alterations in CREB signaling and synaptic plasticity may underlie certain nicotine withdrawal phenotypes. However, the structure of the hippocampus possesses dorsal and ventral subregions, each differing in behavioral, anatomic and gene expression characteristics. This study examines the effects of CREB deletion specifically in the ventral or dorsal hippocampus of animals chronically treated with saline, nicotine, or undergoing 24 h withdrawal. After region-specific viral injections of AAV-GFP or AAV-CRE in CREBloxP/loxP animals, behavioral testing measured anxiety levels, using the Novelty-Induced Hypophagia test, and cognition, using a contextual fear conditioning paradigm. Deletion of CREB in the ventral, but not dorsal, hippocampus resulted in amelioration of nicotine withdrawal-induced anxiety-like behavior in the Novelty-Induced Hypophagia test. In contrast, CREB deletion in the dorsal hippocampus resulted in learning and memory deficits in fear conditioning, whereas CREB deletion in the ventral hippocampus showed an enhancement in learning. Gene expression analysis showed differential treatment- and region-dependent alterations of several CREB target genes that are well-known markers of neuroplasticity within the hippocampus. Collectively, these data provide persuasive evidence towards the distinct roles of CREB within the dorsal and ventral hippocampus separately in mediating select nicotine withdrawal phenotypes.

Differences in Behavioral Responding in Adult and Aged Rats Following Chronic Ethanol Exposure.

Research suggests symptoms of chronic alcoholism, and withdrawal may be more severe in elderly compared with younger adults. However, examination of the effects of long-term ethanol (EtOH) consumption and withdrawal is limited in aged rodents. We thus investigated EtOH withdrawal and potential deficits in cognitive and motor behavior in young adult and aged rats. We also examined the effects of acute allopregnanolone as a potential mechanism contributing to age-related differences in EtOH's cognitive-impairing effects. Male young adult (postnatal days 70 to 72) and aged (approximately 18months) Sprague-Dawley rats were treated with liquid EtOH diet in a modified chronic intermittent EtOH (modified-CIE) paradigm. The severity of EtOH withdrawal was determined using a 4-item rating scale, and withdrawal-induced anxiety-like behavior was assessed in the elevated plus maze (EPM) and open field. After a 14-day EtOH-free period, spatial performance was assessed in the Morris water maze (MWM) during sober acquisition and in response to a subsequent EtOH and allopregnanolone challenge. Modified-CIE adults consumed more EtOH during treatment and exhibited robust EtOH withdrawal using a behavioral rating scale compared to aged rats. In the EPM, adult and aged modified-CIE groups spent increased time in the closed arms, while aged animals also made significantly more closed arm entries, fewer open arm entries, and spent less time in the open arms during withdrawal compared to controls. Modified-CIE decreased MWM performance of adult and aged rats, but did not result in motor impairments in either age group. Finally, acute allopregnanolone increased time to the MWM platform in adults but not aged animals. The elderly may be vulnerable to EtOH withdrawal as modified-CIE aged rats displayed anxiety-like behavior compared to controls during withdrawal despite achieving lower blood EtOH concentrations during treatment than younger adults. Our data also indicate that modified-CIE and EtOH withdrawal cause persistent cognitive impairments in both age groups. The results from this study provide further evidence indicating the elderly may be sensitive to the effects of alcohol.

Enkephalin analog, cyclo[Nε,Nβ-carbonyl-D-Lys2,Dap5] enkephalinamide (cUENK6), inhibits the ethanol withdrawal-induced anxiety-like behavior in rats

An analog of enkephalin, cyclo[Nε,Nβ-carbonyl-D-Lys2,Dap5] enkephalinamide (cUENK6), is predominantly a functional agonist of μ-opioid receptors (MOPr) and, to a lesser extent, of δ-opioid receptors (DOPr) in vitro. The aim of the present study was to determine whether cUENK6 could affect ethanol withdrawal-induced anxiety-like behavior in the elevated plus maze (EPM) test in rats. An anxiety-like effect of withdrawal was predicted to occur in the EPM test 24 h after the last ethanol administration (2 g/kg, intraperitoneally [i.p.]; 15% w/v once daily for 9 days). Ethanol withdrawal decreased the percent of time spent by rats in the open arms and the percent of open-arms entries. cUENK6 (0.25 nmol), given by intracerebroventricular (i.c.v.) injection, significantly reversed these anxiety-like effects of ethanol withdrawal and elevated the percent of time spent by rats in the open arms and the percent of open-arms entries. These effects of cUENK6 were significantly inhibited by the DOPr antagonist naltrindole (NTI) (5 nmol, i.c.v.), but not by the MOPr antagonist β-funaltrexamine (β-FNA) (5 nmol, i.c.v.). The preferential DOPr agonist [Leu5]-enkephalin (LeuEnk) (2.7 and 5.4 nmol, i.c.v.) and the MOPr agonist morphine (6.5 and 13 nmol, i.c.v.) reduced the anxiety-like effects of ethanol withdrawal. cUENK6 at the dose of 0.25 nmol did not disturb locomotor activity in the EPM, in contrast to cUENK6 at the dose of 0.5 nmol, and morphine at 6.5 and 13 nmol. However, similarly to LeuEnk, cUENK6 induced the anxiolytic-like effects in naïve rats. Thus, our study suggests that cUENK6 reduced ethanol withdrawal-induced anxiety-like behavior by activation of δ-opioid receptors rather than μ-opioid receptors.

Involvement of the central melanocortin system in the effects of caffeine on anxiety-like behavior in mice

AimsTo investigate the role of the melanocortin (MC) system in the framework of the central nucleus of the amygdala (CeA) in the differential effects of the adenosine receptor blocker caffeine on anxiety-like behavior, using the social interaction (SI) test. Main methodsCaffeine was injected intraperitoneally, alone or in combination with alpha-melanocyte stimulating hormone (α-MSH), the MC4 receptor agonist RO27-3225 or the antagonist HS014 via the intra-CeA route. The effects of chronic (21days) caffeine, given alone or concurrently with α-MSH, or RO27-3225, were investigated. The effects of withdrawal of these treatments on SI time were also evaluated. Furthermore, the acute effects of HS014 were investigated in different sets of caffeine-withdrawn mice. Key findingsAcute injection of caffeine, RO27-3225, or α-MSH produced anxiety-like behavior. Prior treatment with α-MSH, or RO27-3225 potentiated the caffeine-induced anxiety-like behavior. Subchronic treatment with HS014 increased the SI time, which was attenuated by caffeine. Chronic administration of caffeine resulted in tolerance to caffeine's anxiogenic effect, while abrupt discontinuation of the treatment produced peak anxiety-like behavior at 72h post-withdrawal. Concurrent administration of α-MSH, or RO27-3225 with chronic caffeine delayed the development of tolerance and prevented withdrawal-induced anxiety-like behavior. Moreover, acute treatment with HS014 at 72h post-withdrawal attenuated the anxiety-like behavior. Significanceα-MSH, possibly via MC4 receptor in the neuroanatomical framework of the CeA, may contribute to the acute, chronic and withdrawal actions of caffeine associated with anxiety-like behavior in the neuroanatomical framework of the CeA.

Enhancement of Behavioral Sensitization, Anxiety-Like Behavior, and Hippocampal and Frontal Cortical CREB Levels Following Cocaine Abstinence in Mice Exposed to Cocaine during Adolescence

Adolescence has been linked to greater risk-taking and novelty-seeking behavior and a higher prevalence of drug abuse and risk of relapse. Decreases in cyclic adenosine monophosphate response element binding protein (CREB) and phosphorylated CREB (pCREB) have been reported after repeated cocaine administration in animal models. We compared the behavioral effects of cocaine and abstinence in adolescent and adult mice and investigated possible age-related differences in CREB and pCREB levels. Adolescent and adult male Swiss mice received one daily injection of saline or cocaine (10 mg/kg, i.p.) for 8 days. On day 9, the mice received a saline injection to evaluate possible environmental conditioning. After 9 days of withdrawal, the mice were tested in the elevated plus maze to evaluate anxiety-like behavior. Twelve days after the last saline/cocaine injection, the mice received a challenge injection of either cocaine or saline, and locomotor activity was assessed. One hour after the last injection, the brains were extracted, and CREB and pCREB levels were evaluated using Western blot in the prefrontal cortex (PFC) and hippocampus. The cocaine-pretreated mice during adolescence exhibited a greater magnitude of the expression of behavioral sensitization and greater cocaine withdrawal-induced anxiety-like behavior compared with the control group. Significant increases in CREB levels in the PFC and hippocampus and pCREB in the hippocampus were observed in cocaine-abstinent animals compared with the animals treated with cocaine in adulthood. Interestingly, significant negative correlations were observed between cocaine sensitization and CREB levels in both regions. These results suggest that the behavioral and neurochemical consequences of psychoactive substances in a still-developing nervous system can be more severe than in an already mature nervous system.

Effects of nitric oxide synthase inhibition in the dorsolateral periaqueductal gray matter on ethanol withdrawal-induced anxiety-like behavior in rats

Nitric oxide (NO)-mediated transmission in the dorsolateral periaqueductal gray matter (dlPAG) has been involved in the expression of anxiety-like behaviors. Ethanol withdrawal sensitizes the dlPAG and results in increased anxiety-like responses. The objective of the study was to test the hypothesis that NO in the dlPAG is involved in the expression of ethanol withdrawal-induced anxiety. Male Wistar rats were implanted with guide cannulae aimed at the dlPAG. The animals were forced to consume a liquid diet containing ethanol 6-8 % (v/v) for 15 days as their only source of diet. Six days after surgery and 24 h after ethanol discontinuation, the animals received microinjections of the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), nonselective nitric oxide synthase inhibitor N (G)-nitro-L-arginine methyl ester (L-NAME), selective neuronal nitric oxide synthase inhibitor 1-(2-[trifluoromethyl]phenyl) imidazole (TRIM), or selective inducible nitric oxide synthase (iNOS) inhibitor N-([3-(aminomethyl)phenyl]methyl) ethanimidamide dihydrochloride (1400W) into the dlPAG. Ten minutes later, the animals were tested in the light/dark box. Carboxy-PTIO (1 nmol), L-NAME (200 nmol), TRIM (20 nmol), and 1400W (0.3 and 1 nmol) decreased the anxiogenic-like effects of ethanol withdrawal in rats in the light/dark box test. The NO precursor L-arginine reversed the effects of L-NAME. NO production in the dlPAG may play a role in the modulation of ethanol withdrawal-induced anxiety-like behavior in rats. Furthermore, iNOS-mediated NO synthesis in the dlPAG is predominantly involved in the behavioral expression of anxiety-like behavior during ethanol withdrawal.

Amygdaloid Corticotropin-releasing Factor is Involved in the Anxiolytic Effect of Acupuncture during Ethanol Withdrawal in Rats

In a previous study, acupuncture at acupoint HT7 attenuated ethanol withdrawal-induced anxiety-like behavior in rats by normalizing amygdaloid catecholamines. In the present study, the involvement of amygdaloid corticotropin-releasing factor (CRF) in the anxiolytic effect of acupuncture was investigated during ethanol withdrawal. Rats were intraperitoneally treated with 3 g /kg/day of ethanol for 28 days, and the CRF mRNA levels in the central nucleus of the amygdala (CEA) were measured by using a RT-PCR analysis 72 hours after the last dose of ethanol. During ethanol withdrawal, the rats were bilaterally treated with acupuncture at acupoints HT7, PC6 or at a non-acupoint (Tail) for one min/day for three days. Also, rats were bilaterally injected with CRF into the CEA five minutes after the third acupuncture treatment, after which followed by the elevated-plus maze (EPM) test and the plasma corticosterone radioimmunoassay (RIA) were administered. The RT-PCR analysis showed a significant increase in the amygdaloid CRF mRNA levels in the ethanol-withdrawn rats compared with both the saline-treated rats and the rats treated with acupuncture at HT7, but neither acupuncture at PC6 nor acupuncture at a non-acupoint significantly inhibited the increased mRNA expression. The EPM test and the RIA also showed that the post-acupuncture infusion of CRF greatly reduced the anxiolytic effect of acupuncture at HT7. These results suggest that during ethanol withdrawal, the anxiolytic effect of acupuncture may be mediated through the modulation of amydaloid CRF during ethanol withdrawal.