Withdrawal Time Points Research Articles

LGG-09. REBOUND GROWTH AFTER MAPKI WITHDRAWAL INVOLVES RAPID MAPK REACTIVATION AND CYTOKINE-MEDIATED MICROGLIA RECRUITMENT IN PEDIATRIC LOW-GRADE GLIOMA MODEL

Abstract Patients with pediatric low-grade gliomas (pLGG), the most common primary brain tumor in children, can often benefit from MAPK inhibitor (MAPKi) treatment. However, rapid tumor regrowth, also referred to as rebound growth, may occur once treatment is stopped, constituting a significant clinical challenge. Four patient-derived pLGG models were investigated to model rebound growth in vitro based on viable cell counts in response to MAPKi treatment and withdrawal. A multi-omics dataset of the rebound model encompassing different MAPKi withdrawal timepoints was generated using RNA sequencing and LC-MS/MS based phospho-/proteomics to investigate possible driving mechanisms of rebound growth. Following in vitro validation, putative rebound driving mechanisms were validated in vivo using the BT-40 orthotopic xenograft model. Of the tested models, BT-40 (BRAFV600E, CDKN2A/Bdel) showed rebound growth upon MAPKi withdrawal, characterized by faster cell regrowth after MAPKi withdrawal compared to standard-of-care chemotherapy. Using this model, we observed MAPK pathway reactivation within hours after withdrawal, associated with a transient overactivation of key MAPK molecules at transcriptional (e.g. FOS) and phosphorylation (e.g. pMEK) levels. Additionally, we observed increased expression and secretion of cytokines (in particular CCL2, CX3CL1, CXCL10 and CCL7) upon MAPKi treatment, maintained during early withdrawal (at least until 24 h). While increased cytokine expression did not affect response to MAPKi or rebound growth upon withdrawal in an autocrine manner, increased attraction of microglia cells mediated by these cytokines was observed. MAPK pathway reactivation during rebound growth and increased expression of CX3CL1 and CXCL10 induced by MAPKi treatment could further be confirmed in vivo. Taken together, these data indicate a rapid MAPK reactivation upon MAPKi withdrawal as a tumor cell intrinsic rebound driving mechanism. Furthermore, increased microglia recruitment during MAPKi treatment and withdrawal, mediated by cytokines, may play a role in response to MAPKi treatment and rebound growth upon withdrawal, warranting further evaluation.

Dorsal hippocampal astrocytes mediate the development of heroin withdrawal-enhanced fear learning

There is a significant co-occurrence of opioid use disorder (OUD) and post-traumatic stress disorder (PTSD) in clinical populations. However, the neurobiological mechanisms linking chronic opioid use, withdrawal, and the development of PTSD are poorly understood. Our previous research has shown that proinflammatory cytokines, expressed primarily by astrocytes in the dorsal hippocampus (DH), play a role in the development of heroin withdrawal-enhanced fear learning (HW-EFL), an animal model of PTSD-OUD comorbidity. Given the role of astrocytes in memory, fear learning, and opioid use, our experiments aimed to investigate their involvement in HW-EFL. Experiment 1 examined the effect of withdrawal from chronic heroin administration on GFAP surface area and volume, and identified increased surface area and volume of GFAP immunoreactivity in the dentate gyrus (DG) following 24-hour heroin withdrawal. Experiment 2 examined astrocyte morphology and synaptic interactions at the 24-hour withdrawal timepoint using an astroglial membrane-bound GFP (AAV5-GfaABC1D-lck-GFP). Although we did not detect significant changes in surface area and volume of GfaABC1D-Lck-GFP labelled astrocytes, we did observe a significant increase in the colocalization of astrocyte membranes with PSD-95 (postsynaptic density protein 95) in the DG. Experiment 3 tested if stimulating astroglial Gi signaling in the DH alters HW-EFL, and our results demonstrate this manipulation attenuates HW-EFL. Collectively, these findings contribute to our current understanding of the effects of heroin withdrawal on astrocytes and support the involvement of astrocytes in the comorbid relationship between opioid use and anxiety disorders.

Cap-assisted colonoscopy can increase the rate of sessile serrated lesion detection at the left lateral decubitus position: A retrospective case-control study.

We investigated the effectiveness of cap-assisted colonoscopy conducted with the patient in the left lateral decubitus position at both the colonoscope's insertion and withdrawal timepoints compared to the effectiveness of colonoscopy without a cap conducted in the supine position at withdrawal. This was a case-control study, based on historical comparisons of patients over 2 time periods. The first group of patients underwent colonoscopies with a transparent cap and the patient was in the left lateral decubitus position at both the insertion and withdrawal timepoints from April to June 2019. The subsequent group underwent colonoscopies without a cap and with the patient in the supine position at withdrawal from July to September 2019. The rates of successful intubation, cecal intubation time, and number, size, shape, and location of the detected adenomas and sessile serrated lesions were compared between the 2 groups. Data from 644 colonoscopies (cap-assisted colonoscopy + left lateral decubitus position, n=320; other colonoscopies, n=324) were analyzed. The demographic characteristics and technical performances were similar. The SSL detection rate was significantly higher with cap-assisted colonoscopy and the left lateral decubitus position than with other colonoscopies (3.4% vs 0.93%, P=.029). The adenoma detection rates in the 2 groups were similar (31% and 28%, respectively, P=.43).Cap-assisted colonoscopy in the left lateral decubitus position may increase the detection rate of sessile serrated lesions compared to colonoscopy without a cap and supine position at withdrawal.

The transcriptional response to acute cocaine is inverted in male mice with a history of cocaine self-administration and withdrawal throughout the mesocorticolimbic system

A large body of work has demonstrated that cocaine-induced changes in transcriptional regulation play a central role in the onset and maintenance of cocaine use disorder. An underappreciated aspect of this area of research, however, is that the pharmacodynamic properties of cocaine can change depending on an organism's previous drug-exposure history. In this study, we utilized RNA sequencing to characterize how the transcriptome-wide effects of acute cocaine exposure were altered by a history of cocaine self-administration and long-term withdrawal (30 days) in the ventral tegmental area (VTA), nucleus accumbens (NAc), and prefrontal cortex (PFC) in male mice. First, we found that the gene expression patterns induced by a single cocaine injection (10 mg/kg) were discordant between cocaine-naïve mice and mice in withdrawal from cocaine self-administration. Specifically, the same genes that were upregulated by acute cocaine in cocaine-naïve mice were downregulated by the same dose of cocaine in mice undergoing long-term withdrawal; the same pattern of opposite regulation was observed for the genes downregulated by initial acute cocaine exposure. When we analyzed this dataset further, we found that the gene expression patterns that were induced by long-term withdrawal from cocaine self-administration showed a high degree of overlap with the gene expression patterns of acute cocaine exposure - even though animals had not consumed cocaine in 30 days. Interestingly, cocaine re-exposure at this withdrawal time point reversed this expression pattern. Finally, we found that this pattern was similar across the VTA, PFC, NAc, and within each brain region the same genes were induced by acute cocaine, re-induced during long-term withdrawal, and reversed by cocaine re-exposure. Together, we identified a longitudinal pattern of gene regulation that is conserved across the VTA, PFC, and NAc, and characterized the genes constituting this pattern in each brain region.

Sex differences in the neuroadaptations associated with incubated cocaine-craving: A focus on the dorsomedial prefrontal cortex.

Women have a shorter course from initial cocaine use to meeting the criteria for cocaine use disorder as compared to men. Preclinical findings similarly indicate that females develop key features of an addiction-like phenotype faster than males, including an enhanced motivation for cocaine and compulsive use, indicating that this phenomenon is biologically based. The goals of this study were to determine whether cocaine-craving, another key feature of addiction, also develops sooner during withdrawal in females than males and to determine whether there are sex differences in the molecular mechanisms associated with its development focusing on markers known to mediate cocaine-craving in males (i.e., dorsomedial prefrontal cortex, dmPFC, expression of brain-derived neurotrophic factor exon-IV, Bdnf-IV, and NMDA receptor subunits, Grin2a, Grin2b, and Grin1). Cocaine-craving was assessed following extended-access cocaine self-administration and 2, 7, or 14 days of withdrawal using an extinction/cue-induced reinstatement procedure. Tissue was obtained from the dmPFC immediately after reinstatement testing and gene expression changes were analyzed using real-time qPCR. In males, cocaine-craving (total extinction and cue-induced reinstatement responding) progressively increased from early to later withdrawal time-points whereas in females, cocaine-craving was already elevated during early withdrawal (after 2 days) and did not further increase at later withdrawal time-points. Levels of cocaine-craving, however, were similar between the sexes. Gene expression changes differed markedly between the sexes such that males showed the expected relapse- and withdrawal-associated changes in Bdnf-IV, Grin2a, Grin2b, and Grin1 expression, but females only showed a modest increase Grin1 expression at the intermediate withdrawal timepoint. These findings indicate that cocaine-craving is similarly expressed in males and females although the time-course for its incubation appears to be accelerated in females; the molecular mechanisms also likely differ in females versus males.

Withdrawal from cocaine conditioning progressively alters AMPA receptor-mediated transmission in the ventral hippocampus.

Drugs of abuse, such as cocaine, produce aberrant changes in synaptic transmission and plasticity that emerge throughout withdrawal. One region of the brain that displays a high degree of synaptic plasticity, as well as connectivity with mesolimbic structures such as the nucleus accumbens, is the ventral hippocampus (vH). Here, we investigated the effects of an escalating cocaine dosing schedule on vH CA1 excitatory transmission by measuring place preference and recording excitatory postsynaptic currents (EPSCs) at three different withdrawal time points: withdrawal day (WD) 2, 9 or 28. Behaviourally, this escalating cocaine-conditioning protocol was capable of producing conditioned place preference that persisted through WD28. Physiologically, cocaine conditioning produced an increase in vH excitatory transmission on WD2 that appeared to be the result of an increase in calcium-impermeable (CI)-AMPA receptor density. Excitatory transmission was still enhanced in cocaine-treated animals on WD9; however, a significant increase in the contribution of calcium-permeable (CP)-AMPA receptors to EPSCs was detected as compared with WD2. By WD28, these CP-AMPA receptors provided a major contribution to vH CA1 excitatory transmission, resulting in synaptic responses distinct from WD2 and WD9. Taken together, these results highlight progressive changes in vH synaptic transmission during withdrawal that may enhance cocaine contextual associations.

Persistence of cerebellar ataxia during chronic ethanol exposure is associated with epigenetic up-regulation of Fmr1 gene expression in rat cerebellum.

Alcohol intoxication produces ataxia by affecting the cerebellum, which coordinates movements. Fragile X mental retardation (FMR) protein is a complex regulator of RNA and synaptic plasticity implicated in fragile X-associated tremor/ataxia syndrome, which features ataxia and increased Fmr1mRNA expression resulting from epigenetic dysregulation of FMRP. We recently demonstrated that acute ethanol-induced ataxia is associated with increased cerebellar Fmr1gene expression via histone modifications in rats, but it is unknown whether similar behavioral and molecular changes occur following chronic ethanol exposure. Here, we investigated the effects of chronic ethanol exposure on ataxia and epigenetically regulated changes in Fmr1 expression in the cerebellum. Male adult Sprague-Dawley rats were trained on the accelerating rotarod and then fed with chronic ethanol or a control Lieber-DeCarli diet while undergoing periodic behavioral testing for ataxia during ethanol exposure and withdrawal. Cerebellar tissues were analyzed for expression of the Fmr1gene and its targets using a real-time quantitative polymerase chain reaction assay. The epigenetic regulation of Fmr1was also investigated using a chromatin immunoprecipitation assay. Ataxic behavior measured by the accelerating rotarod behavioral test developed during chronic ethanol treatment and persisted at both the 8-h and 24-h withdrawal time points compared to control diet-fed rats. In addition, chronic ethanol treatment resulted in up-regulated expression of Fmr1mRNA and increased activating epigenetic marks H3K27 acetylation and H3K4 trimethylation at 2 sites within the Fmr1 promoter. Finally, measurement of the expression of relevant FMRP mRNA targets in the cerebellum showed that chronic ethanol up-regulated cAMP response element binding (CREB) Creb1, Psd95, Grm5, and Grin2b mRNA expression without altering Grin2a, Eaa1, or histone acetyltransferases CREB binding protein (Cbp) or p300mRNA transcripts. These results suggest that epigenetic regulation of Fmr1 and subsequent FMRP regulation of target mRNA transcripts constitute neuroadaptations in the cerebellum that may underlie the persistence of ataxic behavior during chronic ethanol exposure and withdrawal.

Pentylenetetrazol-like stimulus is not produced following naloxone-precipitated mitragynine withdrawal in rats.

Kratom (Mitragyna speciosa Korth), a native medicinal plant of Southeast Asia, is proposed to exhibit potential therapeutic value as an opioid substitute. However, studies of its negative emotional states resulting from withdrawal particularly of its main psychoactive compound, mitragynine (MG), are limited. Using the pentylenetetrazol (PTZ) discrimination assay, this study aims to investigate the effects of MG in responding to the PTZ stimulus and to assess the generalisation effects of withdrawal from MG to the PTZ stimulus. Rats (n = 20) were trained on a tandem (FR-10, VI-15) schedule of food reinforcement to press one lever after administration of the anxiogenic compound PTZ (16mg/kg, i.p.) and an alternate lever after vehicle. Following acute tests, training was suspended, and rats were chronically treated with MG or morphine at 8-h intervals for 9days and withdrawal was precipitated on the tenth day using naloxone (1mg/kg, i.p.). The rats were tested for generalisation to PTZ at 2, 8 and 24h after the last dose of MG or morphine administration. Unlike morphine that produced dose-related PTZ-like stimulus, MG at 3, 10, 30 and 45mg/kg doses showed no substitution to the PTZ discriminative stimulus. In contrast to morphine which produced a time-dependent generalisation to the PTZ stimulus, naloxone did not precipitate withdrawal effects in MG-treated rats as they selected the vehicle lever at three withdrawal time points. These results demonstrate that MG produces a very different response to morphine withdrawal that is not associated with anxiogenic-like subjective symptoms. These characteristics of MG may provide further support for use as a novel pharmacotherapeutic intervention for managing opioid use disorder.

Prediction of the intolerance or non-responder to Janus kinase inhibitors in patients with rheumatoid arthritis: a preliminary retrospective study with integrative cluster analysis.

To identify the subpopulation of rheumatoid arthritis (RA) non-responders to Janus kinase inhibitors (JAKis) using cluster analysis. This retrospective study enrolled RA patients who had been treated with JAKis (tofacitinib or baricitinib) between July 2013 and September 2019 in six centres. The endpoint was set as inadequate response to JAKis (JAKis-IR), defined as either non-response to JAKis or their intolerance. Non-response to JAKis was defined as achieving neither American College of Rheumatology 20% response nor Disease Activity Score (ΔDAS28-CRP) >1.2 at 12 weeks. Withdrawal time point included earlier than after 12 weeks from baseline. A hierarchical cluster analysis was performed with variables related with clinical and serological parameters at baseline. The 132 RA patients enrolled were classified into four groups (Group A-D). Groups consisted of three components defined at baseline, as seropositivity, advanced joint destruction, interstitial lung disease presumably associated with RA (RA-ILD). Group A (n=32): seronegative, presence of advanced joint destruction, absence of RA-ILD. Group B (n=35): seropositive, absence of advanced joint destruction and RA-ILD. Group C (n=20): seropositive, absence of advanced joint destruction, presence of RA-ILD. Group D (n=45): seropositive, presence of advanced joint destruction and RA-ILD. The rate of JAKis-IR in four groups was as follows: A, 34.3%; B, 17.1%; C, 20.0%; and D, 8.9%. The difference in JAKis-IR rate between group A and D was statistically significant. A subpopulation of RA patients with a combination of the following three components, seronegativity, advanced joint destruction and absence of RA-ILD, was identified as being prone to JAKis-IR.

Investigations on the excretion of antibiotics in goats' milk following therapeutic application

Data on the excretion of antibiotic residues following therapeutic drug dosages in lactating goats with clinical signs of bacterial infections are currently lacking. Therefore, this study aimed at monitoring the drug residue excretion of a subset of frequently used antibiotics in the milk of dairy goats following their therapeutic administration. Over a period of 4 months, milk samples (udder halves) were collected in 2 goat milk farms from animals treated with antibiotics in routine practice based on the diagnosis of a bacterial infection. The samples were examined up to 3 days following the withdrawal time point. The animals were classified in 3 groups depending on their clinical symptoms and treatment. Goats in group 1 (afebrile goats with various bacterial infections excluding the udder) were treated with intramuscular amoxicillin injection (n = 5). Animals in group 2 (mastitis catarrhalis) were treated with intramammary injection of oxacillin and ampicillin (n = 6). Group 3 consisted of a single goat diagnosed with mastitis. This individual was treated with cefquinome in accordance with the results of the antibiogram. Milk samples were examined qualitatively by using a receptor assay (Betastar®) as well as a microbiological inhibitor assay (Brilliant black reduction test, BRT). The latter assay was also used to semiquanti-tatively analyse drug residue levels in samples from group 2. Following intramuscular treatment with amoxicillin, drug residue levels were estimated to be very similar in both udder halfs. Elimination was complete 3 days after the end of the treatment period. Animals in group 2 showed significant differences between treated and untreated udder halves. However, the untreated halves still exhibited residue levels exceeding the maximum residue limits during the treatment period. In both group 2 and 3, all milk samples were tested negative for drug residues before the end of the withdrawal period. In the present study, no evidence of prolonged residue excretion into milk of goats following therapeutic administration of antibiotics was detected. Both the receptor test and the BRT represent suitable methods for an efficient antibiotic drug residue testing in goat milk. Reliable testing was merely not attainable in cases of milk samples possessing highly altered organoleptic characteristics.

Impact of intermittent voluntary ethanol consumption during adolescence on the expression of endocannabinoid system and neuroinflammatory mediators

The adolescent brain displays high vulnerability to the deleterious effects of ethanol, including greater risk of developing alcohol use disorder later in life. Here, we characterized the gene expression of the endocannabinoid system (ECS) and relevant signaling systems associated with neuroinflammation and emotional behaviors in the brain of young adult control and ethanol-exposed (EtOH) rats. We measured mRNA levels of candidate genes using quantitative real time PCR in the medial prefrontal cortex (mPFC), amygdala and hippocampus. EtOH rats were generated by maintenance on an intermittent and voluntary ethanol consumption during adolescence using the two-bottle choice paradigm (4 days/week for 4 weeks) followed by 2 week-withdrawal, a time-point of withdrawal with no physical symptoms. Mean differences and effect sizes were calculated using t-test and Cohen's d values. In the mPFC and hippocampus, EtOH rats had significantly higher mRNA expression of endocannabinoid-signaling (mPFC: Ppara, Dagla, Daglb and Napepld; and hippocampus: Cnr2, Dagla and Mgll) and neuroinflammation-associated genes (mPFC: Gfap; and hippocampus: Aif1) than in controls. Moreover, EtOH rats had significantly higher mRNA expression of neuropeptide Y receptor genes (Npy1r, Npy2r and Npy5r) in the hippocampus. Finally, EtOH rats also displayed higher plasma endocannabinoid levels than controls. In conclusion, these results suggest that adolescent ethanol exposure can lead to long-term alterations in the gene expression of the ECS and other signaling systems involved in neuroinflammation and regulation of emotional behaviors in key brain areas for the development of addiction.

Biomarkers of infection in the optimization of antibacterial therapy: justified expectations

Despite the continuous improvement of approaches to antimicrobial therapy and the emergence of new highly effective antibiotics, severe bacterial infections being a significant cause of morbidity and mortality remain a top of mind issue for clinicians. Immediate initiation of the effective antibiotic therapy is an essential component of the successful treatment of serious bacterial infections, and therefore, special attention should be paid to the timely diagnosis. Measurements of biomarkers of inflammation (C-reactive protein, procalcitonin, presepsin, proadrenomedullin) in combination with clinical evaluation are important at first for the diagnosis of bacterial infection, and after that these can help to assess the clinical response to therapy and determine the time-point of antibiotics withdrawal. This review presents the characteristics of the main markers of inflammation, discusses the situations when determination of biomarkers is appropriate, and also provides modern clinical recommendations and algorithms regarding the use of these diagnostic markers in the management of patients with bacterial infections.

MGlu5 Receptor Blockade Within the Nucleus Accumbens Shell Reduces Behavioral Indices of Alcohol Withdrawal-Induced Anxiety in Mice.

Withdrawal from binge-drinking increases negative affect, coinciding with increased expression of the metabotropic glutamate receptor 5 (mGlu5) within the shell of the nucleus accumbens (AcbSh). Supporting a causal-effect relationship, systemic treatment with the mGlu5 receptor antagonist MTEP [3-((2-Methyl-4-thiazolyl)ethynyl)pyridine] is anxiolytic in binge-drinking adult and adolescent mice. Here, we employed neuropharmacological approaches to examine the functional relevance of AcbSh mGlu5 for behavioral indices of alcohol withdrawal-induced hyper-anxiety. Adult (PND 56) and adolescent (PND 28) male C57BL/6J mice consumed alcohol under modified Drinking-in-the-Dark procedures (10, 20, and 40% alcohol v/v) for 14 days. At an alcohol withdrawal time-point when mice manifest robust behavioral signs of hyper-anxiety (1 and 28 days withdrawal for adults and adolescents, respectively), mice were infused intra-AcbSh with 0, 1 or 10 μg MTEP and then affect was assayed in the light-dark shuttle box, marble-burying and forced swim tests. Brain tissue was collected to evaluate changes in Egr1 (early growth response protein 1) induction to index AcbSh neuronal activity. As expected, alcohol-experienced mice exhibited behavioral signs of hyper-emotionality. The anxiolytic effects of intra-AchSh MTEP were modest, but dose-dependent, and varied with age of drinking-onset. In adult-onset mice, only the 1 μg MTEP dose reduced withdrawal-induced hyper-anxiety, whereas only the higher dose was effective in adolescent-onset animals. MTEP reduced Egr1 expression within the AcbSh, irrespective of alcohol drinking history or age of drinking-onset. However, only the high MTEP dose reduced Egr1 expression in adolescent-onset binging mice. These results implicate AcbSh mGlu5 in modulating alcohol withdrawal-induced negative affect and suggest age differences in the neurobiological effects of alcohol withdrawal and behavioral responsiveness to mGlu5 blockade within the AcbSh.

Agmatine inhibits nicotine withdrawal induced cognitive deficits in inhibitory avoidance task in rats: Contribution of α2-adrenoceptors

Nicotine abstinence following chronic exposure is associated with impairments in memory and variety of cognitive functions. Daily nicotine (2 mg/kg, sc, four times daily) administration for 14 days and its abrupt withdrawal significantly impaired avoidance learning in inhibitory avoidance task as indicated by a significant decrease in the step through latency. Animals injected with agmatine (10–40 μg/rat, icv) from day 7 to 14 before the first daily dose of nicotine (2 mg/kg, sc) showed increased step through latencies during retrieval test. Similarly Intracerebroventricular injection of l-arginine (25–100 μg/rat), a biosynthetic precursor of agmatine and arcaine (50 μg −100 μg/rat), an agmatinase inhibitor, also increased the step through latency during retrieval test in nicotine withdrawn animals. In separate experiments, α2-adrenoceptor agonist, clonidine (0.5–1 μg/rat, icv) not only demonstrated significant increase in the step through latency as in nicotine withdrawn rats but also potentiated the pharmacological effect of agmatine. In contrast, pre-treatment of α2-adrenoceptor antagonist, yohimbine (0.5 μg/rat, icv) antagonized the memory enhancing effect of agmatine (20 μg/rat, icv) in nicotine withdrawn rats. In addition, brain agmatine analysis carried out at 72 h time point of nicotine withdrawal showed marked decrease in basal brain agmatine content as compared to control. Overall, the data indicate that agmatine attenuates nicotine withdrawal induced memory impairment through modulation of α2adrenergic receptors. Thus, agmatine might have therapeutic implications in the treatment of cognitive deficits following nicotine withdrawal.

Neuropeptide CART prevents memory loss attributed to withdrawal of nicotine following chronic treatment in mice.

Although chronic nicotine administration does not affect memory, its withdrawal causes massive cognitive deficits. The underlying mechanisms, however, have not been understood. We test the role of cocaine- and amphetamine-regulated transcript peptide (CART), a neuropeptide known for its procognitive properties, in this process. The mice on chronic nicotine treatment/withdrawal were subjected to novel object recognition task. The capability of the animal to discriminate between the novel and familiar objects was tested and represented as discrimination index (DI); reduction in the index suggested amnesia. Nicotine for 49days had no effect on DI, but 8-hour withdrawal caused a significant reduction, followed by full recovery at 24-hour withdrawal timepoint. Bilateral CART infusion in dorsal hippocampus rescued deficits in DI at 8-hours, whereas CART-antibody infusion into the dorsal hippocampus attenuated the recovery at 24-hours. Commensurate changes were observed in the CART as well as CART mRNA profiles in the hippocampus. CART mRNA expression and the peptide immunoreactivity did not change significantly following chronic nicotine treatment. However, there was a significant reduction at 8-hour withdrawal, followed by a drastic increase in CART immunoreactivity as well as CART mRNA at 24-hour withdrawal, compared with 8-hour withdrawal. Distinct α7-nicotinic receptor immunoreactivity was detected on the hippocampal CART neurons, suggesting cholinergic inputs. An increase in the synaptophysin immunoreactive elements around CART cells in the dentate gyrus, cornu ammonis 3 and subiculum at 24-hour post-withdrawal timepoint suggested neuronal plasticity. CART circuit dynamics in the hippocampus seems to modulate short-term memory associated with nicotine withdrawal.

Cost of medication overuse headache in Italian patients at the time-point of withdrawal: a retrospective study based on real data.

The objective was to assess the cost of Medication Overuse Headache (MOH) at the time-point of withdrawal treatment. We implemented a protocol in which both direct and indirect cost were directly gathered from patients and referred to the previous three months. Direct costs were calculated by medications for acute treatment and prophylaxis, diagnostic procedures, visits, complementary treatments, informal care. Indirect costs were referred to missed workdays and workdays with reduced productivity: we asked patients to refer their salaries and to rate the overall level of performance in days worked with reduced productivity, and we calculated indirect costs on this basis. A total of 135 patients were enrolled: direct costs were around 415€/month; indirect costs were 530€/month, and were mostly due to presenteeism (350€, 66.3%) rather than to absenteeism (160€, 33.7%). Our data showed higher cost than those of a previous study: this is likely due to a different approach to cost definition, to the inclusion of direct non-medical cost, and of non-pharmacological treatments.

Cue-induced nicotine-seeking behavior after withdrawal with or without extinction in rats.

Exposure to smoking-associated environmental cues during smoke cessation elicits self-reported urge/craving to smoke, which precipitates relapse even after prolonged abstinence. Incubation of cue-induced cigarettes craving during abstinence has been observed in human smokers recently. The present studies assessed cue-induced nicotine-seeking behavior under different withdrawal conditions in rats with a history of nicotine self-administration. We found that non-reinforced operant responding during cue-induced nicotine seeking after different periods of withdrawal from nicotine exhibited an inverted U-shaped curve, with higher levels of responding after 7-21 days of withdrawal than those after 1-day withdrawal. Cue-induced nicotine-seeking responding is long lasting and persists even after 42 days of forced withdrawal in the home cages. Interestingly, repeated testing of cue-induced nicotine seeking at different withdrawal time points (1, 7, 14, 21 and 42 days) in the same individual alleviated responding as compared with the between-subjects assessment. Furthermore, extinction training during nicotine withdrawal significantly decreased cue-induced reinstatement of nicotine-seeking behavior. Together, profound time-dependent incubation of cue-induced craving in nicotine-experienced rats were observed. In addition, repeated cue exposure or extinction training decreases cue-induced craving. The demonstration of incubation of nicotine craving phenomenon in both rat and human studies provides support for the translational potential of therapeutic targets for relapse uncovered through mechanism studies in rats.

Varenicline, the clinically effective smoking cessation agent, restores probabilistic response reversal performance during withdrawal from nicotine.

There is recognition that cognitive problems can contribute to renewed drug taking in former addicts. Our previous work has indicated that current smokers show reduced performance on a probabilistic reversal learning (PRL) task, relative to former smokers. To further explore PRL performance and its relevance to smoking, in addition to the role of nicotine, we developed a model of nicotine withdrawal-induced deficits in rodents. A second goal was to test varenicline, an α4β2 partial agonist, for its ability to restore any cognitive impairment. Acute effects of nicotine and varenicline on PRL performance in non-dependent animals were minimal and confined to speed of responding. When rats were made dependent on nicotine via osmotic minipumps implanted for 7 days (3.16 mg/kg/day), repeated tests at specified withdrawal time points revealed PRL disruption peaking at 12 and 24 hours following surgical removal of minipumps. Withdrawal was characterized by significant deficits in the number of reversals (P < 0.05), speed of responding (P < 0.01) and increases in omissions (P < 0.05). Nicotine (0.2 mg/kg SC) or varenicline (0.3 and 1.0 mg/kg SC) administered 10-minute prior to PRL test sessions during withdrawal, relieved the performance deficits. At 24-hour withdrawal, nicotine and varenicline (1 mg/kg) prevented decrements in reversals, in addition to ameliorating slower speed of responding. The high dose of varenicline only reduced omissions. These results confirm the role of nicotine in withdrawal-induced disruption of PRL performance and suggest that the model may be useful for investigating efficacy of potential new treatments for smoking cessation.

Chronic Intermittent Ethanol Exposure Alters Stress Effects on (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP) Immunolabeling of Amygdala Neurons in C57BL/6J Mice.

The GABAergic neuroactive steroid (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP, allopregnanolone) is decreased in various brain regions of C57BL/6J mice following exposure to an acute stressor or chronic intermittent ethanol (CIE) exposure and withdrawal. It is well established that there are complex interactions between stress and ethanol drinking, with mixed literature regarding the effects of stress on ethanol intake. However, there is little research examining how chronic ethanol exposure alters stress responses. The present work examined the impact of CIE exposure and withdrawal on changes in brain levels of 3α,5α-THP, as well as hormonal and behavioral responses to forced swim stress (FSS). Adult male C57BL/6J mice were exposed to four cycles of CIE to induce ethanol dependence. Following 8 h or 72 h withdrawal, mice were subjected to FSS for 10 min, and 50 min later brains were collected for immunohistochemical analysis of cellular 3α,5α-THP. Behavioral and circulating corticosterone responses to FSS were quantified. Following 8 h withdrawal, ethanol exposure potentiated the corticosterone response to FSS. Following 72 h withdrawal, this difference was no longer observed. Following 8 h withdrawal, stress-exposed mice showed no differences in immobility, swimming or struggling behavior. However, following 72 h withdrawal, ethanol-exposed mice showed less immobility and greater swimming behavior compared to air-exposed mice. Interestingly, cellular 3α,5α-THP levels were increased in the lateral amygdala 8 h and 72 h post-withdrawal in stressed ethanol-exposed mice compared to ethanol-exposed/non-stressed mice. In the paraventricular nucleus of the hypothalamus, stress exposure decreased 3α,5α-THP levels compared to controls following 72 h withdrawal, but no differences were observed 8 h post-withdrawal. There were no differences in cellular 3α,5α-THP levels in the nucleus accumbens shell at either withdrawal time point. These data suggest that there are different mechanisms mediating hormonal, behavioral, and brain responses to stress following CIE exposure. The lateral amygdala appears to be an extremely sensitive brain region exhibiting changes in cellular 3α,5α-THP levels following CIE and exposure to swim stress. It is likely that these changes in cellular 3α,5α-THP levels in the lateral amygdala contribute to the behavioral effects observed following 72 h withdrawal.

Amphetamine Withdrawal Differentially Increases the Expression of Organic Cation Transporter 3 and Serotonin Transporter in Limbic Brain Regions.

Amphetamine withdrawal increases anxiety and stress sensitivity related to blunted ventral hippocampus (vHipp) and enhances the central nucleus of the amygdala (CeA) serotonin responses. Extracellular serotonin levels are regulated by the serotonin transporter (SERT) and organic cation transporter 3 (OCT3), and vHipp OCT3 expression is enhanced during 24 hours of amphetamine withdrawal, while SERT expression is unaltered. Here, we tested whether OCT3 and SERT expression in the CeA is also affected during acute withdrawal to explain opposing regional alterations in limbic serotonergic neurotransmission and if respective changes continued with two weeks of withdrawal. We also determined whether changes in transporter expression were confined to these regions. Male rats received amphetamine or saline for two weeks followed by 24 hours or two weeks of withdrawal, with transporter expression measured using Western immunoblot. OCT3 and SERT expression increased in the CeA at both withdrawal timepoints. In the vHipp, OCT3 expression increased only at 24 hours of withdrawal, with an equivalent pattern seen in the dorsomedial hypothalamus. No changes were evident in any other regions sampled. These regionally specific changes in limbic OCT3 and SERT expression may partially contribute to the serotonergic imbalance and negative affect during amphetamine withdrawal.