Days Of Forced Abstinence Research Articles

Sex-specific behavioral and thalamo-accumbal circuit adaptations after oxycodone abstinence.

Opioid use disorder is marked by a progressive change in the motivation to administer the drug even in the presence of negative consequences. After long periods of abstinence, the urge to return to taking the drug intensifies over time, known as incubation of craving. Conditioned responses to drug-related stimuli, can acquire motivational properties and exert control over motivated behaviors leading to relapse. Although, preclinical data suggest that the behavioral expression of opioid use is similar between male and female rodents, we do not have conclusive results on sex differences on craving and relapse across abstinence periods. Here, we investigated the effects of abstinence from oxycodone self-administration on neurotransmission in the paraventricular thalamus (PVT) to nucleus accumbens shell (NAcSh) pathway in male and female rats. Using optogenetics and ex vivo electrophysiology, we assessed synaptic strength and glutamate release probability in this pathway, as well as NAcSh medium spiny neurons (MSN) intrinsic excitability, in slices from rats which were subjected to either 1 (acute) or 14 (prolonged) days of forced abstinence after self-administration. Our results revealed no sex differences in oxycodone self-administration or somatic withdrawal symptoms following acute abstinence. However, we found a sex-specific enhancement in cue-induced relapse after prolonged, but not acute, abstinence from oxycodone self-administration, with females exhibiting higher relapse rates. Notably, prolonged abstinence led to similar increases in synaptic strength at PVT-NAcSh inputs compared to saline controls in both sexes, which was not observed after acute abstinence. Thus, prolonged abstinence results in a time-dependent increase in PVT-NAcSh synaptic strength and sex-specific effects on cue-induced relapse rates. These findings suggest that prolonged abstinence leads to significant synaptic changes, contributing to heightened relapse vulnerability, highlighting the need for targeted therapeutic strategies in opioid use disorder.

Neuroplasticity-related genes correlate with individual differences in distinct phases of oxycodone self-administration in male rats

Opioid use disorder (OUD) is a chronic condition associated with long-lasting molecular and behavioral changes. Animals with prolonged access to opioids develop behaviors similar to human OUD. Identifying associated molecular changes can provide insight to underpinnings that lead to or maintain OUD. In pilot studies, we identified several miRNA targets that are altered by the administration of oxycodone. We selected mir182 for follow up as it was recently shown to be dysregulated in plasma of men administered oxycodone. In addition, mir182 is increased in reward-related brain regions of male rats following exposure to various addictive substances. The present study utilizes a long-access oxycodone self-administration paradigm to examine changes in mir182 and its mRNA targets associated with neuroplasticity, which may be involved in the maintenance of OUD-like phenotype in rats. Male rats were trained to self-administer oxycodone (0.1 mg/kg/infusion, i. v.) for 6 h daily sessions for 12 days. Each animal had a yoked saline control that received matched saline infusions. Animals were then tested on a progressive ratio schedule to measure motivation to obtain a single infusion of oxycodone. Drug seeking was measured following 28 days of forced abstinence using a 90-min cued/test. RTqPCR was utilized to measure mir182 and mRNA targets related to neuroplasticity (wnt3, plppr4, pou3f3, tle4, cacna2d, and bdnf) from the nucleus accumbens. Data revealed that animals responded on a continuum for oxycodone. When divided into two groups termed high- and low responders, animals diverged during self-administration acquisition and maintained differences in behavior and gene expression throughout the study. mir182 was upregulated in the nucleus accumbens of both high and low responders and negatively correlated with tle4, which showed a strong negative correlation with reinstatement behavior. mRNA target levels were correlated with behaviors associated with increased severity of OUD behavior in male rats.

Incubation of methamphetamine craving in punishment-resistant individuals is associated with activation of specific gene networks in the rat dorsal striatum

Methamphetamine use disorder (MUD) is characterized by loss of control over compulsive drug use. Here, we used a self-administration (SA) model to investigate transcriptional changes associated with the development of early and late compulsivity during contingent footshocks. Punishment initially separated methamphetamine taking rats into always shock-resistant (ASR) rats that continued active lever pressing and shock-sensitive (SS) rats that reduced their lever pressing. At the end of the punishment phase, rats underwent 15 days of forced abstinence at the end of which they were re-introduced to the SA paradigm followed by SA plus contingent shocks. Interestingly, 36 percent of the initial SS rats developed delayed shock-resistance (DSR). Of translational relevance, ASR rats showed more incubation of methamphetamine craving than DSR and always sensitive (AS) rats. RNA sequencing revealed increased striatal Rab37 and Dipk2b mRNA levels that correlated with incubation of methamphetamine craving. Interestingly, Bdnf mRNA levels showed HDAC2-dependent decreased expression in the AS rats. The present SA paradigm should help to elucidate the molecular substrates of early and late addiction-like behaviors.

Effectiveness of pharmacotherapies for diabetes on nicotine, food, and water intake in insulin-resistant rats.

The intersectionality between diabetes medications and nicotine consumption was assessed in female and male rats. Briefly, the rats were fed a high-fat diet (HFD) or regular diet (RD) for 4weeks. Then separate groups received vehicle or a low dose of streptozotocin (STZ; 25mg/kg). Three days later, insulin resistance was assessed by measuring plasma glucose levels for 180min following an injection of insulin (0.75U/kg). The rats were then prepared with jugular catheters, and they were given 23h access to nicotine intravenous self-administration (IVSA) in 4days cycles with 3days of forced abstinence in their home cages where they consumed their respective diet. During the IVSA sessions, operant responses for food and water and changes in body weight were recorded. Prior to administration of the pharmacotherapies, the rats were given access to two doses of nicotine (0.015 then 0.03mg/kg for the remainder of the study). Then, daily injections of the pharmacotherapies were given at the onset of dark cycle (6p.m.) in the following order: 1) dapagliflozin (3.0 then 10.0mg/kg), 2) insulin (0.75U/kg twice), and 3) bromocriptine (3.0 then 10.0mg/kg). The results suggest that our HFD+STZ regiment induced insulin resistance in female and male rats. Also, the HFD-fed rats displayed higher nicotine intake than RD controls, regardless of sex. Administration of insulin, but not dapagliflozin or bromocriptine, normalized nicotine intake in HFD-fed rats to control levels. These results have clinical implications regarding the potential efficacy of insulin to control excessive nicotine intake in persons with diabetes.

MicroRNA-mediated translational pathways are regulated in the orbitofrontal cortex and peripheral blood samples during acute abstinence from heroin self-administration.

Opioid misuse in the United States contributes to >70% of annual overdose deaths. To develop additional therapeutics that may prevent opioid misuse, further studies on the neurobiological consequences of opioid exposure are needed. Here we sought to characterize molecular neuroadaptations involving microRNA (miRNA) pathways in the brain and blood of adult male rats that self-administered the opioid heroin. miRNAs are ∼18-24 nucleotide RNAs that regulate protein expression by preventing mRNA translation into proteins. Manipulation of miRNAs and their downstream pathways can critically regulate drug seeking behavior. We performed small-RNA sequencing of miRNAs and proteomics profiling on tissue from the orbitofrontal cortex (OFC), a brain region associated with heroin seeking, following 2days of forced abstinence from self-administration of 0.03mg/kg/infusion heroin or sucrose. Heroin self-administration resulted in a robust shift of the OFC miRNA profile, regulating 77 miRNAs, while sucrose self-administration only regulated 9 miRNAs that did not overlap with the heroin-induced profile. Conversely, proteomics revealed dual regulation of seven proteins by both heroin and sucrose in the OFC. Pathway analysis determined that heroin-associated miRNA pathways are predicted to target genes associated with the term "prion disease," a term that was also enriched in the heroin-induced protein expression dataset. Lastly, we confirmed that a subset of heroin-induced miRNA expression changes in the OFC are regulated in peripheral serum and correlate with heroin infusions. These findings demonstrate that peripheral blood samples may have biomarker utility for assessment of drug-induced miRNA pathway alterations that occur in the brain following chronic drug exposure.

HIV gp120 impairs nucleus accumbens neuroimmune function and dopamine D3 receptor-mediated inhibition of cocaine seeking in male rats

Cocaine Use Disorders (CUDs) are associated with an increased risk of human immunodeficiency virus (HIV) infection. Cocaine and the HIV envelope protein gp120 each induce distinct deficits to mesocorticolimbic circuit function and motivated behavior; however, little is known regarding how they interact to dysregulate these functions or how such interactions impact pharmacotherapeutic efficacy. We have previously shown that the selective, weak partial agonist of the dopamine D3 receptor (D3R), MC-25-41, attenuates cocaine-seeking behavior in male rats. Here, we sought to characterize changes in striatal neuroimmune function in gp120-exposed rats across abstinence from operant access to cocaine (0.75 mg/kg, i.v.) or sucrose (45 mg/pellet), and to examine the impact of gp120 exposure on MC-25-41-reduced cocaine seeking. After establishing a history of cocaine or sucrose self-administration, rats received intracerebroventricular gp120 infusions daily the first 5 days of abstinence and were sacrificed either on day 6 or after 21 days of forced abstinence and a cue-induced cocaine seeking test. We demonstrated that MC-25-41 treatment attenuated cue-induced cocaine seeking among control rats but not gp120-exposed rats. Moreover, postmortem analysis of nucleus accumbens (NAc) core neuroimmune function indicated cocaine abstinence- and gp120-induced impairments, and the expression of several immune factors within the NAc core significantly correlated with cocaine-seeking behavior. We conclude that cocaine abstinence dysregulates striatal neuroimmune function and interacts with gp120 to inhibit the effectiveness of a D3R partial agonist in reducing cocaine seeking. These findings highlight the need to consider comorbidities, such as immune status, when evaluating the efficacy of novel pharmacotherapeutics.

Glutamine and GABA alterations in cingulate cortex may underlie alcohol drinking in a rat model of co-occurring alcohol use disorder and schizophrenia: an 1H-MRS study

Alcohol use disorder commonly occurs in patients with schizophrenia and significantly worsens the clinical course of the disorder. The neurobiological underpinnings of alcohol drinking are not well understood. Magnetic resonance spectroscopy (MRS) has been used to assess the neurochemical substrates that may be associated with alcohol drinking in patients; however, the causal impact of these findings remains elusive, highlighting the need for studies in animal models. This study performed MRS in the neonatal ventral hippocampal lesioned (NVHL) rat model, a model of co-occurring schizophrenia and substance use disorders. NVHL lesions (or sham surgeries) were performed on post-natal day 7 and animals were given brief exposure to alcohol during adolescence (10% v/v in a 2-bottle choice design). Animals were re-exposed to alcohol during adulthood (20% v/v) until a stable drinking baseline was established, and then forced into abstinence to control for the effects of differential alcohol drinking. Animals were scanned for MRS after one month of abstinence. NVHL rats consumed significantly more alcohol than sham rats and in the cingulate cortex showed significantly higher levels of GABA and glutamine. Significantly lower GABA levels were observed in the nucleus accumbens. No differences between the NVHL and sham animals were observed in the hippocampus. Correlation analysis revealed that GABA and glutamine concentrations in the cingulate cortex significantly correlated with the rats’ alcohol drinking prior to 30 days of forced abstinence. These findings suggest that a potential dysfunction in the glutamate/GABA–glutamine cycle may contribute to alcohol drinking in a rat model of schizophrenia, and this dysfunction could be targeted in future treatment-focused studies.

Transcriptomics in the nucleus accumbens shell reveal sex- and reinforcer-specific signatures associated with morphine and sucrose craving.

Incubation of craving is a well-documented phenomenon referring to the intensification of drug craving over extended abstinence. The neural adaptations that occur during forced abstinence following chronic drug taking have been a topic of intense study. However, little is known about the transcriptomic changes occurring throughout this window of time. To define gene expression changes associated with morphine consumption and extended abstinence, male and female rats underwent 10 days of morphine self-administration. Separate drug-naive rats self-administered sucrose in order to compare opioid-induced changes from those associated with natural, non-drug rewards. After one or 30 days of forced abstinence, rats were tested for craving, or nucleus accumbens shell tissue was dissected for RNA sequencing. Morphine consumption was predictive of drug seeking after extended (30 days) but not brief (1 day) abstinence in both sexes. Extended abstinence was also associated with robust sex- and reinforcer-specific changes in gene expression, suggesting sex differences underlying incubation of morphine and sucrose seeking respectively. Importantly, these changes in gene expression occurred without re-exposure to drug-paired cues, indicating that chronic morphine causes long-lasting changes in gene expression that prime the system for increased craving. These findings lay the groundwork for identifying specific therapeutic targets for curbing opioid craving without impacting the natural reward system in males and females.

Roflumilast treatment during forced abstinence reduces relapse to methamphetamine seeking and taking.

Methamphetamine (METH) is a psychostimulant with high abuse potential. Currently, there are no pharmacological treatments specific for METH abuse or stimulant use disorder generally. Although phosphodiesterase inhibitors have shown some promise, current animal models have not examined their use in abstinence from stimulant abuse. We employed a METH self-administration model in the rat followed by a forced abstinence period during which roflumilast, a phosphodiesterase 4 inhibitor, was administered. A detailed behavioral analysis of chronic treatment with roflumilast during 7 days of forced abstinence showed that roflumilast reduced METH seeking and METH taking upon subsequent relapse test. Roflumilast treatment during 7 days of forced abstinence did not affect sucrose seeking and sucrose taking behaviors. These data suggest that roflumilast may be a treatment for METH use disorder that is effective when administered only during abstinence.

5-HT1B receptor agonist attenuates cocaine self-administration after protracted abstinence and relapse in rats.

The 5-HT1B receptor (5-HT1BR) agonist, CP94253, enhances cocaine intake during maintenance of self-administration (SA) but attenuates intake after 21 days of forced abstinence in male rats. We examined whether CP94253 attenuates cocaine intake in female rats after a period of abstinence, and if these attenuating effects persist or revert to enhancing cocaine intake during resumption (i.e. relapse) of daily cocaine SA. Male and female rats trained to lever press on a fixed ratio 5 schedule of cocaine reinforcement underwent ⩾21 days of forced abstinence. They were then tested for the effects of CP94253 (5.6 mg/kg, SC) or vehicle on cocaine SA. During the test session, rats had 1-h access to the training dose of cocaine (0.75 mg/kg, IV) followed by 1-h access to a lower cocaine dose (0.075 mg/kg, IV). Rats then resumed cocaine SA for 15 days to mimic relapse and were retested as done previously. Subsequently, rats underwent abstinence again (21-60 days) and were tested for CP94253 effects on locomotion and cue reactivity (i.e. responding for light/tone cues previously paired with cocaine infusions). Regardless of sex, CP94253 decreased cocaine intake after abstinence and during resumption of SA and decreased cue reactivity while having no effect on locomotion. CP94253 decreases cocaine intake and cocaine seeking in both males and females even after resumption of cocaine SA. These findings suggest that the inhibitory effects of CP94253 observed after abstinence are long-lasting, and therefore, 5-HT1BR agonists may have clinical efficacy as anti-relapse medications for cocaine use disorders.

Gut and brain profiles that resemble pre-motor and early-stage Parkinson’s disease in methamphetamine self-administering rats

IntroductionMethamphetamine is a potent psychomotor stimulant, and methamphetamine abusers are up to three times more likely to develop Parkinson’s disease (PD) later in life. Prodromal PD may involve gut inflammation and the accumulation of toxic proteins that are transported from the enteric nervous system to the central nervous system to mediate, in part, the degeneration of dopaminergic projections. We hypothesized that self-administration of methamphetamine in rats produces a gut and brain profile that mirrors pre-motor and early-stage PD. MethodsRats self-administered methamphetamine in daily 3 h sessions for two weeks. Motor function was assessed before self-administration, during self-administration and throughout the 56 days of forced abstinence. Assays for pathogenic markers (tyrosine hydroxylase, glial fibrillary acidic protein (GFAP), α-synuclein) were conducted on brain and gut tissue collected at one or 56 days after cessation of methamphetamine self-administration. ResultsMotor deficits emerged by day 14 of forced abstinence and progressively worsened up to 56 days of forced abstinence. In the pre-motor stage, we observed increased immunoreactivity for GFAP and α-synuclein within the ganglia of the myenteric plexus in the distal colon. Increased α-synuclein was also observed in the substantia nigra pars compacta. At 56 days, GFAP and α-synuclein normalized in the gut, but the accumulation of nigral α-synuclein persisted, and the dorsolateral striatum exhibited a significant loss of tyrosine hydroxylase. ConclusionThe pre-motor profile is consistent with gut inflammation and gut/brain α-synuclein accumulation associated with prodromal PD and the eventual development of the neurological disease.

Sex-Specific Role for Egr3 in Nucleus Accumbens D2-Medium Spiny Neurons Following Long-Term Abstinence From Cocaine Self-administration

BackgroundWe previously showed that the transcription factor Egr3 (early growth response 3) is oppositely regulated in nucleus accumbens (NAc) cell subtypes 24 hours following cocaine exposure and bidirectionally mediates cocaine-related behaviors in male rodents. Overexpressing Egr3 in D2 receptor–containing medium spiny neurons (D2-MSNs) before drug exposure reduces the rewarding and psychomotor sensitization effects of cocaine. However, it is unknown if Egr3 plays a role in long-term neuroadaptations in the NAc and relapse to cocaine seeking. MethodsWe measured EGR3 protein levels in the NAc following 20 days of forced abstinence from intravenous cocaine self-administration in 10-week-old Sprague Dawley rats and C57BL/6 mice. In 8- to 10-week-old A2A-Cre mice, we used virally mediated Egr3 overexpression in NAc D2-MSNs to test the role of Egr3 on operant responding during seeking, extinction, and drug-induced reinstatement of cocaine self-administration. To evaluate if Egr3 contributed to sex differences to cocaine relapse, we conducted these procedures in both male and female rodents. ResultsWe found that EGR3 expression was reduced only in female rodents after 20 days of forced abstinence. Additionally, we showed that our self-administration paradigm in mice recapitulated the sex differences in cocaine intake and relapse demonstrated in humans and rats. Finally, whereas Egr3 overexpression in D2-MSNs during forced abstinence facilitated extinction and blunted drug-induced reinstatement in female mice, it had the opposite effect in male mice. ConclusionsWe showed that the immediate early gene Egr3 has long-term effects on drug-related behaviors. Our work suggests that changes in Egr3 expression in D2-MSNs contributes to sex differences in cocaine relapse.

Cholinergic Receptor Blockade in the VTA Attenuates Cue-Induced Cocaine-Seeking and Reverses the Anxiogenic Effects of Forced Abstinence

Drug relapse after periods of abstinence is a common feature of substance abuse. Moreover, anxiety and other mood disorders are often co-morbid with substance abuse. Cholinergic receptors in the ventral tegmental area (VTA) are known to mediate drug-seeking and anxiety-related behavior in rodent models. However, it is unclear if overlapping VTA cholinergic mechanisms mediate drug relapse and anxiety-related behaviors associated with drug abstinence. We examined the effects of VTA cholinergic receptor blockade on cue-induced cocaine seeking and anxiety during cocaine abstinence. Male Sprague–Dawley rats were trained to self-administer intravenous cocaine (~0.5 mg/kg/infusion, FR1 schedule) for 10 days, followed by 14 days of forced abstinence. VTA infusion of the non-selective nicotinic acetylcholine receptor antagonist mecamylamine (0, 10, and 30 μg/side) or the non-selective muscarinic receptor antagonist scopolamine (0, 2.4 and 24 μg /side) significantly decreased cue-induced cocaine seeking. In cocaine naïve rats, VTA mecamylamine or scopolamine also led to dose-dependent increases in open arm time in the elevated plus maze (EPM). In contrast, rats that received I.V. cocaine, compared to received I.V. saline rats, displayed an anxiogenic response on day 14 of abstinence as reflected by decreased open arm time in the EPM. Furthermore, low doses of VTA mecamylamine (10 μg /side) or scopolamine (2.4 μg /side), that did not alter EPM behavior in cocaine naive rats, were sufficient to reverse the anxiogenic effects of cocaine abstinence. Together, these data point to an overlapping role of VTA cholinergic mechanisms to regulate relapse and mood disorder-related responses during cocaine abstinence.

Effect of menthol on nicotine intake and relapse vulnerability in a rat model of concurrent intravenous menthol/nicotine self-administration.

Epidemiological data suggest that menthol may increase vulnerability to cigarette/nicotine use and relapse. While menthol's sensory properties are often attributed as the underlying cause of the enhanced vulnerability, an alternative possibility is that they are mediated via pharmacological interactions with nicotine. This study addressed the possibility that menthol enhances nicotine intake and relapse vulnerability via pharmacological interactions with nicotine using a concurrent intravenous menthol/nicotine self-administration procedure. Following acquisition, adolescent rats were given 23-h/day access to nicotine (0.01mg/kg/infusion), nicotine plus menthol (0.16, 0.32, or 0.64mg/kg/infusion), or menthol alone (0.16, 0.32, 0.64mg/kg/infusion) for a total of 10days. Nicotine-seeking was assessed using an extinction/cue-induced reinstatement procedure following 10days of forced abstinence. We also assessed the effect of menthol (0.32mg/kg/infusion) on progressive ratio responding for nicotine (0.01mg/kg/infusion). Menthol decreased PR responding for nicotine but did not affect self-administration under extended access conditions. The low dose of menthol tended to decrease subsequent extinction responding, and was not different from menthol alone, whereas the high dose decreased reinstatement responding. Although not significant, the highest levels of extinction responding were observed in a minority of rats in the moderate and high menthol-nicotine groups; rats in these groups also took longer to extinguish. Taken together, these results demonstrate that pharmacological interactions of menthol with nicotine reduce, rather than increase, nicotine's reinforcing effects and some measures of relapse vulnerability. Importantly, however, moderate and high menthol doses may increase some aspects of relapse vulnerability in a minority of individuals.

Dopamine D1 Receptor Within Basolateral Amygdala Is Involved in Propofol Relapse Behavior Induced by Cues.

Propofol has been proven to be potentially abused by humans and laboratory animals; however, studies that have examined propofol relapse behavior are limited, and its underlying mechanism remains unclear. In this study, we examined whether basolateral amygdala-specific or systematic administration of the dopamine receptor antagonist alters cue-induced propofol-seeking behaviors in a rat model. Male Sprague-Dawley rats first received 14 days of propofol self-administration training, where active nose poke resulted in the delivery of propofol infusion paired with a tone and light cues. After 1-30 days of forced abstinence, the cue-induced propofol-seeking behaviors were tested in the operant chamber. We demonstrated, for the first time, after a few days of withdrawal from intravenous bolus administration of propofol, propofol-related cues could induce robust reinstatement of drug-seeking behavior. Systematic administration of dopamine D1 receptor antagonist (SCH-23390) or dopamine D2 receptor antagonist (spiperone) inhibited propofol relapse behavior induced by drug-related cues. Furthermore, we show that microinfusion of SCH-23390 into basolateral amygdala dose-dependently attenuated cue-induced propofol drug-seeking behavior, whereas infusion of spiperone had no effect on the propofol relapse behavior. Our results reveal the involvement of dopamine receptors within the basolateral amygdala in the cue-induced propofol relapse behavior in rats.

The L-type calcium channel blocker, isradipine, attenuates cue-induced cocaine-seeking by enhancing dopaminergic activity in the ventral tegmental area to nucleus accumbens pathway.

Previous preclinical and clinical investigations have focused on the L-type calcium channel (LTCC) as a potential therapeutic target for substance abuse. While some clinical studies have examined the ability of LTCC blockers to alter cocaine's subjective effects, very few LTCC studies have examined cocaine relapse. Here, we examined whether ventral tegmental area (VTA)-specific or systemic administration of the LTCC inhibitor, isradipine, altered cocaine-seeking behavior in a rat model. Male Sprague-Dawley rats first received 10 days of cocaine self-administration training (2 h sessions), where active lever depression resulted in delivery of a ∼0.5 mg/kg cocaine infusion paired with a tone + light cue. Rats then underwent 10 days of forced abstinence, without access to cocaine or cocaine cues. Rats were then returned to the opertant chamber for the cue-induced cocaine-seeking test, where active lever depression in the original training context resulted in tone + light cue presentation. We found VTA specific or systemic isradipine administration robustly attenuated cocaine-seeking, without altering cocaine-taking nor natural reward seeking. Dopamine (DA) signaling in the nucleus accumbens (NAc) core is necessary and sufficient for cue-induced drug-seeking. Surprisingly in our study, isradipine enhanced tonic and phasic DA signaling in cocaine abstinent rats, with no change in sucrose abstinent nor naïve rats. Strikingly, isradipine's behavioral effects were dependent upon NAc core DA receptor activation. Together, our findings reveal a novel mechanism by which the FDA-approved drug, isradipine, could act to decrease cocaine relapse.

The effect of cocaine cue re‐exposure after prolonged cocaine self‐administration and forced abstinence on DNA methylation and mRNA expression in the rat dmPFC

Cocaine addiction is a chronic relapsing disorder whereby cocaine craving in response to drug related cues persists, and even intensifies through protracted drug‐free abstinence periods. Studies employing animal models observe cocaine‐seeking patterns that are similar to human addicts. Additionally, there are long term changes in neuron structure, receptor function, and neurotransmission associated with abstinence from cocaine in humans and animals. DNA methylation is an epigenetic modification to the DNA structure that mediates mRNA expression to confer different cell types, but has recently been implicated in learning and memory mechanisms. The long‐term control that DNA methylation has over gene expression in animals makes it a prime candidate for controlling gene expression over the course of abstinence in animals with previous drug experience. To test this, I trained rats to self‐administer either saline (1 h per day) or cocaine (6 h per day) for 15 days, then exposed them to 60 days of forced abstinence. After abstinence half of the rats were put back in self‐administration chambers and re‐exposed to cues for 2 h, while the other half were left in their home cages. After the cue manipulation, all rats were sacrificed, the dmPFC was dissected out, and DNA and mRNA were extracted. Methylated DNA was purified using methylated DNA immuno‐precipitation, and mRNA was reverse‐transcribed into cDNA; levels of each were then quantified via digital PCR procedures. Interestingly, I observed cue‐induced changes in DNA methylation within Homer2, Dlg4, Npas4, and Grin1 promoter regions. These context‐specific changes indicate that DNA methylation is much more dynamic than previously shown. Additionally, I saw changes in mRNA expression for Homer2, Dlg4, and Npas4 that appear to be highly reflective of the changes in DNA methylation. These results indicate re‐exposure to cocaine cues induce gene‐specific mRNA expression via rapid changes in DNA methylation.Support or Funding InformationThis research was supported by NIH grants DA‐027115 and DA‐027525 (TEK) and DA024038 (KKS), as well as funding from and the W.M. Keck Foundation (TEK).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Generalization of effects of environmental enrichment on seeking for different classes of drugs of abuse

BackgroundAddiction is a chronic disease characterized by persistent vulnerability to relapse during abstinence. In animal models of addiction, accumulating evidence suggests that exposure to environmental enrichment (EE) during periods of abstinence can have curative effects on addiction and reduce the risks of relapse. However, until present most studies have mainly focused on cocaine. In this study, we investigated whether EE could have beneficial effects on cue-induced seeking for several psychoactive drugs belonging to different pharmacological classes such as methamphetamine (METH), heroin (HER) and nicotine (NIC). MethodsAfter self-administration training of METH, HER and NIC, rats were housed in enriched (EE) or standard environments (SE) for 21–28 days of forced abstinence and then drug-seeking behavior was assessed in the absence of the drug. ResultsWe found that, compared to SE housing, exposure to EE reduced drug seeking behavior for all drugs tested. ConclusionsThese findings suggest that the anti-craving effects of EE are general for a wide variety of drugs and support the hypothesis that environmental stimulation may be a general intervention for attenuating relapse in humans.

Effects of prolonged abstinence from METH on the hippocampal BDNF levels, neuronal numbers and apoptosis in methamphetamine-sensitized rats

Methamphetamine (METH) use is associated with neuronal damage in various regions of brain, while effects of prolonged abstinence on METH-induced damage are not quite clear. This study evaluated serum and hippocampal BDNF levels, neuronal numbers and apoptosis in METH-sensitized and abstinent rats. Rats were sensitized to METH (2mg/kg, daily/18 days, s.c.). All rats were evaluated for neuron counting, the TUNEL test and serum and hippocampal BDNF levels after 30 days of forced abstinence from METH. The results showed that increased BDNF levels in the hippocampus and serum of METH-sensitized rats returned to control level after 30 days of abstinence. The number of neurons in the DG and CA1 of hippocampus and also, the total hippocampal perimeter and area in METH-sensitized rats were significantly lower than the saline rats. While, the number of neurons was not significantly increased in the hippocampus after prolonged abstinence from METH. Also, METH-sensitized rats showed a significant increase in TUNEL-positive cells, whereas METH-abstinent rats showed a slight but significant decrease in TUNEL-positive cells in the DG and CA3 of hippocampus. These results suggest that despite the reduction in BDNF levels, reducing the number of neurons, perimeter and area of the hippocampus were stable after abstinence. Thus, the degenerative effects of METH have been sustained even after prolonged abstinence in the hippocampus.

Systemic injection of the DAD1 antagonist SCH 23390 reduces saccharin seeking in rats

Conditioned cues can elicit drug- and sucrose-seeking behaviors that have been shown to depend on dopamine (DA) D1 receptors. If DAD1 receptors are also involved in seeking behavior in general, blocking these receptors should reduce seeking behavior for a non-caloric, non-drug of abuse reinforcer such as saccharin. Forty-six male Long-Evans rats lever pressed for 0.3% saccharin solution 1 h/day for 10 days. A lever response also activated a tone plus a white stimulus light. This compound stimulus lasted for 5 s. After 1 day of forced abstinence, rats received systemic (0, 1, or 10 μg/kg IP; n = 15–16 per group) injections of SCH 23390 15 min prior to extinction testing. Systemic SCH 23390 reduced saccharin seeking evidenced by a significant reduction in active lever responding and a significant reduction in the number of active lever-contingent deliveries of the tone + light cue following pretreatment with 10 μg/kg SCH 23390. The slope of responding across the Test session in this group was also significantly steeper, indicating that SCH 23390 may have reduced the persistence of saccharin seeking. The results indicate that DAD1 receptors are involved in saccharin seeking and generalize the previously demonstrated anti-seeking effects of DAD1 antagonism to a non-caloric, non-drug of abuse reinforcer.