Reinstatement Test Research Articles

Cortisol before extinction generalization alters its neural correlates during retrieval

While generalization of fear seems to be naturally acquired as frequently observed in fear-related disorders, extinction learning appears to be stimulus-specific. Thus, treatments aiming to generalize extinction learning comprise the chance to overcome stimulus-specificity and consequently reduce relapse. One suggested candidate is the timing-dependent administration of the stress hormone cortisol.In the present pre-registered, three-day fear conditioning study, we aimed to create a generalized extinction memory trace in 60 healthy men and women using multiple sizes of one conditioned stimulus (CS+G; generalized) during extinction training, whereas the other CS (CS+N; non-generalized) and the CS- were solely presented in their original sizes. Extinction training took place either after pharmacological administration of 20 mg cortisol or placebo. Following successful fear acquisition on day one, generalization effects during extinction training and retrieval were investigated in the comparison of CS+G and CS+N. Insula and dorsal anterior cingulate cortex (dACC) activation for CS+G as compared to CS+N extending to the second half of extinction training indicated prolonged fear processing during extinction training for the CS+G on day two. During retrieval on day three, an activation of the anterior hippocampus occurred for CS+N minus CS+G in the cortisol but not in the placebo group. Additionally, a more posterior hippocampal activation (compared to the other hippocampal activation) was observed for the contrast CS+G minus CS+N. In accordance with our hypotheses, amygdala and dACC responding during reinstatement test was reduced for the CS+G as compared to CS+N. However, cortisol did not modulate amygdala responding, but abolished the CS+G/CS+N differentiation in the dACC relative to placebo. Generalization and cortisol effects were not mirrored in skin conductance responses. In conclusion, extinction generalization processes appear to rely on prolonged fear processing still present in the second half of extinction training that in turn leads to reduced fear-related processing after reinstatement. Cortisol administration prior to extinction training, however, selectively reduced fear-related activation for standard extinction but did not further reduce fear-related activation for extinction generalization.

Can contingency rehearsal during the interval between a retrieval cue and extinction training change the effects of post-retrieval extinction?

Return of fear may be prevented by post-retrieval extinction (PRE), a procedure consisting of presenting a stimulus that was present during conditioning (retrieval cue) prior to extinction training. However, recent evidence suggests that there might be circumstances under which PRE is not effective to prevent the return of fear (boundary conditions), but some of these conditions remain unknown. We explored if rehearsing the CS, US or CS-US contingency during the interval between the retrieval cue and extinction training might change the effects of PRE. One day after differential fear conditioning, healthy human participants (n=83) underwent either standard extinction (control condition, n=31) or two different PRE procedures, one in which participants rehearsed the CS-US contingency during the interval between the retrieval cue and extinction (rehearsal condition, n=25), or another in which they underwent a verbal fluency task directing their attention away from the experimental contingencies during this interval (nonrehearsal condition, n=27). Return of fear in a reinstatement test was observed in both control and rehearsal conditions, whereas in the nonrehearsal condition there was a generalized increase in response to the CS+ and CS-. Differential response in the rehearsal condition had values slightly smaller than the control group with no significant differences from both control and nonrehearsal conditions. These results suggest that the overt behavior of participants during the interval between a retrieval cue and extinction training might change the effects of PRE in healthy human participants, but further manipulations of these variables are needed to confirm these findings.

Outcome-selective reinstatement is predominantly context-independent, and associated with c-Fos activation in the posterior dorsomedial striatum

Research from human and animal studies has found that after responding has been successfully reduced following treatment it can return upon exposure to certain contexts. An individual in recovery from alcohol use disorder, for example, might relapse to drinking upon visiting their favourite bar. However, most of these data have been derived from experiments involving a single (active) response, and the context-dependence of returned responding in situations involving choice between multiple actions and outcomes is less well-understood. We thus investigated how outcome-selective reinstatement – a procedure involving choice between two actions and outcomes – was affected by altering the physical context in rats. In Experiment 1, rats were trained over 6 days to press a left lever for one food outcome (pellets or sucrose) and a right lever for the other outcome. Then, rats received an extinction session in either the same context (A) as lever press training, or in a different context (B). Rats were tested immediately (5 min) after extinction in Context A or B such that there were four groups in total: AAA, ABB, ABA, and AAB. Reinstatement testing consisted of one food outcome being delivered ‘freely’ (i.e. unearned by lever pressing and unsignalled by cues) to the food magazine every 4 min in the following order: Sucrose, Pellet, Pellet, Sucrose. Selective reinstatement was considered intact if pellet delivery increased pressing selectively on the pellet lever, and sucrose delivery selectively increased pressing on the sucrose lever. This result (Reinstated > Nonreinstated) was observed for rats in group AAA and ABB, but not rats in groups ABA and AAB. Experiment 2 was conducted identically, except that rats received two extinction sessions over two days and tested one day later. This time, all groups demonstrated intact outcome-selective reinstatement regardless of context. Analysis of c-Fos expression in several brain regions revealed that only c-Fos expression in the posterior dorsomedial striatum (pDMS) was related to intact reinstatement performance. Overall, these results suggest that outcome-selective reinstatement is predominantly context-independent, and that intact reinstatement is related to neuronal activity in the pDMS.

Secure attachment priming protects against relapse of fear in Young adults

Previous studies have shown that activating the attachment system attenuates fear learning. This study aimed to explore whether attachment priming can also impact on fear extinction processes, which underpin the management of anxiety disorders. In this study, 81 participants underwent a standard fear conditioning and extinction protocol on day 1 and returned 24 h later for an extinction recall and reinstatement test. Half the participants were primed to imagine their closest attachment figure prior to undergoing extinction training, while the other half were instructed to imagine a positive situation. Fear-potentiated startle and subjective expectancies of shock were measured as the primary indicators of fear. Attachment priming led to less relapse during the reinstatement test at the physiological but not subjective levels. These findings have translational potential to imply that activating awareness of attachment figures might augment long-term safety memories acquired in existing treatments to reduce relapse of fear.

A Novel Assay Allowing Drug Self-Administration, Extinction, and Reinstatement Testing in Head-Restrained Mice.

Multiphoton microscopy is one of several new technologies providing unprecedented insight into the activity dynamics and function of neural circuits. Unfortunately, some of these technologies require experimentation in head-restrained animals, limiting the behavioral repertoire that can be integrated and studied. This issue is especially evident in drug addiction research, as no laboratories have coupled multiphoton microscopy with simultaneous intravenous drug self-administration, a behavioral paradigm that has predictive validity for treatment outcomes and abuse liability. Here, we describe a new experimental assay wherein head-restrained mice will press an active lever, but not inactive lever, for intravenous delivery of heroin or cocaine. Similar to freely moving animals, we find that lever pressing is suppressed through daily extinction training and subsequently reinstated through the presentation of relapse-provoking triggers (drug-associative cues, the drug itself, and stressors). Finally, we show that head-restrained mice will show similar patterns of behavior for oral delivery of a sucrose reward, a common control used for drug self-administration experiments. Overall, these data demonstrate the feasibility of combining drug self-administration experiments with technologies that require head-restraint, such as multiphoton imaging. The assay described could be replicated by interested labs with readily available materials to aid in identifying the neural underpinnings of substance use disorder.

Repeated oxytocin treatment during abstinence inhibited context- or restraint stress-induced reinstatement of methamphetamine-conditioned place preference and promoted adult hippocampal neurogenesis in mice

Propensity to relapse, even after long-term abstinence, is a crucial feature of methamphetamine (METH) abuse. We and other laboratories have reported that acute treatment of oxytocin (OXT), a hormone and neuropeptide, could inhibit reinstatement of METH seeking in animal studies. However, the effects of repeated OXT treatment on METH reinstatement as well as underlying mechanisms are still unclear. In the present study, the effects of repeated OXT treatment during abstinence on context- or restraint stress-induced reinstatement were investigated using the mice conditioned place preference (CPP) paradigm. After three intermittent injections of METH (2 mg/kg, i.p.) to induce CPP, mice received a daily bilateral intra-hippocampus injection of OXT (0.625, 1.25 or 2.5 μg) for 8 consecutive days before the context- or restraint stress-induced reinstatement test. Meanwhile, adult hippocampal neurogenesis (AHN) level was detected using immunostaining. To further clarify the role of AHN underlying OXT's effects on METH-CPP reinstatement, temozolomide (TMZ, 25 mg/kg, i.p.) was employed to deplete AHN prior to OXT treatment. The data showed that repeated OXT treatment (1.25 and 2.5 μg, intra-hippocampus) significantly inhibited both context- and restraint stress-induced METH-CPP reinstatement and concomitantly promoted AHN in a dose-dependent manner. Notably, TMZ pre-treatment markedly abolished all the above-mentioned effects of OXT, suggesting that AHN was closely involved in OXT's inhibition on reinstatement induced by both triggers. Taken together, the present study indicated that repeated OXT treatment during abstinence could inhibit both context- and restraint stress-induced METH-CPP reinstatement possibly by promoting AHN in mice, which provided a better understanding for OXT's beneficial effects on METH addiction.

Gender difference in retrieval-extinction of conditioned fear memory

<p id="C2">For nearly half a century, the memory reconsolidation theory, which believes that even memories that have been consolidated would be reactivated during the retrieval process and temporarily return to an unstable state, has been continuously developed. Based on this understanding, the researchers proposed the retrieval-extinction paradigm. That is, after successfully learning the pairing rule between a conditioned stimulus (CS) and unconditioned stimulus (US), the original memory was un-stabilized by presenting a single CS, and then traditional extinction training was performed. This paradigm has been proven in some studies to weaken the expression of non-adaptive fear memory effectively. <br/>However, other studies have reached different results, indicating that this paradigm failed to suppress the fear recovery. Current research mainly focused on the boundary conditions of memory conditions and retrieval conditions to explain the difference between studies, such as prediction error, acquisition time, the intensity of acquisition, and individual differences. Gender difference, as an essential variable of individual differences, is an influential factor in the study of boundary conditions. In the standard extinction paradigm, research has concluded that women easily acquire fear memory but that such fear memory is more difficult to extinct than that in men. However, it is relatively rare to explore gender differences in the research of the retrieval-extinction paradigm. Therefore, this study considered gender as a variable, using geometric figures as CS, wrist electric shock as the US, and skin conductance response as an indicator of fear response, to explore whether there is a gender difference in the fear extinction effect. <br/>The results showed no gender difference in the spontaneous fear recovery of the retrieval-extinction paradigm because all subjects successfully suppressed the spontaneous fear recovery. Nevertheless, there was a significant gender difference in the reinstatement test, in which men showed increased skin conductance responses (SCR), whereas women did not show any increased SCR. Besides, there were gender differences in the spontaneous fear recovery and reinstatement test of the standard extinction paradigm. After training by this paradigm, women showed increased SCR in spontaneously fear recovery and a tendency to fear generalization. In contrast, men showed suppressed spontaneous fear recovery but showed increased SCR in the reinstatement test. <br/>Our study proved a gender difference in the extinction effect of the retrieval-extinction paradigm (women showed the best extinction effect) and verified that the extinction effect of the retrieval-extinction paradigm was better than that of the standard extinction paradigm. From the results, we could demonstrate that the retrieval-extinction paradigm is an effective intervention method, especially for women. It will be beneficial to further verify evidence about the effectiveness of the retrieval-extinction paradigm and clarify its object and scope in clinical applications. Our study suggests that the retrieval-extinction paradigm in clinical transformation needs to provide more personalized treatment plans for male and female patients, thus improving clinical applicability and treatment effectiveness.

Ibogaine Blocks Cue- and Drug-Induced Reinstatement of Conditioned Place Preference to Ethanol in Male Mice

Ibogaine is a psychedelic extracted from the plant Tabernanthe iboga Baill. (Apocynaceae), natural from Africa, and has been proposed as a potential treatment for substance use disorders. In animal models, ibogaine reduces ethanol self-administration. However, no study to date has investigated the effects of ibogaine on ethanol-induced conditioned place preference (CPP). The present study aimed to investigate the effects of repeated treatment with ibogaine on the reinstatement of CPP to ethanol in male mice. The rewarding effects of ethanol (1.8 g/kg, i. p.) or ibogaine (10 or 30 mg/kg, p. o.) were investigated using the CPP model. Furthermore, we evaluated the effects of repeated treatment with ibogaine (10 or 30 mg/kg, p. o.) on the reinstatement of ethanol-induced CPP. Reinstatement was evaluated under two conditions: 1) during a priming injection re-exposure test in which animals received a priming injection of ethanol and had free access to the CPP apparatus; 2) during a drug-free test conducted 24 h after a context-paired re-exposure, in which subjects received an injection of ethanol and were confined to the compartment previously conditioned to ethanol. Our results show that ethanol, but not ibogaine, induced CPP in mice. Treatment with ibogaine after conditioning with ethanol blocked the reinstatement of ethanol-induced CPP, both during a drug priming reinstatement test and during a drug-free test conducted after re-exposure to ethanol in the ethanol-paired compartment. Our findings add to the literature suggesting that psychedelics, in particular ibogaine, may have therapeutic properties for the treatment of alcohol use disorder at doses that do not have rewarding effects per se.

Stimulation and transient inactivation of ventral tegmental area modify reinstatement of acquisition phase of morphine-induced conditioned place preference in male rats

For processing the development of psychological dependency, opioid reinforcement, and opiate-related associative reward, learning, and memory in the brain, the ventral tegmental area (VTA) is considered the key zone. As the responsible region for the morphine role in conditioned place preference (CPP), this area has an important role. So, the present research was conducted to investigate the effects of different intensities of electrical stimulation on VTA utilizing CPP, with two morphine doses. Reversible inactivation of VTA was performed via bilateral microinjection of Lidocaine into this area with two implanted separate cannulas. Our findings indicated that 5 mg/kg morphine-induced CPP was suppressed by 150 μA VTA electrical stimulation. The results also showed that bilateral Intra-VTA administration of Lidocaine significantly decreased the 5 mg/kg morphine-induced CPP acquisition phase in comparison with their respective sham group, which reversed in the reinstatement test. It should be concluded that these findings are important for the detection of mesolimbic nervous system ties and could help to find new ways to attenuate the rewarding action of morphine.

Treatment with dopamine \u03b2-hydroxylase (DBH) inhibitors prevents morphine use and relapse-like behavior in rats

BackgroundOpioid use disorders are serious contributors to the harms associated with the drug use. Unfortunately, therapeutic interventions for opioid addicts after detoxification have been limited and not sufficiently effective. Recently, several studies have led to promising results with disulfiram (DSF), a dopamine β-hydroxylase (DBH) inhibitor, showing that it is a potent agent against not only alcohol but also addiction to various drugs.Materials and methodsThis study was designed to examine whether DSF and nepicastat (NEP; another DBH inhibitor) modify morphine intake and reinstatement of seeking-behavior using the rat model of intravenous morphine self-administration. Additionally, we intended to estimate the effects of both inhibitors on the locomotor activity as well as on extracellular dopamine and its metabolite levels in the nucleus accumbens using microdialysis in naive rats.ResultsWe demonstrated that both DBH inhibitors reduced responding to morphine self-administration. Moreover, DSF and NEP administered acutely before reinstatement test sessions consistently attenuated the reinforcing effects of morphine and a morphine-associated conditioned cue. The observed effects for lower doses (6.25–25 mg/kg; ip) of both DBH inhibitors seem to be independent of locomotor activity reduction and dopamine level in the nucleus accumbens. Neither DSF nor NEP administered daily during morphine abstinence with extinction training sessions had any effect on active lever-responding and changed the reinstatement induced by morphine priming doses. Reinstatement of drug-seeking behavior induced by a conditioned cue previously associated with morphine delivery was attenuated following repeated administration of DSF or NEP during the abstinence period.ConclusionThese results seem to point to the significance of DBH inhibition as a potential pharmacotherapy against morphine use disorders.Graphic abstract

The effects of quercetin on nicotine-induced reward effects in mice

Tobacco smoking remains the primary cause of preventable mortality and morbidity in the world. The complexity of the nicotine dependency process included the withdrawal effect that triggers recurrence being the main problem. Quercetin, known as an antioxidant, binds free radicals and modulates endogenous antioxidants through Nrf2 activations is expected as a potential agent to reduce the risk of nicotine dependence. This research aims to evaluate quercetin's effects on reducing the risk of nicotine addiction. Conditioned Place Preference (CPP) with a biased design was used to evaluate nicotine's reward effects in male Balb/C mice. Preconditioning test was performed on day 1; conditioning test was done twice daily on day 2-4 by administering quercetin (i.p.) 50mg/kg along with nicotine (s.c.) 0.5mg/kg or Cigarette Smoke Extract (CSE) (s.c.) contained nicotine 0.5mg/kg; and postconditioning test was performed on day 5 continue with extinction test on day 6, 8, 10, 12, and reinstatement test on day 13. The duration spent in each compartment was recorded and analyzed. Nicotine 0.5mg/kg and CSE 0.5mg/kg significantly induced reward effects (p<0.05). There was no decrease of reward effect during the extinction-reinstatement stage of the postconditioning phase (p>0.05), while quercetin 50mg/kg both induced along with nicotine or CSE was able to inhibit the reward effect of nicotine (p>0.05). Quercetin reduced the risk of nicotine dependence and has a potential effect to use as a therapy for nicotine dependence, especially as a preventive agent.

Environmental enrichment reduces heroin seeking following incubation of craving in both male and female rats

BackgroundContemporary treatments for heroin use disorder demonstrate only limited efficacy when the goals are long term abstinence and prevention of relapse. We have demonstrated that environmental enrichment (EE) reduces cue-induced heroin reinstatement in male rats. The present study is an attempt to extend the “anti-relapse” effects of EE to female rats and to periods where incubation of craving is hypothesized to occur. MethodsThis experiment implemented a 3-phase procedure. In Phase 1, male and female rats were trained to self-administer heroin for 15 days. Phase 2 consisted of a 3- or 15-day forced abstinence (FA) period. In Phase 3 half of the rats were placed into EE and the other half in non-EE housing and subsequently tested for responding in extinction (no heroin or cues) for 15 days followed by a cue-induced reinstatement test. ResultsWe found that rats in the 15 days FA condition showed significantly enhanced drug seeking during extinction, irrespective of sex. We also found that EE significantly reduced this effect. During reinstatement, EE significantly reduced drug seeking in male and female rats and in both 3- and 15-day FA groups. ConclusionsEE, with or without prolonged FA, effectively reduced heroin seeking in male and female rats. These findings indicate that EE can reduce drug-seeking in males and females and when putative incubation of craving (i.e., prolonged abstinence period) has occurred and suggest that it may aid in the development of future long-term behavioral treatments for individuals at risk for heroin relapse.

Cell Adhesion Factors in the Orbitofrontal Cortex Control Cue-Induced Reinstatement of Cocaine Seeking and Amygdala-Dependent Goal Seeking.

Repeated cocaine exposure causes dendritic spine loss in the orbitofrontal cortex, which might contribute to poor orbitofrontal cortical function following drug exposure. One challenge, however, has been verifying links between neuronal structural plasticity and behavior, if any. Here we report that cocaine self-administration triggers the loss of dendritic spines on excitatory neurons in the orbitofrontal cortex of male and female mice (as has been reported in rats). To understand functional consequences, we locally ablated neuronal β1-integrins, cell adhesion receptors that adhere cells to the extracellular matrix and thus support dendritic spine stability. Degradation of β1-integrin tone: (1) caused dendritic spine loss, (2) exaggerated cocaine-seeking responses in a cue-induced reinstatement test, and (3) impaired the ability of mice to integrate new learning into familiar routines, a key function of the orbitofrontal cortex. Stimulating Abl-related gene kinase, overexpressing Proline-rich tyrosine kinase, and inhibiting Rho-associated coiled-coil containing kinase corrected response strategies, uncovering a β1-integrin-mediated signaling axis that controls orbitofrontal cortical function. Finally, use of a combinatorial gene silencing/chemogenetic strategy revealed that β1-integrins support the ability of mice to integrate new information into established behaviors by sustaining orbitofrontal cortical connections with the basolateral amygdala.SIGNIFICANCE STATEMENT Cocaine degenerates dendritic spines in the orbitofrontal cortex, a region of the brain involved in interlacing new information into established behaviors. One challenge has been verifying links between cellular structural stability and behavior, if any. In this second of two related investigations, we study integrin family receptors, which adhere cells to the extracellular matrix and thereby stabilize dendritic spines (see also DePoy et al., 2019). We reveal that β1-integrins in the orbitofrontal cortex control food- and cocaine-seeking behaviors. For instance, β1-integrin loss amplifies cocaine-seeking behavior and impairs the ability of mice to integrate new learning into familiar routines. We identify likely intracellular signaling partners by which β1-integrins support orbitofrontal cortical function and connectivity with the basolateral amygdala.

Neural adaptation of cingulate and insular activity during delayed fear extinction: A replicable pattern across assessment sites and repeated measurements

Adapting threat-related memories towards changing environments is a fundamental ability of organisms. One central process of fear reduction is suggested to be extinction learning, experimentally modeled by extinction training that is repeated exposure to a previously conditioned stimulus (CS) without providing the expected negative consequence (unconditioned stimulus, US). Although extinction training is well investigated, evidence regarding process-related changes in neural activation over time is still missing. Using optimized delayed extinction training in a multicentric trial we tested whether: 1) extinction training elicited decreasing CS-specific neural activation and subjective ratings, 2) extinguished conditioned fear would return after presentation of the US (reinstatement), and 3) results are comparable across different assessment sites and repeated measures. We included 100 healthy subjects (measured twice, 13-week-interval) from six sites. 24 h after fear acquisition training, extinction training, including a reinstatement test, was applied during fMRI. Alongside, participants had to rate subjective US-expectancy, arousal and valence. In the course of the extinction training, we found decreasing neural activation in the insula and cingulate cortex as well as decreasing US-expectancy, arousal and negative valence towards CS+. Re-exposure to the US after extinction training was associated with a temporary increase in neural activation in the anterior cingulate cortex (exploratory analysis) and changes in US-expectancy and arousal ratings. While ICCs-values were low, findings from small groups suggest highly consistent effects across time-points and sites. Therefore, this delayed extinction fMRI-paradigm provides a solid basis for the investigation of differences in neural fear-related mechanisms as a function of anxiety-pathology and exposure-based treatment.

MC-100093, a Novel β-Lactam Glutamate Transporter-1 Enhancer Devoid of Antimicrobial Properties, Attenuates Cocaine Relapse in Rats.

Cocaine use disorder currently lacks Food and Drug Administration-approved treatments. In rodents, the glutamate transporter-1 (GLT-1) is downregulated in the nucleus accumbens after cocaine self-administration, and increasing the expression and function of GLT-1 reduces the reinstatement of cocaine seeking. The β-lactam antibiotic ceftriaxone upregulates GLT-1 and attenuates cue- and cocaine-induced cocaine seeking without affecting motivation for natural rewards. Although ceftriaxone shows promise for treating cocaine use disorder, it possesses characteristics that limit successful translation from bench to bedside, including poor brain penetration, a lack of oral bioavailability, and a risk of bacterial resistance when used chronically. Thus, we aimed to develop novel molecules that retained the GLT-1-enhancing effects of ceftriaxone but displayed superior drug-like properties. Here, we describe a new monocyclic β-lactam, MC-100093, as a potent upregulator of GLT-1 that is orally bioavailable and devoid of antimicrobial properties. MC-100093 was synthesized and tested in vitro and in vivo to determine physiochemical, pharmacokinetic, and pharmacodynamic properties. Next, adult male rats underwent cocaine self-administration and extinction training. During extinction training, rats received one of four doses of MC-100093 for 6-8 days prior to a single cue-primed reinstatement test. Separate cohorts of rats were used to assess nucleus accumbens GLT-1 expression and MC-100093 effects on sucrose self-administration. We found that 50 mg/kg MC-100093 attenuated cue-primed reinstatement of cocaine seeking while upregulating GLT-1 expression in the nucleus accumbens core. This dose did not produce sedation, nor did it decrease sucrose consumption or body weight. Thus, MC-100093 represents a potential treatment to reduce cocaine relapse. SIGNIFICANCE STATEMENT: Increasing GLT-1 activity reliably reduces drug-seeking across classes of drugs; however, existing GLT1-enhancers have side effects and lack oral bioavailability. To address this issue, novel GLT-1 enhancers were synthesized, and the compound with the most favorable pharmacokinetic and pharmacodynamic properties, MC-100093, was selected for further testing. MC-100093 attenuated cued cocaine seeking without reducing food seeking or locomotion and upregulated GLT-1 expression in the nucleus accumbens.

Escalation and reinstatement of fentanyl self-administration in male and female rats.

Escalation of drug intake and craving are two DSM-5 hallmark symptoms of opioid use disorder (OUD). This study determined if escalation of intake as modeled by long access (LgA) self-administration (SA) and craving measured by reinstatement are related. Adult male and female Sprague-Dawley rats were trained to self-administer fentanyl across 7 daily 1-h short access (ShA) sessions, followed by 21 SA sessions of either 1- or 6-h duration (ShA or LgA). Following 14 1-h extinction sessions, Experiment 1 assessed reinstatement induced by either fentanyl (10 or 30µg/kg) or yohimbine (1 or 2mg/kg), and Experiment 2 assessed reinstatement induced by a drug-associated cue light. Females acquired fentanyl SA faster than males. When shifted to LgA sessions, LgA rats escalated fentanyl intake, but ShA rats did not; no reliable sex difference in the rate of escalation was observed. In extinction, compared to ShA rats, LgA rats initially responded less and showed less decay of responding across sessions. A priming injection of fentanyl induced reinstatement, with LgA rats reinstating more than ShA rats at the 30µg/kg dose. Yohimbine (1mg/kg) also induced reinstatement, but there was no effect of access group or sex. With cue-induced reinstatement, LgA females reinstated less than LgA males and ShA females. Among the different reinstatement tests assessed, escalation of fentanyl SA increased only drug-primed reinstatement, suggesting a limited relationship between escalation of drug intake and craving (reinstatement) for OUD.

Estradiol Regulation of the Prelimbic Cortex and the Reinstatement of Cocaine Seeking in Female Rats.

Relapse susceptibility in women with substance use disorders (SUDs) has been linked to the estrogen, 17β-estradiol (E2). Our previous findings in female rats suggest that the influence of E2 on cocaine seeking can be localized to the prelimbic prefrontal cortex (PrL-PFC). Here, we investigated the receptor mechanisms through which E2 regulates the reinstatement of extinguished cocaine seeking. Sexually mature female rats underwent intravenous cocaine self-administration (0.5 mg/inf; 14 × 2 h daily) and extinction, and then were ovariectomized before reinstatement testing. E2 (10 µg/kg, i.p.) alone did not reinstate cocaine seeking, but it potentiated reinstatement when combined with an otherwise subthreshold priming dose of cocaine. A similar effect was observed following intra-PrL-PFC microinfusions of E2 and by systemic or intra-PrL-PFC administration of the estrogen receptor (ER)β agonist, DPN, but not agonists at ERα or the G-protein-coupled ER1 (GPER1). By contrast, E2-potentiated reinstatement was prevented by intra-PrL-PFC microinfusions of the ERβ antagonist, MPP, or the GPER1 antagonist, G15, but not an ERα antagonist. Whole-cell recordings in PrL-PFC layer (L)5/6 pyramidal neurons revealed that E2 decreases the frequency, but not amplitude, of GABAA-dependent miniature IPSCs (mIPSC). As was the case with E2-potentiated reinstatement, E2 reductions in mIPSC frequency were prevented by ERβ and GPER1, but not ERα, antagonists and mimicked by ERβ, but not GPER1, agonists. Altogether, the findings suggest that E2 activates ERβ and GPER1 in the PrL-PFC to attenuate the GABA-mediated constraint of key outputs that mediate cocaine seeking.

Augmentation of fear extinction by theta-burst transcranial magnetic stimulation of the prefrontal cortex in humans.

BackgroundFear extinction alone does not erase the original fear memory. Interventions that enhance extinction can be beneficial for the treatment of fear-related disorders. Repetitive transcranial magnetic stimulation has been shown to improve memory performance. The present study examined the effects of intermittent theta-burst stimulation (iTBS) on fear extinction and the return of fear memory in humans.MethodsNinety-one young healthy volunteers underwent 3 experiments using a randomized controlled experimental design. Participants first acquired fear conditioning, after which they received 30 Hz iTBS before and after extinction training. The iTBS was applied to 1 of 2 targets: the left dorsolateral prefrontal cortex (dlPFC) and the vertex (control). Fear responses were measured 24 hours later and 1 month later.ResultsDuring the spontaneous recovery and reinstatement tests, iTBS of the left dlPFC before and after extinction significantly reduced fear response, whereas iTBS of the vertex had no effect on fear memory performance. This combined approach had a relatively long-lasting effect (i.e., at least 1 month).LimitationsWe did not explore the effect of iTBS of the dlPFC on the expression of fear without extinction training. The neural mechanisms of iTBS with fear extinction to inhibit the fear response are unclear. Our results are preliminary and should be interpreted with caution.ConclusionThe present results showed that 30 Hz iTBS of the left dlPFC enhanced retention of fear extinction. Our study introduces a new intervention for fear memory and suggests that the left dlPFC may be a treatment target for fear-related disorders.

Enhancement of cue-induced reinstatement of alcohol seeking by acute total sleep restriction in male Wistar rats

Clinical studies suggest that sleep impairment is a barrier to successful treatment in alcohol use disorder (AUD) patients, with sleep disruption associated with relapse to alcohol taking. To date, no preclinical study has evaluated the relationship between impaired sleep and alcohol relapse. In the present study, we used a self-administration model to investigate the effects of sleep restriction on reinstatement induced by alcohol-paired environmental cues. Using a sucrose fading protocol, male Wistar rats (N = 8) were trained to self-administer alcohol under a fixed-ratio 2 schedule of alcohol delivery such that completion of every second response resulted in the delivery of the alcohol solution and activation of the alcohol-paired cue light. Once self-administration was stable, behavior was extinguished by omitting delivery of the alcohol solution and the alcohol-paired cues. When responding reached low, stable levels, alcohol seeking was induced by re-presentation of the alcohol-paired cues but with no alcohol solution available for self-administration. To evaluate the effects of sleep restriction on cue-induced alcohol seeking, reinstatement tests were conducted after 6-h of total (slow wave + rapid eye movement [REM]) sleep restriction using the gentle handling method or after 6-h of REM sleep-only restriction using the flower pot method. Relevant control conditions also were evaluated. The results showed that acute restriction of total sleep, but not REM sleep primarily, significantly augmented cue-induced reinstatement of alcohol seeking. This increase was specific to total sleep restriction conditions and cannot be attributed to differences in alcohol intake, responding, or days to extinction. Our findings imply that acute slow wave sleep restriction is necessary and/or sufficient for the enhancement of cue-induced alcohol seeking and, further, suggest that decreased slow wave sleep in AUD patients places individuals at a unique risk for relapse.

Deep Brain Stimulation of the Lateral Hypothalamus Facilitates Extinction and Prevents Reinstatement of Morphine Place Preference in Rats

ObjectivesWe have previously shown that high-frequency (HF) deep brain stimulation (DBS) of the lateral hypothalamus (LH) during the acquisition phase of morphine-induced conditioned place preference (CPP) abolished the development of morphine reward. In the present study, we investigated the effect of DBS in the LH during the extinction phase of morphine CPP. Materials and MethodsRats were implanted with electrodes in the LH and went through conditioning trials for morphine CPP (40 min each, for three days), followed by extinction trials (20 min, for nine days). DBS-like stimulation (square pulses at 13 or 130 Hz, 200 μA, 100 μsec) was applied during the extinction trials. ResultsRats that received HF-DBS (130 Hz) accomplished extinction of morphine place preference by day 5 of the phase, whereas those in sham-stimulation or low-frequency-DBS (LF-DBS, 13 Hz) groups reached the criterion for extinction at day 8. One day later, rats received a priming injection of morphine (2 mg/kg) to reinstate the extinguished preference. While rats in the sham-DBS and LF-DBS relapsed into the state of preferring morphine-associated context, those in the HF-DBS group did not show such preference. Rats were then proceeded into an additional phase of extinction training (20 min, once daily, three to five days) with DBS, followed by restraint stress-induced reinstatement test. Again, sham-DBS and LF-DBS had no effect on relapse to the morphine place preferring state, but HF-DBS completely prevented the relapse. ConclusionHF-DBS facilitated extinction of morphine place preference and disrupted drug priming- and stress-induced renewal of morphine place preference.